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Novel differential gene expression in human cirrhosis detected by suppression subtractive hybridization
Nicholas A Shackel1, Peter H McGuinness, Catherine A Abbott
1A.W. Morrow Gastroenterology and Liver Centre, Centenary Institute of Cancer Medicine and Cell Biology, Royal Prince Alfred Hospital, Sydney, Australia.
Abstract:
Pathogenic molecular pathways in cirrhotic liver diseases such as hepatitis C virus (HCV), autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) are poorly characterized. Differentially expressed genes are often important in disease pathogenesis. Suppression subtractive hybridization (SSH) is a genome-wide approach that enriches for differentially expressed mRNA transcripts. We aimed to make novel observations of differential gene expression in cirrhosis using SSH combined with quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Liver transcriptomes in HCV cirrhosis, AIH cirrhosis, PBC, and nondiseased liver tissue were examined by SSH. Resulting complementary DNA (cDNA) clones were rescreened for differential expression by dot-blot hybridization and then sequenced. Selected gene expression was quantified by real-time RT-PCR. Following SSH, 694 clones were rescreened for differential gene expression, of which 145 were sequenced and found to derive from 89 different genes. Seven clones were homologous only with expressed sequence tag (EST) sequences encoding genes having no known function. Up-regulated expression of four genes was confirmed by real-time RT-PCR: transmembrane 4 superfamily member 3 (tetraspanin CO-029) in all forms of cirrhosis, hedgehog interacting protein (HIP) in AIH cirrhosis and chitinase 3-like-1 (HC gp-39 or ykl-40) and arginine-glutamic acid repeat (RERE) in HCV cirrhosis. RERE gene polymorphisms and splice variants were observed in all tissues examined. Tetraspanin CO-029 up-regulation was primarily localized to bile ductular cells. In conclusion, novel observations of differential gene expression in human cirrhosis were made using SSH as the primary discovery tool. In particular, further studies of the RERE gene and its products in HCV associated liver disease are warranted.
Insights
This study identified novel differentially expressed genes in liver cirrhosis using suppression subtractive hybridization. Key genes like tetraspanin CO-029, HIP, chitinase 3-like-1, and RERE showed altered expression, offering new insights into cirrhosis pathogenesis.
Area of Science:
- Hepatology
- Molecular Biology
- Genomics
Background:
- Pathogenic molecular pathways in cirrhotic liver diseases like hepatitis C virus (HCV), autoimmune hepatitis (AIH), and primary biliary cirrhosis (PBC) remain poorly understood.
- Differentially expressed genes are crucial in disease pathogenesis, necessitating advanced techniques for their identification.
Purpose of the Study:
- To identify novel differentially expressed genes in human liver cirrhosis using suppression subtractive hybridization (SSH) combined with quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR).
- To investigate gene expression patterns in HCV cirrhosis, AIH cirrhosis, PBC, and non-diseased liver tissues.
Main Methods:
- Suppression subtractive hybridization (SSH) was employed to enrich for differentially expressed mRNA transcripts from liver tissues.
- Complementary DNA (cDNA) clones were sequenced, and differential gene expression was confirmed using dot-blot hybridization and real-time RT-PCR.
- Analysis included liver transcriptomes from HCV cirrhosis, AIH cirrhosis, PBC, and healthy controls.
Main Results:
- SSH identified 89 unique differentially expressed genes across various cirrhosis types.
- Up-regulation of four genes was confirmed: tetraspanin CO-029 (all cirrhosis types), hedgehog interacting protein (HIP) (AIH cirrhosis), chitinase 3-like-1 (HCV and AIH cirrhosis), and arginine-glutamic acid repeat (RERE) (HCV cirrhosis).
- RERE gene polymorphisms and splice variants were detected, and tetraspanin CO-029 upregulation was localized to bile ductular cells.
Conclusions:
- SSH is an effective tool for discovering novel differentially expressed genes in human liver cirrhosis.
- Further investigation into the RERE gene and its role in HCV-associated liver disease is warranted.
- Identifying specific gene expression changes provides new molecular targets for understanding and potentially treating liver cirrhosis.