Function And Mechanism Of Mirnas In The Process Of Immune Tolerance Of HBV-positive Hepatocellular Carcinoma

ObjectThe hepatitis B virus (HBV) is a widespread pathogen all over the world, and particularly in China, where there is over25%infected patients. According to statistics, there are more than90%of liver diseases were related to HBV infection in China. Chronic HBV infection is the major risk factor for hepatocellular carcinoma (HCC). Because chronic HBV infection could not only cause serious liver tumor but also lead to changes of tumor microenviroment and lower immune response to cancer cells (immune tolerance). Understanding the function of HBV infection in HCC tumorigenesis and the mechanism involved in this procession is good for providing better therapy to HCC. Finding specific factors or signal pathways that participated in the procession and then further breaking the Immune tolerance, possibly new insights of therapeutic relevance will be yielded.Hepatitis B virus (HBV) belongs to a family of small, enveloped DNA virus and causes acute or persistent infection in liver. Infection of the human liver with HBV induces the development of chronic hepatitis, cirrhosis and terrible hepatocellular carcinoma (HCC). Chronic and persistent HBV infection could lead to immune tolerance, which is meaning the failure to mount an immune response to an antigen. In this condition, HCC cancer cells could not be cleanup by host immune system and more harmful to host. Human leucocyte antigen-G (HLA-G) belongs to a non-classic MHC-I family and was considered to be an immune tolerance molecule, which could bind to immunosuppressive receptors and induced immunosuppressive signaling. Preliminary study found that HLA-G was up-expressed in gastric carcinoma and lung cancer cells and contributed to tumor immune escape. HLA-G might become a new marker of tumor.MicroRNA (miRNA) is an endogenous, small, non-coding RNA which can bind to specific target complementary mRNA and inhibit translation. MicroRNA plays an important role in various biological processes, including cellular death, proliferation and differentiation, and is also involved in various diseases such as cancer. Recently, there numerous studies highlighted miRNAs were significantly differentially expressed in HCC tumorigenesis and the expression was tissue-spectific, indicating that miRNAs may cause great epigenetic changes in HCC tumorigenesis. However, most studies focused on the porpuse to investigate the relationship between the expression of miRNAs with tumor progression and prognosis, by testing the expression of miRNAs in different style/stage of liver diseases or in lesion tissues and blood. Only few studies focused on the porpuse to investigate the function of miRNAs in HCC tumorigenesis, which was important to HCC therapy and diagnosis.In this study, we first investigated the expression of HLA-G and miRNAs in HCC tissues and HCC cell lines, especially in HBV infected HCC tissues. We found that the expression of HLA-G was up-regulated by HBV infection and was related to the expression of miR-152. Then we further tried to find out the relationship between HBV infection and HLA-G/miRNA expression in HCC cell lines in vitro, finding that miR-152was down-regulated by HBV infection and then leding to high expression of HLA-G which suppressed the cytotoxity of NK cells against cancer cells by "HLA-G-ILT" interaction.Methods1. The expression of HLA-G in clinic Hepatocellular carcinoma (HCC) tissues and adjacent no-tumor tissues was detected by Immunohistochemistry;2. RT-PCR and Real-time PCR were used to analyse the expression of miR-148a, miR-148b, miR-152and U6in clinic Hepatocellular carcinoma tissues;3. Real-time PCR, FACS and Western Blot were used to analyse the expression of HLA-G in HepG2.2.15and HepG2cells;4. MTT was used to determine the cytotoxity of NK-92cells against HepG2and HepG2.2.15cells;5. Real-time PCR was used to analyse the expression of miR-148a, miR-148b and miR-152in HepG2.2.15and HepG2cells;6. HBV DNA sequence was transfered into HepG2cells by Lipofactamine2000using a pSuper plasmid vector; Real-time PCR and Western Blot were used to analyse the expression of HLA-G; Real-time PCR was used to analyse the expression of miR-148a, miR-148b and miR-152; MTT was used to determine the cytotoxity of NK-92cells against HBV+HepG2cells;7. miR-148a/148b/152mimics were transfered into HepG2.2.15cells by Lipofactamine2000; Real-time PCR and Western Blot were used to analyse the expression of HLA-G; MTT was used to determine the cytotoxity of NK-92cells against HBV+HepG2cells.Results1. The expression of HLA-G protein in tissues of patient with HBV positive HCC was significantly higher than that of with negative HCC by Immunohistochemistry. And the expression of miRNA-152in HBV positive HCC tissues was lower than in HBV negative HCC tissues by real-time PCR, there was a significant difference between them (P<0.05);2. The expression of HLA-G in HBV+HepG2.2.15cells was significantly higher than in HBV-HepG2cells, both in mRNA and protein levels;3. The cytotoxicity of NK-92against HepG2.2.15cells was lower than against HepG2cells in vitro;4. The expression of miR-148a/148b/152in HBV+HepG2.2.15cells was significantly lower than in HBV-HepG2cells;5. The expression of HLA-G was up-regulated in HepG2cells by ectopic expression of HBV gene while the expression of miR-148a/148b/152were down-regulated, and the cytotoxicity of NK-92against HepG2cells was decreased;6. The expression of HLA-G was down-regulated in HepG2.2.15cells by over-expression of miR-148a/148b/152mimics respectively, and the cytotoxicity of NK-92against HepG2cells was decreased.Conclusion In this study, we focus on the alteration of HLA-G and miRNAs expression inHBV related HCC tumorigenesis and the regulate mechanism between HBV and HLA-G, trying to find out the mechanism of HBV related immune tolerance.From results above, we found that chronic HBV infection could cause immune tolerance of HCC partly by a microRNA-dependent manner, including miR-148a, miR-148b and miR-152. HLA-G was one target of these miRNAs, which could be down-regulated by these miRNAs. As an immunosuppressive factor, HLA-G was contributed to HCC immune tolerance. This study discussed a new mechanism from miRNA expression about HCC immune inhibition due to HBV infection, and analysed the relationship between key moleculars of HBV involved in HCC and miRNA. There may be an important significance to understand further the process of HBV related HCC tumorigenesis and the performance of immune tolerance.Taken together, this understanding between miRNAs and HBV interaction need further investigation and HLA-G and miRNA might be new potentially biomarkers for diagnosis and therapy of HBV infection and HBV-positive HCC.