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

Figure 2.

Figure 2

HBV enhances hepatoma cell migration and invasion by upregulating miR-520c-3p

(A) miR-520c-3p promotes HCC migration and invasion. Huh7 cells were transfected with miR-520c-3p mimic or NC-mimic. The resulting cells were subjected to wound healing assay to measure the migration of the cells (left, top, representative photos of the assay; bottom, quantification of the assay) or Matrigel invasion assay to measure the invasiveness of the cells (right, top, representative photos of the assay; bottom, quantification of the assay). (B) Anti-miR-520c-3p inhibits HCC migration and invasion. Huh7 cells were transfected with miR-520c-3p inhibitor or NC-inhibitor. The cells were then assayed as in (A). (C) miR-520c-3p regulates the expression of proteins involved in EMT. NC-mimic or miR-520c-3p-mimic and NC-inhibitor or miR-520c-3p inhibitor (150 nM) were transfected into Huh7 cells, and the levels of Vimentin, E-cadherin, and MMP9 were examined by western blot. Note that MMP9 and Vimentin were positively regulated by miR-520c-3p while E-cadherin was negatively regulated by miR-520c-3p, suggesting that miR-520c-3p promotes EMT. (D) miR-520c-3p mediates HBV-induced cell migration and invasion. Huh7 cells were transfected with pUC18 or pHBV1.3 plasmids and NC-inhibitor or miR-520c-3p inhibitor before assaying for cell migration and invasion as in (A). (E) miR-520c-3p is required for enhanced EMT by HBV. Huh7 cells were transfected as in (D), and the levels of Vimentin, E-cadherin, and MMP9 were examined by western blot. (F) miR-520c-3p promotes metastasis in vivo. (Left) The levels of miR-520c-3p in LM3-miR-520c stably transfected cells or control cells were detected by qPCR. (Middle and Right) Huh7-miR-520c stably transfected cells were injected intravenously into male BALB/c nude. Eight weeks later, the animals were subjected to live imaging (middle) and the relative fluorescence intensity of the mice was calculated by using ImageJ. The scatter diagram was drawn by using GraphPad Prism software (right). (G) Representative HE images of mouse liver (left) and lung (right) tissues. (Three independent experiments were performed, and representative data are shown.) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 in (A), (B), (D) and (F).