Original Article
Copyright ©2011 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Jun 14, 2011; 17(22): 2748-2773
Published online Jun 14, 2011. doi: 10.3748/wjg.v17.i22.2748
miRNA studies in in vitro and in vivo activated hepatic stellate cells
Gunter Maubach, Michelle Chin Chia Lim, Jinmiao Chen, Henry Yang, Lang Zhuo
Gunter Maubach, Michelle Chin Chia Lim, Lang Zhuo, Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos #04-01, Singapore 138669, Singapore
Gunter Maubach, Institute of Experimental Internal Medicine, Leipziger Strasse 44, Magdeburg 39120, Germany
Jinmiao Chen, Henry Yang, Bioinformatics Lab, Singapore Immunology Network, 8A Biomedical Grove, Singapore 138648, Singapore
Author contributions: Maubach G was involved in the conceptualization of the study, the design and carrying out of the experiments, and writing of the manuscript; Lim MCC performed the experiments and was also involved in editing the manuscript; Chen J and Yang H performed the analysis of the microarray data and edited the manuscript; Zhuo L engaged in the design of the study and writing of the manuscript.
Supported by Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore)
Correspondence to: Dr. Lang Zhuo, Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos #04-01, Singapore 138669, Singapore. lzhuo@ibn.a-star.edu.sg
Telephone: +65-68247114 Fax: +65-64789080
Received: May 19, 2010
Revised: September 14, 2010
Accepted: September 21, 2010
Published online: June 14, 2011

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


Citation: Maubach G, Lim MCC, Chen J, Yang H, Zhuo L. miRNA studies in in vitro and in vivo activated hepatic stellate cells. World J Gastroenterol 2011; 17(22): 2748-2773
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
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
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 nameFold changeP value
Upregulated compared to day 0
rno-let-7b4.700.0242
rno-let-7c3.750.0236
rno-let-7e2.770.0340
rno-miR-125b11.980.0113
rno-miR-1321.970.0184
rno-miR-14317.050.0014
rno-miR-1452.290.0483
rno-miR-1523.010.0255
rno-miR-199a3.460.0415
rno-miR-215.730.0142
rno-miR-2102.340.0186
rno-miR-21418.440.0011
rno-miR-223.110.0392
rno-miR-22110.090.0007
rno-miR-2222.670.0317
rno-miR-318.910.0013
Downregulated compared to day 0
rno-let-7f-2.170.0327
rno-miR-10a-3.530.0417
rno-miR-122a-349.630.00002
rno-miR-125a-2.360.0474
rno-miR-126-170.320.0003
rno-miR-146a-16.830.0352
rno-miR-150-9.810.0325
rno-miR-151*-3.720.0345
rno-miR-161-4.380.0366
rno-miR-181a1-4.500.0346
rno-miR-192-6.080.0206
rno-miR-194-6.080.0168
rno-miR-195-14.500.0130
rno-miR-207-1.930.0449
rno-miR-26a-5.170.0163
rno-miR-26b-4.810.0300
rno-miR-296-1.930.0292
rno-miR-29a1-2.380.0644
rno-miR-30a-5p-5.350.0327
rno-miR-30b-10.510.0075
rno-miR-30c-9.690.0138
rno-miR-30d-8.680.0093
rno-miR-335-3.740.0500
rno-miR-422b1-8.490.0455
rno-miR-483-2.490.0451
rno-miR-99a-2.970.0383
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
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), p70S6K, 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
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
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
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 IDRepresentative public IDGene symbolGene titleLog2Fold changeP-value
1383164_atAW5243665.06585433.494553.49E-05
1379902_atBE1081704.56845323.726920.000154
1382211_atAI6025424.40170521.13710.008197
1394456_atAW5257223.90581814.988850.000414
1373740_atAA8513853.77760313.714240.002694
1393437_atAW1426083.41776510.686853.36E-05
1389579_atBI2843723.41251110.648010.010102
1374065_atBG3789203.32719510.036580.000137
1393018_atAI0719843.32374310.012590.000201
1392105_atAW5275333.229469.3791660.005625
1373062_atBM3886503.1749099.0311430.00033
1373776_atAI4063413.1274838.739093.23E-06
1378457_atAI1794502.9930317.961450.001547
1391428_atAI6391622.9590797.7762740.003728
1393314_atBI2898402.8267217.09468.14E-05
1379382_atAI1448652.8103397.0144920.01755
1391481_atBE1044242.7427236.6933240.000235
1376435_atBI3033402.6882296.4452170.00028
1395327_atAW5223412.6514766.2830980.017329
1371506_atAA8912072.6116236.1119110.013546
1394833_atBE1209302.6047066.0826763.53E-06
1375230_atAA8001922.5129365.7078060.000375
1374811_atAA8587052.5052325.6774060.035302
1377934_atBF3872892.4616915.5086190.005853
1383240_atBE1107532.3740725.1840230.004602
1372921_atAI0732192.3557295.1185290.036703
1376800_atAA8924962.3210594.996990.000124
1384137_atAI0303182.2654114.8079150.000711
1398597_atAI0446992.2001114.5951480.00022
1393782_atBF3967902.190074.5632750.003901
1382330_atBE1168382.1887254.5590230.000179
1398657_atAI0458962.1644824.4830535.91E-05
1376617_atBE1074822.1093464.3149570.001578
1378111_atAI5760022.1045234.3005550.023
1379936_atAA8751322.0966454.2771360.005892
1377946_atBF4200432.0717664.204013.19E-05
1377675_atAI1777432.0631364.1789360.005112
1380940_atBF4026032.046824.1319420.007683
1381335_atBE3496582.0467254.1316697.30E-05
1374971_atAA8189542.0361714.1015560.000603
1397781_atBF4147511.9683853.9132990.004705
1391841_atBE1035371.9480963.8586490.003749
1388546_atAI0133281.9185443.7804150.005524
1379444_atBF2836941.8361663.5705990.00094
1392627_x_atBI2821141.8288183.5524599.63E-05
1374432_atBE1182511.8204233.5318470.023594
1389239_atBM3843771.8200563.5309493.85E-06
1394578_atBI2997611.7831173.4416890.019487
1376734_atBI2790301.7760633.4249030.021384
1392820_atBI2850641.7631193.3943120.000366
1382212_atAI3852011.7618833.3914050.008303
1374273_atBG6654331.7403163.3410830.001848
1390471_atBM3834111.7321123.3221380.003308
1396009_atBE1082581.7264463.3091172.68E-05
1382294_atAI5761111.6984523.2455260.004164
1390459_atBG6702471.6838323.2128020.000503
1381577_atAI1701311.6747063.1925449.98E-05
1388720_atBM3907131.6691883.1803560.000841
1371394_x_atBG6648271.6608463.1620180.000385
1379719_atAI4083861.6298393.0947848.76E-06
1392876_atBG3750981.5972973.0257580.000425
1377881_atAA9970271.5832952.9965340.000249
1392924_atBG3715911.5739842.9772582.15E-05
1378152_atAI1703491.5721572.973490.000818
1390987_atAI4068581.5396112.9071610.005124
1390300_atBM3836351.5393172.9065680.001988
1381996_atBG6667121.5247932.8774540.007289
1392893_a_atAA9262391.4926442.8140421.82E-05
1378462_atBE1073961.4844392.7980830.027693
1391028_atAI5111261.4805972.7906410.002716
1385978_atAI0727881.480072.7896220.01557
1392813_atAI5489941.4727772.7755560.028907
1379903_atAI0598531.4564592.7443390.005294
1382291_atAI4543321.4472512.7268790.021889
1396539_atBE1192211.4446272.7219240.00325
1395381_atBF5422391.4284282.6915320.018331
1394709_atAI4069671.4267542.6884120.013879
1378780_atBF4103251.420942.6775980.009832
1374172_atAI0108831.4072562.6523210.005074
1377551_atBE1185801.4069212.6517060.000613
1378172_atAI0081191.3902072.6211649.29E-05
1389744_atAW5271941.3717562.5878540.029149
1393728_atAA9645411.3619782.5703730.00664
1389284_atBI2757471.3525252.5535860.009308
1377309_atAA9630851.3445392.539490.000217
1394012_atBI3039331.3250722.5054539.03E-06
1396886_atBF3878691.3196442.4960450.000671
1389172_atAI1793911.3187252.4944565.03E-06
1376011_atAI4113591.315432.4887650.013276
1374171_atAI1705071.3139562.4862240.003317
1382802_x_atAW9208281.3131692.4848670.0032
1395211_s_atBE1185571.2943662.4526920.014857
1391727_atBG6627101.2943392.4526460.001351
1373079_atBI2964271.2919692.4486210.040034
1375005_atBF4038241.2917272.448210.001959
