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( Chia-yen Dai ),( Shu-chi Wang ),( Meng-hsuan Hsieh ),( Cheng-fu Yang ),( Ching-i Huang ),( Chung-feng Huang ),( Ming-lun Yeh ),( Jee-fu Huang ),( Wang-long Chung ),( Ming-lung Yu ) 대한간학회 2017 춘·추계 학술대회 (KASL) Vol.2017 No.1
Aims: The different hepatitis C virus (HCV) replication has been reported among individual hepatocytes in chronic HCV infection by identifying hepatocytes with different HCV RNA levels. We have previously established a fluorescence-activated cell sorting (FACS) protocol to study the effects of different intracellular viral loads in HCV-infected cells. The present study aimed to further study the gene expression on different hepatocellular carcinoma (HCC) cells with different HCV viral load. Methods: The JFH1-EYFP viral florescence intensity was used to sort the high and low viral load cells after 5 days infection in vitro which has been shown in our previous study that infected cells efficiently and accurately discriminated between high- and low-viral load cell populations. The next generation sequence-RNA sequence was used to clarify the mRNA and miRNA gene network between HCV-high and HCV-low infected cells of the HCC cell line. Venn diagram summarizing the probe sets that were differentially expressingbetween the Huh7.5.1 versus each differential viral load cell population and miRDB and miRTar databases were used to predict HVL and LVL/S2 unique miRNA target genes. Results: By analyzing the NGS dataset and miRNA microarray dataset, of the significant transcripts, three miRNA were unique for the LVL/S2 cells and nine miRNA unique for the HVL. Twenty-three miRNA were common for all 3 viral load groups. We verified them by q-PCR and data confirmed the array data expression level. We found that high viral loads were associated with cell inflammation- and cell death-associated pathway; and the low viral loads were associated many stress response- and cell adhesion molecular (CAMs)-related genes. Conclusions: With the established cell sorting protocol, we have demonstrated that different gene network between HCV-high and HCV-low infected cells in JFH1-EYFP infectious cells exists. Our results may provide a boarder gene regulation map between high and low viral load cell populations.