miRNA studies in in vitro and in vivo activated hepatic stellate cells

Abstract

AIM: To understand which and how different miRNAs are implicated in the process of hepatic stellate cell (HSC) activation.

METHODS: We used microarrays to examine the differential expression of miRNAs during in vitro activation of primary HSCs (pHSCs). The transcriptome changes upon stable transfection of rno-miR-146a into an HSC cell line were studied using cDNA microarrays. Selected differentially regulated miRNAs were investigated by quantitative real-time polymerase chain reaction during in vivo HSC activation. The effect of miRNA mimics and inhibitor on the in vitro activation of pHSCs was also evaluated.

RESULTS: We found that 16 miRNAs were upregulated and 26 were downregulated significantly in 10-d in vitro activated pHSCs in comparison to quiescent pHSCs. Overexpression of rno-miR-146a was characterized by marked upregulation of tissue inhibitor of metalloproteinase-3, which is implicated in the regulation of tumor necrosis factor-α activity. Differences in the regulation of selected miRNAs were observed comparing in vitro and in vivo HSC activation. Treatment with miR-26a and 29a mimics, and miR-214 inhibitor during in vitro activation of pHSCs induced significant downregulation of collagen type I transcription.

CONCLUSION: Our results emphasize the different regulation of miRNAs in in vitro and in vivo activated pHSCs. We also showed that miR-26a, 29a and 214 are involved in the regulation of collagen type I mRNA.

Key Words: Hepatic stellate cells; miRNA; miR-146a; Nuclear factor-κB

INTRODUCTION

Liver fibrosis, characterized by an overproduction of extracellular matrix (ECM), is a common outcome of different chronic liver diseases[1]. Hepatic stellate cells (HSCs) are one of the major cell types responsible for the production of ECM molecules like collagens, laminin, proteoglycans and fibronectin[2]. The production of different ECM molecules is increased upon transdifferentiation (activation) of HSCs from a quiescent to an activated myofibroblast-like state[3,4]. Consequently, the regulation of the complex process of HSC activation is of great interest to the research community. Understanding this process should lead to the discovery of therapeutic strategies for liver fibrosis. Due to the complexity of the activation of HSCs, the number of regulatory steps is expected to be overwhelming[5], and requires addressing many different targets at the same time, either with different compounds or with one compound that is able to work on many different targets.

miRNAs are small approximately 23-nt non-coding RNAs, which are able to regulate hundreds of different proteins. The versatility of miRNAs is attributed to the imperfect binding (seed region) to the 3’-UTR of mRNAs, which results in, contrary to siRNA, many binding partners. The regulation by miRNAs is also different to siRNAs because it leads to a translational repression and/or mRNA destabilization[6,7]. That miRNAs fulfill regulatory functions has been established by their involvement in many different processes and diseases[8,9]. Therefore, it is tempting to use these molecules in order to treat liver fibrosis; a condition that is caused by a deregulation of biological processes. To succeed in this attempt, we need to identify the miRNAs, which are differentially regulated in the normal and diseased liver, and more specifically in the HSCs; one cell type that is responsible for the fibrotic process.

The purpose of this study was to identify differentially regulated miRNAs in in vitro activated HSCs, in order to study them in an in vivo animal model, and finally, to determine their role in the activation process.

MATERIALS AND METHODS

Isolation of rat primary HSCs and cell culture conditions

Wistar rats were used to isolate primary HSCs (pHSCs) according to a published pronase/collagenase in situ perfusion protocol[10]. The isolation protocol was approved by the Institutional Animal Care and Use Committee under #080389. For in vitro activation, the cells were seeded into 75-cm² culture flasks and harvested after 3, 5, 7 or 10 d. Primary cells and the HSC-2 cell line were cultured in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a 5% CO₂ humidified incubator.

HSC-2 is a spontaneous immortalized cell line derived from the pHSCs of a male Wistar rat. The primary cells were passaged several times before clonal selection by limiting dilution[11].

The purity of pHSCs from rats on normal and choline-deficient ethionine supplemented (CDE) diet was assessed using vitamin A autofluorescence or real-time polymerase chain reaction (PCR), respectively (Figure 1A). All cell culture reagents were purchased from Invitrogen (Carlsbad, CA, USA).

Figure 1 Primary hepatic stellate cells and over-expression of miR-146a in hepatic stellate cell-2 cell line. A: Bright-field image of 1 d cultivated primary hepatic stellate cells and the corresponding vitamin A autofluorescence image are shown. Scale bar represents 100 μm. Real-time polymerase chain reaction (PCR) for in vivo activated hepatic stellate cells from rats on normal (n = 2) and choline-deficient ethionine supplemented (CDE) diet (n = 4). The mean ± SE for each diet model is shown; B: A representative image for the over-expression of miR-146a as visualized by the reporter GFP is shown. Real-time PCR for three independent clones confirmed the expression of miR-146a. The data represent the mean ± SE of triplicate reactions. SMAA: Smooth muscle α-actin.

In vivo activation of rat HSCs

Six- to eight-week-old male Wistar rats were fed the CDE diet (CDE model) (MP Biomedicals, Solon, OH, USA, #0296021410) for 4 wk (Figure 2). Livers were isolated, perfused with PBS and fixed in neutralized formalin (paraffin embedding) or in vivo activated pHSCs were isolated.

Figure 2 Histological and immunohistochemical analysis of livers from rats receiving choline-deficient ethionine supplemented diet for 4 wk. A: HE staining shows the structural changes between control and choline-deficient ethionine supplemented (CDE) diet livers. No severe steatosis is observed; B: The Sirius Red staining depicts the deposition of collagen around the portal area and the whole liver; C: The increase in smooth muscle α-actin (SMAA) staining reflects the increasing number of myofibroblasts seen in patches throughout the liver. Scale bar represents 200 μm; D: The Western blotting data confirm the increase in SMAA and ColI.

Isolation of miRNA for microarray and analysis

miRNA was extracted from quiescent (freshly isolated) and 10-d in vitro-activated pHSCs using the PureLink purification kit (K1570-01; Invitrogen). The miRNA microarray (NCode Multi-Species miRNA microarray V2) was performed according to the manufacturer’s manual (MIRLS-20; Invitrogen). For each experiment, a dye swap was performed. The arrays were scanned using a GenePix 4200AL array scanner. The raw datasets were deposited under #GSE19463 at the Gene Expression Omnibus (GEO) repository[12]. For two-color miRNA arrays, averaging of dye-swapped arrays was performed to minimize the dye effects prior to normalization using the Cross-Correlation method[13]. The targets of differentially regulated miRNAs (Table 1) were predicted by three different methods, TargetScan 5.1[14], mirBASE target[15], and miRNA Viewer[16] using default parameters. Targets predicted by at least two tools were selected and grouped into upregulated and downregulated miRNAs, respectively. These two groups of targets were subjected to pathway analysis using Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, CA, USA). A ratio was calculated whereby the number of predicted targets in a given pathway was divided by the total number of molecules in that pathway. The Fisher’s exact test was used by the software to calculate a P value. This P value represented the probability that the association between the predicted targets and the pathway could not be explained by chance alone. The P value cutoff was set at P≤ 0.001. The x axis was the negative logarithm of P value with a base of 10 (-log₁₀P value).

Table 1 Differentially regulated miRNAs as identified by miRNA microarray.

miRNA name	Fold change	P value
Upregulated compared to day 0
rno-let-7b	4.70	0.0242
rno-let-7c	3.75	0.0236
rno-let-7e	2.77	0.0340
rno-miR-125b	11.98	0.0113
rno-miR-132	1.97	0.0184
rno-miR-143	17.05	0.0014
rno-miR-145	2.29	0.0483
rno-miR-152	3.01	0.0255
rno-miR-199a	3.46	0.0415
rno-miR-21	5.73	0.0142
rno-miR-210	2.34	0.0186
rno-miR-214	18.44	0.0011
rno-miR-22	3.11	0.0392
rno-miR-221	10.09	0.0007
rno-miR-222	2.67	0.0317
rno-miR-31	8.91	0.0013
Downregulated compared to day 0
rno-let-7f	-2.17	0.0327
rno-miR-10a	-3.53	0.0417
rno-miR-122a	-349.63	0.00002
rno-miR-125a	-2.36	0.0474
rno-miR-126	-170.32	0.0003
rno-miR-146a	-16.83	0.0352
rno-miR-150	-9.81	0.0325
rno-miR-151*	-3.72	0.0345
rno-miR-161	-4.38	0.0366
rno-miR-181a1	-4.50	0.0346
rno-miR-192	-6.08	0.0206
rno-miR-194	-6.08	0.0168
rno-miR-195	-14.50	0.0130
rno-miR-207	-1.93	0.0449
rno-miR-26a	-5.17	0.0163
rno-miR-26b	-4.81	0.0300
rno-miR-296	-1.93	0.0292
rno-miR-29a1	-2.38	0.0644
rno-miR-30a-5p	-5.35	0.0327
rno-miR-30b	-10.51	0.0075
rno-miR-30c	-9.69	0.0138
rno-miR-30d	-8.68	0.0093
rno-miR-335	-3.74	0.0500
rno-miR-422b1	-8.49	0.0455
rno-miR-483	-2.49	0.0451
rno-miR-99a	-2.97	0.0383

¹Data from two experiments.

Real-time PCR

The verification of the microarray data and subsequent miRNA assessments were performed for let-7b, let-7c, miR-16, 26a, 29a, 31, 125b, 143, 146a, 150 and 214 by using the respective Taqman MicroRNA assays (P/N 4427975, Applied Biosystems, Foster City, CA, USA). The U6 snRNA assay (ID 001973) served as a normalization control. Total RNA was isolated using the NucleoSpin RNAII kit (Macherey-Nagel, Germany). Total RNA and miRNA were isolated using the same kit but with a small modification. Briefly, the cell lysate was adjusted to contain 35% ethanol and passed through the RNAII column to bind the total RNA. The ethanol concentration of the flow through was then adjusted to > 70% and passed through the same column in order to bind the miRNA. The Cells-to-Ct kit (Invitrogen, P/N 4391848) was used for some experiments to quantify the miRNA expression with the respective miRNA assays. The reverse transcription and real-time PCR were performed according to the assays protocol using the ABI 7500 Fast Real Time PCR System (Applied Biosystems). Taqman assays used were smooth muscle α-actin (SMAA) (Rn01759928_g1), Col1a1 (Rn01463849_g1), interleukin (IL)-6 (Rn00561420_m1), cyclooxygenase-2 (Cox-2) (Rn00568225_m1), RelA (Rn01502266_m1), CD31 (Rn01467259_m1), Albumin (Rn01413833_m1), CD68 (Rn01495643_g1) and tissue inhibitor of metalloproteinase (TIMP)-3 (Rn00441826_m1).

Nuclear factor-κB siRNA transfection

HSC-2 cells were seeded at a density of 10⁶ per 100 mm cell culture dish and incubated at 37°C. The siRNA was mixed at a final concentration of 10 nmol/L with 1 mL DMEM without serum and 120 μL HiPerfect transfection reagent (Qiagen, Germany) and incubated for 10 min. The mixture was added drop-wise to the cells and incubated for 48 h. For the mock control, only the HiPerfect reagent was used. The ON-Targetplus nuclear factor (NF)-κB siRNAs used were J-080033-11 and J-080033-12 (Dharmacon, Lafayette, CO, USA). These conditions were tested for transfection efficiency using FITC-labeled siRNA and FACS analysis.

Overexpression of miR-146a in an HSC cell line

The vector was constructed by amplification of a 487-bp fragment containing the rno-miR-146a from rat genomic DNA using the following primer pair: sense 5'-AAGCTTGCCACCAGTCCCATCCTTCACC-3' (HindIII), anti-sense 5'-GGATCCTTCCTCTGTGCTGGGATTACAGGGTG-3' (BamHI). After sub-cloning, the rno-miR-146a was excised using BamHI/EcoRV and cloned into pcDNA6.2/GW EmGFP-miR (Invitrogen). The HSC-2 cells were stably transfected with the construct using Lipofectamine 2000 (Invitrogen) and selected in cell culture medium supplemented with 10 μg/mL Blasticidin. The clonal selection was achieved using FACS.

Gene expression array and analysis

Total RNA from HSC-2 cells overexpressing miR-146a and control cells (two different passages) were used to study the transcriptome changes using the GeneChip Rat Genome 230 2.0 (Affymetrix, USA). The preparation of the samples was performed according to the technical manual P/N 702232 Rev. 3 (Affymetrix) using one-cycle cDNA and target labeling. The chips were scanned using a Genechip Scanner 3000 (Affymetrix). The raw datasets were deposited under #GSE19463 at the GEO repository[12].

The microarray probe set data was summarized using the Robust Multi-Array Average expression measure method, and pre-processed to correct unreliable (small) intensities for each array. The pre-processed data were then normalized using the Cross-Correlation method[13]. For each gene, a fold change value was calculated for samples vs control. Differentially expressed genes (DEGs) were selected based on the criterion of fold change > 2. The P values of DEGs were obtained using one-tailed Student’s t test. Pathway analysis was carried out on the DEGs using Ingenuity Pathway Analysis (Ingenuity Systems).

Transfection of miRNA mimics and hairpin-inhibitor

Cells were seeded at 20 000 per well in 48-well plates 24 h prior to transfection. The miRNA mimics or hairpin-inhibitor were added at the required final concentration (miR-26a, 146a, controls and quadruple transfection: 50 nmol/L each; miR-29a and 214: 200 nmol/L each) to 750 μL DMEM without serum, followed by 10 μL HiPerfect transfection reagent. The mixture was incubated for 10 min. The medium from each well was aspirated and replaced by 250 μL of the mixture. The transfection was performed in triplicate. Controls were either HiPerfect reagent only (mock) or control miRNAs for the mimic and/or inhibitor.

SDS-PAGE and Western blotting

Cells were lysed in ProteoJet lysis buffer (#K0301; Fermentas, Glen Burnie, MD, USA) and the protein concentration was estimated using the BCA method (Thermo Scientific, USA). The samples were separated in 4%-12% Bis-Tris NuPage gels (Invitrogen) and transferred onto nitrocellulose membranes. The membranes were blocked for 1 h at room temperature using 5% non-fat milk in TBS-Tween (TBS-T). The primary antibodies were applied in the following dilutions: interleukin receptor associated kinase 1 (IRAK1) (sc-7883; Santa Cruz Biotechnology, Santa Cruz, CA, USA,) 1:400; tumor necrosis factor receptor associated factor 6 (TRAF6) (sc-7221; Santa Cruz Biotechnology) 1:400; IκBα (#4814; Cell Signaling, Danvers, MA, USA) 1:1000; pIκBα (#2859; Cell Signaling) 1:750; Cox-2 (sc-1747; Santa Cruz Biotechnology) 1:5000; and β-actin (ab-8227; Abcam, Cambridge, UK) 1:5000. After three washes in TBS-T, the appropriate HRP-conjugated secondary antibody was given at 1:2000 dilution in blocking solution. After three washes in TBS-T, the membrane was developed using the chemiluminiscence substrate (Millipore, Billerica, MA, USA). Primary and secondary antibodies were incubated at 4°C overnight and 1 h at room temperature, respectively.

Electrophoretic mobility shift assay

Nuclear protein extract from rno-miR-146a-overexpressing clones was obtained using the NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Scientific). The electrophoretic mobility shift assay (EMSA) was performed using the NF-κB(I) EMSA kit according to its protocol (AY1030; Panomics, USA), as described previously[17]. The samples were separated in a 6% non-denaturing polyacrylamide gel (Invitrogen) and transferred to a nylon membrane.

