Figure 5.

miR-101-3p Inhibitor Reversed the Inhibitory Effect of RC3H2 Knockdown on Malignant Behaviors of OSCC Cells

(A) miRWalk v2.0 predicts the sequence of EZH2 with highly conserved putative miR-101-3p binding sites and construction of the EZH2-Mut vector, which changed GTACTGTG into AGGACAC from nucleotides 59 to 65. (B) Downregulation of the reporter gene with the wild-type region from EZH2-WT was apparent, whereas no effect on the EZH2-Mut was detected. (C) RC3H2 overexpression partially reversed miR-101-3p mimic-induced reduction of the luciferase activity of the EZH2-WT luciferase reporter vector compared with negative control. (D) The downregulation of EZH2 and H3K27Me3 protein levels by RC3H2 silencing was partially reversed through miR-101-3p inhibitor in HN4 and Cal27 cells. (E and F) The cell proliferation of HN4 (E) and Cal27 (F) cells co-transfected with si-Scramble plus in-NC, si-RC3H2 plus in-NC, si-Scramble plus in-miR-101-3p, or si-RC3H2 plus in-miR-101-3p were evaluated by CCK-8 assays. (G) The colony-forming abilities of HN4 and Cal27 cells co-transfected with si-Scramble plus in-NC, si-RC3H2 plus in-NC, si-Scramble plus in-miR-101-3p, or si-RC3H2 plus in-miR-101-3p were determined by colony-formation assays. (H) The invasion abilities of HN4 and Cal27 cells co-transfected with si-Scramble plus in-NC, si-RC3H2 plus in-NC, si-Scramble plus in-miR-101-3p, or si-RC3H2 plus in-miR-101-3p were performed by Transwell assays. Data are presented as mean ± SD from three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).