The Expression Of Connexin32 Gene And Its Methylation Regulation In Hepatocarcinoma Cell Line

Background: Hepatocellular carcinoma(HCC) is one of the most common tumors in human. Many studies have showed that there are multiple genetic and epigenetic alterations involved in the carcinogenesis of HCC. But its molecular mechanisms are poorly understood. So for, several HCC-related genes have been found. Connexin is such a gene has been recognized as a tumor suppressor gene family. The Loss of connexins expression has been reported in many types of tumors including HCC. However ,the molecular mechanism of its inactivation is not very clear.Object: to investigate the role of methylation in connexin32 gene silencing in hepatcarcinoma cell line—HepG2 cell line,so as to restore the normal expression and regain the normal GJIC, to supply some theory foundation for the diagnose and treatment of hepatocellular carcinoma.Method: culture HepG2 cell line in three groups: one was cultured in media without 5-Aza-2'-CdR,the other two were cultured in media with diffirent concentration of 5-Aza-2'-CdR(1uM,5uM).To investigate the expression of Cx32 gene of the three groups by immunohistochemical method; apply methylation- specific PCR(MS-PCR) to probe their methylation status in Cx32 gene promotor region.Results: The total positive rate of the Cx32 expression in the three groups was 18.5% (+17%,++1.5%),77% (+65.5%,++11.5%),88 % (+58 %,++30% ) respectively ,there was significant diffirence( P < 0.01). The location of Cx32 protain was still aberrant, after demethylation, both the cell growth and malignancy decreased. The results of the MS-PCR were hypermethylation, partial demethylation and total demethylation, respectively.Conclusion: 1.The Cx32 gene of HepG2 was hypermethylation and the expression of Cx32 was down-regulated, which indicated the Cx32 expression of HCC was abnormal; 2.After demethylation, the Cx32 expression of HepG2 cells was increased, the growth of the cell line was repressed, indicated that the Cx32 expression was regulated by methylation. 3. The location of the increased Cx32 protein was still aberrant after demethylation, maybe there were other regulations.