Immunohistochemistry and staining of liver sections

Slides were de-paraffinized and the antigen retrieved by heat exposure in the Target Retrieval Solution pH 9 (S2367; Dako, Glostrup, DK) using a 2100-Retriever retrieval steamer for 45 min. The endogenous peroxidase was blocked with 3% H₂O₂ in methanol for 15 min. Protein was blocked in 10% normal goat serum in PBS for 20 min. The slides were incubated with mouse anti-human SMAA (M0851; Dako) at 1:100 dilution for 1 h, washed and incubated with an anti-mouse HRP-conjugated antibody (K4001; Dako) for 30 min, and developed with DAB (K3468; Dako). All incubations were carried out at room temperature. Nuclei were counter stained with hematoxylin. Hematoxylin and eosin and Sirius Red staining was performed according to standard protocols on paraffin sections. Bright-field images were taken with the LEICA RMB-DM epifluorescence microscope (LEICA, Germany).

Statistics

All quantitative data were presented as mean ± SE. Experimental data were analyzed using the two-tailed Student’s t test assuming equal variances. P≤ 0.05 was considered significant. The time-dependent changes during in vitro HSC activation were tested for significance at the 0.05 level using one-way ANOVA and Bonferroni’s post-hoc test. The array data were normalized and analyzed as described in the respective sections above.

RESULTS

Identification of differentially regulated miRNAs in in vitro activated pHSCs and comparison to in vivo activated pHSCs

In 10-d in vitro activated pHSCs, 16 miRNAs were upregulated and 26 were downregulated significantly in comparison to quiescent pHSCs (Table 1). We included miR-29a, although the P value was above the threshold of 0.05, for further studies because of its predicted targets, which consisted of a number of collagens. The microarray data were confirmed for a number of chosen miRNAs (let-7b, 7c, miR-16, 26a, 29a, 31, 125b, 143, 146a, 150 and 214) using real-time PCR in three additional experiments (Figure 3A). Using isolated in vivo activated pHSCs from rats on CDE diet, we found that only miRNAs let-7b, 7c, miR-31, 143 and 214 showed the same regulation as observed for the in vitro activated pHSCs (Figure 3B).

Figure 3 Verification of microarray data by real-time polymerase chain reaction of 11 differentially regulated miRNAs and their regulation upon in vivo activation of hepatic stellate cells. A: The graph depicts the changes in the miRNA expression of 11 miRNAs detected by real-time polymerase chain reaction, comparing quiescent with 10-d culture activated primary hepatic stellate cells (pHSCs). The data represent the mean ± SE of three independent experiments (^aP≤ 0.05, ^bP≤ 0.005); B: The graph illustrates the relative expression levels of miRNAs in isolated in vivo activated pHSCs (n = 4, choline-deficient ethionine supplemented diet) compared to normal diet (n = 2) (^aP≤ 0.05, ^bP≤ 0.005).

Pathway analysis for differentially regulated miRNAs in in vitro activated pHSCs

We performed a pathway analysis using the predicted targets of the differentially regulated miRNAs. The enrichment of genes in single pathways is shown as the -log of the P value (P≤ 0.001). Signaling pathways which were affected include endothelin-1, cyclin-dependent kinase 5, extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), p70^S6K, chemokine, bone morphogenetic protein (BMP) and IL-6 for the upregulated miRNAs, as well as ERK/MAPK, production of NO and reactive oxygen species (ROS), AMP activated protein kinase (AMPK), transforming growth factor (TGF)-β, integrin, cAMP-mediated signaling and phosphatase and tensin homolog (PTEN) for the downregulated miRNAs (Figure 4A and B).

Figure 4 Predicted targets of all differentially regulated miRNAs during in vitro activation of primary hepatic stellate cells (Table 1) were analyzed. The two charts represent the enrichment of molecules in affected pathways for the upregulated (A) and downregulated (B) miRNAs. Only pathways with P≤ 0.001 are shown. IGF-1: Insulin-like growth factor-1; ERK: Extracellular signal-regulated kinase; MAPK: Mitogen-activated protein kinase; BMP: Bone morphogenetic protein; IL: Interleukin; ROS: Reactive oxygen species; AMPK: AMP activated protein kinase; TGF: Transforming growth factor; PTEN: Phosphatase and tensin homolog; LXR: Liver X receptor; RXR: Retinoid X receptor; PPAR: Peroxisome proliferator-activated receptor; HGF: Hepatocyte growth factor.

Overexpression of miR-146a in HSC-2 and transcriptome analysis

Studies have shown that miR-146a is linked to inflammation and the NF-κB pathway through the two known targets IRAK1 and TRAF6[18,19]. In order to study the function of miR-146a in activated HSCs in vitro, we overexpressed this miRNA in a HSC cell line HSC-2[11]. The level of miR-146a in this cell line is very low, making it suitable for the overexpression. The expression of the reporter green fluorescent protein and the real-time PCR validation of the miR-146a expression (Figure 1B) provided evidence for the successful overexpression of miR-146a in three different clones (S1, S4 and S5).

IRAK1 and TRAF6 are direct targets of miR-146a with two target sites for each mRNA (Figure 5A). We were able to show downregulation of these proteins in all three clones (Figure 5B). The functional consequence of this downregulation can be seen by suppression of the phosphorylation of IκB at Ser32 (Figure 5B). The reduced phosphorylation of IκB in turn should lead to the retention of NF-κB in the cytoplasm. Indeed, our EMSA illustrated that there was reduced nuclear binding activity of NF-κB to an NF-κB probe in all clones (Figure 5C). One of the genes regulated by NF-κB is Cox-2, which is functionally related to HSCs due to its pro-apoptotic effect on HSCs[20,21]. Therefore, we investigated the protein level of Cox-2 in the miR-146a-overexpressing clones, and found the expected downregulation (Figure 5D). Surprisingly, further investigation revealed that the mRNAs of NF-κB and Cox-2 were upregulated (Figure 5E). In contrast, we observed a significant downregulation of IL-6 mRNA, another target of NF-κB, in the clones S1, S4 and S5 (Figure 5E). We also found a significant upregulation of SMAA and collagen I (ColI) mRNAs, a HSC activation and a fibrotic marker, respectively (Figure 5E).

Figure 5 Changes during overexpression of rno-miR-146a in the hepatic stellate cell-2 cell line. A: Depicted are two putative binding sites of miR-146a to the 3’-UTR of rat tumor necrosis factor receptor associated factor 6 (TRAF6) and rat interleukin receptor associated kinase 1 (IRAK1), respectively; B: The Western blotting data show the suppression of TRAF6 and IRAK1, resulting in the decreased phosphorylation of IκB, although the expression of IκB remained unchanged. A representative Western blotting for two independent experiments is shown; C: Electrophoretic mobility shift assay (EMSA) results demonstrated a decrease in nuclear factor (NF)-κB DNA binding activity due to the overexpression of miR-146a. TATA binding protein (TBP) showed equal loading of samples. A representative EMSA experiment is shown out of three independent samples for each clone; D: miR-146a-overexpressing clones showed a reduced level of cyclooxygenase-2 (Cox-2) protein. The Western blotting shown is representative of two independent experiments; E: The relative fold change in mRNA expression between hepatic stellate cell (HSC)-2 and miR-146a-overexpressing HSC-2 cells for five different targets [NF-κB (RelA), Cox-2, smooth muscle α-actin, ColI, interleukin-6] is shown. The data represent the mean ± SE of two independent experiments (^aP≤ 0.005).

In order to establish a link between the regulation of miR-146a and NF-κB activity, as proposed by Taganov et al[18], we transfected NF-κB siRNAs into HSC-2 cells. The efficiency of the transfection was shown by the downregulation of NF-κB in total cell lysates and nuclear extracts, which resulted in a decrease in NF-κB DNA binding activity (Figure 6A and B, respectively). We also found downregulation of miR-146a in NF-κB siRNA-transfected cells, thereby confirming a regulation of miR-146a by NF-κB in HSCs (Figure 6C). Surprisingly, we noticed an increase in the Cox-2 protein expression (Figure 6D), which implied a yet unclear involvement of miR-146a in the regulation of this enzyme.

Figure 6 Regulation of miR-146a by nuclear factor-κB. A: Knock-down experiments using nuclear factor (NF)-κB siRNAs showed a reduced level of cellular NF-κB (RelA) protein; B: The nuclear level of NF-κB (RelA) was decreased and showed a diminished DNA binding activity. Depicted is a representative Western blotting and electrophoretic mobility shift assay from three independent experiments; C: Downregulation of NF-κB (RelA) mRNA due to NF-κB siRNA transfection was accompanied by a decrease in miR-146a after 24 h. The data represent the mean ± SE of two independent experiments (^aP≤ 0.05, ^bP≤ 0.01, ^cP≤ 0.001); D: Cyclooxygenase-2 (Cox-2) protein was upregulated after NF-κB siRNA transfection. Shown are a representative Western blotting and the densitometric analysis of six independent experiments (^aP≤ 0.05).

The differences in the NF-κB-dependent regulation of Cox-2 and IL-6 have already hinted at the intricacy of the influence of the miR-146a overexpression has on the gene expression in activated HSCs. In order to get an overview of the transcriptome changes, we performed a gene expression analysis of the three miR-146a-overexpressing clones, and compared them with control cells using a cDNA microarray. The analysis yielded 485 up- and 309 downregulated transcripts (Supplementary Tables 1 and 2), which satisfied a P value ≤ 0.05 and at least twofold change. Among the upregulated genes were Lmcd1, CD81, FGF13, Col4a1, Cadherin 11 and BMP-4. The highly downregulated genes included Col15a1, MMP-2, Thy-1, IL-1RL1 and Cadherin 13.

Supplementary Table 1 Upregulated genes in miR-146a-transfected hepatic stellate cell-2.

