| Background:Cholangiocarcinoma is the second most common primary liver cancer, and accounts for approximately 10% -25% of all hepatobiliary malignancies. It is classified into three types:intrahepatic cholangiocarcinoma, hilar cholangiocarcinoma and extrahepatic cholangiocarcinoma. The incidence of cholangiocarcinoma varies substantially in different geographic conditions and populations. Established risk factors for this disease include parasitic infections, primary sclerosing cholangitis, bile duct cysts, intrahepatic bile duct stones and toxins.This study was aimed to search for the feature genes relating to malignant biliary strictures (cholangiocarcinoma) and to annotate biological function of the genes. The study also validated the genes by experimental approaches and investigated their possible roles in cholangiocarcinoma.Methods:We downloaded the gene expression profile GSE34166 from Gene Expression Omnibus database which includes 10 samples (4 benign bile duct samples and 6 cholangiocarcinoma samples). Differentially expressed genes (DEGs) were identified between normal and disease samples using LIMMA package in R language. With these DEGs, protein-protein interaction network was constructed with String software, from which the hub gene was identified with the highest degree. Subsequently, a predicted network of hub gene within all human genes (score>0.9) was built, followed by pathway functional analysis of the genes in the network. RT-PCR was used to detect the expression of important dysregulated DEGs in cholangiocarcinoma cells, which were identified by analysis of gene expression profile GSE34166, in comparsion with that in normal intrahepatic bile duct epithelial cells (NIBEC). PCDNA3.1-GSTA1 plasmid was constructed and transfected into HCCC-9810 cells for overexpression of GSTA1. Annexin V-PI apopotsis assay, flow cytometery and MTT assay were adopted to detect the impact of GSTA1 overexpression and EPHB2 silence on apoptosis, cell cycle and cell viability.Results:1. Totally,378 DEGs were identified between normal and malignant biliary strictures samples, including 212 up-regulated DEGs and 165 down-regulated DEGs. Among these genes, HOXB6, HOXA10 and EPHB2 were significantly up-regulated with a larger fold change, while SLC4A4 and GSTA1 were significantly down-regulated with a larger fold change.2. The protein-protein interaction network was built with 209 pairs of proteins encoded by DEGs. In the network, GSTA1 was selected as hub gene with highest degree. In the predicted network based on the hub gene GSTA1, and GSTA3 were primarily connected with a great number of CYP genes. Result of functional analysis shows that genes in the network were significantly enriched in three pathway:metabolism of xenobiotics by cytochrome P450, drug metabolism and glutathione metabolism. The finding from text mining is that gene GSTA1 might be under the modulation of 4 transcription factors:Sp-1, Ap-1, c-Jun and c-Fos.3. Experimental studies showed that the expression of HOXB6, HOXA10 and SLC4A4 was inconsistent with the predicted results, while the expression of EPHB2 and GSTA1 was consistent with the predicted results.4. Overexpression of GSTA1 exerted no obvious effect on the cell viability, apoptosis and cell cycle of HCCC-9810 cells.5. EPHB2 silence had no obvious effect on apoptosis of RBE cells, while an obvious effect on cell cycle and cell viability.Conclusion:These findings suggested that EPHB2 and GSTA1 gene did differentially expressed in cholangiocarcinoma, and EPHB2 had a notable effect on cell cycle and viability. These potential key genes were expected to be used as new biomarkers on diagnosis and treatment of cholangiocarcinoma. This study may provide new information for the molecular mechanisms of cholangiocarcinoma pathogenesis, provide the theoretical basis and experimental basis for new treatment strategies. |
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