Figure 2

miR-375 directly targets and represses E6AP. (A) Sequence of putative miR-375 binding site in the 3′UTR of E6AP. E6AP 3′UTR-mut was generated by disrupting base paring of four nucleotides, positions 2 to 5. (B) Relative FL repression effects of miR-375 on the FL-E6AP reporter. Results were compared with FL-E6AP co-transfected with NS control for statistical analysis. (C) E6AP protein level changes in SiHa cells transfected with miR-375 inhibitor, -mimic, or NS control were analyzed by Western blot. Tubulin expression was used as internal control. 25%, 50%, 100% amounts of untreated cell lysates were included to calibrate the semiquantitative analysis. (D) In parallel, relative endogenous mRNA levels of E6AP from SiHa cells transfected with miR-375 inhibitor, -mimic, or NS control were measured using qRT-PCR. (E) E6AP protein level in HeLa cells transfected with miR-375 inhibitor, -mimic, or NS control was analyzed by Western blot. (F) Endogenous E6AP mRNA level in HeLa cells were measured 48 h post-transfection. Results are expressed as mean ± SD from at least three independent experiments. **p < 0.01.