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. 2022 May 20;29:47–63. doi: 10.1016/j.omtn.2022.05.031

Figure 4.

Figure 4

miR-520c-3p targets PTEN to mediate HBV-induced cell migration and invasion

(A) Bioinformatics identification of the miR-520c-3p binding site in PTEN 3′-UTR and its validation with a reporter assay. PTEN 3′-UTR luciferase reporter plasmid (PTEN-wild type) or its mutant plasmid (PTEN-Mut) were transfected into Huh7 cells in the presence of miR-520c-3p mimic or scrambled control, and relative luciferase expression is shown as the mean ± standard deviation. (B) miR-520c-3p regulates the level of PTEN in cells. Huh7 cells were transfected with miR-520c-3p mimic/inhibitor or NC miRNA. At 48 h after transfection, the levels of PTEN were examined by qPCR (top) and western blot (bottom). (C) PTEN mRNA and protein levels are much lower in HBV-infected HCC tissues compared with their adjacent normal tissues and the reduction in PTEN level is accompanied by increased miR-520c-3p level in human patients. (Top) qPCR analysis of PTEN levels in HCC (T) or adjacent normal (N) tissues. (Middle) Western blot analysis of PTEN levels in HCC (T) or adjacent normal (N) tissues on randomly selected 18 out of patient tissues, the level of PTEN expression was significantly lower in HCC tumor tissues compared with their adjacent normal tissues by western blot. (Bottom) PTEN mRNA expression was negatively correlated with miR-520c-3p mRNA expression in HCC tumor tissues (R = −0.5000, p = 0.0016). (D) PTEN mRNA and protein levels were decreased by HBV in PHHs and Huh7 cells. (Left) HBV virus-infected PHHs; (right) HBV plasmids transfected into Huh7 cells. At 48 h after infection/transfection, PTEN mRNA (top) and protein (bottom) levels were determined. (E) Anti-miR-520c-3p prevented the downregulation of PTEN protein levels by HBV. Huh7 cells were co-transfected with HBV and/or miR-520c-3p-inhibitor or controls and subjected to western blot analysis of the level of PTEN. (F) PTEN is important for HBV-induced migration, invasion, and EMT via miR-520c-3p. Huh7 cells were transfected with pUC18 or pHBV1.3, NC-inhibitor, or miR-520c-3p inhibitor, and siNC or siPTEN and were then assayed for migration and invasion as in Figure 2A (left). The levels of the EMT-associated proteins Vimentin, E-cadherin, and MMP9 were determined by western blot analyses (right). Three independent experiments were performed, and representative data are shown. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 in (A) and (B) and (D–F).