Figure 4.

MKL2 as a microRNA (miR)‐532‐5p target and biological effects of miR‐532‐5p introduction compared with KRAS or MKL2 knockdown in lung adenocarcinoma cells. (a) Quantitative RT‐PCR and Western blot analyses of MKL2 in miR‐532‐5p‐introduced NCI‐H23 and NCI‐H1299 cells. Rel. exp., relative expression. (b) Dual luciferase assay using reporter constructs carrying wild‐type (wt) or mutant (mut 1, mut 2, mut 1 + 2) sequences of the MKL2 3′‐UTR in NCI‐H23 cells transiently transfected with miR‐532‐5p mimics or negative control (NC). (c) Colony formation assay for the same four lung adenocarcinoma cell lines with and without KRAS mutations. Treatment with miR‐532‐5p and siMKL2 markedly inhibited colony formation in all four cell lines, whereas siKRAS treatment significantly reduced the number of colonies only in NCI‐H23 and ACC‐LC‐94 cells. (d) Flow cytometry analysis was used to determine sub‐G₁ populations in the same panel of four lung adenocarcinoma cell lines. Sub‐G₁ populations were markedly increased in all four cell lines when treated with either miR‐532‐5p or siMKL2, whereas siKRAS treatment significantly induced apoptosis only in NCI‐H23 and ACC‐LC‐94 cells. All experiments were carried out in triplicate and data shown represent mean ± SD. *P < 0.05; **P < 0.01, two‐tailed Student's t‐test. n.s., not significant.