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Figure 3 | Molecular Cancer

Figure 3

From: miR-375 activates p21 and suppresses telomerase activity by coordinately regulating HPV E6/E7, E6AP, CIP2A, and 14-3-3ζ

Figure 3

miR-375 increases p21, p53, and RB in HPV16- and 18-positive cancer. (A) Protein levels of p21, p53, and RB in SiHa cells transfected with miR-375 inhibitor, -mimic, or NS control were measured by Western blot analysis. 25%, 50%, 100% amounts of untreated cell lysates were included to calibrate the semiquantitative measurement. (B) Relative endogenous mRNA levels of p21, p53, and RB were measured in SiHa cells transfected with miR-375 inhibitor, -mimic, or NS control using qRT-PCR. (C) One hundred thousand SiHa cells were seeded on 24-well plate and the number of cells was counted by trypan blue exclusion staining assay 48 h post-transfection. (D) Protein levels of p21, p53, and RB in HeLa cells transfected with miR-375 inhibitor, -mimic, or NS control were measured by Western blot analysis. (E) Relative p21 mRNA levels were measured in HeLa cells transfected with miR-375 inhibitor, -mimic, or NS control. (F) Trypan blue exclusion staining assay was used to analyze the proliferation rate of HeLa cells 48 h post-transcfection. (G) Flow cytometry analysis demonstrates G1 arrest of SiHa cells 48 h after transfection with miR-375-mimic compared to miR-375 inhibitor or NS control. (H) Protein levels of CIP2A, p53, and p21 in SiHa cells transfected with si-CIP2A and/or si-p53 were measured by Western blot analysis. (I) Protein levels of CIP2A, p53, and p21 in SiHa cells transfected with si-p53 and/or miR-375-mimic were measured by Western blot analysis. (J) mRNA levels of p21 in SiHa cells transfected with si-p53 and/or miR-375-mimic were measured by qRT-PCR. The concentrations of siRNA or miRNA used in panels H, I, and J were 10 nM and 25 nM, respectively. Results are expressed as mean ± SD from three independent experiments. *p < 0.05, ***p < 0.001, and ****p < 0.0001.

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