Restoration of Microrna-126 Expression Reduces Apoptosis in Hepatitis B Virus Infected Hepatocellular Carcinoma Cells

Abstract

Background and Aim: Hepatocellular carcinoma (HCC) is third leading cause of cancer related mortality worldwide. Chronic hepatitis B (CHB) is one of the reasons for HCC development. India has over 40 million chronic HBV infected individuals, but estimation of HCC is grim. The reasons of the under report may be due to its asymptomatic appearance, late detection in the advanced stages and poor awareness. Extrapolating our previous findings of miR-126 as biomarker for HBV related HCC, here we have elucidated the pathophysiological role of overexpressed miR-126 in hepatocarcinogenesis. Methods: Targets of miR-126 were identified using TargetScan, microRNA.org and miRecords. premiR-126 was cloned, overexpressed in HepG2 cell line and expression of miR-126 and its targets were verified by qRT-PCR and western blot analysis. Trancriptional regulation of miR-126 was decided using ENCODE Chip seq database and verified by ChIP analysis. Cellular apoptosis was determined by Annexin V/PI method. Results: To validate our previous oveservation, miR-126 expression was verified in HBV transfected cell line along with two transactivator proteins HBx and HBc independently. Similar to the data in HCC samples, miR-126 was overexpressed in HBV-DNA transfected cells, but no effect of HBc and HBx was noted. Considering Bioinformatics analysis and ENCODE ChIP-seq data, binding of MYC as major transcription factor for miR-126 was validated by ChIP analysis with anti-MYC antibody and also verified after overexpression of Myc-cDNA. This data correlated with MYC overexpression in HCC samples compared to liver cirrhosis. In silico analysis showed that miR-126 could target several proteins involved in apoptosis and inflammosome pathways (CASP1/CASP3/APAF1/CARD16). Furthermore, apoptosis assay and western blot analysis also showed that miR-126 reduces apoptotic cell death by reducing CASP3 expression. Conclusion: Thus, our data highlights that miR-126 may induce uncontrolled proliferation of cancer cells and inhibition of mir-126 may be an important therapeutic strategy for HBV-related HCC. The authors have none to declare.