1382174_atAI2279961.2906732.4464210.006458
1380088_atAW5330211.2841682.4354155.37E-05
1383910_atBF3982201.280892.4298890.003117
1381498_atAA9561161.2261832.3394730.001176
1391936_a_atBI2891101.2125112.3174060.041362
1379089_atBM3828381.208372.3107640.000681
1372993_atBI2996211.2044922.3045610.000924
1390671_atAI0446661.2034242.3028560.001722
1394916_atAW5267141.2027242.3017390.001978
1389397_atAI2340121.2024262.3012630.000195
1376637_atAI1024011.1980022.2942170.027098
1386552_atBF2840271.1971452.2928560.003348
1377792_atAW5248911.1950752.2895680.013872
1384952_atAI0289681.1844532.2727720.001744
1376768_atBM3868071.1826822.2699840.008644
1389127_atBF5529081.178772.2638370.001451
1392140_atBF4195841.1741262.2565610.010974
1375707_atAA8179931.1716682.252720.017511
1377994_atAI5012371.1659862.2438662.56E-05
1372027_atAI0097131.1650362.2423880.004861
1374290_atAI4081911.1618342.2374160.000514
1372820_atBE1091021.1571672.230196.76E-05
1395629_atBE1053361.1527542.2233790.001122
1373628_atAA8183421.1487022.2171430.003268
1383697_atAW5309051.147532.2153420.000199
1384269_atBF3868871.1449962.2114550.039498
1379733_atBF3964741.1443262.2104280.000479
1379682_atBI2816681.1380582.2008460.00129
1393334_atAW5284481.1349572.196120.000111
1372583_atAI0090941.1348342.1959320.000776
1390193_atBF3898841.1323712.1921880.007448
1373114_atAI4084421.1320062.1916320.026188
1395586_atBF5459301.1307562.1897350.009671
1389085_atBI2963591.1220112.1765020.033565
1378898_atBE1092931.1171042.1691110.018502
1384433_atAI0721531.1160052.1674590.008374
1392778_atAA8916341.1123872.162030.000734
1372515_atBI2811771.1097942.1581480.000373
1394727_atAI4079421.1042672.1498960.000884
1391617_atAI1711031.1032782.1484220.00173
1384680_atAA9243361.1031742.1482681.20E-05
1374246_atBF4023921.1008072.1447460.001207
1395350_atAW9191901.1000622.143640.02672
1397343_atBE1132581.0988152.1417870.000702
1384051_atBF3900661.0968842.1389230.00037
1381654_atBF3986371.0869822.1242920.008757
1392619_atBE1181071.0813792.1160580.010153
1376747_atBE1070751.0795542.1133820.014697
1380763_atAI1011941.0784762.1118040.008553
1393653_atBM3848311.0772412.1099970.029779
1379844_atAW5310721.0739842.1052390.00402
1394948_atBI3035271.0733842.1043640.028955
1393911_atAI5023001.0699422.0993490.001054
1378006_atAI2338321.0691452.098190.00023
1379451_atAI5490811.0691222.0981550.025226
1391643_atBI2907581.0658582.0934150.009922
1390136_atBE1092741.0622742.088220.003245
1389256_atBG3812561.0614092.0869690.02736
1376465_atBI2952401.0558422.0789310.025628
1390515_atAA9983831.052462.0740630.003395
1390205_atBE1088761.0521832.0736650.000145
1374728_atBG6717861.0506052.0713990.043671
1394883_atAI1796161.0504052.0711110.038398
1372104_atBF2890021.0437452.0615730.000585
1379252_atAW5228331.0415292.0584080.000451
1377151_atAI1028331.0402282.0565530.00364
1377880_atAI1706331.0385062.0540990.00023
1389908_atBE1071671.0352232.0494310.01567
1373082_atAA8937431.0339042.0475580.017691
1373537_atBE1131751.0283072.0396290.005797
1392578_atAI0708751.0210812.0294390.01047
1377705_atBF5499711.0199222.0278090.001107
1371854_atBG3744511.0197592.027580.012677
1375647_atBG6719431.0061592.0085560.017266
1385903_atAA8596271.0043382.0060230.000426
1398265_atNM_013040Abcc9ATP-binding cassette, sub-family C (CFTR/MRP), member 91.437832.709130.001785
1387287_a_atD83598Abcc9ATP-binding cassette, sub-family C (CFTR/MRP), member 91.2230922.3344650.004574
1397375_atBM384537Acsl5Acyl-CoA synthetase long-chain family member 51.1533182.2242490.01568
1386926_atNM_053607Acsl5Acyl-CoA synthetase long-chain family member 51.0535922.0756920.015907
1370857_atBI282702Acta2Smooth muscle α-actin2.2295144.689760.000129
1398294_atNM_031005Actn1Actinin, α 11.1319252.1915090.001329
1368223_atNM_024400Adamts1ADAM metallopeptidase with thrombospondin type 1 motif, 12.0485654.1369420.028144
1376481_atBF416285Adamts9A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 92.3926345.2511540.000223
1374535_atBI283881Afap1l2Actin filament associated protein 1-like 22.2030324.604460.000634
1368869_atBG663107Akap12A kinase (PRKA) anchor protein 121.5297132.8872840.019479
1368868_atNM_057103Akap12A kinase (PRKA) anchor protein 121.0104752.0145750.013205
1387493_atNM_133515Akap5A kinase (PRKA) anchor protein 51.5695152.9680490.003304
1370043_atNM_031753AlcamActivated leukocyte cell adhesion molecule1.6038633.0395610.010096
1383469_atBG377269Aldh1a3Aldehyde dehydrogenase 1 family, member A31.1168472.1687250.00291
1370638_atAF069525Ank3Ankyrin 3, epithelial1.8184413.5269980.00833
1367664_atNM_013220Ankrd1Ankyrin repeat domain 1 (cardiac muscle)1.6689733.1798810.020907
1367665_atL81174Ankrd1Ankyrin repeat domain 1 (cardiac muscle)1.5035012.83530.035713
1372069_atBF284716Ankrd15Ankyrin repeat domain 151.3336182.5203390.003331
1367974_atNM_012823Anxa3Annexin A33.37217610.354437.22E-05
1367975_atBF283732Anxa3Annexin A31.9147773.7705540.000113
1395313_s_atAI179982Anxa3Annexin A31.7171913.2879570.000365
1373654_atBM389254Anxa8Annexin A81.142592.207770.000179
1392815_atBE114489Arap2ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 21.5010882.8305620.000231
1387018_atNM_053770Argbp2Arg/Abl-interacting protein ArgBP21.5133782.8547780.039786
1373315_atAI176425Arnt2Aryl hydrocarbon receptor nuclear translocator 22.0411254.1156620.023949
1378134_atBI291629Atp8b1ATPase, Class I, type 8B, member 11.0617252.0874267.68E-05
1368485_atNM_024401AvilAdvillin2.1215684.3516670.012237
1370823_atAF387513BambiBMP and activin membrane-bound inhibitor, homolog (Xenopus laevis)1.5260892.8800410.003285
1372613_atAI232784Bdh23-hydroxybutyrate dehydrogenase, type 21.1774342.2617420.013257
1387232_atNM_012827Bmp4Bone morphogenetic protein 43.0745388.4241890.000456
1380459_atAI555023Btbd14aBTB (POZ) domain containing 14A1.357672.5627093.53E-06
1386995_atBI288701Btg2B-cell translocation gene 2, anti-proliferative1.2280552.3425090.004008
1377086_atAI233530C1qtnf3C1q and tumor necrosis factor related protein 32.5131685.7087230.002654
1376657_atBE117767Cadm1Cell adhesion molecule 11.040912.0575250.001137
1393452_atBM391835Car9Carbonic anhydrase 92.223634.6706710.00011
1390101_atAI170609Ccdc107Coiled-coil domain containing 1071.1529652.2237040.000166
1398827_atNM_013087Cd81Cd81 molecule3.90109514.939860.00033
1388936_atBI296340Cdh11Cadherin 113.49367911.264250.016743
1370371_a_atU23056Ceacam1 /// Ceacam10Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) /// carcinoembryonic antigen-related cell adhesion molecule 102.3507285.1008140.001261
1393142_atBF562621Cep70Centrosomal protein 70 kDa1.4143082.6653185.96E-05
1368675_atNM_032084Chn2Chimerin (chimaerin) 21.1112482.1603240.00013
1389368_atAW253242Cnksr3Cnksr family member 31.1156932.1669910.002136
1376868_atBM389293Cobll1Cobl-like 12.8078577.0024350.002758
1372439_atAI176393Col4a1Collagen, type IV, α 13.58705812.017454.28E-05
1373245_atBE111752Col4a1Collagen, type IV, α 13.1486318.8681349.98E-05
1388494_atBI281705Col4a2Collagen, type IV, α 22.8005956.9672760.000478
1393891_atBE128699Col8a1Collagen, type VIII, α 11.151472.2214010.003468
1367782_atNM_012812Cox6a2Cytochrome c oxidase, subunit VIa, polypeptide 22.2457344.7427850.001312
1386921_atNM_013128CpeCarboxypeptidase E2.5648235.9168230.021482
1382037_atAI600057Crim1Cysteine rich transmembrane BMP regulator 1 (chordin like)2.0410474.115440.00285
1391448_atBI289620Crim1Cysteine rich transmembrane BMP regulator 1 (chordin like)1.91563.7727060.000387
