The Molecular Mechanism of lncRNA H19 RegulatingmiR-146a to Inhibit HBV Replication
ZENG Rong1, ZHANG Zili1, LIU Jiamin1, YU Ling1, YE Qing2, PAN Wanlong1*
This study aims to investigate the molecular mechanism of lncRNA H19 regulating HBV (hepatitis B virus) replication through miR-146a, and lay the foundation for exploring the molecular mechanisms of HBVreplication. The relative expression levels of H19 in HepG2 and HBV stable replicating cell line HepG2.2.15 were detected by RT-qPCR (real time quantitative polymerase chain reaction), and the expression levels of H19 in livertissues of HBV (–) and HBV (+) patients were detected by fluorescence in situ hybridization. After overexpressionor knockdown of H19, miR-146a and FEN1 (flap endonuclease 1) in HepG2.2.15 cells, the expression levels ofH19, miR-146a and its downstream target genes IRAK1 (interleukin-1 receptor-associated kinase 1)/TRAF6 (TNFreceptor associated factor 6) and FEN1 were detected by RT-qPCR, the copy number of HBV DNA was detectedby q-PCR (quantitative polymerase chain reaction), and the expression level of FEN1 protein was detected by WB(Western blot). At the same time, 10 cases of clinical HBV (+) liver tissues and five cases of HBV (–) liver tissueswere collected to test the above indexes, and WB and immunohistochemistry were used to test the protein expression level of FEN1 in the two groups of specimens, respectively. In HepG2.2.15 cells, H19 decreased by 99%compared to the control group. Fluorescence in situ hybridization demonstrated that the expression of H19 in HBV(+) liver tissue was lower than that in HBV (–) liver tissue (P<0.01). After overexpressing of H19 in HepG2.2.15cells, HBV DNA copies decreased by (1.13±0.05)×105 compared with the control group, and miR-146a and FEN1decreased by 63% and 62%, respectively; after overexpression of miR-146a and FEN1 in HepG2.2.15 cells, HBVDNA copies increased by (1.45±0.05)×105 and (0.90±0.13)×105, respectively, and H19 decreased by 51% and 54%compared with the control group (P<0.05). The results of clinical specimens showed that compared with HBV (–)liver tissues, RT-qPCR revealed that H19 decreased by 81% in HBV (+) liver tissue, miR-146a, FEN1 increased21.92 and 5.14 times, respectively. Meanwhile, both WB and immunohistochemistry showed upregulated FEN1protein (P<0.05). lncRNA H19 acts on the downstream target gene IRAK1/TRAF6 by down-regulating the expression of miR-146a, thereby reducing the expression of FEN1 and inhibiting the replication of HBV DNA, laying thefoundation for exploring the molecular mechanisms of HBV replication.
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