Association of differentially expressed genes with activation of mouse hepatic stellate cells by high-density cDNA mircoarray

Table 1 Primer sequences for RT-PCR

Gene name	Primer sequence	Expected product size
Betaine homocysteine	Left 5’–GCC TAT AGC GGC TAC CAT GT-3’	399 bp
methyltransferase (BHMT)	Right 5’-CTC TGC AAT GGC CCT GAT GT-3’
Defender against cell	Left 5’-TTC GGC TAC TGT CTC CTC GT -3’	197 bp
death protein 1 (DAD1)	Right 5’-ACG AGG TGC AGG ATA GTG CT-3’
Annexin VI	Left 5’-TAC CCC GGA GTA TTT TGC TG-3’	220 bp
	Right 5’-GTC CCC TCC ACA TAG CTT CA-3’
Neural cadherin	Left 5’-ATA CAG TGT CAC TGG GCC AG- 3’	499 bp
(N-Cadherin)	Right 5’-CGT AAG TGG GAT TGC CTT CC- 3’
Macrophage migration	Left 5’-TTT TAG TGG CAC GAG CGA CC-3’	199 bp
inhibitory factor (MIF)	Right 5’-AAG CGA AGG TGG AAC CGT TC- 3’
GAPDH	Left 5’-ACC ACA GTC CAT GCC ATC AC-3’	452 bp
	Right 5’-TCC ACC ACC CTG TTG CTG TA-3’

Table 2 Part of the previously reported up-regulated genes in activation of HSC

GenBank accession number	Gene name	Ratio (Cy5/Cy3)	Potential gene function
M73741	Alpha-B2-crystallin, complete cds	155.619	Small heat shock protein gene, an early marker for HSC activation
X52046	Procollagen, type III, alpha 1	119.092	Extracellular matrix (ECM), over- expressed in hepatic fibrosis
M18194	Fibronectin mRNA	29.958	ECM, over expressed in hepatic fibrosis
J02870	Laminin receptor mRNA	6.62	ECM receptor (integrin), cell adhesion
L08115	CD9 antigen	6.094	Cell membrane glycoprotein, involved in cell activation and adhesion
U16163	Prolyl 4-hydroxylase alpha (II) - subunit mRNA, complete cds	4.555	An enzyme which is essential for the collagen synthesis in HSC
X62622	TIMP-2 mRNA for tissue inhibitor of metalloproteinase, type 2	3.732	Inhibition for ECM degradation
X04017	mRNA for cysteine-rich glycoprotein SPARC	3.435	Regulation of cell shape, adhesion, migration and proliferation
AF070470	SPARC-related protein (SRG) mRNA, complete cds	3.181	Regulation of cell shape, adhesion, migration
M21495	Cytoskeletal gamma-actin mRNA,	3.092	Relate skeleton structure of cell
AF188297	TGF-beta receptor binding protein (Trip1) mRNA, complete cds	2.976	TGF-beta mediated signaling pathway
AJ245923	alpha-tubulin 8 (Tuba 8 gene) mRNA	2.901	Relate skeleton structure of cell
AF053454	Tetraspan TM4SF (Tspan-6) mRNA complete cds,	2.896	Cell development, differentiation, motility
AF013262	Lumican gene, complete cds	2.862	Small, leucine-rich proteoglycan which involves in the regulation of collagen figril assembly
L02526	Protein kinase (MEK) mRNA	2.714	Signaling molecule in MAPK family
Y00769	mRNA for integrin beta subunit	2.521	Signaling molecular in cell adhesion, proliferation and migration