1398622_atAI703807Crim1Cysteine rich transmembrane BMP regulator 1 (chordin like)1.808393.5025120.000527
1376457_atAI175861Crispld2Cysteine-rich secretory protein LCCL domain containing 21.377772.5986640.018183
1387922_atAF109674Crispld2Cysteine-rich secretory protein LCCL domain containing 21.1955182.290270.003604
1368059_atNM_053955CrymCrystallin, mu1.2884022.4425740.000395
1383590_atAA963863Csgalnact1Chondroitin sulfate N-acetylgalactosaminyltransferase 13.3040079.8765470.022018
1370057_atNM_017148Csrp1Cysteine and glycine-rich protein 11.0304072.0426010.001628
1388583_atBF283398Cxcl12Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)2.0921114.2637160.042845
1387655_atAF189724Cxcl12Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)1.6431363.1234410.028532
1369633_atAI171777Cxcl12Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)1.5674762.9638580.045339
1368290_atNM_031327Cyr61Cysteine-rich, angiogenic inducer, 611.2616512.3976990.022554
1371436_atAI176924Ddah2Dimethylarginine dimethylaminohydrolase 21.7101733.2720010.006371
1368013_atNM_080399Ddit4lDNA-damage-inducible transcript 4-like1.0562352.0794980.002531
1389894_atBF399476Dlc1Deleted in liver cancer 11.0683362.0970130.002194
1377835_atBM390876Dock8Dedicator of cytokinesis 82.2303194.6923770.005526
1388506_atAW144509DspDesmoplakin1.8307213.5571489.48E-05
1368146_atU02553Dusp1Dual specificity phosphatase 11.5602992.9491490.003438
1368949_atNM_053820Ebf1Early B-cell factor 11.3164662.4905530.000315
1369519_atNM_012548Edn1Endothelin 11.3660232.5775910.009786
1368541_atNM_053719EmbEmbigin1.0640322.0907670.031817
1377752_atBE112998Emp2Epithelial membrane protein 21.3925992.6255120.000189
1373617_atAA818807Emp2Epithelial membrane protein 21.3472382.5442460.000131
1377311_atAI045616Emx2Empty spiracles homeobox 21.0320952.0449920.001019
1369096_atNM_134331Epha7Eph receptor A71.4277982.6903580.001357
1385788_atAW534949Ephb3Eph receptor B31.3216652.4995440.000783
1369182_atNM_013057F3Coagulation factor III (thromboplastin, tissue factor)2.9451697.7016578.99E-05
1377940_atBF398271Fam101bFamily with sequence similarity 101, member B1.0691872.098250.047471
1384507_atAA817708Fam105aFamily with sequence similarity 105, member A1.1694052.249190.00595
1389146_atBF283267Fam107bFamily with sequence similarity 107, member B1.016682.0232570.000409
1393910_atBF563961Fam13a1Family with sequence similarity 13, member A14.49028422.475530.003831
1379625_atBG664461Fam164aFamily with sequence similarity 164, member A1.5504212.9290260.001732
1384648_atAA963844Fam164aFamily with sequence similarity 164, member A1.2192812.3283070.000456
1391944_atBI296237Fam184a /// RGD1560557Family with sequence similarity 184, member A /// similar to minichromosome maintenance protein 8 isoform 11.268562.4092090.001168
1373286_atAA875261Fblim1Filamin binding LIM protein 11.0559772.0791260.000141
1376500_atAI639044Fbxo23F-box only protein 231.0642182.0910360.001619
1386614_atBG671466Fbxo23F-box only protein 231.0169122.0235830.010871
1368114_atNM_053428Fgf13Fibroblast growth factor 133.65462312.593640.004204
1370106_atNM_019199Fgf18Fibroblast growth factor 182.489425.6155220.016971
1369313_atNM_031677Fhl2Four and a half LIM domains 21.4917132.8122270.011052
1371951_atAA800031Fhl2Four and a half LIM domains 21.3191642.4952150.010936
1372825_atBI290551Fnbp1Formin binding protein 11.4818082.7929850.003539
1376784_atBI274481Fnbp1Formin binding protein 11.4075722.6529030.007946
1369471_atNM_138914Fnbp1Formin binding protein 11.1424822.2076040.012674
1377342_s_atBE105446Fnbp1Formin binding protein 11.0439052.0618010.023433
1370829_atM69056FntbFarnesyltransferase, CAAX box, β2.001254.0034680.001272
1368711_atNM_012743Foxa2Forkhead box A23.1255138.7271650.00498
1380387_atBE105492Foxp2Forkhead box P21.8081793.5019990.000372
1383721_atAI556075Fzd8Frizzled homolog 8 (Drosophila)1.8916683.7106418.54E-05
1372016_atBI287978Gadd45bGrowth arrest and DNA-damage-inducible, β1.2871022.4403740.007019
1369735_atNM_057100Gas6Growth arrest specific 62.0309064.0866140.000694
1367627_atNM_031031GatmGlycine amidinotransferase (L-arginine:glycine amidinotransferase)1.7049893.2602640.001931
1390557_atBF394809GcaGrancalcin1.2948022.4534330.01519
1379031_atBM390697GcaGrancalcin1.1328242.1928760.01681
1374903_atAI234819Gcnt2Glucosaminyl (N-acetyl) transferase 2, I-branching enzyme1.7803913.4351920.00076
1370375_atJ05499Gls2Glutaminase 2 (liver, mitochondrial)1.3818862.6060880.000222
1392888_atAI071251Gpc4Glypican 41.1498332.2188830.017197
1373773_atBF394166Gpm6aGlycoprotein m6a1.492992.8147170.004487
1370389_atAB036421Gpm6bGlycoprotein m6b1.7296873.3165580.041126
1382955_atBI284296Gpr126G protein-coupled receptor 1261.6919143.2308510.002139
1373693_atBF414143Gprc5cG protein-coupled receptor, family C, group 5, member C1.1725962.2541690.000245
1368618_atNM_031623Grb14Growth factor receptor bound protein 142.2630624.8000930.001165
1368401_atM85035Gria2Glutamate receptor, ionotropic, AMPA 21.1855772.2745430.038866
1383897_atBE117477H2afy2H2A histone family, member Y21.4674482.7653220.003118
1384541_atBM391441Hapln1Hyaluronan and proteoglycan link protein 12.7202246.5897530.021358
1370125_atNM_019189Hapln1Hyaluronan and proteoglycan link protein 12.5323685.7852060.010848
1368983_atNM_012945HbegfHeparin-binding EGF-like growth factor1.0579622.0819880.018982
1376867_atBE095833Hspc159Galectin-related protein2.8212427.0677070.000688
1373515_atBI275737Hspc159Galectin-related protein2.640046.2334870.000522
1387028_a_atM86708Id1Inhibitor of DNA binding 11.0978222.1403130.005605
1390507_atBI296097Isg20Interferon stimulated exonuclease 202.2729054.8329530.040035
1394824_atBF398348Itga11Integrin, α 111.8857283.6953950.000279
1393558_atAI137931Itga6Integrin, α 61.3411122.5334640.001146
1382439_atAI070686Itgb6Integrin, β 61.316972.4914240.005869
1387907_atJ05510Itpr1Inositol 1,4,5-triphosphate receptor, type 11.7092043.2698030.001386
1368725_atNM_019147Jag1Jagged 11.0458782.0646220.010981
1398124_atAI071356Jazf1JAZF zinc finger 11.3251352.5055641.36E-05
1396701_atBE110052KalrnKalirin, RhoGEF kinase1.0505122.0712650.00053
1369144_a_atNM_031739Kcnd3Potassium voltage gated channel, Shal-related family, member 31.9788343.9417440.01975
1394039_atBM382886Klf5Kruppel-like factor 51.1682712.2474220.001471
1368363_atNM_053394Klf5Kruppel-like factor 51.107292.1544060.000169
1388932_atBI274917Lama5Laminin, α 51.499222.8268980.008496
1367880_atNM_012974Lamb2Laminin, β 21.0554872.078420.000777
1370993_atAA997129Lamc1Laminin, γ 11.1444162.2105660.001299
1388422_atBI275904Lims2LIM and senescent cell antigen like domains 23.2263899.3592240.000228
1376632_atAI602501Lmcd1LIM and cysteine-rich domains 13.91942415.130880.000893
1381798_atBE114958Lmo7LIM domain 71.6489183.1359830.000383
1375726_atBI284480Lmo7LIM domain 71.2230482.3343930.000945
1381190_atAI598833Lmo7LIM domain 71.0512482.0723210.001218
1375523_atBE108178LOC294446Similar to Myristoylated alanine-rich C-kinase substrate (MARCKS) (ACAMP-81)1.2863312.439070.000713
1370948_a_atM59859LOC294446 /// LOC681252 /// MarcksSimilar 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 substrate1.1130142.1629713.48E-05
1370949_atM59859LOC294446 /// LOC681252 /// MarcksSimilar 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 substrate1.0782132.1114195.02E-05
1381434_s_atAW253721LOC302022Similar to nidogen 2 protein1.4622232.7553260.002301
