Figure 7. NKIRAS2 was directly targeted by miR-4492 in OS cells. (A) The miR-4492 was predicted to bind to the NKIRAS2 3′-UTR by Targetscan. Plasmids containing mutant or putative NKIRAS2 3'-UTR-luciferase reporters were transfected into U2OS and HOS cells. (B) The qRT-PCR confirmed the decreased expression of NKIRAS2 in OS cell lines. Results are shown as means±SD. n=5; **P<0.01 by the t-test. (C) The expression of NKIRAS2 in mimic NC, mimic miR-4492, inhibitor NC and inhibitor miR-4492 groups was measured by qRT-PCR. n=5. (D, E) Luciferase activity in U2OS and HOS cells was detected and is shown as mean±SD. n=5; *P<0.05, **P<0.01 by the t-test. (F) Proteins were extracted from OS cells and the expression level of NKIRAS2 was analyzed by western blot. (G) qRT-PCR measured the transfection efficiency of NKIRAS2 in U2OS and HOS cells. n=5; ***P<0.001 by the t-test. (H) Cell viability of OS cells was assessed by CCK-8 assays. n=5; * P<0.05, **P<0.01. (I) Colony formation by HOS and U2OS cell lines was inhibited by the overexpression of NKIRAS2 and was obviously enhanced by NKIRAS2 silencing. The number of clones was shown in the violin plot. n=5; **P<0.01, ***P<0.001 by the t-test. (J) Representative images of invading OS cells. Scale bar: 200 μm.