Probe ID	Representative public ID	Gene symbol	Gene title	Log2	Fold change	P-value
1383164_at	AW524366			5.065854	33.49455	3.49E-05
1379902_at	BE108170			4.568453	23.72692	0.000154
1382211_at	AI602542			4.401705	21.1371	0.008197
1394456_at	AW525722			3.905818	14.98885	0.000414
1373740_at	AA851385			3.777603	13.71424	0.002694
1393437_at	AW142608			3.417765	10.68685	3.36E-05
1389579_at	BI284372			3.412511	10.64801	0.010102
1374065_at	BG378920			3.327195	10.03658	0.000137
1393018_at	AI071984			3.323743	10.01259	0.000201
1392105_at	AW527533			3.22946	9.379166	0.005625
1373062_at	BM388650			3.174909	9.031143	0.00033
1373776_at	AI406341			3.127483	8.73909	3.23E-06
1378457_at	AI179450			2.993031	7.96145	0.001547
1391428_at	AI639162			2.959079	7.776274	0.003728
1393314_at	BI289840			2.826721	7.0946	8.14E-05
1379382_at	AI144865			2.810339	7.014492	0.01755
1391481_at	BE104424			2.742723	6.693324	0.000235
1376435_at	BI303340			2.688229	6.445217	0.00028
1395327_at	AW522341			2.651476	6.283098	0.017329
1371506_at	AA891207			2.611623	6.111911	0.013546
1394833_at	BE120930			2.604706	6.082676	3.53E-06
1375230_at	AA800192			2.512936	5.707806	0.000375
1374811_at	AA858705			2.505232	5.677406	0.035302
1377934_at	BF387289			2.461691	5.508619	0.005853
1383240_at	BE110753			2.374072	5.184023	0.004602
1372921_at	AI073219			2.355729	5.118529	0.036703
1376800_at	AA892496			2.321059	4.99699	0.000124
1384137_at	AI030318			2.265411	4.807915	0.000711
1398597_at	AI044699			2.200111	4.595148	0.00022
1393782_at	BF396790			2.19007	4.563275	0.003901
1382330_at	BE116838			2.188725	4.559023	0.000179
1398657_at	AI045896			2.164482	4.483053	5.91E-05
1376617_at	BE107482			2.109346	4.314957	0.001578
1378111_at	AI576002			2.104523	4.300555	0.023
1379936_at	AA875132			2.096645	4.277136	0.005892
1377946_at	BF420043			2.071766	4.20401	3.19E-05
1377675_at	AI177743			2.063136	4.178936	0.005112
1380940_at	BF402603			2.04682	4.131942	0.007683
1381335_at	BE349658			2.046725	4.131669	7.30E-05
1374971_at	AA818954			2.036171	4.101556	0.000603
1397781_at	BF414751			1.968385	3.913299	0.004705
1391841_at	BE103537			1.948096	3.858649	0.003749
1388546_at	AI013328			1.918544	3.780415	0.005524
1379444_at	BF283694			1.836166	3.570599	0.00094
1392627_x_at	BI282114			1.828818	3.552459	9.63E-05
1374432_at	BE118251			1.820423	3.531847	0.023594
1389239_at	BM384377			1.820056	3.530949	3.85E-06
1394578_at	BI299761			1.783117	3.441689	0.019487
1376734_at	BI279030			1.776063	3.424903	0.021384
1392820_at	BI285064			1.763119	3.394312	0.000366
1382212_at	AI385201			1.761883	3.391405	0.008303
1374273_at	BG665433			1.740316	3.341083	0.001848
1390471_at	BM383411			1.732112	3.322138	0.003308
1396009_at	BE108258			1.726446	3.309117	2.68E-05
1382294_at	AI576111			1.698452	3.245526	0.004164
1390459_at	BG670247			1.683832	3.212802	0.000503
1381577_at	AI170131			1.674706	3.192544	9.98E-05
1388720_at	BM390713			1.669188	3.180356	0.000841
1371394_x_at	BG664827			1.660846	3.162018	0.000385
1379719_at	AI408386			1.629839	3.094784	8.76E-06
1392876_at	BG375098			1.597297	3.025758	0.000425
1377881_at	AA997027			1.583295	2.996534	0.000249
1392924_at	BG371591			1.573984	2.977258	2.15E-05
1378152_at	AI170349			1.572157	2.97349	0.000818
1390987_at	AI406858			1.539611	2.907161	0.005124
1390300_at	BM383635			1.539317	2.906568	0.001988
1381996_at	BG666712			1.524793	2.877454	0.007289
1392893_a_at	AA926239			1.492644	2.814042	1.82E-05
1378462_at	BE107396			1.484439	2.798083	0.027693
1391028_at	AI511126			1.480597	2.790641	0.002716
1385978_at	AI072788			1.48007	2.789622	0.01557
1392813_at	AI548994			1.472777	2.775556	0.028907
1379903_at	AI059853			1.456459	2.744339	0.005294
1382291_at	AI454332			1.447251	2.726879	0.021889
1396539_at	BE119221			1.444627	2.721924	0.00325
1395381_at	BF542239			1.428428	2.691532	0.018331
1394709_at	AI406967			1.426754	2.688412	0.013879
1378780_at	BF410325			1.42094	2.677598	0.009832
1374172_at	AI010883			1.407256	2.652321	0.005074
1377551_at	BE118580			1.406921	2.651706	0.000613
1378172_at	AI008119			1.390207	2.621164	9.29E-05
1389744_at	AW527194			1.371756	2.587854	0.029149
1393728_at	AA964541			1.361978	2.570373	0.00664
1389284_at	BI275747			1.352525	2.553586	0.009308
1377309_at	AA963085			1.344539	2.53949	0.000217
1394012_at	BI303933			1.325072	2.505453	9.03E-06
1396886_at	BF387869			1.319644	2.496045	0.000671
1389172_at	AI179391			1.318725	2.494456	5.03E-06
1376011_at	AI411359			1.31543	2.488765	0.013276
1374171_at	AI170507			1.313956	2.486224	0.003317
1382802_x_at	AW920828			1.313169	2.484867	0.0032
1395211_s_at	BE118557			1.294366	2.452692	0.014857
1391727_at	BG662710			1.294339	2.452646	0.001351
1373079_at	BI296427			1.291969	2.448621	0.040034
1375005_at	BF403824			1.291727	2.44821	0.001959
1382174_at	AI227996			1.290673	2.446421	0.006458
1380088_at	AW533021			1.284168	2.435415	5.37E-05
1383910_at	BF398220			1.28089	2.429889	0.003117
1381498_at	AA956116			1.226183	2.339473	0.001176
1391936_a_at	BI289110			1.212511	2.317406	0.041362
1379089_at	BM382838			1.20837	2.310764	0.000681
1372993_at	BI299621			1.204492	2.304561	0.000924
1390671_at	AI044666			1.203424	2.302856	0.001722
1394916_at	AW526714			1.202724	2.301739	0.001978
1389397_at	AI234012			1.202426	2.301263	0.000195
1376637_at	AI102401			1.198002	2.294217	0.027098
1386552_at	BF284027			1.197145	2.292856	0.003348
1377792_at	AW524891			1.195075	2.289568	0.013872
1384952_at	AI028968			1.184453	2.272772	0.001744
1376768_at	BM386807			1.182682	2.269984	0.008644
1389127_at	BF552908			1.17877	2.263837	0.001451
1392140_at	BF419584			1.174126	2.256561	0.010974
1375707_at	AA817993			1.171668	2.25272	0.017511
1377994_at	AI501237			1.165986	2.243866	2.56E-05
1372027_at	AI009713			1.165036	2.242388	0.004861
1374290_at	AI408191			1.161834	2.237416	0.000514
1372820_at	BE109102			1.157167	2.23019	6.76E-05
1395629_at	BE105336			1.152754	2.223379	0.001122
1373628_at	AA818342			1.148702	2.217143	0.003268
1383697_at	AW530905			1.14753	2.215342	0.000199
1384269_at	BF386887			1.144996	2.211455	0.039498
1379733_at	BF396474			1.144326	2.210428	0.000479
1379682_at	BI281668			1.138058	2.200846	0.00129
1393334_at	AW528448			1.134957	2.19612	0.000111
1372583_at	AI009094			1.134834	2.195932	0.000776
1390193_at	BF389884			1.132371	2.192188	0.007448
1373114_at	AI408442			1.132006	2.191632	0.026188
1395586_at	BF545930			1.130756	2.189735	0.009671
1389085_at	BI296359			1.122011	2.176502	0.033565
1378898_at	BE109293			1.117104	2.169111	0.018502
1384433_at	AI072153			1.116005	2.167459	0.008374
1392778_at	AA891634			1.112387	2.16203	0.000734
1372515_at	BI281177			1.109794	2.158148	0.000373
1394727_at	AI407942			1.104267	2.149896	0.000884
1391617_at	AI171103			1.103278	2.148422	0.00173
1384680_at	AA924336			1.103174	2.148268	1.20E-05
1374246_at	BF402392			1.100807	2.144746	0.001207
1395350_at	AW919190			1.100062	2.14364	0.02672
1397343_at	BE113258			1.098815	2.141787	0.000702
1384051_at	BF390066			1.096884	2.138923	0.00037
1381654_at	BF398637			1.086982	2.124292	0.008757
1392619_at	BE118107			1.081379	2.116058	0.010153
1376747_at	BE107075			1.079554	2.113382	0.014697
1380763_at	AI101194			1.078476	2.111804	0.008553
1393653_at	BM384831			1.077241	2.109997	0.029779
1379844_at	AW531072			1.073984	2.105239	0.00402
1394948_at	BI303527			1.073384	2.104364	0.028955
1393911_at	AI502300			1.069942	2.099349	0.001054
1378006_at	AI233832			1.069145	2.09819	0.00023
1379451_at	AI549081			1.069122	2.098155	0.025226
1391643_at	BI290758			1.065858	2.093415	0.009922
1390136_at	BE109274			1.062274	2.08822	0.003245
1389256_at	BG381256			1.061409	2.086969	0.02736
1376465_at	BI295240			1.055842	2.078931	0.025628
1390515_at	AA998383			1.05246	2.074063	0.003395
1390205_at	BE108876			1.052183	2.073665	0.000145
1374728_at	BG671786			1.050605	2.071399	0.043671
1394883_at	AI179616			1.050405	2.071111	0.038398
1372104_at	BF289002			1.043745	2.061573	0.000585
1379252_at	AW522833			1.041529	2.058408	0.000451
1377151_at	AI102833			1.040228	2.056553	0.00364
1377880_at	AI170633			1.038506	2.054099	0.00023
1389908_at	BE107167			1.035223	2.049431	0.01567
1373082_at	AA893743			1.033904	2.047558	0.017691
1373537_at	BE113175			1.028307	2.039629	0.005797
1392578_at	AI070875			1.021081	2.029439	0.01047
1377705_at	BF549971			1.019922	2.027809	0.001107
1371854_at	BG374451			1.019759	2.02758	0.012677
1375647_at	BG671943			1.006159	2.008556	0.017266
1385903_at	AA859627			1.004338	2.006023	0.000426
1398265_at	NM_013040	Abcc9	ATP-binding cassette, sub-family C (CFTR/MRP), member 9	1.43783	2.70913	0.001785
1387287_a_at	D83598	Abcc9	ATP-binding cassette, sub-family C (CFTR/MRP), member 9	1.223092	2.334465	0.004574
1397375_at	BM384537	Acsl5	Acyl-CoA synthetase long-chain family member 5	1.153318	2.224249	0.01568
1386926_at	NM_053607	Acsl5	Acyl-CoA synthetase long-chain family member 5	1.053592	2.075692	0.015907
1370857_at	BI282702	Acta2	Smooth muscle α-actin	2.229514	4.68976	0.000129
1398294_at	NM_031005	Actn1	Actinin, α 1	1.131925	2.191509	0.001329
1368223_at	NM_024400	Adamts1	ADAM metallopeptidase with thrombospondin type 1 motif, 1	2.048565	4.136942	0.028144
1376481_at	BF416285	Adamts9	A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 9	2.392634	5.251154	0.000223
1374535_at	BI283881	Afap1l2	Actin filament associated protein 1-like 2	2.203032	4.60446	0.000634
1368869_at	BG663107	Akap12	A kinase (PRKA) anchor protein 12	1.529713	2.887284	0.019479
1368868_at	NM_057103	Akap12	A kinase (PRKA) anchor protein 12	1.010475	2.014575	0.013205
1387493_at	NM_133515	Akap5	A kinase (PRKA) anchor protein 5	1.569515	2.968049	0.003304
1370043_at	NM_031753	Alcam	Activated leukocyte cell adhesion molecule	1.603863	3.039561	0.010096
1383469_at	BG377269	Aldh1a3	Aldehyde dehydrogenase 1 family, member A3	1.116847	2.168725	0.00291
1370638_at	AF069525	Ank3	Ankyrin 3, epithelial	1.818441	3.526998	0.00833
1367664_at	NM_013220	Ankrd1	Ankyrin repeat domain 1 (cardiac muscle)	1.668973	3.179881	0.020907
1367665_at	L81174	Ankrd1	Ankyrin repeat domain 1 (cardiac muscle)	1.503501	2.8353	0.035713
1372069_at	BF284716	Ankrd15	Ankyrin repeat domain 15	1.333618	2.520339	0.003331
1367974_at	NM_012823	Anxa3	Annexin A3	3.372176	10.35443	7.22E-05
1367975_at	BF283732	Anxa3	Annexin A3	1.914777	3.770554	0.000113
1395313_s_at	AI179982	Anxa3	Annexin A3	1.717191	3.287957	0.000365
1373654_at	BM389254	Anxa8	Annexin A8	1.14259	2.20777	0.000179
1392815_at	BE114489	Arap2	ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 2	1.501088	2.830562	0.000231
1387018_at	NM_053770	Argbp2	Arg/Abl-interacting protein ArgBP2	1.513378	2.854778	0.039786
1373315_at	AI176425	Arnt2	Aryl hydrocarbon receptor nuclear translocator 2	2.041125	4.115662	0.023949
1378134_at	BI291629	Atp8b1	ATPase, Class I, type 8B, member 1	1.061725	2.087426	7.68E-05
1368485_at	NM_024401	Avil	Advillin	2.121568	4.351667	0.012237
1370823_at	AF387513	Bambi	BMP and activin membrane-bound inhibitor, homolog (Xenopus laevis)	1.526089	2.880041	0.003285
1372613_at	AI232784	Bdh2	3-hydroxybutyrate dehydrogenase, type 2	1.177434	2.261742	0.013257
1387232_at	NM_012827	Bmp4	Bone morphogenetic protein 4	3.074538	8.424189	0.000456
1380459_at	AI555023	Btbd14a	BTB (POZ) domain containing 14A	1.35767	2.562709	3.53E-06
1386995_at	BI288701	Btg2	B-cell translocation gene 2, anti-proliferative	1.228055	2.342509	0.004008
1377086_at	AI233530	C1qtnf3	C1q and tumor necrosis factor related protein 3	2.513168	5.708723	0.002654
1376657_at	BE117767	Cadm1	Cell adhesion molecule 1	1.04091	2.057525	0.001137
1393452_at	BM391835	Car9	Carbonic anhydrase 9	2.22363	4.670671	0.00011
1390101_at	AI170609	Ccdc107	Coiled-coil domain containing 107	1.152965	2.223704	0.000166
1398827_at	NM_013087	Cd81	Cd81 molecule	3.901095	14.93986	0.00033
1388936_at	BI296340	Cdh11	Cadherin 11	3.493679	11.26425	0.016743
1370371_a_at	U23056	Ceacam1 /// Ceacam10	Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) /// carcinoembryonic antigen-related cell adhesion molecule 10	2.350728	5.100814	0.001261
1393142_at	BF562621	Cep70	Centrosomal protein 70 kDa	1.414308	2.665318	5.96E-05
1368675_at	NM_032084	Chn2	Chimerin (chimaerin) 2	1.111248	2.160324	0.00013
1389368_at	AW253242	Cnksr3	Cnksr family member 3	1.115693	2.166991	0.002136
1376868_at	BM389293	Cobll1	Cobl-like 1	2.807857	7.002435	0.002758
1372439_at	AI176393	Col4a1	Collagen, type IV, α 1	3.587058	12.01745	4.28E-05
1373245_at	BE111752	Col4a1	Collagen, type IV, α 1	3.148631	8.868134	9.98E-05