1373232_atAI008975LOC302022Similar to nidogen 2 protein1.1539042.2251520.007223
1390158_atBI290752LOC304903Similar to Pappalysin-2 precursor (Pregnancy-associated plasma protein-A2) (PAPP-A2) (Pregnancy-associated plasma protein-E1) (PAPP-E)1.0894622.1279470.01314
1384907_atAI411835LOC306096Similar to Dachshund homolog 1 (Dach1)2.3659975.1550860.015526
1383888_atAA998264LOC307495Similar to biliverdin reductase B (flavin reductase (NADPH))1.3453012.5408326.23E-05
1379465_atAW527596LOC311134Hypothetical LOC3111341.691933.2308850.004884
1392074_atAA926082LOC500046Similar to hypothetical protein FLJ219861.8865853.6975890.042067
1392592_atAI137045LOC679869Similar to transcription factor 7-like 2, T-cell specific, HMG-box1.0985482.1413910.000254
1394497_atAI535239LOC679869 /// LOC683733Similar 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)1.0555052.0784450.000768
1373088_atBI295811LOC682888Hypothetical protein LOC6828881.2438312.3682651.11E-06
1388447_atAA800701LOC683626Similar to limb-bud and heart1.1936942.2873770.00702
1379815_atAI713959LOC683733Similar to Transcription factor 7-like 2 (HMG box transcription factor 4) (T-cell-specific transcription factor 4) (TCF-4) (hTCF-4)1.4068982.6516641.15E-05
1377156_atBI273936LOC683733Similar to Transcription factor 7-like 2 (HMG box transcription factor 4) (T-cell-specific transcription factor 4) (TCF-4) (hTCF-4)1.332042.5175830.001391
1383488_atAA817785LOC687536Similar 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)1.5341252.8961270.003143
1386120_atBF393607LOC689147Hypothetical protein LOC6891471.7537853.3724220.003982
1393414_atAW142650LOC689176Similar to transmembrane protein 641.201662.3000420.047653
1376691_atAI103213LOC689176Similar to transmembrane protein 641.1504532.2198360.044249
1374016_atAI502597Lpar1Lysophosphatidic acid receptor 11.299752.4618630.003803
1370048_atNM_053936Lpar1Lysophosphatidic acid receptor 11.2680342.4083310.001243
1389913_atBI276990Lrrfip1Leucine rich repeat (in FLII) interacting protein 11.2604082.3956350.000794
1368448_atNM_021586Ltbp2Latent transforming growth factor β binding protein 22.0719054.2044150.00222
1367768_atNM_031655LxnLatexin1.3816432.6056490.000525
1374933_atBI277043McamMelanoma cell adhesion molecule1.008052.0111910.034411
1369218_atNM_031517MetMet proto-oncogene1.2538672.3847980.000472
1384617_atAI385260MGC72614Hypothetical LOC3105401.3185172.4940960.001888
1398387_atAI009530MGC72614Hypothetical LOC3105401.2637462.4011840.001149
1367568_a_atNM_012862MgpMatrix Gla protein3.35343610.22080.010514
1384150_atAA901038Mid1Midline 11.0625842.0886690.000603
1370072_atNM_012608MmeMembrane metallo endopeptidase3.64630812.521260.003799
1372457_atBF284182Mtus1Mitochondrial tumor suppressor 11.629143.0932855.84E-06
1380321_atBI287786Mtus1Mitochondrial tumor suppressor 11.4022132.6430660.000159
1378970_atAW252385MybphlMyosin binding protein H-like1.0802832.1144510.043933
1370158_atAA946388Myh10Myosin, heavy chain 10, non-muscle1.1923922.2853140.005978
1388298_atBI279044Myl9Myosin, light chain 9, regulatory1.3683692.5817860.025217
1389507_atAI072446Nedd4lNeural precursor cell expressed, developmentally down-regulated 4-like1.0102442.0142520.002318
1369679_a_atAB060652NfiaNuclear factor I/A1.0181882.0253740.008943
1388618_atBM389302Nid2Nidogen 21.7406443.3418430.002285
1368883_atNM_030868NovNephroblastoma overexpressed gene1.9149443.7709930.010007
1371412_a_atBE107450NrepNeuronal regeneration related protein1.7403463.3411520.003311
1369783_a_atU02319Nrg1Neuregulin 11.3412732.5337480.00085
1370607_a_atU02323Nrg1Neuregulin 11.3222.5001240.000298
1371211_a_atU02315Nrg1Neuregulin 11.3046532.4702420.000467
1382814_atAW521702Odz3Odz, odd Oz/ten-m homolog 3 (Drosophila)2.654926.2981150.004174
1377702_atBG380173P2ry5Purinergic receptor P2Y, G-protein coupled, 51.0731592.1040360.000211
1367687_a_atM25719PamPeptidylglycine α-amidating monooxygenase1.1927162.2858260.021615
1398487_atBF419639Pbx1Pre-B-cell leukemia homeobox 11.0925332.1324810.006868
1393966_atAW530825Pbx1Pre-B-cell leukemia homeobox 11.0189722.0264740.000957
1370490_atL43592Pcdhb12Protocadherin β 121.403582.6455720.026763
1377042_atBI288196Pcgf5Polycomb group ring finger 51.1899042.2813760.002754
1392773_atAA859578Pcsk5Proprotein convertase subtilisin/kexin type 51.5893483.0091340.002877
1393467_atBF549923Pcsk5Proprotein convertase subtilisin/kexin type 51.3325972.5185560.000461
1387812_atNM_012999Pcsk6Proprotein convertase subtilisin/kexin type 63.74865413.441790.001431
1382345_atAA955299Pctk2PCTAIRE protein kinase 21.0208742.0291475.29E-05
1374157_atAA858930Pde4bPhosphodiesterase 4B, cAMP specific2.4920885.6259180.004862
1369044_a_atAF202733Pde4bPhosphodiesterase 4B, cAMP specific1.3070882.4744150.025797
1374616_atBM384311PdgfrlPlatelet-derived growth factor receptor-like1.8721223.6607071.96E-06
1368703_atNM_053326Pdlim5PDZ and LIM domain 51.3604652.567680.000723
1386913_atNM_019358PdpnPodoplanin2.7063856.5268420.01551
1374969_atAA799832Pgm5Phosphoglucomutase 51.91233.7640874.83E-05
1368860_atNM_017180Phlda1Pleckstrin homology-like domain, family A, member 11.2362792.3559010.031298
1378106_atAI029402Phlda2Pleckstrin homology-like domain, family A, member 21.0635742.0901040.007195
1388539_atBE113268Pkp2Plakophilin 21.2163332.3235530.004529
1382659_atBF289229Pla2r1Phospholipase A2 receptor 11.819953.530695.56E-05
1387122_atNM_012760Plagl1Pleiomorphic adenoma gene-like 16.37702283.114140.001611
1386962_atNM_024353Plcb4Phospholipase C, β 41.727993.312660.008873
1370489_a_atU57836Plcb4Phospholipase C, β 41.4792772.788090.007828
1369029_atNM_057194Plscr1Phospholipid scramblase 11.3213452.498990.002555
1370247_a_atAA943163Pmp22Peripheral myelin protein 221.3126532.4839790.000418
1372531_atBE106488Ppfibp2PTPRF interacting protein, binding protein 2 (liprin β 2)1.7988873.4795170.023491
1393082_atAI044747Ppp1r14cProtein phosphatase 1, regulatory (inhibitor) subunit 14c1.2636392.4010060.009828
1368716_atNM_133425Ppp1r14cProtein phosphatase 1, regulatory (inhibitor) subunit 14c1.0859182.1227250.010686
1370012_atNM_031557PtgisProstaglandin I2 (prostacyclin) synthase1.8783283.6764860.007631
1368527_atU03389Ptgs2Prostaglandin-endoperoxide synthase 21.7237963.3030440.03712
1377427_atBE104739Ptpn14Protein tyrosine phosphatase, non-receptor type 141.1401422.2040281.10E-05
1374774_atBF552241Ptpn14Protein tyrosine phosphatase, non-receptor type 141.0651122.0923323.73E-05
1368035_a_atX83505PtprfProtein tyrosine phosphatase, receptor type, F2.1964634.5835430.005199
1384227_atAI044031PtprkProtein tyrosine phosphatase, receptor type, K, extracellular region2.1725194.5080974.14E-05
1390034_atBF393945Ralgps2Ral GEF with PH domain and SH3 binding motif 21.1928822.2860890.025134
1367791_atNM_031645Ramp1Receptor (G protein-coupled) activity modifying protein 11.907323.7511160.017043
1368660_atNM_021690Rapgef3Rap guanine nucleotide exchange factor (GEF) 31.0189532.0264480.003623
1390159_atAA819332Rasgrp3RAS guanyl releasing protein 3 (calcium and DAG-regulated)1.4338322.7016330.016442
1383322_atBG375198Rasl11bRAS-like family 11 member B1.757783.3817750.008884
1387581_atNM_022959Rassf9Ras association (RalGDS/AF-6) domain family (N-terminal) member 92.5801195.979892.33E-07
1383247_a_atBI291029rCG_35099Spinster homolog 21.2708312.4130040.006005
1388791_atBI275911RGD1309930Similar to 2810022L02Rik protein1.3275692.5097940.01678
