Figure 4

miR-375 control on CIP2A-MYC pathway also contributes to p21 elevation. (A) p21, p53, RB, CIP2A, and MYC protein levels in MCF7 cells transfected with miR-375 inhibitor, -mimic, or NS control were measured by Western blot analysis. Tubulin expression was used as internal control. 25%, 50%, 100% amounts of untreated cell lysates were included to calibrate the semiquantitative measurement. (B) Transfection with miR-375-mimic significantly upregulated p21 mRNA in MCF7. Relative endogenous p21 mRNA levels were measured in MCF7 cells transfected with miR-375 inhibitor, -mimic, or NS control for 48 h using qRT-PCR. (C) CIP2A and MYC protein levels were effectively silenced by si-CIP2A transfection with 1 and 10 nM concentrations for 48 h. Increased p21 protein levels were detected in si-CIP2A dose-dependent manner. 10 nM of si-GFP was used as a control. (D) Protein levels of CIP2A, p53, and p21 in MCF7 cells transfected with si-p53 and/or miR-375-mimic were measured by Western blot analysis. (E) mRNA levels of p21 in MCF7 cells transfected with si-p53 and/or miR-375-mimic were measured by qRT-PCR. (F) Flow cytometry analysis demonstrates G1 arrest of MCF7 cells 48 h after transfection with miR-375-mimic compared to miR-375 inhibitor or NS control. The concentrations of siRNA or miRNA used in panels D, E, and F were 10 nM and 25 nM, respectively. (G) Schematic depiction of miR-375-mediated repression of CIP2A, E6, E6AP, and E7 in HPV16-positive cells that simultaneously increases tumor suppressor p53, p21, and RB, and causes cell cycle arrest. Results are expressed as mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.