1388494_at	BI281705	Col4a2	Collagen, type IV, α 2	2.800595	6.967276	0.000478
1393891_at	BE128699	Col8a1	Collagen, type VIII, α 1	1.15147	2.221401	0.003468
1367782_at	NM_012812	Cox6a2	Cytochrome c oxidase, subunit VIa, polypeptide 2	2.245734	4.742785	0.001312
1386921_at	NM_013128	Cpe	Carboxypeptidase E	2.564823	5.916823	0.021482
1382037_at	AI600057	Crim1	Cysteine rich transmembrane BMP regulator 1 (chordin like)	2.041047	4.11544	0.00285
1391448_at	BI289620	Crim1	Cysteine rich transmembrane BMP regulator 1 (chordin like)	1.9156	3.772706	0.000387
1398622_at	AI703807	Crim1	Cysteine rich transmembrane BMP regulator 1 (chordin like)	1.80839	3.502512	0.000527
1376457_at	AI175861	Crispld2	Cysteine-rich secretory protein LCCL domain containing 2	1.37777	2.598664	0.018183
1387922_at	AF109674	Crispld2	Cysteine-rich secretory protein LCCL domain containing 2	1.195518	2.29027	0.003604
1368059_at	NM_053955	Crym	Crystallin, mu	1.288402	2.442574	0.000395
1383590_at	AA963863	Csgalnact1	Chondroitin sulfate N-acetylgalactosaminyltransferase 1	3.304007	9.876547	0.022018
1370057_at	NM_017148	Csrp1	Cysteine and glycine-rich protein 1	1.030407	2.042601	0.001628
1388583_at	BF283398	Cxcl12	Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)	2.092111	4.263716	0.042845
1387655_at	AF189724	Cxcl12	Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)	1.643136	3.123441	0.028532
1369633_at	AI171777	Cxcl12	Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)	1.567476	2.963858	0.045339
1368290_at	NM_031327	Cyr61	Cysteine-rich, angiogenic inducer, 61	1.261651	2.397699	0.022554
1371436_at	AI176924	Ddah2	Dimethylarginine dimethylaminohydrolase 2	1.710173	3.272001	0.006371
1368013_at	NM_080399	Ddit4l	DNA-damage-inducible transcript 4-like	1.056235	2.079498	0.002531
1389894_at	BF399476	Dlc1	Deleted in liver cancer 1	1.068336	2.097013	0.002194
1377835_at	BM390876	Dock8	Dedicator of cytokinesis 8	2.230319	4.692377	0.005526
1388506_at	AW144509	Dsp	Desmoplakin	1.830721	3.557148	9.48E-05
1368146_at	U02553	Dusp1	Dual specificity phosphatase 1	1.560299	2.949149	0.003438
1368949_at	NM_053820	Ebf1	Early B-cell factor 1	1.316466	2.490553	0.000315
1369519_at	NM_012548	Edn1	Endothelin 1	1.366023	2.577591	0.009786
1368541_at	NM_053719	Emb	Embigin	1.064032	2.090767	0.031817
1377752_at	BE112998	Emp2	Epithelial membrane protein 2	1.392599	2.625512	0.000189
1373617_at	AA818807	Emp2	Epithelial membrane protein 2	1.347238	2.544246	0.000131
1377311_at	AI045616	Emx2	Empty spiracles homeobox 2	1.032095	2.044992	0.001019
1369096_at	NM_134331	Epha7	Eph receptor A7	1.427798	2.690358	0.001357
1385788_at	AW534949	Ephb3	Eph receptor B3	1.321665	2.499544	0.000783
1369182_at	NM_013057	F3	Coagulation factor III (thromboplastin, tissue factor)	2.945169	7.701657	8.99E-05
1377940_at	BF398271	Fam101b	Family with sequence similarity 101, member B	1.069187	2.09825	0.047471
1384507_at	AA817708	Fam105a	Family with sequence similarity 105, member A	1.169405	2.24919	0.00595
1389146_at	BF283267	Fam107b	Family with sequence similarity 107, member B	1.01668	2.023257	0.000409
1393910_at	BF563961	Fam13a1	Family with sequence similarity 13, member A1	4.490284	22.47553	0.003831
1379625_at	BG664461	Fam164a	Family with sequence similarity 164, member A	1.550421	2.929026	0.001732
1384648_at	AA963844	Fam164a	Family with sequence similarity 164, member A	1.219281	2.328307	0.000456
1391944_at	BI296237	Fam184a /// RGD1560557	Family with sequence similarity 184, member A /// similar to minichromosome maintenance protein 8 isoform 1	1.26856	2.409209	0.001168
1373286_at	AA875261	Fblim1	Filamin binding LIM protein 1	1.055977	2.079126	0.000141
1376500_at	AI639044	Fbxo23	F-box only protein 23	1.064218	2.091036	0.001619
1386614_at	BG671466	Fbxo23	F-box only protein 23	1.016912	2.023583	0.010871
1368114_at	NM_053428	Fgf13	Fibroblast growth factor 13	3.654623	12.59364	0.004204
1370106_at	NM_019199	Fgf18	Fibroblast growth factor 18	2.48942	5.615522	0.016971
1369313_at	NM_031677	Fhl2	Four and a half LIM domains 2	1.491713	2.812227	0.011052
1371951_at	AA800031	Fhl2	Four and a half LIM domains 2	1.319164	2.495215	0.010936
1372825_at	BI290551	Fnbp1	Formin binding protein 1	1.481808	2.792985	0.003539
1376784_at	BI274481	Fnbp1	Formin binding protein 1	1.407572	2.652903	0.007946
1369471_at	NM_138914	Fnbp1	Formin binding protein 1	1.142482	2.207604	0.012674
1377342_s_at	BE105446	Fnbp1	Formin binding protein 1	1.043905	2.061801	0.023433
1370829_at	M69056	Fntb	Farnesyltransferase, CAAX box, β	2.00125	4.003468	0.001272
1368711_at	NM_012743	Foxa2	Forkhead box A2	3.125513	8.727165	0.00498
1380387_at	BE105492	Foxp2	Forkhead box P2	1.808179	3.501999	0.000372
1383721_at	AI556075	Fzd8	Frizzled homolog 8 (Drosophila)	1.891668	3.710641	8.54E-05
1372016_at	BI287978	Gadd45b	Growth arrest and DNA-damage-inducible, β	1.287102	2.440374	0.007019
1369735_at	NM_057100	Gas6	Growth arrest specific 6	2.030906	4.086614	0.000694
1367627_at	NM_031031	Gatm	Glycine amidinotransferase (L-arginine:glycine amidinotransferase)	1.704989	3.260264	0.001931
1390557_at	BF394809	Gca	Grancalcin	1.294802	2.453433	0.01519
1379031_at	BM390697	Gca	Grancalcin	1.132824	2.192876	0.01681
1374903_at	AI234819	Gcnt2	Glucosaminyl (N-acetyl) transferase 2, I-branching enzyme	1.780391	3.435192	0.00076
1370375_at	J05499	Gls2	Glutaminase 2 (liver, mitochondrial)	1.381886	2.606088	0.000222
1392888_at	AI071251	Gpc4	Glypican 4	1.149833	2.218883	0.017197
1373773_at	BF394166	Gpm6a	Glycoprotein m6a	1.49299	2.814717	0.004487
1370389_at	AB036421	Gpm6b	Glycoprotein m6b	1.729687	3.316558	0.041126
1382955_at	BI284296	Gpr126	G protein-coupled receptor 126	1.691914	3.230851	0.002139
1373693_at	BF414143	Gprc5c	G protein-coupled receptor, family C, group 5, member C	1.172596	2.254169	0.000245
1368618_at	NM_031623	Grb14	Growth factor receptor bound protein 14	2.263062	4.800093	0.001165
1368401_at	M85035	Gria2	Glutamate receptor, ionotropic, AMPA 2	1.185577	2.274543	0.038866
1383897_at	BE117477	H2afy2	H2A histone family, member Y2	1.467448	2.765322	0.003118
1384541_at	BM391441	Hapln1	Hyaluronan and proteoglycan link protein 1	2.720224	6.589753	0.021358
1370125_at	NM_019189	Hapln1	Hyaluronan and proteoglycan link protein 1	2.532368	5.785206	0.010848
1368983_at	NM_012945	Hbegf	Heparin-binding EGF-like growth factor	1.057962	2.081988	0.018982
1376867_at	BE095833	Hspc159	Galectin-related protein	2.821242	7.067707	0.000688
1373515_at	BI275737	Hspc159	Galectin-related protein	2.64004	6.233487	0.000522
1387028_a_at	M86708	Id1	Inhibitor of DNA binding 1	1.097822	2.140313	0.005605
1390507_at	BI296097	Isg20	Interferon stimulated exonuclease 20	2.272905	4.832953	0.040035
1394824_at	BF398348	Itga11	Integrin, α 11	1.885728	3.695395	0.000279
1393558_at	AI137931	Itga6	Integrin, α 6	1.341112	2.533464	0.001146
1382439_at	AI070686	Itgb6	Integrin, β 6	1.31697	2.491424	0.005869
1387907_at	J05510	Itpr1	Inositol 1,4,5-triphosphate receptor, type 1	1.709204	3.269803	0.001386
1368725_at	NM_019147	Jag1	Jagged 1	1.045878	2.064622	0.010981
1398124_at	AI071356	Jazf1	JAZF zinc finger 1	1.325135	2.505564	1.36E-05
1396701_at	BE110052	Kalrn	Kalirin, RhoGEF kinase	1.050512	2.071265	0.00053
1369144_a_at	NM_031739	Kcnd3	Potassium voltage gated channel, Shal-related family, member 3	1.978834	3.941744	0.01975
1394039_at	BM382886	Klf5	Kruppel-like factor 5	1.168271	2.247422	0.001471
1368363_at	NM_053394	Klf5	Kruppel-like factor 5	1.10729	2.154406	0.000169
1388932_at	BI274917	Lama5	Laminin, α 5	1.49922	2.826898	0.008496
1367880_at	NM_012974	Lamb2	Laminin, β 2	1.055487	2.07842	0.000777
1370993_at	AA997129	Lamc1	Laminin, γ 1	1.144416	2.210566	0.001299
1388422_at	BI275904	Lims2	LIM and senescent cell antigen like domains 2	3.226389	9.359224	0.000228
1376632_at	AI602501	Lmcd1	LIM and cysteine-rich domains 1	3.919424	15.13088	0.000893
1381798_at	BE114958	Lmo7	LIM domain 7	1.648918	3.135983	0.000383
1375726_at	BI284480	Lmo7	LIM domain 7	1.223048	2.334393	0.000945
1381190_at	AI598833	Lmo7	LIM domain 7	1.051248	2.072321	0.001218
1375523_at	BE108178	LOC294446	Similar to Myristoylated alanine-rich C-kinase substrate (MARCKS) (ACAMP-81)	1.286331	2.43907	0.000713
1370948_a_at	M59859	LOC294446 /// LOC681252 /// Marcks	Similar to Myristoylated alanine-rich C-kinase substrate (MARCKS) (ACAMP-81) /// similar to Myristoylated alanine-rich C-kinase substrate (MARCKS) (Protein kinase C substrate 80 kDa protein) /// myristoylated alanine rich protein kinase C substrate	1.113014	2.162971	3.48E-05
1370949_at	M59859	LOC294446 /// LOC681252 /// Marcks	Similar to Myristoylated alanine-rich C-kinase substrate (MARCKS) (ACAMP-81) /// similar to Myristoylated alanine-rich C-kinase substrate (MARCKS) (Protein kinase C substrate 80 kDa protein) /// myristoylated alanine rich protein kinase C substrate	1.078213	2.111419	5.02E-05
1381434_s_at	AW253721	LOC302022	Similar to nidogen 2 protein	1.462223	2.755326	0.002301
1373232_at	AI008975	LOC302022	Similar to nidogen 2 protein	1.153904	2.225152	0.007223
1390158_at	BI290752	LOC304903	Similar to Pappalysin-2 precursor (Pregnancy-associated plasma protein-A2) (PAPP-A2) (Pregnancy-associated plasma protein-E1) (PAPP-E)	1.089462	2.127947	0.01314
1384907_at	AI411835	LOC306096	Similar to Dachshund homolog 1 (Dach1)	2.365997	5.155086	0.015526
1383888_at	AA998264	LOC307495	Similar to biliverdin reductase B (flavin reductase (NADPH))	1.345301	2.540832	6.23E-05
1379465_at	AW527596	LOC311134	Hypothetical LOC311134	1.69193	3.230885	0.004884
1392074_at	AA926082	LOC500046	Similar to hypothetical protein FLJ21986	1.886585	3.697589	0.042067
1392592_at	AI137045	LOC679869	Similar to transcription factor 7-like 2, T-cell specific, HMG-box	1.098548	2.141391	0.000254
1394497_at	AI535239	LOC679869 /// LOC683733	Similar to transcription factor 7-like 2, T-cell specific, HMG-box /// similar to Transcription factor 7-like 2 (HMG box transcription factor 4) (T-cell-specific transcription factor 4) (TCF-4) (hTCF-4)	1.055505	2.078445	0.000768
1373088_at	BI295811	LOC682888	Hypothetical protein LOC682888	1.243831	2.368265	1.11E-06
1388447_at	AA800701	LOC683626	Similar to limb-bud and heart	1.193694	2.287377	0.00702
1379815_at	AI713959	LOC683733	Similar to Transcription factor 7-like 2 (HMG box transcription factor 4) (T-cell-specific transcription factor 4) (TCF-4) (hTCF-4)	1.406898	2.651664	1.15E-05
1377156_at	BI273936	LOC683733	Similar to Transcription factor 7-like 2 (HMG box transcription factor 4) (T-cell-specific transcription factor 4) (TCF-4) (hTCF-4)	1.33204	2.517583	0.001391
1383488_at	AA817785	LOC687536	Similar to Forkhead box protein F1 (Forkhead-related protein FKHL5) (Forkhead-related transcription factor 1) (FREAC-1) (Hepatocyte nuclear factor 3 forkhead homolog 8) (HFH-8)	1.534125	2.896127	0.003143
1386120_at	BF393607	LOC689147	Hypothetical protein LOC689147	1.753785	3.372422	0.003982
1393414_at	AW142650	LOC689176	Similar to transmembrane protein 64	1.20166	2.300042	0.047653
1376691_at	AI103213	LOC689176	Similar to transmembrane protein 64	1.150453	2.219836	0.044249
1374016_at	AI502597	Lpar1	Lysophosphatidic acid receptor 1	1.29975	2.461863	0.003803
1370048_at	NM_053936	Lpar1	Lysophosphatidic acid receptor 1	1.268034	2.408331	0.001243
1389913_at	BI276990	Lrrfip1	Leucine rich repeat (in FLII) interacting protein 1	1.260408	2.395635	0.000794
1368448_at	NM_021586	Ltbp2	Latent transforming growth factor β binding protein 2	2.071905	4.204415	0.00222
1367768_at	NM_031655	Lxn	Latexin	1.381643	2.605649	0.000525
1374933_at	BI277043	Mcam	Melanoma cell adhesion molecule	1.00805	2.011191	0.034411
1369218_at	NM_031517	Met	Met proto-oncogene	1.253867	2.384798	0.000472
1384617_at	AI385260	MGC72614	Hypothetical LOC310540	1.318517	2.494096	0.001888
1398387_at	AI009530	MGC72614	Hypothetical LOC310540	1.263746	2.401184	0.001149
1367568_a_at	NM_012862	Mgp	Matrix Gla protein	3.353436	10.2208	0.010514
1384150_at	AA901038	Mid1	Midline 1	1.062584	2.088669	0.000603
1370072_at	NM_012608	Mme	Membrane metallo endopeptidase	3.646308	12.52126	0.003799
1372457_at	BF284182	Mtus1	Mitochondrial tumor suppressor 1	1.62914	3.093285	5.84E-06
1380321_at	BI287786	Mtus1	Mitochondrial tumor suppressor 1	1.402213	2.643066	0.000159
1378970_at	AW252385	Mybphl	Myosin binding protein H-like	1.080283	2.114451	0.043933
1370158_at	AA946388	Myh10	Myosin, heavy chain 10, non-muscle	1.192392	2.285314	0.005978
1388298_at	BI279044	Myl9	Myosin, light chain 9, regulatory	1.368369	2.581786	0.025217