1395336_atBE098691RGD1309930Similar to 2810022L02Rik protein1.3097612.4790050.004198
1374898_atAW527473RGD1311422Similar to CG8841-PA1.0398472.0560090.002294
1373584_atBE113205RGD1559643Similar to hypothetical protein A430031N041.019522.0272450.00071
1372380_atAI231308RGD1561067Similar to RNA binding protein gene with multiple splicing3.35087510.202670.002486
1375898_atAW252379RGD1561067Similar to RNA binding protein gene with multiple splicing3.0919728.5266060.004328
1376619_atAI412803RGD1561090Similar to protein tyrosine phosphatase, receptor type, D2.033854.0949621.02E-06
1374591_atAI409042RGD1561090Similar to protein tyrosine phosphatase, receptor type, D1.9731523.9262512.32E-05
1376919_atBG665267RGD1562317Similar to expressed sequence AW2123941.3763782.5961570.000204
1388879_atBG669292RGD1562717Similar to ABI gene family, member 3 (NESH) binding protein1.7962983.4732770.026049
1388906_atBM389311RGD1564174Similar to novel protein similar to Tensin Tns1.1575112.2307224.32E-05
1383398_atAI059150RGD1564327Similar to integrin α 81.6493883.1370050.000121
1385354_atBE120766RGD1564327Similar to integrin α 81.4721112.7742769.05E-07
1376546_atBE120498RGD1565432Similar to hypothetical protein1.9212953.7876290.009661
1396347_atBF395640RGD1565926RGD15659261.0209522.0292580.010695
1371731_atAI408151RGD1566215Similar to Coatomer γ-2 subunit (γ-2 coat protein) (γ-2 COP)1.9284013.8063325.12E-05
1380425_atAI012859RnaselRibonuclease L (2',5'-oligoisoadenylate synthetase-dependent)1.6714013.1852370.002887
1377116_atBI301478RnaselRibonuclease L (2',5'-oligoisoadenylate synthetase-dependent)1.6269173.0885230.001458
1381533_atAI144754Rnd1Rho family GTPase 11.1965792.2919550.000495
1379693_atAI409154Robo2Roundabout, axon guidance receptor, homolog 2 (Drosophila)1.2458772.3716260.001543
1390632_atBE107414Rspo3R-spondin 3 homolog (Xenopus laevis)1.0491282.0692790.005955
1388356_atAI406499S100a16S100 calcium binding protein A161.4046362.647510.00415
1368379_atNM_054001Scarb2Scavenger receptor class B, member 22.1246244.3608930.001346
1393338_atAW528719ScxScleraxis1.6449993.1274760.007845
1368394_atAF140346Sfrp4Secreted frizzled-related protein 41.6100043.0525270.003747
1367802_atNM_019232Sgk1Serum/glucocorticoid regulated kinase 11.2659472.4048490.007546
1389779_atAA800626Sh2d4aSH2 domain containing 4A1.417922.6720.032404
1392301_atAI237897Sh3tc1SH3 domain and tetratricopeptide repeats 11.2147382.3209870.006644
1392556_atBF410961Shroom3Shroom family member 31.4544962.7406079.57E-05
1376040_atBI290044Sipa1l2Signal-induced proliferation-associated 1 like 21.1856192.2746090.012637
1368565_atNM_019225Slc1a3Solute carrier family 1 (glial high affinity glutamate transporter), member 31.9263173.8008360.003174
1376165_atBE098153Slc24a3Solute carrier family 24 (sodium/potassium/calcium exchanger), member 31.1455112.2122440.000291
1398295_atNM_031684Slc29a1Solute carrier family 29 (nucleoside transporters), member 11.2224262.3333870.003869
1369074_atNM_130748Slc38a4Solute carrier family 38, member 42.257814.782650.00054
1392349_atBE116021Slc5a3Solute carrier family 5 (inositol transporters), member 31.0316722.0443920.000967
1387968_atL22022Slc6a15Solute carrier family 6 (neutral amino acid transporter), member 152.5815345.9857590.018733
1368920_atNM_031321Slit3Slit homolog 3 (Drosophila)1.6525223.1438280.03084
1384437_atAI576309Smarca1SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 11.832943.5626234.14E-05
1377695_atBF281135Smtnl2Smoothelin-like 22.0186334.0519960.047065
1375349_atBI295776Sorbs1Sorbin and SH3 domain containing 12.5085325.6904090.000169
1372728_atBE103745Sort1Sortilin 11.3195172.4958250.006034
1371004_atAI070124Sort1Sortilin 11.1179232.1703430.00142
1379611_atBF416979Spsb1splA/ryanodine receptor domain and SOCS box containing 11.0250122.0349760.009283
1373554_atBE349698Spsb1splA/ryanodine receptor domain and SOCS box containing 11.0109722.0152690.008275
1389142_atAI013361Sqrdlsulfide quinone reductase-like (yeast)2.2784734.8516420.001121
1368109_atNM_031337St3gal5ST3 β-galactoside α-2,3-sialyltransferase 51.6830943.2111585.13E-05
1370907_atM83143St6gal1ST6 β-galactosamide α-2,6-sialyltranferase 11.2943252.4526220.000575
1389420_atBI279446Stap2Signal transducing adaptor family member 21.2087812.3114230.019358
1370680_atAF483620Stau2Staufen, RNA binding protein, homolog 2 (Drosophila)1.0156152.0217640.000276
1372602_atBI295979Stbd1Starch binding domain 12.7614996.7810062.53E-05
1373590_atBI295949StomStomatin1.2673822.4072430.003323
1379732_atAW920037Stx11Syntaxin 111.9767243.9359839.77E-05
1368771_atNM_134378Sulf1Sulfatase 12.2383264.7184920.000659
1376572_a_atAI045848SvilSupervillin1.3207082.4978870.019303
1367570_atNM_031549TaglnTransgelin1.6967333.241660.00228
1367859_atNM_013174Tgfb3Transforming growth factor, β 31.5098322.8477680.003273
1375951_atAA818521ThbdThrombomodulin1.3114042.4818290.020049
1370474_atJ03819ThrbThyroid hormone receptor β2.2192054.6563680.002376
1387983_atJ03933ThrbThyroid hormone receptor β1.1847292.2732060.003624
1383623_atBM383909Thyn1Thymocyte nuclear protein 11.0438262.0616880.002044
1375138_atAA893169Timp3TIMP metallopeptidase inhibitor 36.26469176.888260.015514
1389836_a_atAI599265Timp3TIMP metallopeptidase inhibitor 35.10526434.422120.013029
1372926_atAI009159Timp3TIMP metallopeptidase inhibitor 34.00494416.054920.025103
1368989_atNM_012886Timp3TIMP metallopeptidase inhibitor 32.2533014.7677250.015049
1373847_atAW435343Tm4sf1Transmembrane 4 L six family member 12.1146334.3307980.000242
1378305_atAI578087Tm4sf1Transmembrane 4 L six family member 11.9385123.83310.000317
1390832_atBI294696Tmcc3Transmembrane and coiled-coil domain family 32.1193914.3451060.005139
1376623_atAI409186Tmem204Transmembrane protein 2041.0850942.1215147.30E-05
1383314_atBE110098Tmem51Transmembrane protein 511.3445622.5395313.99E-05
1371361_atBI278826Tns1Tensin 11.1585952.2323990.000276
1370288_a_atAF372216Tpm1Tropomyosin 1, α2.0371814.1044280.000937
1395794_atBF395218Tpm1Tropomyosin 1, α1.9971383.9920730.008961
1371241_x_atAF370889Tpm1Tropomyosin 1, α1.6635663.1679850.013541
1370287_a_atM23764Tpm1Tropomyosin 1, α1.590743.0120395.95E-05
1368724_a_atNM_019131Tpm1Tropomyosin 1, α1.0548912.0775610.0189
1372219_atAA012755Tpm2Tropomyosin 21.0645632.0915372.16E-05
1398759_atNM_013043Tsc22d1TSC22 domain family, member 11.0513682.0724940.000844
1377630_atAI408602Tspan13Tetraspanin 131.4816872.7927520.019678
1375057_atBG377313Tspan18Tetraspanin 181.4540712.7398020.03087
1398476_atAW527349VclVinculin1.2444742.3693210.000134
1372905_atAW433888VclVinculin1.1363212.1981973.11E-05
1369098_atNM_013155VldlrVery low density lipoprotein receptor1.5053962.8390270.001107
1387455_a_atNM_013155VldlrVery low density lipoprotein receptor1.4710282.7721940.001621
1389611_atAA849857VldlrVery low density lipoprotein receptor1.4359452.7055930.000947
1368854_atAI227991Vsnl1Visinin-like 12.4726955.5507960.007104
1368853_atNM_012686Vsnl1Visinin-like 12.0778064.2216470.004903
1387873_atBI279661Wfdc1WAP four-disulfide core domain 11.4492922.7307390.003744
1370221_atBF419320Wisp1WNT1 inducible signaling pathway protein 11.0585672.0828610.000154
1393613_atBE117871Zfp462Zinc finger protein 4621.0883872.1263610.004351
1383462_atBF566263Znf294Zinc finger protein 2941.0102462.0142550.000352
Supplementary Table 2 Downregulated genes in miR-146a-transfected hepatic stellate cell-2.
