Figure 3.

KLF4 promotes miR-544 transcription by activating miR-544 promoter. A and B. qRT-PCR and western blot analysis of KLF4 expression in CaSki and HeLa cells transfected with pcDNA-KLF4. C. Relative luciferase activity of the miR-544 promoter in HeLa cells transfected with pcDNA-KLF4. Luciferase constructs containing the miR-544 promoter (pGL3-Promoter) and the unmodified construct (pRL-TK) were co-transfected with pcDNA3.1 or pcDNA-KLF4 into HeLa cells. Firefly luciferase activity was normalized to Renilla luciferase activity. D. qRT-PCR analysis of miR-544 expression in CaSki and HeLa cells with KLF4 overexpression. E and F. qRT-PCR and western blot analysis of YWHAZ expression in CaSki and HeLa cells with KLF4 overexpression. G. qRT-PCR analysis of KLF4 mRNA expression in 20 paired tumor tissues (Tumor) and adjacent normal cervical tissues (Normal) and cervical cancer cell lines (CaSki and HeLa). H. ChIP qRT-PCR assay were performed to detect the in vivo binding of transcription factor KLF4 to miR-544 promoter. HeLa cell chromatin fragments were immunoprecipitated with the specific anti-KLF4 antibody or normal IgG. The immunoprecipitated DNA samples were analyzed by qRT-PCR using primers specific to the miR-544 promoter. I. Relative luciferase activity of the mutant miR-544 promoter constructs in HeLa cells transfected with pcDNA-KLF4. Firefly luciferase activity was normalized to Renilla luciferase activity. The data represent the mean ± SD from three independent experiments. *P < 0.05.