1389507_at	AI072446	Nedd4l	Neural precursor cell expressed, developmentally down-regulated 4-like	1.010244	2.014252	0.002318
1369679_a_at	AB060652	Nfia	Nuclear factor I/A	1.018188	2.025374	0.008943
1388618_at	BM389302	Nid2	Nidogen 2	1.740644	3.341843	0.002285
1368883_at	NM_030868	Nov	Nephroblastoma overexpressed gene	1.914944	3.770993	0.010007
1371412_a_at	BE107450	Nrep	Neuronal regeneration related protein	1.740346	3.341152	0.003311
1369783_a_at	U02319	Nrg1	Neuregulin 1	1.341273	2.533748	0.00085
1370607_a_at	U02323	Nrg1	Neuregulin 1	1.322	2.500124	0.000298
1371211_a_at	U02315	Nrg1	Neuregulin 1	1.304653	2.470242	0.000467
1382814_at	AW521702	Odz3	Odz, odd Oz/ten-m homolog 3 (Drosophila)	2.65492	6.298115	0.004174
1377702_at	BG380173	P2ry5	Purinergic receptor P2Y, G-protein coupled, 5	1.073159	2.104036	0.000211
1367687_a_at	M25719	Pam	Peptidylglycine α-amidating monooxygenase	1.192716	2.285826	0.021615
1398487_at	BF419639	Pbx1	Pre-B-cell leukemia homeobox 1	1.092533	2.132481	0.006868
1393966_at	AW530825	Pbx1	Pre-B-cell leukemia homeobox 1	1.018972	2.026474	0.000957
1370490_at	L43592	Pcdhb12	Protocadherin β 12	1.40358	2.645572	0.026763
1377042_at	BI288196	Pcgf5	Polycomb group ring finger 5	1.189904	2.281376	0.002754
1392773_at	AA859578	Pcsk5	Proprotein convertase subtilisin/kexin type 5	1.589348	3.009134	0.002877
1393467_at	BF549923	Pcsk5	Proprotein convertase subtilisin/kexin type 5	1.332597	2.518556	0.000461
1387812_at	NM_012999	Pcsk6	Proprotein convertase subtilisin/kexin type 6	3.748654	13.44179	0.001431
1382345_at	AA955299	Pctk2	PCTAIRE protein kinase 2	1.020874	2.029147	5.29E-05
1374157_at	AA858930	Pde4b	Phosphodiesterase 4B, cAMP specific	2.492088	5.625918	0.004862
1369044_a_at	AF202733	Pde4b	Phosphodiesterase 4B, cAMP specific	1.307088	2.474415	0.025797
1374616_at	BM384311	Pdgfrl	Platelet-derived growth factor receptor-like	1.872122	3.660707	1.96E-06
1368703_at	NM_053326	Pdlim5	PDZ and LIM domain 5	1.360465	2.56768	0.000723
1386913_at	NM_019358	Pdpn	Podoplanin	2.706385	6.526842	0.01551
1374969_at	AA799832	Pgm5	Phosphoglucomutase 5	1.9123	3.764087	4.83E-05
1368860_at	NM_017180	Phlda1	Pleckstrin homology-like domain, family A, member 1	1.236279	2.355901	0.031298
1378106_at	AI029402	Phlda2	Pleckstrin homology-like domain, family A, member 2	1.063574	2.090104	0.007195
1388539_at	BE113268	Pkp2	Plakophilin 2	1.216333	2.323553	0.004529
1382659_at	BF289229	Pla2r1	Phospholipase A2 receptor 1	1.81995	3.53069	5.56E-05
1387122_at	NM_012760	Plagl1	Pleiomorphic adenoma gene-like 1	6.377022	83.11414	0.001611
1386962_at	NM_024353	Plcb4	Phospholipase C, β 4	1.72799	3.31266	0.008873
1370489_a_at	U57836	Plcb4	Phospholipase C, β 4	1.479277	2.78809	0.007828
1369029_at	NM_057194	Plscr1	Phospholipid scramblase 1	1.321345	2.49899	0.002555
1370247_a_at	AA943163	Pmp22	Peripheral myelin protein 22	1.312653	2.483979	0.000418
1372531_at	BE106488	Ppfibp2	PTPRF interacting protein, binding protein 2 (liprin β 2)	1.798887	3.479517	0.023491
1393082_at	AI044747	Ppp1r14c	Protein phosphatase 1, regulatory (inhibitor) subunit 14c	1.263639	2.401006	0.009828
1368716_at	NM_133425	Ppp1r14c	Protein phosphatase 1, regulatory (inhibitor) subunit 14c	1.085918	2.122725	0.010686
1370012_at	NM_031557	Ptgis	Prostaglandin I2 (prostacyclin) synthase	1.878328	3.676486	0.007631
1368527_at	U03389	Ptgs2	Prostaglandin-endoperoxide synthase 2	1.723796	3.303044	0.03712
1377427_at	BE104739	Ptpn14	Protein tyrosine phosphatase, non-receptor type 14	1.140142	2.204028	1.10E-05
1374774_at	BF552241	Ptpn14	Protein tyrosine phosphatase, non-receptor type 14	1.065112	2.092332	3.73E-05
1368035_a_at	X83505	Ptprf	Protein tyrosine phosphatase, receptor type, F	2.196463	4.583543	0.005199
1384227_at	AI044031	Ptprk	Protein tyrosine phosphatase, receptor type, K, extracellular region	2.172519	4.508097	4.14E-05
1390034_at	BF393945	Ralgps2	Ral GEF with PH domain and SH3 binding motif 2	1.192882	2.286089	0.025134
1367791_at	NM_031645	Ramp1	Receptor (G protein-coupled) activity modifying protein 1	1.90732	3.751116	0.017043
1368660_at	NM_021690	Rapgef3	Rap guanine nucleotide exchange factor (GEF) 3	1.018953	2.026448	0.003623
1390159_at	AA819332	Rasgrp3	RAS guanyl releasing protein 3 (calcium and DAG-regulated)	1.433832	2.701633	0.016442
1383322_at	BG375198	Rasl11b	RAS-like family 11 member B	1.75778	3.381775	0.008884
1387581_at	NM_022959	Rassf9	Ras association (RalGDS/AF-6) domain family (N-terminal) member 9	2.580119	5.97989	2.33E-07
1383247_a_at	BI291029	rCG_35099	Spinster homolog 2	1.270831	2.413004	0.006005
1388791_at	BI275911	RGD1309930	Similar to 2810022L02Rik protein	1.327569	2.509794	0.01678
1395336_at	BE098691	RGD1309930	Similar to 2810022L02Rik protein	1.309761	2.479005	0.004198
1374898_at	AW527473	RGD1311422	Similar to CG8841-PA	1.039847	2.056009	0.002294
1373584_at	BE113205	RGD1559643	Similar to hypothetical protein A430031N04	1.01952	2.027245	0.00071
1372380_at	AI231308	RGD1561067	Similar to RNA binding protein gene with multiple splicing	3.350875	10.20267	0.002486
1375898_at	AW252379	RGD1561067	Similar to RNA binding protein gene with multiple splicing	3.091972	8.526606	0.004328
1376619_at	AI412803	RGD1561090	Similar to protein tyrosine phosphatase, receptor type, D	2.03385	4.094962	1.02E-06
1374591_at	AI409042	RGD1561090	Similar to protein tyrosine phosphatase, receptor type, D	1.973152	3.926251	2.32E-05
1376919_at	BG665267	RGD1562317	Similar to expressed sequence AW212394	1.376378	2.596157	0.000204
1388879_at	BG669292	RGD1562717	Similar to ABI gene family, member 3 (NESH) binding protein	1.796298	3.473277	0.026049
1388906_at	BM389311	RGD1564174	Similar to novel protein similar to Tensin Tns	1.157511	2.230722	4.32E-05
1383398_at	AI059150	RGD1564327	Similar to integrin α 8	1.649388	3.137005	0.000121
1385354_at	BE120766	RGD1564327	Similar to integrin α 8	1.472111	2.774276	9.05E-07
1376546_at	BE120498	RGD1565432	Similar to hypothetical protein	1.921295	3.787629	0.009661
1396347_at	BF395640	RGD1565926	RGD1565926	1.020952	2.029258	0.010695
1371731_at	AI408151	RGD1566215	Similar to Coatomer γ-2 subunit (γ-2 coat protein) (γ-2 COP)	1.928401	3.806332	5.12E-05
1380425_at	AI012859	Rnasel	Ribonuclease L (2',5'-oligoisoadenylate synthetase-dependent)	1.671401	3.185237	0.002887
1377116_at	BI301478	Rnasel	Ribonuclease L (2',5'-oligoisoadenylate synthetase-dependent)	1.626917	3.088523	0.001458
1381533_at	AI144754	Rnd1	Rho family GTPase 1	1.196579	2.291955	0.000495
1379693_at	AI409154	Robo2	Roundabout, axon guidance receptor, homolog 2 (Drosophila)	1.245877	2.371626	0.001543
1390632_at	BE107414	Rspo3	R-spondin 3 homolog (Xenopus laevis)	1.049128	2.069279	0.005955
1388356_at	AI406499	S100a16	S100 calcium binding protein A16	1.404636	2.64751	0.00415
1368379_at	NM_054001	Scarb2	Scavenger receptor class B, member 2	2.124624	4.360893	0.001346
1393338_at	AW528719	Scx	Scleraxis	1.644999	3.127476	0.007845
1368394_at	AF140346	Sfrp4	Secreted frizzled-related protein 4	1.610004	3.052527	0.003747
1367802_at	NM_019232	Sgk1	Serum/glucocorticoid regulated kinase 1	1.265947	2.404849	0.007546
1389779_at	AA800626	Sh2d4a	SH2 domain containing 4A	1.41792	2.672	0.032404
1392301_at	AI237897	Sh3tc1	SH3 domain and tetratricopeptide repeats 1	1.214738	2.320987	0.006644
1392556_at	BF410961	Shroom3	Shroom family member 3	1.454496	2.740607	9.57E-05
1376040_at	BI290044	Sipa1l2	Signal-induced proliferation-associated 1 like 2	1.185619	2.274609	0.012637
1368565_at	NM_019225	Slc1a3	Solute carrier family 1 (glial high affinity glutamate transporter), member 3	1.926317	3.800836	0.003174
1376165_at	BE098153	Slc24a3	Solute carrier family 24 (sodium/potassium/calcium exchanger), member 3	1.145511	2.212244	0.000291
1398295_at	NM_031684	Slc29a1	Solute carrier family 29 (nucleoside transporters), member 1	1.222426	2.333387	0.003869
1369074_at	NM_130748	Slc38a4	Solute carrier family 38, member 4	2.25781	4.78265	0.00054
1392349_at	BE116021	Slc5a3	Solute carrier family 5 (inositol transporters), member 3	1.031672	2.044392	0.000967
1387968_at	L22022	Slc6a15	Solute carrier family 6 (neutral amino acid transporter), member 15	2.581534	5.985759	0.018733
1368920_at	NM_031321	Slit3	Slit homolog 3 (Drosophila)	1.652522	3.143828	0.03084
1384437_at	AI576309	Smarca1	SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 1	1.83294	3.562623	4.14E-05
1377695_at	BF281135	Smtnl2	Smoothelin-like 2	2.018633	4.051996	0.047065
1375349_at	BI295776	Sorbs1	Sorbin and SH3 domain containing 1	2.508532	5.690409	0.000169
1372728_at	BE103745	Sort1	Sortilin 1	1.319517	2.495825	0.006034
1371004_at	AI070124	Sort1	Sortilin 1	1.117923	2.170343	0.00142
1379611_at	BF416979	Spsb1	splA/ryanodine receptor domain and SOCS box containing 1	1.025012	2.034976	0.009283
1373554_at	BE349698	Spsb1	splA/ryanodine receptor domain and SOCS box containing 1	1.010972	2.015269	0.008275
1389142_at	AI013361	Sqrdl	sulfide quinone reductase-like (yeast)	2.278473	4.851642	0.001121
1368109_at	NM_031337	St3gal5	ST3 β-galactoside α-2,3-sialyltransferase 5	1.683094	3.211158	5.13E-05
1370907_at	M83143	St6gal1	ST6 β-galactosamide α-2,6-sialyltranferase 1	1.294325	2.452622	0.000575
1389420_at	BI279446	Stap2	Signal transducing adaptor family member 2	1.208781	2.311423	0.019358
1370680_at	AF483620	Stau2	Staufen, RNA binding protein, homolog 2 (Drosophila)	1.015615	2.021764	0.000276
1372602_at	BI295979	Stbd1	Starch binding domain 1	2.761499	6.781006	2.53E-05
1373590_at	BI295949	Stom	Stomatin	1.267382	2.407243	0.003323
1379732_at	AW920037	Stx11	Syntaxin 11	1.976724	3.935983	9.77E-05
1368771_at	NM_134378	Sulf1	Sulfatase 1	2.238326	4.718492	0.000659
1376572_a_at	AI045848	Svil	Supervillin	1.320708	2.497887	0.019303
1367570_at	NM_031549	Tagln	Transgelin	1.696733	3.24166	0.00228
1367859_at	NM_013174	Tgfb3	Transforming growth factor, β 3	1.509832	2.847768	0.003273
1375951_at	AA818521	Thbd	Thrombomodulin	1.311404	2.481829	0.020049
1370474_at	J03819	Thrb	Thyroid hormone receptor β	2.219205	4.656368	0.002376
1387983_at	J03933	Thrb	Thyroid hormone receptor β	1.184729	2.273206	0.003624
1383623_at	BM383909	Thyn1	Thymocyte nuclear protein 1	1.043826	2.061688	0.002044
1375138_at	AA893169	Timp3	TIMP metallopeptidase inhibitor 3	6.264691	76.88826	0.015514
1389836_a_at	AI599265	Timp3	TIMP metallopeptidase inhibitor 3	5.105264	34.42212	0.013029
1372926_at	AI009159	Timp3	TIMP metallopeptidase inhibitor 3	4.004944	16.05492	0.025103
1368989_at	NM_012886	Timp3	TIMP metallopeptidase inhibitor 3	2.253301	4.767725	0.015049
1373847_at	AW435343	Tm4sf1	Transmembrane 4 L six family member 1	2.114633	4.330798	0.000242
1378305_at	AI578087	Tm4sf1	Transmembrane 4 L six family member 1	1.938512	3.8331	0.000317
1390832_at	BI294696	Tmcc3	Transmembrane and coiled-coil domain family 3	2.119391	4.345106	0.005139
1376623_at	AI409186	Tmem204	Transmembrane protein 204	1.085094	2.121514	7.30E-05
1383314_at	BE110098	Tmem51	Transmembrane protein 51	1.344562	2.539531	3.99E-05
1371361_at	BI278826	Tns1	Tensin 1	1.158595	2.232399	0.000276
1370288_a_at	AF372216	Tpm1	Tropomyosin 1, α	2.037181	4.104428	0.000937
1395794_at	BF395218	Tpm1	Tropomyosin 1, α	1.997138	3.992073	0.008961
1371241_x_at	AF370889	Tpm1	Tropomyosin 1, α	1.663566	3.167985	0.013541
1370287_a_at	M23764	Tpm1	Tropomyosin 1, α	1.59074	3.012039	5.95E-05
1368724_a_at	NM_019131	Tpm1	Tropomyosin 1, α	1.054891	2.077561	0.0189
1372219_at	AA012755	Tpm2	Tropomyosin 2	1.064563	2.091537	2.16E-05
1398759_at	NM_013043	Tsc22d1	TSC22 domain family, member 1	1.051368	2.072494	0.000844
1377630_at	AI408602	Tspan13	Tetraspanin 13	1.481687	2.792752	0.019678
1375057_at	BG377313	Tspan18	Tetraspanin 18	1.454071	2.739802	0.03087
1398476_at	AW527349	Vcl	Vinculin	1.244474	2.369321	0.000134
1372905_at	AW433888	Vcl	Vinculin	1.136321	2.198197	3.11E-05
1369098_at	NM_013155	Vldlr	Very low density lipoprotein receptor	1.505396	2.839027	0.001107
1387455_a_at	NM_013155	Vldlr	Very low density lipoprotein receptor	1.471028	2.772194	0.001621
1389611_at	AA849857	Vldlr	Very low density lipoprotein receptor	1.435945	2.705593	0.000947
1368854_at	AI227991	Vsnl1	Visinin-like 1	2.472695	5.550796	0.007104
1368853_at	NM_012686	Vsnl1	Visinin-like 1	2.077806	4.221647	0.004903
1387873_at	BI279661	Wfdc1	WAP four-disulfide core domain 1	1.449292	2.730739	0.003744
1370221_at	BF419320	Wisp1	WNT1 inducible signaling pathway protein 1	1.058567	2.082861	0.000154
1393613_at	BE117871	Zfp462	Zinc finger protein 462	1.088387	2.126361	0.004351
1383462_at	BF566263	Znf294	Zinc finger protein 294	1.010246	2.014255	0.000352