Probe IDRepresentative public IDGene symbolGene titleLog2Fold changeP-value
1374345_atAI111707-3.40046-10.55941.80E-07
1397317_atBI296984-3.25476-9.545133.34E-07
1397400_atBM391846-3.11991-8.693355.36E-05
1382027_atBI296880-3.05526-8.312373.85E-07
1380245_atAI411847-2.90249-7.477158.46E-07
1381129_atBF392367-2.52142-5.741470.018162
1383211_atBE109736-2.46677-5.528050.006284
1380057_atBE097091-2.33498-5.045430.000685
1381048_atBF398435-2.3159-4.979160.000443
1381064_atAI137604-2.26573-4.808961.32E-07
1373583_atBF396317-2.2618-4.795891.23E-07
1377240_atAW526305-2.19645-4.583510.001977
1394468_atBF287020-2.12335-4.357050.000157
1377678_atBI283757-2.12273-4.355190.000117
1375101_atBI292651-2.05823-4.164760.000442
1380712_atAI406475-2.04987-4.140680.006175
1372110_atBE113148-1.95331-3.872610.000463
1394517_atAW522148-1.88952-3.705130.006671
1390530_atAI169239-1.87482-3.667550.00151
1376463_atAA955579-1.87022-3.655870.001028
1384743_atBF418132-1.86231-3.635892.42E-14
1377114_atAI410861-1.8391-3.577888.88E-06
1377161_atBG378317-1.83484-3.567310.039081
1383220_atBE114231-1.73514-3.329110.000179
1393143_atAI045866-1.69968-3.248280.005065
1376324_atBF406329-1.68113-3.206790.004765
1390429_atBF398114-1.62771-3.090216.83E-05
1382431_atAI103530-1.6169-3.067161.15E-06
1391251_atBI290666-1.60415-3.040161.73E-05
1383580_atAA859643-1.58751-3.00530.000305
1372011_atBI292028-1.5749-2.979150.000487
1377232_atBF406608-1.54704-2.922170.003334
1398457_atAI146156-1.50417-2.836620.005925
1381161_a_atBI301117-1.47549-2.780780.008417
1382142_atAI029975-1.45644-2.744313.98E-05
1382472_atAI502459-1.44328-2.719398.76E-05
1375266_atBG380633-1.43613-2.705950.010349
1381862_atAW524296-1.39645-2.632530.004828
1393235_atAI059968-1.39173-2.623920.000172
1385181_atAI029337-1.39139-2.623321.30E-07
1377686_atAA859337-1.37593-2.595353.53E-07
1372449_atAW253616-1.37158-2.587530.00912
1377556_atAW535380-1.36924-2.583340.004323
1372637_atAI169241-1.36639-2.578250.000419
1375957_atAW434654-1.30351-2.468284.46E-06
1383936_atBM386842-1.29935-2.461181.09E-05
1384724_atAA850766-1.28208-2.43193.20E-05
1379510_atBF546306-1.20859-2.311129.04E-05
1382296_atBF291041-1.20836-2.310751.53E-05
1382544_atAI058746-1.20774-2.309753.79E-06
1376816_atBF284903-1.19854-2.295070.000155
1383019_atBF558478-1.18776-2.277980.000454
1374558_atAI010316-1.17751-2.261868.04E-06
1389250_atAW915115-1.16975-2.249729.96E-05
1377328_atBI290012-1.16378-2.240440.009615
1380602_atAI764190-1.16264-2.238670.001006
1384812_atAI229409-1.15815-2.231720.006986
1396217_atBF542447-1.1372-2.199540.000345
1397668_atH34328-1.13718-2.19953.26E-07
1374932_atBI282731-1.13055-2.189430.001048
1385381_atAA996491-1.12644-2.18320.000161
1394047_atBE107848-1.10947-2.157670.035398
1397452_atAI112776-1.10848-2.156180.000172
1393730_atBI277836-1.10819-2.155750.03382
1381975_atBG371767-1.10049-2.144270.001469
1380699_atBE110761-1.09332-2.133640.000497
1374920_atAI228955-1.07203-2.102392.65E-05
1375473_atBI296644-1.05345-2.075480.001418
1373914_atBM389075-1.05092-2.071860.001887
1383436_atBG376768-1.04917-2.069330.000656
1390405_atAA942765-1.0454-2.063940.003275
1376911_atBM386385-1.04165-2.058580.000165
1384183_atAA996869-1.03133-2.043910.004095
1377469_atAI103161-1.0216-2.030160.006072
1393030_atBE115641-1.01825-2.025470.000652
1377113_atBF415786-1.0168-2.023430.002724
1382960_atBE108047-1.01496-2.020849.24E-05
1377955_atAI137602-1.01226-2.017074.55E-07
1391853_atAA998997-1.00175-2.002430.000388
1384086_atBG671196-1.00024-2.000330.031992
1385235_atAA818804A2bp1Ataxin 2 binding protein 1-1.24533-2.370734.71E-05
1383130_atBF555795A2bp1Ataxin 2 binding protein 1-1.07712-2.109830.000947
1394490_atAI502114Abca1ATP-binding cassette, sub-family A (ABC1), member 1-1.80354-3.490763.72E-07
1384381_atBF284523Abca1ATP-binding cassette, sub-family A (ABC1), member 1-1.27132-2.413820.000215
1383355_atAW918387Abca1ATP-binding cassette, sub-family A (ABC1), member 1-1.21129-2.315455.71E-07
1369928_atNM_019212Acta1Actin, α 1, skeletal muscle-3.10676-8.614494.42E-05
1370856_atAA800705Actc1Actin, α, cardiac muscle 1-1.26863-2.409332.02E-05
1394483_atAW535310Adamts5ADAM metallopeptidase with thrombospondin type 1 motif, 5-1.53094-2.889751.96E-05
1390383_atBI285616AdfpAdipose differentiation related protein-2.49834-5.650373.30E-08
1382680_atBG673602AdfpAdipose differentiation related protein-2.22186-4.664961.20E-05
1387395_atNM_017161Adora2bAdenosine A2B receptor-2.08666-4.247630.000258
1395695_atBE126420Aebp1AE binding protein 1-1.5972-3.025550.005916
1372301_atBI278482Aebp1AE binding protein 1-1.55735-2.943130.01167
1368342_atNM_031544Ampd3Adenosine monophosphate deaminase 3-1.65434-3.147790.004546
1377783_atBI294141Angpt4Angiopoietin 4-1.64644-3.13060.001964
1397579_x_atBI294552Apc2Adenomatosis polyposis coli 2-1.91631-3.774550.00151
1395461_atBI294552Apc2Adenomatosis polyposis coli 2-1.30834-2.476560.002146
1391083_atBM384457Arhgap22Rho GTPase activating protein 22-1.09826-2.140960.007774
1377385_atBE100015Arhgap27Rho GTPase activating protein 27-1.30775-2.475566.57E-07
1387959_atAB009372AspgAsparaginase homolog (S. cerevisiae)-1.29918-2.460890.000534
1380726_atBI290633AspnAsporin-2.13124-4.380920.032454
1381504_atAI639412AspnAsporin-1.90669-3.749470.02918
1368477_atNM_012914Atp2a3ATPase, Ca++ transporting, ubiquitous-1.60464-3.041190.000593
1369664_atNM_053019Avpr1aArginine vasopressin receptor 1A-2.07931-4.226051.62E-05
1375941_atBI292120Baiap2l1BAI1-associated protein 2-like 1-1.64069-3.118161.70E-06
1369807_atNM_030851Bdkrb1Bradykinin receptor B1-1.11926-2.172360.000155
1391345_atBI293047BmperBMP-binding endothelial regulator-1.85131-3.608283.59E-06
1387540_atNM_012514Brca1Breast cancer 1-1.02475-2.034610.000898
1381995_atAW530502Brunol4Bruno-like 4, RNA binding protein (Drosophila)-2.20211-4.601527.21E-08
1387893_atD88250C1sComplement component 1, s subcomponent-1.26395-2.401520.048179
1375569_atBM386267Ccdc92Coiled-coil domain containing 92-1.03224-2.04520.000197
1367973_atNM_031530Ccl2Chemokine (C-C motif) ligand 2-2.02826-4.079120.002595
1379935_atBF419899Ccl7Chemokine (C-C motif) ligand 7-1.07909-2.112710.006482
1370810_atL09752Ccnd2Cyclin D2-1.31667-2.490910.005098
1389490_atBI274335Cd248CD248 molecule, endosialin-3.64068-12.47251.26E-06
1389755_atBM391858Cdca7lCell division cycle associated 7 like-1.4418-2.716610.000141
1369425_atNM_138889Cdh13Cadherin 13-3.4441-10.88374.56E-08
1375719_s_atBG381748Cdh13Cadherin 13-3.2068-9.233012.00E-08
1373102_atBI282750Cdh13Cadherin 13-3.03097-8.17366.92E-06
1373054_atAA801076Cdw92CDW92 antigen-1.00321-2.004450.000362
1396034_atBF402373Ces7Carboxylesterase 7-2.24147-4.728797.58E-05
1389179_atBF284899CideaCell death-inducing DNA fragmentation factor, α subunit-like effector A-1.13966-2.203291.12E-05
1367740_atM14400CkbCreatine kinase, brain-1.40456-2.647380.000293
1392672_atAI576758Clec11aC-type lectin domain family 11, member a-1.45028-2.732620.004619
1368571_atNM_021997Clip2CAP-GLY domain containing linker protein 2-1.09569-2.137154.77E-07
1372584_atBG672517Cnrip1Cannabinoid receptor interacting protein 1-1.11448-2.165171.59E-08
1379345_atBM386752Col15a1Collagen, type XV, α 1-6.09695-68.44853.18E-06
1388939_atAA800298Col15a1Collagen, type XV, α 1-4.48588-22.40712.71E-05
1384969_atBE109107Col24a1Collagen, type XXIV, α 1-1.8271-3.548240.002725
1371349_atAI598402Col6a1Collagen, type VI, α 1-1.66097-3.162280.001251
1371369_atBI287851Col6a2Collagen, type VI, α 2-1.80919-3.504459.31E-05
1372818_atBI284441Colec12Collectin sub-family member 12-2.38156-5.2113.25E-07
1372774_atAI170570Coq6Coenzyme Q6 homolog (yeast)-1.57329-2.975831.94E-06
1369964_atNM_130411Coro1aCoronin, actin binding protein 1A-1.6641-3.169167.57E-05
