miR-133a positively regulated p53/p21 pathway.

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miR-133a positively regulated p53/p21 pathway. miR-133a positively regulated p53/p21 pathway. A, representative images of the G0–G1 phase arrest by miR-133a overexpression. Cell-cycle distribution was analyzed by fluorescence-activated cell sorting after G2 trapping assay. Briefly, cells were transfected with miR-133a or miR-Ctrl. Nocodazole (25 ng/mL) was added 48 hours after transfection for another 14 hours. B, miR-133a caused accumulation of cells at G0–G1 phase. C, miR-133a increased the protein level of p21 and p53 in HCT116 and LoVo cells. c-myc protein was unchanged after treated with miR-133a in both cell lines. D, ectopic expression of miR-133a induced p21 mRNA expression in HCT116 and LoVo. E, ectopic expression of miR-133a showed no significant effects on p53 mRNA level in HCT116 and LoVo. F, ectopic expression of miR-133a activated p53/p21 pathway in HCT116 cells. G, ectopic expression of miR-133a activated p53/p21 pathway in LoVo cells. p53-binding activities and p21 promoter transcription activities were determined by dual luciferase assay system at 48 hours after transfection (n = 3, mean ± SD). H, si-p53 antagonized miR-133a–mediated p21 induction. HCT116 and LoVo cells were transfected with the miR-133a, siRNA against p53, or the corresponding RNA control in the indicated combination for 24 hours. The protein levels of the indicated gene were determined by Western blotting. I and J, si-p53 antagonized miR-133a–mediated growth-inhibitory effect in MTT assay. HCT116 (I) and LoVo (J) cells were transfected with the miR-133a, siRNA against p53, or the corresponding RNA control in the indicated combination. NS, not significant. Yujuan Dong et al. Mol Cancer Res 2013;11:1051-1060 ©2013 by American Association for Cancer Research