Supplementary Table 2 Downregulated genes in miR-146a-transfected hepatic stellate cell-2.

Probe ID	Representative public ID	Gene symbol	Gene title	Log2	Fold change	P-value
1374345_at	AI111707			-3.40046	-10.5594	1.80E-07
1397317_at	BI296984			-3.25476	-9.54513	3.34E-07
1397400_at	BM391846			-3.11991	-8.69335	5.36E-05
1382027_at	BI296880			-3.05526	-8.31237	3.85E-07
1380245_at	AI411847			-2.90249	-7.47715	8.46E-07
1381129_at	BF392367			-2.52142	-5.74147	0.018162
1383211_at	BE109736			-2.46677	-5.52805	0.006284
1380057_at	BE097091			-2.33498	-5.04543	0.000685
1381048_at	BF398435			-2.3159	-4.97916	0.000443
1381064_at	AI137604			-2.26573	-4.80896	1.32E-07
1373583_at	BF396317			-2.2618	-4.79589	1.23E-07
1377240_at	AW526305			-2.19645	-4.58351	0.001977
1394468_at	BF287020			-2.12335	-4.35705	0.000157
1377678_at	BI283757			-2.12273	-4.35519	0.000117
1375101_at	BI292651			-2.05823	-4.16476	0.000442
1380712_at	AI406475			-2.04987	-4.14068	0.006175
1372110_at	BE113148			-1.95331	-3.87261	0.000463
1394517_at	AW522148			-1.88952	-3.70513	0.006671
1390530_at	AI169239			-1.87482	-3.66755	0.00151
1376463_at	AA955579			-1.87022	-3.65587	0.001028
1384743_at	BF418132			-1.86231	-3.63589	2.42E-14
1377114_at	AI410861			-1.8391	-3.57788	8.88E-06
1377161_at	BG378317			-1.83484	-3.56731	0.039081
1383220_at	BE114231			-1.73514	-3.32911	0.000179
1393143_at	AI045866			-1.69968	-3.24828	0.005065
1376324_at	BF406329			-1.68113	-3.20679	0.004765
1390429_at	BF398114			-1.62771	-3.09021	6.83E-05
1382431_at	AI103530			-1.6169	-3.06716	1.15E-06
1391251_at	BI290666			-1.60415	-3.04016	1.73E-05
1383580_at	AA859643			-1.58751	-3.0053	0.000305
1372011_at	BI292028			-1.5749	-2.97915	0.000487
1377232_at	BF406608			-1.54704	-2.92217	0.003334
1398457_at	AI146156			-1.50417	-2.83662	0.005925
1381161_a_at	BI301117			-1.47549	-2.78078	0.008417
1382142_at	AI029975			-1.45644	-2.74431	3.98E-05
1382472_at	AI502459			-1.44328	-2.71939	8.76E-05
1375266_at	BG380633			-1.43613	-2.70595	0.010349
1381862_at	AW524296			-1.39645	-2.63253	0.004828
1393235_at	AI059968			-1.39173	-2.62392	0.000172
1385181_at	AI029337			-1.39139	-2.62332	1.30E-07
1377686_at	AA859337			-1.37593	-2.59535	3.53E-07
1372449_at	AW253616			-1.37158	-2.58753	0.00912
1377556_at	AW535380			-1.36924	-2.58334	0.004323
1372637_at	AI169241			-1.36639	-2.57825	0.000419
1375957_at	AW434654			-1.30351	-2.46828	4.46E-06
1383936_at	BM386842			-1.29935	-2.46118	1.09E-05
1384724_at	AA850766			-1.28208	-2.4319	3.20E-05
1379510_at	BF546306			-1.20859	-2.31112	9.04E-05
1382296_at	BF291041			-1.20836	-2.31075	1.53E-05
1382544_at	AI058746			-1.20774	-2.30975	3.79E-06
1376816_at	BF284903			-1.19854	-2.29507	0.000155
1383019_at	BF558478			-1.18776	-2.27798	0.000454
1374558_at	AI010316			-1.17751	-2.26186	8.04E-06
1389250_at	AW915115			-1.16975	-2.24972	9.96E-05
1377328_at	BI290012			-1.16378	-2.24044	0.009615
1380602_at	AI764190			-1.16264	-2.23867	0.001006
1384812_at	AI229409			-1.15815	-2.23172	0.006986
1396217_at	BF542447			-1.1372	-2.19954	0.000345
1397668_at	H34328			-1.13718	-2.1995	3.26E-07
1374932_at	BI282731			-1.13055	-2.18943	0.001048
1385381_at	AA996491			-1.12644	-2.1832	0.000161
1394047_at	BE107848			-1.10947	-2.15767	0.035398
1397452_at	AI112776			-1.10848	-2.15618	0.000172
1393730_at	BI277836			-1.10819	-2.15575	0.03382
1381975_at	BG371767			-1.10049	-2.14427	0.001469
1380699_at	BE110761			-1.09332	-2.13364	0.000497
1374920_at	AI228955			-1.07203	-2.10239	2.65E-05
1375473_at	BI296644			-1.05345	-2.07548	0.001418
1373914_at	BM389075			-1.05092	-2.07186	0.001887
1383436_at	BG376768			-1.04917	-2.06933	0.000656
1390405_at	AA942765			-1.0454	-2.06394	0.003275
1376911_at	BM386385			-1.04165	-2.05858	0.000165
1384183_at	AA996869			-1.03133	-2.04391	0.004095
1377469_at	AI103161			-1.0216	-2.03016	0.006072
1393030_at	BE115641			-1.01825	-2.02547	0.000652
1377113_at	BF415786			-1.0168	-2.02343	0.002724
1382960_at	BE108047			-1.01496	-2.02084	9.24E-05
1377955_at	AI137602			-1.01226	-2.01707	4.55E-07
1391853_at	AA998997			-1.00175	-2.00243	0.000388
1384086_at	BG671196			-1.00024	-2.00033	0.031992
1385235_at	AA818804	A2bp1	Ataxin 2 binding protein 1	-1.24533	-2.37073	4.71E-05
1383130_at	BF555795	A2bp1	Ataxin 2 binding protein 1	-1.07712	-2.10983	0.000947
1394490_at	AI502114	Abca1	ATP-binding cassette, sub-family A (ABC1), member 1	-1.80354	-3.49076	3.72E-07
1384381_at	BF284523	Abca1	ATP-binding cassette, sub-family A (ABC1), member 1	-1.27132	-2.41382	0.000215
1383355_at	AW918387	Abca1	ATP-binding cassette, sub-family A (ABC1), member 1	-1.21129	-2.31545	5.71E-07
1369928_at	NM_019212	Acta1	Actin, α 1, skeletal muscle	-3.10676	-8.61449	4.42E-05
1370856_at	AA800705	Actc1	Actin, α, cardiac muscle 1	-1.26863	-2.40933	2.02E-05
1394483_at	AW535310	Adamts5	ADAM metallopeptidase with thrombospondin type 1 motif, 5	-1.53094	-2.88975	1.96E-05
1390383_at	BI285616	Adfp	Adipose differentiation related protein	-2.49834	-5.65037	3.30E-08
1382680_at	BG673602	Adfp	Adipose differentiation related protein	-2.22186	-4.66496	1.20E-05
1387395_at	NM_017161	Adora2b	Adenosine A2B receptor	-2.08666	-4.24763	0.000258
1395695_at	BE126420	Aebp1	AE binding protein 1	-1.5972	-3.02555	0.005916
1372301_at	BI278482	Aebp1	AE binding protein 1	-1.55735	-2.94313	0.01167
1368342_at	NM_031544	Ampd3	Adenosine monophosphate deaminase 3	-1.65434	-3.14779	0.004546
1377783_at	BI294141	Angpt4	Angiopoietin 4	-1.64644	-3.1306	0.001964
1397579_x_at	BI294552	Apc2	Adenomatosis polyposis coli 2	-1.91631	-3.77455	0.00151
1395461_at	BI294552	Apc2	Adenomatosis polyposis coli 2	-1.30834	-2.47656	0.002146
1391083_at	BM384457	Arhgap22	Rho GTPase activating protein 22	-1.09826	-2.14096	0.007774
1377385_at	BE100015	Arhgap27	Rho GTPase activating protein 27	-1.30775	-2.47556	6.57E-07
1387959_at	AB009372	Aspg	Asparaginase homolog (S. cerevisiae)	-1.29918	-2.46089	0.000534
1380726_at	BI290633	Aspn	Asporin	-2.13124	-4.38092	0.032454
1381504_at	AI639412	Aspn	Asporin	-1.90669	-3.74947	0.02918
1368477_at	NM_012914	Atp2a3	ATPase, Ca++ transporting, ubiquitous	-1.60464	-3.04119	0.000593
1369664_at	NM_053019	Avpr1a	Arginine vasopressin receptor 1A	-2.07931	-4.22605	1.62E-05
1375941_at	BI292120	Baiap2l1	BAI1-associated protein 2-like 1	-1.64069	-3.11816	1.70E-06
1369807_at	NM_030851	Bdkrb1	Bradykinin receptor B1	-1.11926	-2.17236	0.000155
1391345_at	BI293047	Bmper	BMP-binding endothelial regulator	-1.85131	-3.60828	3.59E-06
1387540_at	NM_012514	Brca1	Breast cancer 1	-1.02475	-2.03461	0.000898
1381995_at	AW530502	Brunol4	Bruno-like 4, RNA binding protein (Drosophila)	-2.20211	-4.60152	7.21E-08
1387893_at	D88250	C1s	Complement component 1, s subcomponent	-1.26395	-2.40152	0.048179
1375569_at	BM386267	Ccdc92	Coiled-coil domain containing 92	-1.03224	-2.0452	0.000197
1367973_at	NM_031530	Ccl2	Chemokine (C-C motif) ligand 2	-2.02826	-4.07912	0.002595
1379935_at	BF419899	Ccl7	Chemokine (C-C motif) ligand 7	-1.07909	-2.11271	0.006482
1370810_at	L09752	Ccnd2	Cyclin D2	-1.31667	-2.49091	0.005098
1389490_at	BI274335	Cd248	CD248 molecule, endosialin	-3.64068	-12.4725	1.26E-06
1389755_at	BM391858	Cdca7l	Cell division cycle associated 7 like	-1.4418	-2.71661	0.000141
1369425_at	NM_138889	Cdh13	Cadherin 13	-3.4441	-10.8837	4.56E-08
1375719_s_at	BG381748	Cdh13	Cadherin 13	-3.2068	-9.23301	2.00E-08
1373102_at	BI282750	Cdh13	Cadherin 13	-3.03097	-8.1736	6.92E-06
1373054_at	AA801076	Cdw92	CDW92 antigen	-1.00321	-2.00445	0.000362
1396034_at	BF402373	Ces7	Carboxylesterase 7	-2.24147	-4.72879	7.58E-05
1389179_at	BF284899	Cidea	Cell death-inducing DNA fragmentation factor, α subunit-like effector A	-1.13966	-2.20329	1.12E-05
1367740_at	M14400	Ckb	Creatine kinase, brain	-1.40456	-2.64738	0.000293
1392672_at	AI576758	Clec11a	C-type lectin domain family 11, member a	-1.45028	-2.73262	0.004619
1368571_at	NM_021997	Clip2	CAP-GLY domain containing linker protein 2	-1.09569	-2.13715	4.77E-07
1372584_at	BG672517	Cnrip1	Cannabinoid receptor interacting protein 1	-1.11448	-2.16517	1.59E-08
1379345_at	BM386752	Col15a1	Collagen, type XV, α 1	-6.09695	-68.4485	3.18E-06
1388939_at	AA800298	Col15a1	Collagen, type XV, α 1	-4.48588	-22.4071	2.71E-05
1384969_at	BE109107	Col24a1	Collagen, type XXIV, α 1	-1.8271	-3.54824	0.002725
1371349_at	AI598402	Col6a1	Collagen, type VI, α 1	-1.66097	-3.16228	0.001251
1371369_at	BI287851	Col6a2	Collagen, type VI, α 2	-1.80919	-3.50445	9.31E-05
1372818_at	BI284441	Colec12	Collectin sub-family member 12	-2.38156	-5.211	3.25E-07
1372774_at	AI170570	Coq6	Coenzyme Q6 homolog (yeast)	-1.57329	-2.97583	1.94E-06
1369964_at	NM_130411	Coro1a	Coronin, actin binding protein 1A	-1.6641	-3.16916	7.57E-05
1392996_at	BG668435	Cpeb1	Cytoplasmic polyadenylation element binding protein 1	-1.42707	-2.68901	3.55E-05
1368293_at	NM_031766	Cpz	Carboxypeptidase Z	-1.17127	-2.2521	0.020505
1376051_at	BI293393	Cryl1	Crystallin, lambda 1	-2.8568	-7.24406	4.30E-05
1383575_at	BG376561	Ctnnd2	Catenin (cadherin-associated protein), delta 2 (neural plakophilin-related arm-repeat protein)	-2.38434	-5.22105	9.56E-05
1369947_at	NM_031560	Ctsk	Cathepsin K	-1.00677	-2.00941	0.001608
1387969_at	U22520	Cxcl10	Chemokine (C-X-C motif) ligand 10	-2.22859	-4.68674	4.90E-05
1368738_at	D11354	Cyp11b1	Cytochrome P450, subfamily 11B, polypeptide 1	-1.731	-3.31958	0.003738
1387305_s_at	NM_012539	Cyp11b1 /// Cyp11b2	Cytochrome P450, subfamily 11B, polypeptide 1 /// cytochrome P450, subfamily 11B, polypeptide 2	-1.73027	-3.3179	4.54E-05
1387276_at	NM_021584	Dclk1	Doublecortin-like kinase 1	-1.05016	-2.07076	0.000907
1384971_at	BI289108	Depdc7	DEP domain containing 7	-1.10481	-2.1507	0.000468
1371732_at	BI285485	Dpt	Dermatopontin	-1.89757	-3.72585	0.009977
1383853_at	BE103067	Dyrk3	Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 3	-1.76949	-3.40934	1.66E-07
1383641_at	BF414702	Ednra	Endothelin receptor type A	-2.19922	-4.5923	0.01467
1378342_at	BF284819	Ednra	Endothelin receptor type A	-1.78129	-3.43733	0.004867
1393415_at	BF548891	Ednra	Endothelin receptor type A	-1.51332	-2.85467	0.00743
1391442_at	AA957585	Ehd3	EH-domain containing 3	-1.71662	-3.28665	3.90E-05
1367905_at	NM_019370	Enpp3	Ectonucleotide pyrophosphatase/phosphodiesterase 3	-1.33397	-2.52096	9.07E-05
1382434_at	AI059015	Entpd5	Ectonucleoside triphosphate diphosphohydrolase 5	-1.63931	-3.11518	2.50E-06
1370503_s_at	AB032828	Epb4.1l3	Erythrocyte protein band 4.1-like 3	-1.9425	-3.84372	0.005443
1368515_at	NM_053927	Epb4.1l3	Erythrocyte protein band 4.1-like 3	-1.52656	-2.88098	0.000173
1369422_at	NM_138850	Fap	Fibroblast activation protein, α	-1.69721	-3.24274	0.001039
1376561_at	AW523739	Fbxo16	F-box protein 16	-1.11142	-2.16058	6.95E-06
1367850_at	NM_053843	Fcgr2a /// LOC498276 /// LOC498277	Fc fragment of IgG, low affinity IIa, receptor (CD32) /// Fc γ receptor II β /// similar to Low affinity immunoglobulin γ Fc region receptor III precursor (IgG Fc receptor III) (Fc-γ RIII) (FcRIII)	-1.23098	-2.34726	0.007866
1392865_at	BG371594	Fgf9	Fibroblast growth factor 9	-2.37371	-5.18274	0.002748
1373882_at	AI170324	Figf	C-fos induced growth factor	-2.48251	-5.58869	0.000198
1387709_at	AY032728	Figf	C-fos induced growth factor	-2.21994	-4.65873	5.44E-07
1374726_at	AI411941	Fndc1	Fibronectin type III domain containing 1	-2.18189	-4.53749	0.005581
1370248_at	AA851939	Fxyd6	FXYD domain-containing ion transport regulator 6	-1.91583	-3.77331	0.004165
1385636_at	AI029226	Fzd3	Frizzled homolog 3 (Drosophila)	-1.30152	-2.46488	6.39E-06
1388395_at	AI406939	G0s2	G0/G1switch 2	-1.69782	-3.2441	6.68E-05
1382314_at	BE096523	G1p2	Interferon, α-inducible protein (clone IFI-15K)	-1.43064	-2.69565	0.012403
1370963_at	AJ131902	Gas7	Growth arrest specific 7	-2.12746	-4.36946	0.001866
1387221_at	NM_024356	Gch1	GTP cyclohydrolase 1	-1.0345	-2.0484	0.001025
1368085_at	NM_133595	Gchfr	GTP cyclohydrolase I feedback regulator	-1.09278	-2.13284	1.23E-05
1368770_at	NM_022276	Gcnt1	Glucosaminyl (N-acetyl) transferase 1, core 2	-1.35748	-2.56237	0.000674
1387659_at	AF245172	Gda	Guanine deaminase	-1.54413	-2.91629	0.000209
1377761_at	BI296057	Gfpt2	Glutamine-fructose-6-phosphate transaminase 2	-2.3887	-5.23685	0.003422
1387007_at	NM_012959	Gfra1	GDNF family receptor α 1	-1.49726	-2.82306	0.000202
1367954_at	U59486	Gfra1	GDNF family receptor α 1	-1.10817	-2.15573	0.0006
1397461_at	BF416400	Glt8d2	Glycosyltransferase 8 domain containing 2	-1.11036	-2.15899	8.28E-06
1386870_at	BI275294	Glul	Glutamate-ammonia ligase (glutamine synthetase)	-1.18385	-2.27182	0.000113
1367632_at	NM_017073	Glul	Glutamate-ammonia ligase (glutamine synthetase)	-1.11961	-2.17288	0.003082
1369302_at	NM_133573	Gper	G protein-coupled estrogen receptor 1	-1.10018	-2.14381	0.001057
1387241_at	NM_031696	Gpr88	G-protein coupled receptor 88	-1.11226	-2.16184	0.002865
1369926_at	NM_022525	Gpx3	Glutathione peroxidase 3	-2.05946	-4.16829	1.35E-06
1374488_at	AI175700	Gramd1b	GRAM domain containing 1B	-1.12881	-2.18678	0.001788
