Figure 3

MiR-3188 functions as an oncogene in HCC by targeting ZHX2 and activating Notch signaling. (a) Schematic diagram of putative binding sites of miR-3188 on ZHX2 3′-UTR. ZHX2 3′-UTR mutant indicated the 3′- UTR of ZHX2 with the mutation in miR-3188 binding sites. (b) Relative luciferase assay in HepG2 cells showed that miR-3188 significantly suppressed luciferase activity of wild-type reporter constructs, whereas miR-3188 KO significantly promoted luciferase activity of wild-type constructs. (c) MiR-3188 overexpression significantly reduced the luciferase activity from wild-type ZHX2 3'-UTR luciferase reporter construct, whereas this profound inhibition was abolished when the putative miR-3188 target sequences in the ZHX2 3′-UTR were mutated in the vector. (d) Overexpression of miR-3188 was inversely correlated with ZHX2 downregulation (R²=0.508, P<0.01, Pearson’s correlation). (e) When HepG2 cells were co-transfected with miR-3188 KO or control and siZHX2 or siRNA-NC using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction, cell viability of transfected cells was determined by using CCK-8 assay at 24, 48, and 72 h. (f) Cell cycle was detected 24 h after transfection by propidium iodide staining flow cytometry. Bar charts indicated the percentage of cells in G0–G1, S, and G2–M cell cycle phases. (g) Cell apoptosis was determined by Annexin-V/phycoerythrin combined labeling flow cytometry 48 h after transfection. Evaluation of apoptosis was determined by the percentage of apoptotic cell number in total cell number. (h) Migration ability was quantified by measuring gap distance at time points 0 and 72 h. (i) Representative images of plate colony formation and anchorage-independent growth in HepG2 cells are shown. (j) Histograms show the colony numbers of the indicated clones. (k) Representative images of cell migration (24 h after transfection) and invasion (48 h after transfection) across a membrane with 8 mm pores with or without Matrigel are exhibited. (l) Histograms indicate the number of cells across the membrane. All experiments were performed in triplicate. *P<0.05, **P<0.01