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. 2022 Feb 21;11(1):616–628. doi: 10.1080/22221751.2022.2037393

Figure 2.

Figure 2.

The miR-192-3p increases HBV replication and transcription level in hepatic cell lines. (A–D) HepG2.2.15 cells were transfected with miR-192-3p mimics or inhibitors at 40 nM; Huh7 cells were co-transfected with pSM2 plasmid and miR-192-3p mimics or inhibitors at 40 nM, and harvested at 72 h post-transfection. Secreted HBsAg (A) and (B) HBeAg levels in culture supernatants were determined by chemiluminescence immunoassay. (C) Encapsidated HBV replicative intermediates were isolated and detected by Southern blotting. (D) Intracellular HBV pgRNA level was determined by quantitative real-time RT-PCR analysis using primers matching to the pgRNA-specific region. (E, F) PHHs with HBV virion infection (MOI = 1000) were transfected with miR-192-3p mimics or inhibitors at 40 nM at 96 h post-HBV infection. At six days post-miRNA transfection, the intracellular encapsidated HBV replicative intermediates and pgRNA levels in PHHs were separately determined as described above. (G) The miR-192-3p levels in HepG2.2.15 and Huh7 cells as well as PHHs were measured by quantitative real-time RT-PCR analysis using specific primers. (H, I) Huh7 cells were co-transfected with miR-192-3p mimics or miNC at 40 nM and pMIR-REPORT plasmids (including pMIR-Luc, -HBV FL, -HBV1, -HBV2, -HBV3, or -HBV 3′UTR) or HBV promoter luciferase reporters containing the regions of pSP1, pSP2, pCP, and pXP) for 48 h with Renilla as an internal control. Dual-Glo luciferase report assay was performed to measure the firefly and Renilla luciferase activities. The results were calculated by fold change and normalized to the miNC samples. (J, K) HepG2.2.15 cells were transfected with miR-192-3p mimics or inhibitors at 40 nM and harvested at 72 h post-transfection. Nuclear receptor FXRα mRNA and protein levels were measured by quantitative real-time RT-PCR and Western blotting analysis, separately. *P < .05; **P < 0.01; ns, no significance. miNC, miRNA negative control; anti-miNC, miRNA inhibitor control; PHH, primary human hepatocyte; RC, relaxed circular DNA; SS, single-stranded DNA.