1368180_s_at	NM_017013	Gsta2 /// LOC494499	Glutathione S-transferase A2 /// LOC494499 protein	-2.31111	-4.96266	6.68E-05
1371298_at	BF284168	H19	H19 fetal liver mRNA	-1.87119	-3.65833	0.000303
1391575_at	BG380566	Hapln4	Hyaluronan and proteoglycan link protein 4	-1.12321	-2.17831	0.002326
1368255_at	NM_017354	Hnt	Neurotrimin	-2.33813	-5.05648	0.0043
1367816_at	NM_133621	Hopx	HOP homeobox	-1.32323	-2.50226	0.003008
1393592_at	AA998087	Hs3st5	Heparan sulfate (glucosamine) 3-O-sulfotransferase 5	-1.19206	-2.28479	0.004675
1368578_at	NM_017265	Hsd3b1	Hydroxy-delta-5-steroid dehydrogenase, 3 β- and steroid delta-isomerase 1	-2.132	-4.38324	0.00025
1387282_at	NM_053612	Hspb8	Heat shock protein 8	-1.47457	-2.77901	2.14E-07
1388721_at	BG380282	Hspb8	Heat shock protein 8	-1.28154	-2.43098	1.12E-06
1376908_at	AW531805	Ifit3	Interferon-induced protein with tetratricopeptide repeats 3	-1.35357	-2.55544	0.019235
1382220_at	AI180454	Igf2bp2	Insulin-like growth factor 2 mRNA binding protein 2	-1.40203	-2.64272	0.00737
1387180_at	NM_053953	Il1r2	Interleukin 1 receptor, type II	-2.39783	-5.27012	0.000126
1387273_at	NM_013037	Il1rl1	Interleukin 1 receptor-like 1	-3.65216	-12.5721	0.013461
1370692_at	U04317	Il1rl1	Interleukin 1 receptor-like 1	-1.22822	-2.34278	0.004082
1387504_at	NM_133575	Il1rl2	Interleukin 1 receptor-like 2	-1.5258	-2.87946	7.08E-05
1377163_at	BM385741	Inhbb	Inhibin β-B	-1.23015	-2.34591	0.014339
1369043_at	NM_012971	Kcna4	Potassium voltage-gated channel, shaker-related subfamily, member 4	-4.1772	-18.091	8.01E-08
1390404_at	BF556962	Lama2	Laminin, α 2	-2.54141	-5.82159	9.68E-05
1370138_at	NM_130429	Lef1	Lymphoid enhancer binding factor 1	-1.10908	-2.15708	0.000657
1378179_a_at	AW524864	Lhfpl2	Lipoma HMGIC fusion partner-like 2	-1.03801	-2.05339	0.00217
1371094_at	L06804	Lhx2	LIM homeobox 2	-1.06797	-2.09648	0.000653
1389885_at	BI294855	Limd2	LIM domain containing 2	-1.05098	-2.07194	6.73E-05
1376871_at	AA891475	LOC680910 /// LOC681069 /// LOC681182 /// LOC681196 /// LOC685030 /// LOC685048 /// LOC685111 /// LOC685262 /// LOC685305 /// LOC686848 /// LOC686899 /// RGD1559588 /// RGD1561143 /// RGD1561730 /// RGD1562525 /// RGD1563400 /// RGD1566006	Similar to paired immunoglobin-like type 2 receptor β /// similar to paired immunoglobin-like type 2 receptor β /// similar to paired immunoglobin-like type 2 receptor β /// similar to paired immunoglobin-like type 2 receptor β /// similar to paired immunoglobin-like type 2 receptor β /// similar to paired immunoglobin-like type 2 receptor β /// similar to paired immunoglobin-like type 2 receptor β /// similar to paired immunoglobin-like type 2 receptor β /// similar to paired immunoglobin-like type 2 receptor β /// similar to paired immunoglobin-like type 2 receptor β /// similar to paired immunoglobin-like type 2 receptor β /// similar to cell surface receptor FDFACT /// similar to cell surface receptor FDFACT /// similar to cell surface receptor FDFACT /// similar to cell surface receptor FDFACT /// similar to paired immunoglobin-like type 2 receptor β /// similar to paired immunoglobin-like type 2 receptor β	-1.06939	-2.09855	2.14E-06
1385047_x_at	AI012782	LOC685048 /// LOC685111 /// RGD1559588 /// Vom2r61	Similar to paired immunoglobin-like type 2 receptor β /// similar to paired immunoglobin-like type 2 receptor β /// similar to cell surface receptor FDFACT /// vomeronasal 2 receptor, 61	-2.82049	-7.064	5.90E-06
1393688_at	AI012782	LOC685048 /// LOC685111 /// RGD1559588 /// Vom2r61	Similar to paired immunoglobin-like type 2 receptor β /// similar to paired immunoglobin-like type 2 receptor β /// similar to cell surface receptor FDFACT /// vomeronasal 2 receptor, 61	-2.75933	-6.7708	2.26E-07
1371293_at	AI103218	LOC688228	Similar to Myosin light polypeptide 4 (Myosin light chain 1, atrial isoform)	-1.30459	-2.47013	6.68E-07
1398732_at	BF553297	LOC688273	Hypothetical protein LOC688273	-3.37961	-10.4079	0.000276
1384540_at	BE101066	Lrfn3	Leucine rich repeat and fibronectin type III domain containing 3	-1.05498	-2.07769	0.00043
1388347_at	AI233210	Ly6e	Lymphocyte antigen 6 complex, locus E	-2.7688	-6.81539	1.82E-05
1376184_at	BG381127	Lynx1	Ly6/neurotoxin 1	-1.98858	-3.96846	8.46E-08
1393645_at	BI288003	Mageb16	Melanoma antigen family B, 16	-1.53459	-2.89706	0.000131
1388152_at	BG374290	Map2	Microtubule-associated protein 2	-1.7733	-3.41836	0.008563
1382046_at	AA963495	Map3k3	Mitogen activated protein kinase kinase kinase 3	-1.24191	-2.36511	1.42E-06
1392547_at	AI716211	MGC105649	Hypothetical LOC302884	-2.31654	-4.98136	0.004157
1388300_at	AA892234	Mgst3	Microsomal glutathione S-transferase 3	-1.007	-2.00973	0.017898
1393836_at	BE097933	Mitf	Microphthalmia-associated transcription factor	-1.0956	-2.13702	0.010323
1368590_at	NM_080776	Mmp16	Matrix metallopeptidase 16	-1.01729	-2.02411	0.000829
1370301_at	U65656	Mmp2	Matrix metallopeptidase 2	-3.98031	-15.7831	0.000568
1382190_at	BF405725	Mrgprf	MAS-related GPR, member F	-3.60572	-12.1739	3.94E-07
1368441_at	NM_031658	Msln	Mesothelin	-1.36403	-2.57403	0.001477
1376648_at	BI275570	Mycn	V-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian)	-1.62357	-3.08136	0.002089
1368415_at	NM_012604	Myh3	Myosin, heavy chain 3, skeletal muscle, embryonic	-1.24039	-2.36262	0.003414
1387787_at	NM_012605	Mylpf	Myosin light chain, phosphorylatable, fast skeletal muscle	-2.12252	-4.35453	0.000567
1398655_at	AA955902	Myod1	Myogenic differentiation 1	-2.39143	-5.24677	9.65E-05
1373839_at	BG372386	Nope	Neighbor of Punc E11	-1.24338	-2.36752	0.009522
1371036_at	BG671431	Nrcam	Neuronal cell adhesion molecule	-1.60969	-3.05187	2.40E-06
1384112_at	BI289470	Nt5e	5' nucleotidase, ecto	-1.1205	-2.17423	0.004069
1392780_at	BF283270	Nxf7	nuclear RNA export factor 7	-3.39721	-10.5357	0.005424
1377497_at	BF419319	Oasl	2'-5'-oligoadenylate synthetase-like	-1.36533	-2.57636	2.34E-05
1369008_a_at	NM_053573	Olfm1	Olfactomedin 1	-3.58878	-12.0318	0.000169
1368940_at	NM_017255	P2ry2	Purinergic receptor P2Y, G-protein coupled 2	-1.21116	-2.31523	0.009814
1383273_a_at	AA956005	Pcbp3	Poly(rC) binding protein 3	-2.10678	-4.30729	4.21E-06
1383274_at	AA956005	Pcbp3	Poly(rC) binding protein 3	-1.8662	-3.64572	0.000552
1385116_at	BF386807	Pcdhb21	Protocadherin β 21	-1.13304	-2.19321	0.007625
1373368_at	BI279680	PCOLCE2	Procollagen C-endopeptidase enhancer 2	-1.805	-3.4943	7.61E-07
1368145_at	NM_013002	Pcp4	Purkinje cell protein 4	-4.28374	-19.4775	0.000401
1370941_at	AI232379	Pdgfra	Platelet derived growth factor receptor, α polypeptide	-1.62538	-3.08523	0.011837
1377100_at	AI172172	Pds5b	PDS5, regulator of cohesion maintenance, homolog B (S. cerevisiae)	-1.14971	-2.21869	1.40E-05
1388634_at	BI277505	Pgm1	Phosphoglucomutase 1	-1.66935	-3.18071	9.06E-09
1369473_at	NM_017033	Pgm1	Phosphoglucomutase 1	-1.50694	-2.84207	3.02E-06
1383749_at	AI112954	Phospho1	Phosphatase, orphan 1	-1.16307	-2.23934	0.003657
1370445_at	D88666	Pla1a	Phospholipase A1 member A	-1.35014	-2.54937	0.001836
1390190_at	BI293691	Plac1	Placenta-specific 1	-2.19087	-4.56582	0.000174
1384558_at	BI276313	Plac9	Placenta-specific 9	-1.07069	-2.10043	0.007797
1367800_at	NM_013151	Plat	Plasminogen activator, tissue	-1.15024	-2.21951	0.004677
1391187_at	BI303019	Ppl	Periplakin	-1.11393	-2.16434	0.000262
1368259_at	NM_017043	Ptgs1	Prostaglandin-endoperoxide synthase 1	-2.42323	-5.3637	3.32E-05
1381806_at	BF418208	Ptgs1	Prostaglandin-endoperoxide synthase 1	-1.06169	-2.08737	5.08E-05
1372084_at	AI104546	Ptp4a3	Protein tyrosine phosphatase 4a3	-1.0212	-2.02961	2.54E-05
1368350_at	NM_013080	Ptprz1	Protein tyrosine phosphatase, receptor-type, Z polypeptide 1	-1.04165	-2.05858	0.022268
1373646_at	BM384841	Rab15	RAB15, member RAS oncogene family	-1.17275	-2.2544	7.21E-05
1374035_at	BI296482	Rem2	RAS (RAD and GEM) like GTP binding 2	-1.00521	-2.00723	0.017185
1368080_at	NM_054008	Rgc32	Response gene to complement 32	-1.00087	-2.00121	0.032818
1392883_at	AI013730	RGD1305269	Similar to hypothetical protein	-1.04588	-2.06463	1.81E-05
1373226_at	BF400995	RGD1308019	Similar to hypothetical protein FLJ20245	-1.10844	-2.15613	0.015649
1381757_at	AA965058	RGD1309501	Hypothetical LOC305552	-1.28517	-2.43712	2.17E-05
1373596_at	AI230766	RGD1310423	Similar to hypothetical protein FLJ31737	-2.01177	-4.03277	6.86E-08
1398577_at	BI297744	RGD1310507	Similar to RIKEN cDNA 1300017J02	-1.72535	-3.30661	0.000821
1390397_at	BF413152	RGD1310753	Similar to chromosome 20 open reading frame 39	-2.10856	-4.31261	5.11E-05
1393191_at	BF554733	RGD1561205	Similar to RIKEN cDNA 2610200G18	-1.39361	-2.62736	0.000183
1376693_at	AA998964	RGD1563091	Similar to OEF2	-1.00045	-2.00063	0.047169
1395145_at	BF544481	Rgl1	Ral guanine nucleotide dissociation stimulator,-like 1	-1.5473	-2.9227	0.000329
1394472_at	BF282814	Rgl1	Ral guanine nucleotide dissociation stimulator,-like 1	-1.20268	-2.30166	0.005425
1391075_at	AI179271	Rgs17	Regulator of G-protein signaling 17	-1.50436	-2.83699	0.004883
1368373_at	NM_019343	Rgs7	Regulator of G-protein signaling 7	-3.29254	-9.79836	1.82E-05
1370142_at	NM_022175	Rhox5	Reproductive homeobox 5	-3.12793	-8.74178	2.44E-09
1383554_at	AW142796	Rnf128	Ring finger protein 128	-1.473	-2.77598	1.47E-05
1389735_at	BE107296	Rps6ka6	Ribosomal protein S6 kinase polypeptide 6	-1.18452	-2.27288	0.013943
1384707_at	AI600020	Scara5	Scavenger receptor class A, member 5 (putative)	-1.46379	-2.75833	9.19E-06
1392856_at	AI549470	Serf1	Small EDRK-rich factor 1	-1.52508	-2.87802	0.00071
1375084_at	BF419780	Serinc2	Serine incorporator 2	-1.64207	-3.12113	0.000591
1377034_at	BF411331	Serpinb1a	Serine (or cysteine) proteinase inhibitor, clade B, member 1a	-1.17504	-2.25799	8.45E-05
1369547_at	NM_130404	Serpinb7	Serine (or cysteine) peptidase inhibitor, clade B, member 7	-1.4094	-2.65626	0.001746
1393620_at	AI113325	Sesn3	Sestrin 3	-1.17758	-2.26198	0.001718
1390119_at	BF396602	Sfrp2	Secreted frizzled-related protein 2	-1.64679	-3.13135	0.011642
1367881_at	NM_013016	Sirpa	Signal-regulatory protein α	-1.88267	-3.68756	1.79E-06
1392789_at	BI296353	Slc25a36	Solute carrier family 25, member 36	-2.25714	-4.78042	4.14E-05
1372341_at	AI233213	Slc25a36	Solute carrier family 25, member 36	-2.07958	-4.22685	0.000633
1369237_at	NM_053996	Slc6a7	Solute carrier family 6 (neurotransmitter transporter, L-proline), member 7	-1.12269	-2.17753	0.00114
1368322_at	NM_012880	Sod3	Superoxide dismutase 3, extracellular	-2.20485	-4.61028	1.15E-05
1368254_a_at	AB049572	Sphk1	Sphingosine kinase 1	-1.09627	-2.13802	0.00473
1368655_at	NM_020074	Srgn	Serglycin	-2.51283	-5.70737	0.006656
1373146_at	AI716240	Ssx2ip	Synovial sarcoma, X breakpoint 2 interacting protein	-1.23239	-2.34956	0.001168
1387174_a_at	AB006007	Star	Steroidogenic acute regulatory protein	-3.29954	-9.84599	0.000194
1368406_at	NM_031558	Star	Steroidogenic acute regulatory protein	-2.92085	-7.57291	7.29E-07
1377672_at	BI300997	Sult1c2	Sulfotransferase family, cytosolic, 1C, member 2	-3.45192	-10.9429	3.19E-06
1369531_at	NM_133547	Sult1c2	Sulfotransferase family, cytosolic, 1C, member 2	-2.77927	-6.86504	2.07E-05
1369627_at	L10362	Sv2b	Synaptic vesicle glycoprotein 2b	-2.39744	-5.26866	0.000178
1385637_at	AI029494	Svep1	Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1	-1.08524	-2.12172	0.000482
1383686_at	BE111537	Syngr1	Synaptogyrin 1	-1.73204	-3.32197	0.00524
1384202_at	BI287326	Tesc	Tescalcin	-1.69209	-3.23124	0.004681
1371913_at	BG379319	Tgfbi	Transforming growth factor, β induced	-1.05453	-2.07704	0.023537
1369652_at	AI145313	Thy1	Thy-1 cell surface antigen	-3.93458	-15.2907	2.03E-07
1369651_at	NM_012673	Thy1	Thy-1 cell surface antigen	-3.67268	-12.7522	7.68E-08
1392980_at	AI716456	Tiam1	T-cell lymphoma invasion and metastasis 1	-1.56449	-2.95774	7.40E-06
1382222_at	BI293607	Tmem163	Transmembrane protein 163	-2.1182	-4.34153	8.73E-06
1376106_at	AI010157	Tmem178	Transmembrane protein 178	-3.28823	-9.76912	0.007054
1377554_at	BF394106	Tnfsf9	Tumor necrosis factor (ligand) superfamily, member 9	-1.95931	-3.88877	1.88E-05
1370332_at	AF159356	Unc13d	Unc-13 homolog D (C. elegans)	-2.36452	-5.1498	0.000313
1368474_at	NM_012889	Vcam1	Vascular cell adhesion molecule 1	-3.24587	-9.48643	1.90E-05
1388142_at	AA850991	Vcan	Versican	-1.54149	-2.91096	0.000288
1388054_a_at	AF072892	Vcan	Versican	-1.53261	-2.89308	0.000536
1371232_a_at	AF084544	Vcan	Versican	-1.47614	-2.78204	0.000764
1388265_x_at	AF084544	Vcan	Versican	-1.45114	-2.73423	0.001864
1389253_at	BI289085	Vnn1	Vanin 1	-1.15392	-2.22518	0.000172
1382283_at	BF283711	Wipf1	WAS/WASL interacting protein family, member 1	-1.01562	-2.02176	3.13E-05
1387227_at	NM_057192	Wipf1	WAS/WASL interacting protein family, member 1	-1.00508	-2.00706	8.16E-05
1389119_at	AI105018	Xirp1	Xin actin-binding repeat containing 1	-1.17137	-2.25226	9.90E-05
1372989_at	BI296586	Zdhhc14	Zinc finger, DHHC-type containing 14	-3.38007	-10.4112	1.23E-07