Table 3 A portion of up-regulated genes in activated HSC

GenBank accession number	Gene name	Ratio (Cy5/Cy3)	Potential gene function
AF061017	UDP-glucose dehydrogenase, mRNA complete cds	15.56	Glycosaminoglycan , hyaluronan and heparin sulfate biosynthesis
X65553	mRNA for poly (A) binding protein	7.405	A regulator of translation initiation
AF029844	Elongation factor 1-beta homology mRNA, complete cds	6.305	Translation factor and has multi-functions
NM_010798	Macrophage migration inhibitory factor (Mif), mRNA	5.847	Proinflammatory peptide and a mediator of growth factor dependent ERK MAP kinase activation and cell cycle progression
M31131	Neural cadherin (N-cadherin) mRNA	5.559	Cell adhesion molecule
AB029930	mRNA for caveolin-1 beta isoform	4.945	Inhibit the eNOS activity
D12618	mRNA for nucleosome assembly protein-1, complete cds	4.528	Histone H2A-H2B shutting protein that promotes histone deposition, and is important for maintaining chromatin structure
M22432	Protein synthesis elongation factor Tu (eEF-Yu, eEf-1-1alpha) mRNA, complete cds	4.344	Translation factor involved in the protein biosynthesis
D31717	mRNA for ribophorin	4.319	Glycoprotein synthesis
NM_012052	Ribosomal protein S3 (Rps3), mRNA	4.297	Protein biosynthesis
X72711	mRNA for replication factor C (140 Kda), long subunit	4.245	DNA synthesis and repair, and a regulator of NF-kappa B
X13460	Annexin VI, p68 (Mouse mRNA for p68 protein of the lipocortin family)	4.218	Calcium binding protein
NM_011290	Ribosomal protein L6 (Rp16), mRNA	4.089	Protein biosynthesis
U12273	Apurinic/apyrimidinic endonuclease (APEX) gene, complete cds	3.488	A DNA repair enzyme and an activator of several transcription factors
D42048	mRNA for squalene epoxidase	3.298	Rate-limiting enzyme of cholesterol biosynthesis
AJ250491	mRNA for receptor activity modifying protein 3 (Ramp3) gene	3.255	Complex with the calcitonin receptor-like receptor, establishing a functional receptor for adrenomedullin
L04280	Ribosomal protein (RPL12) mRNA	3.172	Protein biosynthesis
K02927	Ribonucleotide reductase subunit M1 mRNA, complete cds	3.156	Rate-limiting enzyme in DNA synthesis and repair
AF087568	Palmitoyl-protein thioesterase precursor mRNA, complete cds	3.059	Cell survival signaling
AF020039	NADP-dependent isocitrate dehydrogenase (Idh) mRNA, complete cds	3.038	A key enzyme of the tricarboxylic acid cycle
X57960	Ribosomal protein L7	2.962	Protein biosynthesis
U09816	GM2 activator protein (Gm2a) mRNA	2.888	Enzymatic hydrolysis of GM2
AB018575	Cdc 7 mRNA, complete cds	2.816	Essential for G1/S transition
AF041054	E1B19K/Bcl-2 binding protein homology (Nip3) mRNA, nuclear gene encoding mitochondria protein, complete cds	2.813	Nips is a pro-apoptotic mitochondria protein
L04128	Ribosomal protein L18 (rpL18) mRNA	2.751	Protein biosynthesis
Z31554	(129/SV) Cctd mRNA for CCT (chaperonin containing TCP-1) delta subunit	2.743	Stabilize the cytoskeletal protein actin and tubulin
D63784	mRNA for MIDA1, complete cds	2.703	Regulate cell growth
AF026481	eIF-1A (eIF-1A) mRNA, complete cds	2.682	Translation initiation factor
U35249	CDK-activating kinase assembly factor p36/MAT1, complete cds	2.645	An assembly factor and a targeting subunit of cyclin-dependent kinase-activating kinase that is involved in cell cycle control, transcription and DNA repair
M76131	Elongation factor (ef-2) mRNA, 3’end	2.614	Protein biosynthesis
X64713	mRNA for cyclin B1	2.576	Cell cycle regulatory protein
D26091	mRNA for mCDC47, complete cds	2.570	A member of the minichromosome maintenance (Mcm) family involve in the DNA replication licensing system
U78085	Ribosomal protein S5 mRNA	2.507	Protein synthesis
AF141322	Caveolin-2 mRNA, complete cds	2.458	Inhibit the eNOS activity
M33934	IMP dehydrogenase mRNA, complete cds	2.429	An enzyme involved in de novo synthesis of guanine nucleotides
U83628	Defender against cell death protein 1 (DAD1) mRNA, complete cds	2.372	Apoptotic suppressor gene

Table 4 Part of down-regulated genes in the activated HSC

GenBank accession number	Gene name	Ratio (Cy5/Cy3)	Potential gene function
D49949	mRNA for IGIF precursor poly-peptide in (Interleukin 18), complete cds	0.065	In the development of Th1 cells and tissue injury inflammatory reaction
U63146	Retinal binding protein (RBP) mRNA	0.125	Retinoid storage and metabolism
AF035644	Potentially prenylated protein tyrosine phosphatase mPRL-2 mRNA, complete cds	0.226	Cellular regulation
X58287	mR-PTPu gene for protein tyrosine phosphatase, receptor-type M	0.280	Cellular regulation
AF033381	Betaine homocysteine methyl transferase (BHMT) mRNA, complete cds	0.299	A key liver enzyme that is important for homocysteine homeostasis
M75720	Alpha-1 serine protease inhibitor 3 mRNA, complete cds	0.308	Anti-inflammatory effect
U20497	P19 protein mRNA, complete cds	0.324	CDK4&CDK6 inhibitor, cell cycle inhibitor
D85596	AMP deaminase H-type, complete cds	0.339	Involved in the biosynthesis, inter-conversion and degradation of purine compounds
Y10138	Gene encoding prostaglandin D synthase	0.361	A PGD producing enzyme and a retinoid transporter
NM_013498	CAMP response element modulator (CREM) mRNA	0.366	Down-regulator of CAMP-induced transcription
U67187	G protein signaling regulator RGS2 (rgs2) mRNA, complete cds	0.369	Negatively regulate G-coupled receptor function
AF023919	PK-120 precursor (itih-4) mRNA, complete cds	0.421	Inter-alpha-trypsin-inhibitor H4, is a potential regulator for ECM proteins
AF077950	Protein inhibitor of activated STAT protein PIAS 1 mRNA, complete cds	0.433	Inhibition of STAT-1-mediated gene activation
M75718	Alpha-1 protease inhibitor 4 mRNA, complete cds	0.437	Anti-inflammatory activity
D38046	mRNA for type II DNA topoisomerase beta isoform, complete cds	0.445	An essential enzyme that alters DNA topology which is important for cell survive and apoptosis
AF073996	myotubularin (Mtm1) mRNA complete cds	0.464	Subfamily of protein tyrosine phosphatases

Table 5 Semi-quantitative analysis of RT-PCR results

Groups	Mean ± SD	N	p value
	(quotient of the detected gene/GAPDH)
MIF from quiescent HSC	0.269 ± 0.016	5
MIF from activated HSC	0.759 ± 0.046	5	< 0.05
N-Cadherin from quiescent HSC	0.177 ± 0.063	5
N-Cadherin from activated HSC	0.776 ± 0.087	5	< 0.05
Annexin VI from quiescent HSC	0.137 ± 0.04	5
Annexin VI from activated HSC	0.478 ± 0.025	5	< 0.05
DAD1 from quiescent HSC	0.174 ± 0.016	5
DAD1 from activated HSC	0.593 ± 0.047	5	< 0.05
BHMT from quiescent HSC	0.602 ± 0.083	5
BHMT from activated HSC	0.134 ± 0.059	5	< 0.05