The Impact Of HCV Core Protein On The Expression Of MRNA,lncRNA And CircRNA In Human Huh7 Hepatoma Cell Line And The Preliminary Carcinogenic Mechanism Study

Research background:Hepatitis C Virus (HCV) has a closely correlation with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). As one of the most important fectors, HCV core protein constructs the capsid of HCV, and it participates in immune escape of HCV, resisting to the drug therapy and the liver diseases like live fibrosis. It is known that HCV infection is related to HCC. As a high fatality rate cancer, HCC morbidity is increasing these years. Dysfunction of cell cycle, apoptosis resistance and the activation of oncogene or related singling pathway are the main molecular mechanisms lead to HCC. Noncoding RNAs (ncRNAs) are general terms of the RNAs which won't be translated to proteins, and long noncoding RNAs (lncRNAs) are one of the biology and medical research hotspot, and they have been indicated to take part in growth, development of organisms, and the occurrence of many diseases like HCC. Besides, circular RNAs (circRNAs), as a new functional ncRNAs, have been drawing more and more attentions due to their unique structure and fuctions, such as absorbing miRNA and participating in cancer pathway. According to some researche reports, there are some lncRNAs have been identified to be related to the HCC caused by Hepatitis B virus (HBV). For instance, HBx protein can down regulate the expression of lncRNA-Dreh to lead the proliferation and metastasis of tumor, but the the roles of lncRNAs in HCC, which are caused by HCV or HCV core, are a little of know now. Given that the similarity of HCV and HBV, we can infer that lncRNAs may involve in HCC that is related to HCV or HCV core protein. Moreover, circRNAs have a vast prospect in the field of disease diagnosis and treatment.Research Purpose:Based on these background mentioned, in this study, we use the HCV core protein to investigate the impact of the core protein of HCV 1b gene type on the expression of lncRNA and circRNA in human Huh7 hepatoma cell line and to explore their preliminary carcinogenic mechanism.Research methods:Construct recombination eukaryotic expression vector PEGFP-core (HCV1b genotype core protein), then this vector and empty vector PEGFP-C1 was transfected to the Huh7 cell lines respectively, and stable transfected cell clones were harvested by G418 selection. Then RT-PCR was performed to detect the expression of core protein. After extracting the total RNA of the core stable expression cell clones and conrrol cell clones respectively, they were send to the company to be analyzed by the lncRNA and circRNA microarray to get the impact of HCVlb gene type core protein on the expression profiles of Huh7 cell lines. Then the genes, which were up-regulated or down-regulated 2 folds in core stable expression cell clones compared to the control cell clones, were selected to be analyzed by bioinformatics tools to find the correlation with the cells'physiology and pathology changes. And by combining literature reading and the molecular target site predict platform, fourteen genes were selected and the possible carcinogenic pathway were predicted (pathway picture can be found in the second section of experiment part of this article), then qPCR were performed to verify the expression differences of the molecules in pathways. At last, recombination eukaryotic expression vectors of some molecules (miR122, miR192, miR204) were constructed and transfected into the Huh7-core cell lines respectively to further explore those two relative pathways.Results:There are two parts in LncRNA microarray, and the lncRNA detection part contains 30586 sites, and mRNA detection part contains 26109 sites. The results showed that 4851 lncRNAs were up-regulated and 3569 lncRNAs were down-regulated more than two folds compared to the control genes; 4785 mRNAs were up-regulated and 3192 mRNAs were down-regulated more than two folds compared with the control genes. Circa microarray (containing 5396 sites) found 823 and 419 circRNAs were up-regulated or down-regulated respectively. By cluster analysis and GO-analysis, we found that these genes participate in metabolism including macromolecule metabolic process and nucleic acid metabolic process, biochemistry reaction like DNA damage response and regulation of voltage-gated calcium channel. Meantime, we used the KEGG pathway analysis, and found that these genes have some connection with the endoplasmic reticulum stress, apoptosis, calcium singling pathway, Wnt singling pathway, HBV, HCV infection pathway and others. Moreover,10 genes were confirmed having higher or lower expression in the predicted pathways. In the end, miR122, miR192, miR204 expression vectors were constructed and transfected into the Huh7-core cell lines successfully, and their regulation to the target genes were verified by qPCR.Conclusion:miR204-HPCAL, HOTTIP-GLS and Circ0001498-miR122-TGFBRAP1 two pathways may involve in the carcinogenic progression of HCVlb gene type core protein that participates in HCV related HCC.