We further analyzed the pathways which were significantly enriched, using a P value ≤ 0.05 as a threshold. Here, we observed enrichment for signaling pathways like integrin-linked kinase, hepatic fibrosis/HSC activation and caveolar-mediated endocytosis, calcium, cAMP-mediated signaling, integrin, endothelin-1 for the upregulated genes (Figure 7A), and hepatic fibrosis/HSC activation, lipopolysaccharide (LPS)/IL-1-mediated inhibition of retinoid X receptor (RXR) function and nitrogen metabolism, and liver X receptor/RXR activation for the downregulated genes (Figure 7B).

Figure 7 Pathway analysis for the differentially expressed genes of miR-146a over-expressing clones. The charts depict the pathways affected by the (A) or (B) of genes upon stable transfection of miR-146a into hepatic stellate cell-2 cells. Only pathways with P≤ 0.05 are shown. ILK: Integrin-linked kinase; FGF: Fibroblast growth factor; IL: Interleukin; LPS: Lipopolysaccharide; LXR: Liver X receptor; RXR: Retinoid X receptor.

The most interesting finding was the robust upregulation of TIMP-3 mRNA (Supplementary Tables 1), verified by real-time PCR (Figure 8A), which is an inhibitor of the tumor necrosis factor-α converting enzyme[22], and has been proposed as a tumor suppressor. Similarly, pHSCs treated with miR-146a mimic also showed induction of TIMP-3 mRNA (Figure 8B).

Figure 8 Relative expression of tissue inhibitor of metalloproteinase-3 mRNA in rno-miR-146a-overexpressing hepatic stellate cell-2 cells. A: The graph depicts the relative changes in tissue inhibitor of metalloproteinase (TIMP)-3 mRNA of three rno-miR-146a-overexpressing clones detected by real-time polymerase chain reaction (PCR). The data represent the mean ± SE of two different passages for each clone (^aP≤ 0.005); B: Primary hepatic stellate cells were treated with 50 nmol/L miR-146a mimic, and the expression of TIMP-3 mRNA was analyzed by real-time PCR and expressed as fold change relative to mock controls. The data represent the mean ± SE of three independent experiments (^bP≤ 0.01). HSC: Hepatic stellate cell.

Time-dependent expression of different miRNAs during in vitro activation of pHSCs

The fact that miR-146a was not downregulated in in vivo activated pHSCs (CDE diet) prompted us to study the time-dependent expression of this miRNA during the in vitro activation of pHSCs, together with miR-26a, 29a and 214. The expression of miR-146a was indeed downregulated at day 3 already, and recovered subsequently until day 10. Although the miR-146a level at day 10 was still lower than that in quiescent pHSCs at day 0, there was still a 10-fold increase between day 3 and 10 (Figure 9A). In contrast, the expression of miR-26a and 29a did not change as dramatically from day 3 to day 10. We also noticed that miR-214 started to increase only from day 5 onwards (Figure 9A).

Figure 9 Time-dependent changes in the expression of different miRNAs during in vitro activation and miRNA mimic and inhibitor transfection of primary hepatic stellate cells. A: The relative changes in the expression level after 3, 5, 7 and 10 d of in vitro primary hepatic stellate cells (pHSCs) activation is shown for miR-146a, 26a, 29a and 214 (^aP≤ 0.05, one-way ANOVA). The data represent one of two independent experiments performed in triplicate; B: pHSCs were transfected with miR-146a, 26a, 29a mimics or miR-214 inhibitor or in combination. The control transfection consisted of control miRNAs for the mimic and/or inhibitor. The smooth muscle α-actin and ColI mRNA expression was analyzed as fold change relative to mock controls. The data represent the mean ± SE of two independent experiments, each performed in triplicate (^aP≤ 0.05, ^bP≤ 0.01, ^cP≤ 0.001). SMAA: Smooth muscle α-actin.

Regulation of SMAA and ColI transcripts in pHSCs by different miRNA mimics and inhibitor

In order to study the effect of different miRNA mimics or inhibitor on the in vitro activation process, we transfected 3-d in vitro activated pHSCs for 3 d with miR-146a, 26a, 29a mimics, miR-214 hairpin inhibitor or all combined. The impact on the HSC activation was followed using real-time PCR to study the changes on the mRNA levels of SMAA and ColI. The high efficiency of transfection was demonstrated by real-time PCR (Figure 10). We found moderate upregulation of the activation marker SMAA by miR-146a (Figure 9B); an observation seen also for the miR-146a-overexpressing clones (Figure 5E and cDNA microarray data). In fact, all cells transfected with the mimics, the inhibitor or combined showed an upwards trend for SMAA mRNA compared to the mock control, although the level did not always change significantly (Figure 9B). For the ColI expression, we noted again an increase caused by miR-146a mimic (not significant) and a decrease by miR-26a, 29a mimic and miR-214 inhibitor. The quadruple transfection led to a suppression of ColI mRNA (Figure 9B).

Figure 10 Transfection of primary hepatic stellate cells with different miRNA mimics and inhibitor. The miRNA expression was analyzed as fold change relative to mock transfected primary hepatic stellate cells (pHSCs). Shown are data for the transfection of miR-146a mimic and miR-214 inhibitor with respective control (A), for the transfection of miR-26a and miR-29a mimic with respective control (B) and for the quadruple transfection of miR-146a, miR-26a, miR-29a mimic and miR-214 inhibitor (C) (^aP≤ 0.05, ^bP≤ 0.005).

DISCUSSION

The aim of this study was to gain a deeper insight into the regulation of miRNAs during the activation process of pHSCs, as well as the influence of up- or downregulation of miRNAs on the gene expression and activation of HSCs.

The expression analysis of miRNAs between quiescent and in vitro activated pHSCs yielded a number of induced and suppressed miRNAs (Table 1), some of which (miR-143, 16, 122, 146a, 92b, 126) confirmed the findings of Guo et al[23]. On the other hand, there were some differences in the regulation of certain miRNAs (miR-328, 207), which could be attributed to the dynamic nature of miRNA regulation and the different use of quiescent pHSCs (day 0 vs day 2).

When evaluating the miRNAs expression profile of the in vitro and in vivo activated pHSCs, a clear distinction was seen in the expression of miR-16, 26a, 29a, 125b, 146a and 150 (compare Figure 3A and B); a phenomenon which could be explained by the distinct HSC activation process. This has been shown at the gene expression level by De Minicis et al[24]. The in vivo activation was performed over a period of 4 wk, whereas the in vitro activation was monitored over 10 d, which could also account for some differences in the miRNA expression, assuming a dynamic regulation.

On the other hand, we found that certain miRNAs (let-7b, 7c and miR-214) were regulated in the same way during in vitro and in vivo activation of pHSCs. It also became clear to us that miR-214 could be a potential candidate for a diagnostic approach, because this miRNA always shows robust upregulation.

Pathway analysis of the miRNA microarray data was performed to obtain information on signaling cascades involving predicted targets of the differentially regulated miRNAs in in vitro activated pHSCs (Figure 4A and B). NO and ROS are known to play a role in the activation process and apoptosis of HSCs[25,26]. The pathways for AMPK, ERK/MAPK, PTEN and TGF-β are also implicated in HSC activation[27-30]. We noticed that a number of pathways were present in the charts for both up- and downregulated miRNAs, which could denote the complexity of regulated targets by each single miRNA, and possibly a cooperative effect between up- and downregulated miRNAs.

A number of publications have shown that miR-146a is involved in inflammatory diseases, regulation of the immune response and NF-κB[19,31-33]. In the early events of liver fibrosis, the activation of HSCs is in part driven by the hepatic inflammatory process, during which different cytokines are secreted by various liver cells, like Kupffer cells, endothelial cells and hepatocytes[34,35]. Involvement of NF-κB in HSC activation has also been shown in several research papers[36,37]. Therefore, we overexpressed miR-146a in an HSC cell line and observed changes consistent with the findings from Bhaumik et al[19]. The detected increase in the NF-κB transcript (Figure 5E) could be explained by a feedback mechanism to the reduced nuclear activity, which leads to the upregulation of the mRNA.

Cox-2 is inducible in activated HSCs by various stimuli and is thought to regulate proliferation[21]. Others have shown that the inhibition of this enzyme has a beneficial antifibrotic effect[20,38,39]. The seemingly discrepant findings of the protein (lower) and transcript (elevated) level for Cox-2 in the miR-146a-overexpressing HSCs (Figure 5D and E) hint at independent pathways for the regulation of Cox-2. These pathways have been shown for intestinal myofibroblasts[40] and during ischemic injury of ileal mucosa[41]. Lasa et al[42] and others have shown that the p38 MAPK signaling cascade is able to stabilize the Cox-2 mRNA[43], which could also explain an elevated transcript level. We also cannot exclude that other mechanisms could be involved in stabilizing the Cox-2 mRNA and/or a regulation of Cox-2 by other miRNAs like miR-26a or 143, which are also present in the cell line HSC-2 and for which Cox-2 is a predicted target.

In contrast, the IL-6 mRNA, another molecule regulated by NF-κB, was downregulated (Figure 5E). This observation implies that IL-6 regulation in HSCs is more tightly associated with NF-κB than that of Cox-2.

We were also interested to know whether downregulation of the NF-κB DNA binding activity triggered by miR-146a overexpression could facilitate a feedback loop in HSCs; a notion supported by the fact that the promoter region of miR-146a contains a number of NF-κB binding sites[18]. As expected, a reduction in the NF-κB DNA binding activity (Figure 6B) leads to a decrease in miR-146a (Figure 6C). The observed upregulation of Cox-2 protein (Figure 6D) was somewhat surprising and again substantiated the speculation that other pathways such as p38 MAPK, C-Jun N-terminal kinase and ERK could participate in the regulation of Cox-2 in HSCs[40,44,45].

The microarray analysis revealed that the transcriptome changes caused by miR-146a overexpression are complex and numerous pathways are affected (Figure 7, Supplementary Tables 1 and 2). We found that several DEGs coincided with data from earlier publications on HSC activation[24,46], suggesting that a number of genes affected by miR-146a overexpression are also involved in the activation process. Pathway analysis of the DEGs (Figure 7) confirms a link between miR-146a and inflammation (LPS/IL-1 mediated inhibition of RXR function, eicosanoid signaling, nitrogen metabolism and NRF2-mediated oxidative stress response pathways). That the miR-146a overexpression in HSC-2 cells leads to changes in the pathway called hepatic fibrosis/HSC activation emphasizes that these changes are specific for the HSCs. The upregulation of TIMP-3 (Supplementary Table 1 and Figure 8) again emphasizes the involvement of miR-146a in inflammatory processes and immunity, by linking it to the TNFα activity[47].

We noticed a robust downregulation of miR-146a during in vitro, but a missing regulation of miR-146a during in vivo activation of pHSCs (CDE diet). We hypothesized that there is a dynamic component in the regulation of miR-146a. We effectively found that there is a time-dependent regulation of miR-146a over 10 d of in vitro activation of pHSCs. From an in vivo perspective, it could be a possibility that miR-146a is decreased following the first insult to the liver, but reaches almost a normal level during the developing fibrosis, as seen for the in vivo activated pHSCs (CDE diet). The mechanism behind this miR-146a regulation is not clear, but the involvement of different transcription factors [NF-IL6, interferon regulatory factor (IRF 3/7)] binding to its promoter region is conceivable[18].

The dynamic nature of miRNA regulation during the in vitro activation of pHSCs could also partially explain the differences in the expression pattern of the miRNAs in vitro and in vivo. The dynamic nature of miRNA expression has been shown for the T-cell development[48], and it makes sense if we consider the multitude of effects a single miRNA can have due to the imperfect complementarity to its target sequence.

The in vivo targets of a miRNA treatment are pHSCs, therefore, we assessed the effects of several miRNAs mimics (miR-26a, 29a, 146a) and inhibitor (miR-214) on the activation state of pHSCs. The transfection with a combination of all mimics and inhibitor was performed so as to examine possible cooperative effects between different miRNAs, as a first step to understand the cooperativity of miRNA expression changes during HSC activation. The miR-26a, 29a mimics and miR-214 inhibitor showed a significant suppression of the ColI mRNA (Figure 9B). This is somewhat surprising because even though a number of collagens are predicted targets for miR-26a and 29a, none has a perfect binding site, which would explain regulation by mRNA degradation. Therefore, we conclude that the mechanism by which miR-26a, 29a and 214 downregulate the ColI mRNA is indirect, as also suggested by van Rooij et al[49] for miR-29a. The downregulation of ColI by the quadruple transfection shows some synergistic effect between the miRNAs.

Our findings showed the differential regulation of miRNAs in in vitro and in vivo activation of pHSCs, and particularly, the involvement of miR-26a, 29a and 214 in the regulation of ColI mRNA. Moreover, miR-146a overexpression or treatment with miR-146a mimic upregulates TIMP-3 mRNA, which suggests an association between miR-146a, TNFα activity and inflammation. In conclusion, our observations help build a global picture of the miRNA regulation during HSC activation in vitro and in vivo, and may have important implications when considering a therapeutic approach for treating liver fibrosis using miRNAs.

COMMENTS

Background

miRNAs are a relatively new and exciting tool to control the expression of multiple genes. During liver injury and subsequent wound healing involving hepatic stellate cells (HSCs), complex regulatory processes occur and have to be tightly regulated in this cell type. miRNAs could be one tool to control these processes, and therefore, it is of interest to the research community to gain information about the expression of miRNAs during liver fibrosis in HSCs.

Research frontiers

Liver fibrosis and subsequently cirrhosis are common outcomes of chronic injuries to the liver. HSCs are involved in liver fibrosis and repair. The tools for the treatment of liver fibrosis are limited and are still under development. In this study, the authors aimed to gain information for the possible role of miRNAs in liver fibrosis and whether they could become a future tool to develop a treatment for liver fibrosis by addressing the changes in HSCs.

Innovations and breakthroughs

Different publications have analyzed the miRNA expression in HSCs in vitro and studied the effect of various differentially regulated miRNAs in HSCs. The authors analyzed the miRNA expression in an in vivo model of hepatic fibrosis, namely choline-deficient ethionine supplemented diet. Furthermore, they studied the transcriptome changes upon overexpression of miR-146a and found that, in particular, tissue inhibitor of metalloproteinase-3 showed robust up-regulation, a hitherto unreported effect, which emphasizes its involvement in inflammation. Another important finding was the dynamics of miRNA regulation during the in vitro activation of HSCs.

Applications

miRNAs are becoming a promising tool for the regulation of gene expression. In order to use this tool, it is necessary to understand the role and regulation of the targeted miRNA. In this study, the authors describe the dynamic regulation of specific miRNAs. The results of this study show clearly that the use of miRNAs as target molecules will have to take this dynamic component into consideration. The same is valid for the use of miRNAs as therapeutic agents.

Terminology

miRNAs are small non-coding RNAs that are about 23 nucleotides long. The versatility of miRNAs depends on the imperfect binding (seed region) to the 3’-UTR of mRNAs. This imperfect binding results in many different binding partners. The regulation by miRNAs leads to a translational repression and/or mRNA destabilization.

Peer review

The field of miRNA research as well as HSC activation mechanisms are very up to date and important areas of research, in order to find new strategies against liver fibrosis. The methods used are comprehensive and convincing. In all, the study was fairly well conducted and interesting.