Validation And Functional Assay On HBV-expressed MiRNA And Analysis Of HBV Effect On Cellular MiRNA Expression In Hepatocellular Carcinoma Cell Lines

Hepatitis B virus (HBV) is the smallest DNA virus infecting human beings. It’s one of the crucial reasons for virus hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). However, there is currently no effective way to eradicate HBV infection. Deep research on HBV pathopoiesis will help to solve the problem. miRNA is an endogenous, regulative non-coding RNA of-22nt in length. It was reported that many DNA viruses have coding sequences for miRNAs. These viral miRNAs interact with host genes and promote viral infection. The first part of my research was the validation and functional analysis of HBV-expressed miRNA. Previous study in our group revealed that several HBV genomic regions may generate stem-loop structure, which is essential for miRNA biogenesis. Through next-generation sequencing of the HepG2.2.15(contain complete HBV genome to secret viral particle) small RNAs, we found a miRNA expressed by HBV (named hbv-miR-BCP). Subsequent experiments verified the expression of hbv-miR-BCP in HepG2.2.15. Based on the sequence of hbv-miR-BCP, three host candidate targets——TRADD, ULBP2, ULBP3were obtained by miRanda software. These genes associate with pathways of eliminating viral infection. TRADD was then validated to be the probable target by luciferase assay. These results provided significant basis for functional study of HBV miRNA and HBV pathopoiesis.The second part of my study was the analysis of HBV effect on cellular miRNA expression in hepatocellular carcinoma cell lines. miRNA profile vary from different stages as well as cells and tissues. We identified distinct miRNA profiles between HepG2and HepG2.2.15by sequencing the total small RNAs of these two cell lines. Among the diversely expressed miRNAs,10miRNAs were validated through qRT-PCR. HBV X protein (HBx) was speculated to contribute to different miRNA profile due to its transcription-modulatory function. We thus investigated the expression pattern of the previous10miRNAs in HBx overexpressed HepG2and regular HepG2cells. Five of them were confirmed to express differently between these two cell lines. We suggest futher research on these miRNAs will help to reveal the pathogenesis of HCC.