1392996_atBG668435Cpeb1Cytoplasmic polyadenylation element binding protein 1-1.42707-2.689013.55E-05
1368293_atNM_031766CpzCarboxypeptidase Z-1.17127-2.25210.020505
1376051_atBI293393Cryl1Crystallin, lambda 1-2.8568-7.244064.30E-05
1383575_atBG376561Ctnnd2Catenin (cadherin-associated protein), delta 2 (neural plakophilin-related arm-repeat protein)-2.38434-5.221059.56E-05
1369947_atNM_031560CtskCathepsin K-1.00677-2.009410.001608
1387969_atU22520Cxcl10Chemokine (C-X-C motif) ligand 10-2.22859-4.686744.90E-05
1368738_atD11354Cyp11b1Cytochrome P450, subfamily 11B, polypeptide 1-1.731-3.319580.003738
1387305_s_atNM_012539Cyp11b1 /// Cyp11b2Cytochrome P450, subfamily 11B, polypeptide 1 /// cytochrome P450, subfamily 11B, polypeptide 2-1.73027-3.31794.54E-05
1387276_atNM_021584Dclk1Doublecortin-like kinase 1-1.05016-2.070760.000907
1384971_atBI289108Depdc7DEP domain containing 7-1.10481-2.15070.000468
1371732_atBI285485DptDermatopontin-1.89757-3.725850.009977
1383853_atBE103067Dyrk3Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 3-1.76949-3.409341.66E-07
1383641_atBF414702EdnraEndothelin receptor type A-2.19922-4.59230.01467
1378342_atBF284819EdnraEndothelin receptor type A-1.78129-3.437330.004867
1393415_atBF548891EdnraEndothelin receptor type A-1.51332-2.854670.00743
1391442_atAA957585Ehd3EH-domain containing 3-1.71662-3.286653.90E-05
1367905_atNM_019370Enpp3Ectonucleotide pyrophosphatase/phosphodiesterase 3-1.33397-2.520969.07E-05
1382434_atAI059015Entpd5Ectonucleoside triphosphate diphosphohydrolase 5-1.63931-3.115182.50E-06
1370503_s_atAB032828Epb4.1l3Erythrocyte protein band 4.1-like 3-1.9425-3.843720.005443
1368515_atNM_053927Epb4.1l3Erythrocyte protein band 4.1-like 3-1.52656-2.880980.000173
1369422_atNM_138850FapFibroblast activation protein, α-1.69721-3.242740.001039
1376561_atAW523739Fbxo16F-box protein 16-1.11142-2.160586.95E-06
1367850_atNM_053843Fcgr2a /// LOC498276 /// LOC498277Fc 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)-1.23098-2.347260.007866
1392865_atBG371594Fgf9Fibroblast growth factor 9-2.37371-5.182740.002748
1373882_atAI170324FigfC-fos induced growth factor-2.48251-5.588690.000198
1387709_atAY032728FigfC-fos induced growth factor-2.21994-4.658735.44E-07
1374726_atAI411941Fndc1Fibronectin type III domain containing 1-2.18189-4.537490.005581
1370248_atAA851939Fxyd6FXYD domain-containing ion transport regulator 6-1.91583-3.773310.004165
1385636_atAI029226Fzd3Frizzled homolog 3 (Drosophila)-1.30152-2.464886.39E-06
1388395_atAI406939G0s2G0/G1switch 2-1.69782-3.24416.68E-05
1382314_atBE096523G1p2Interferon, α-inducible protein (clone IFI-15K)-1.43064-2.695650.012403
1370963_atAJ131902Gas7Growth arrest specific 7-2.12746-4.369460.001866
1387221_atNM_024356Gch1GTP cyclohydrolase 1-1.0345-2.04840.001025
1368085_atNM_133595GchfrGTP cyclohydrolase I feedback regulator-1.09278-2.132841.23E-05
1368770_atNM_022276Gcnt1Glucosaminyl (N-acetyl) transferase 1, core 2-1.35748-2.562370.000674
1387659_atAF245172GdaGuanine deaminase-1.54413-2.916290.000209
1377761_atBI296057Gfpt2Glutamine-fructose-6-phosphate transaminase 2-2.3887-5.236850.003422
1387007_atNM_012959Gfra1GDNF family receptor α 1-1.49726-2.823060.000202
1367954_atU59486Gfra1GDNF family receptor α 1-1.10817-2.155730.0006
1397461_atBF416400Glt8d2Glycosyltransferase 8 domain containing 2-1.11036-2.158998.28E-06
1386870_atBI275294GlulGlutamate-ammonia ligase (glutamine synthetase)-1.18385-2.271820.000113
1367632_atNM_017073GlulGlutamate-ammonia ligase (glutamine synthetase)-1.11961-2.172880.003082
1369302_atNM_133573GperG protein-coupled estrogen receptor 1-1.10018-2.143810.001057
1387241_atNM_031696Gpr88G-protein coupled receptor 88-1.11226-2.161840.002865
1369926_atNM_022525Gpx3Glutathione peroxidase 3-2.05946-4.168291.35E-06
1374488_atAI175700Gramd1bGRAM domain containing 1B-1.12881-2.186780.001788
1368180_s_atNM_017013Gsta2 /// LOC494499Glutathione S-transferase A2 /// LOC494499 protein-2.31111-4.962666.68E-05
1371298_atBF284168H19H19 fetal liver mRNA-1.87119-3.658330.000303
1391575_atBG380566Hapln4Hyaluronan and proteoglycan link protein 4-1.12321-2.178310.002326
1368255_atNM_017354HntNeurotrimin-2.33813-5.056480.0043
1367816_atNM_133621HopxHOP homeobox-1.32323-2.502260.003008
1393592_atAA998087Hs3st5Heparan sulfate (glucosamine) 3-O-sulfotransferase 5-1.19206-2.284790.004675
1368578_atNM_017265Hsd3b1Hydroxy-delta-5-steroid dehydrogenase, 3 β- and steroid delta-isomerase 1-2.132-4.383240.00025
1387282_atNM_053612Hspb8Heat shock protein 8-1.47457-2.779012.14E-07
1388721_atBG380282Hspb8Heat shock protein 8-1.28154-2.430981.12E-06
1376908_atAW531805Ifit3Interferon-induced protein with tetratricopeptide repeats 3-1.35357-2.555440.019235
1382220_atAI180454Igf2bp2Insulin-like growth factor 2 mRNA binding protein 2-1.40203-2.642720.00737
1387180_atNM_053953Il1r2Interleukin 1 receptor, type II-2.39783-5.270120.000126
1387273_atNM_013037Il1rl1Interleukin 1 receptor-like 1-3.65216-12.57210.013461
1370692_atU04317Il1rl1Interleukin 1 receptor-like 1-1.22822-2.342780.004082
1387504_atNM_133575Il1rl2Interleukin 1 receptor-like 2-1.5258-2.879467.08E-05
1377163_atBM385741InhbbInhibin β-B-1.23015-2.345910.014339
1369043_atNM_012971Kcna4Potassium voltage-gated channel, shaker-related subfamily, member 4-4.1772-18.0918.01E-08
1390404_atBF556962Lama2Laminin, α 2-2.54141-5.821599.68E-05
1370138_atNM_130429Lef1Lymphoid enhancer binding factor 1-1.10908-2.157080.000657
1378179_a_atAW524864Lhfpl2Lipoma HMGIC fusion partner-like 2-1.03801-2.053390.00217
1371094_atL06804Lhx2LIM homeobox 2-1.06797-2.096480.000653
1389885_atBI294855Limd2LIM domain containing 2-1.05098-2.071946.73E-05
1376871_atAA891475LOC680910 /// LOC681069 /// LOC681182 /// LOC681196 /// LOC685030 /// LOC685048 /// LOC685111 /// LOC685262 /// LOC685305 /// LOC686848 /// LOC686899 /// RGD1559588 /// RGD1561143 /// RGD1561730 /// RGD1562525 /// RGD1563400 /// RGD1566006Similar 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-1.06939-2.098552.14E-06
1385047_x_atAI012782LOC685048 /// LOC685111 /// RGD1559588 /// Vom2r61Similar 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, 61-2.82049-7.0645.90E-06
1393688_atAI012782LOC685048 /// LOC685111 /// RGD1559588 /// Vom2r61Similar 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, 61-2.75933-6.77082.26E-07
1371293_atAI103218LOC688228Similar to Myosin light polypeptide 4 (Myosin light chain 1, atrial isoform)-1.30459-2.470136.68E-07
1398732_atBF553297LOC688273Hypothetical protein LOC688273-3.37961-10.40790.000276
1384540_atBE101066Lrfn3Leucine rich repeat and fibronectin type III domain containing 3-1.05498-2.077690.00043
1388347_atAI233210Ly6eLymphocyte antigen 6 complex, locus E-2.7688-6.815391.82E-05
1376184_atBG381127Lynx1Ly6/neurotoxin 1-1.98858-3.968468.46E-08
1393645_atBI288003Mageb16Melanoma antigen family B, 16-1.53459-2.897060.000131
1388152_atBG374290Map2Microtubule-associated protein 2-1.7733-3.418360.008563
1382046_atAA963495Map3k3Mitogen activated protein kinase kinase kinase 3-1.24191-2.365111.42E-06
1392547_atAI716211MGC105649Hypothetical LOC302884-2.31654-4.981360.004157
1388300_atAA892234Mgst3Microsomal glutathione S-transferase 3-1.007-2.009730.017898
1393836_atBE097933MitfMicrophthalmia-associated transcription factor-1.0956-2.137020.010323
1368590_atNM_080776Mmp16Matrix metallopeptidase 16-1.01729-2.024110.000829
1370301_atU65656Mmp2Matrix metallopeptidase 2-3.98031-15.78310.000568
1382190_atBF405725MrgprfMAS-related GPR, member F-3.60572-12.17393.94E-07
1368441_atNM_031658MslnMesothelin-1.36403-2.574030.001477
1376648_atBI275570MycnV-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian)-1.62357-3.081360.002089
1368415_atNM_012604Myh3Myosin, heavy chain 3, skeletal muscle, embryonic-1.24039-2.362620.003414
