Figure 4.

R-2HG/ERα can decrease YAP1 expression via altering miR16-5P expression

(A) Real-time PCR showed YAP1 mRNA expression of shERα in QBC939 cells and oeERα in HuCCT1 cells. (B) Real-time PCR detected YAP1 expression in QBC939 and HuCCT1 cells treated w/wo R-2HG. (C) RIP assay to detect the YAP1 mRNA levels in the Argonaute 2 (Ago2) complex in QBC939 cells transfected with pLKO or shERα (left) and in HuCCT1 cells transfected with pWPI or oeERα (right). (D) Quantitative real-time PCR was used to screen seven potential miRNAs that might be able to regulate YAP1 in QBC939 cells transfected with ERα-shRNA or pLKO (left) and HuCCT1 cells transfected with oeERα or pWPI (right). (E) Western blot was used to examine YAP1 expression from the four groups of QBC939 cells as indicated (with shERα+miR16-5p inhibitor, pLKO+miR16-5p inhibitor, shERα+Ctrl, or pLKO+Ctrl) (left), and another four groups in HuCCT1 cells as indicated (oemiR16-5p+pWPI, oemiR16-5p+oeERα, pLKO+pWPI, pLKO+oeERα) (right). (F) MTT (left) and colony formation (middle) assays were performed in QBC939 cells transfected w/wo shERα and w/wo miR16-5p inhibitor as indicated (left) and in four groups of HuCCT1 cells as indicated (middle). (G) MTT (left) and colony formation (middle) assays were performed in 4 groups of HuCCT1 cells as indicated. For (E, F, and G), quantitations are at the right and presented as mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01; NS, not significant.