Figure 1.

miR-544 downregulates YWHAZ expression by binding to the YWHAZ 3’UTR. A. Predicted binding sites of miR-544 to the 3’UTR of YWHAZ. The wild type (WT), mutated (Mut), and deleted (Del) YWHAZ 3’UTR binding sites are shown. The mutant nucleotides of the miR-544 binding sites are indicated in red. B. Relative luciferase activity of the YWHAZ WT, Mut, and Del reporter in the presence of 30 nM miR-544 mimic or miRNA mimic NC. Renilla luciferase was normalized to firefly luciferase. C. qRT-PCR analysis of YWHAZ mRNA from CaSki and HeLa cells transfected with miR-544 mimic (30 nM) or miRNA mimic NC (30 nM). GAPDH mRNA was used as an internal control. D. Western blot analysis of YWHAZ expression in CaSki and HeLa cells transfected with miR-544 mimic, inhibitor or NC. Tubulin was used as a loading control. E. qRT-PCR analysis of mature miR-544 expression in 20 paired tumor tissues (Tumor) and adjacent normal cervical tissues (Normal) and cervical cancer cell lines (CaSki and HeLa). RNU6B was used as an internal control. F. qRT-PCR analysis of YWHAZ mRNA expression in 20 paired tumor tissues (Tumor) and adjacent normal cervical tissues (Normal) and cervical cancer cell lines (CaSki and HeLa). GAPDH mRNA was used as an internal control. G. Western blot analysis of YWHAZ protein levels in 20 paired tumor tissues (T) and adjacent normal cervical tissues (N). Tubulin was used as a loading control. Two paired samples showing no differences in YWHAZ expression are labeled with boxes. The data represent the mean ± SD from three independent experiments. *P < 0.05.