1387787_atNM_012605MylpfMyosin light chain, phosphorylatable, fast skeletal muscle-2.12252-4.354530.000567
1398655_atAA955902Myod1Myogenic differentiation 1-2.39143-5.246779.65E-05
1373839_atBG372386NopeNeighbor of Punc E11-1.24338-2.367520.009522
1371036_atBG671431NrcamNeuronal cell adhesion molecule-1.60969-3.051872.40E-06
1384112_atBI289470Nt5e5' nucleotidase, ecto-1.1205-2.174230.004069
1392780_atBF283270Nxf7nuclear RNA export factor 7-3.39721-10.53570.005424
1377497_atBF419319Oasl2'-5'-oligoadenylate synthetase-like-1.36533-2.576362.34E-05
1369008_a_atNM_053573Olfm1Olfactomedin 1-3.58878-12.03180.000169
1368940_atNM_017255P2ry2Purinergic receptor P2Y, G-protein coupled 2-1.21116-2.315230.009814
1383273_a_atAA956005Pcbp3Poly(rC) binding protein 3-2.10678-4.307294.21E-06
1383274_atAA956005Pcbp3Poly(rC) binding protein 3-1.8662-3.645720.000552
1385116_atBF386807Pcdhb21Protocadherin β 21-1.13304-2.193210.007625
1373368_atBI279680PCOLCE2Procollagen C-endopeptidase enhancer 2-1.805-3.49437.61E-07
1368145_atNM_013002Pcp4Purkinje cell protein 4-4.28374-19.47750.000401
1370941_atAI232379PdgfraPlatelet derived growth factor receptor, α polypeptide-1.62538-3.085230.011837
1377100_atAI172172Pds5bPDS5, regulator of cohesion maintenance, homolog B (S. cerevisiae)-1.14971-2.218691.40E-05
1388634_atBI277505Pgm1Phosphoglucomutase 1-1.66935-3.180719.06E-09
1369473_atNM_017033Pgm1Phosphoglucomutase 1-1.50694-2.842073.02E-06
1383749_atAI112954Phospho1Phosphatase, orphan 1-1.16307-2.239340.003657
1370445_atD88666Pla1aPhospholipase A1 member A-1.35014-2.549370.001836
1390190_atBI293691Plac1Placenta-specific 1-2.19087-4.565820.000174
1384558_atBI276313Plac9Placenta-specific 9-1.07069-2.100430.007797
1367800_atNM_013151PlatPlasminogen activator, tissue-1.15024-2.219510.004677
1391187_atBI303019PplPeriplakin-1.11393-2.164340.000262
1368259_atNM_017043Ptgs1Prostaglandin-endoperoxide synthase 1-2.42323-5.36373.32E-05
1381806_atBF418208Ptgs1Prostaglandin-endoperoxide synthase 1-1.06169-2.087375.08E-05
1372084_atAI104546Ptp4a3Protein tyrosine phosphatase 4a3-1.0212-2.029612.54E-05
1368350_atNM_013080Ptprz1Protein tyrosine phosphatase, receptor-type, Z polypeptide 1-1.04165-2.058580.022268
1373646_atBM384841Rab15RAB15, member RAS oncogene family-1.17275-2.25447.21E-05
1374035_atBI296482Rem2RAS (RAD and GEM) like GTP binding 2-1.00521-2.007230.017185
1368080_atNM_054008Rgc32Response gene to complement 32-1.00087-2.001210.032818
1392883_atAI013730RGD1305269Similar to hypothetical protein-1.04588-2.064631.81E-05
1373226_atBF400995RGD1308019Similar to hypothetical protein FLJ20245-1.10844-2.156130.015649
1381757_atAA965058RGD1309501Hypothetical LOC305552-1.28517-2.437122.17E-05
1373596_atAI230766RGD1310423Similar to hypothetical protein FLJ31737-2.01177-4.032776.86E-08
1398577_atBI297744RGD1310507Similar to RIKEN cDNA 1300017J02-1.72535-3.306610.000821
1390397_atBF413152RGD1310753Similar to chromosome 20 open reading frame 39-2.10856-4.312615.11E-05
1393191_atBF554733RGD1561205Similar to RIKEN cDNA 2610200G18-1.39361-2.627360.000183
1376693_atAA998964RGD1563091Similar to OEF2-1.00045-2.000630.047169
1395145_atBF544481Rgl1Ral guanine nucleotide dissociation stimulator,-like 1-1.5473-2.92270.000329
1394472_atBF282814Rgl1Ral guanine nucleotide dissociation stimulator,-like 1-1.20268-2.301660.005425
1391075_atAI179271Rgs17Regulator of G-protein signaling 17-1.50436-2.836990.004883
1368373_atNM_019343Rgs7Regulator of G-protein signaling 7-3.29254-9.798361.82E-05
1370142_atNM_022175Rhox5Reproductive homeobox 5-3.12793-8.741782.44E-09
1383554_atAW142796Rnf128Ring finger protein 128-1.473-2.775981.47E-05
1389735_atBE107296Rps6ka6Ribosomal protein S6 kinase polypeptide 6-1.18452-2.272880.013943
1384707_atAI600020Scara5Scavenger receptor class A, member 5 (putative)-1.46379-2.758339.19E-06
1392856_atAI549470Serf1Small EDRK-rich factor 1-1.52508-2.878020.00071
1375084_atBF419780Serinc2Serine incorporator 2-1.64207-3.121130.000591
1377034_atBF411331Serpinb1aSerine (or cysteine) proteinase inhibitor, clade B, member 1a-1.17504-2.257998.45E-05
1369547_atNM_130404Serpinb7Serine (or cysteine) peptidase inhibitor, clade B, member 7-1.4094-2.656260.001746
1393620_atAI113325Sesn3Sestrin 3-1.17758-2.261980.001718
1390119_atBF396602Sfrp2Secreted frizzled-related protein 2-1.64679-3.131350.011642
1367881_atNM_013016SirpaSignal-regulatory protein α-1.88267-3.687561.79E-06
1392789_atBI296353Slc25a36Solute carrier family 25, member 36-2.25714-4.780424.14E-05
1372341_atAI233213Slc25a36Solute carrier family 25, member 36-2.07958-4.226850.000633
1369237_atNM_053996Slc6a7Solute carrier family 6 (neurotransmitter transporter, L-proline), member 7-1.12269-2.177530.00114
1368322_atNM_012880Sod3Superoxide dismutase 3, extracellular-2.20485-4.610281.15E-05
1368254_a_atAB049572Sphk1Sphingosine kinase 1-1.09627-2.138020.00473
1368655_atNM_020074SrgnSerglycin-2.51283-5.707370.006656
1373146_atAI716240Ssx2ipSynovial sarcoma, X breakpoint 2 interacting protein-1.23239-2.349560.001168
1387174_a_atAB006007StarSteroidogenic acute regulatory protein-3.29954-9.845990.000194
1368406_atNM_031558StarSteroidogenic acute regulatory protein-2.92085-7.572917.29E-07
1377672_atBI300997Sult1c2Sulfotransferase family, cytosolic, 1C, member 2-3.45192-10.94293.19E-06
1369531_atNM_133547Sult1c2Sulfotransferase family, cytosolic, 1C, member 2-2.77927-6.865042.07E-05
1369627_atL10362Sv2bSynaptic vesicle glycoprotein 2b-2.39744-5.268660.000178
1385637_atAI029494Svep1Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1-1.08524-2.121720.000482
1383686_atBE111537Syngr1Synaptogyrin 1-1.73204-3.321970.00524
1384202_atBI287326TescTescalcin-1.69209-3.231240.004681
1371913_atBG379319TgfbiTransforming growth factor, β induced-1.05453-2.077040.023537
1369652_atAI145313Thy1Thy-1 cell surface antigen-3.93458-15.29072.03E-07
1369651_atNM_012673Thy1Thy-1 cell surface antigen-3.67268-12.75227.68E-08
1392980_atAI716456Tiam1T-cell lymphoma invasion and metastasis 1-1.56449-2.957747.40E-06
1382222_atBI293607Tmem163Transmembrane protein 163-2.1182-4.341538.73E-06
1376106_atAI010157Tmem178Transmembrane protein 178-3.28823-9.769120.007054
1377554_atBF394106Tnfsf9Tumor necrosis factor (ligand) superfamily, member 9-1.95931-3.888771.88E-05
1370332_atAF159356Unc13dUnc-13 homolog D (C. elegans)-2.36452-5.14980.000313
1368474_atNM_012889Vcam1Vascular cell adhesion molecule 1-3.24587-9.486431.90E-05
1388142_atAA850991VcanVersican-1.54149-2.910960.000288
1388054_a_atAF072892VcanVersican-1.53261-2.893080.000536
1371232_a_atAF084544VcanVersican-1.47614-2.782040.000764
1388265_x_atAF084544VcanVersican-1.45114-2.734230.001864
1389253_atBI289085Vnn1Vanin 1-1.15392-2.225180.000172
1382283_atBF283711Wipf1WAS/WASL interacting protein family, member 1-1.01562-2.021763.13E-05
1387227_atNM_057192Wipf1WAS/WASL interacting protein family, member 1-1.00508-2.007068.16E-05
1389119_atAI105018Xirp1Xin actin-binding repeat containing 1-1.17137-2.252269.90E-05
1372989_atBI296586Zdhhc14Zinc finger, DHHC-type containing 14-3.38007-10.41121.23E-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
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
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
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
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.

Footnotes

Peer reviewers: Dr. Katja Breitkopf, Department of Medicine II, University Hospital Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany; Richard A Rippe, Professor of Medicine, Division of Gastroenterology and Hepatology, Department of Medicine, University of North Carolina, Chapel Hill, NC 27599-7032, United States

S- Editor Wang JL L- Editor Kerr C E- Editor Zheng XM

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