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. 2021 Mar 1;23(1):21–31. doi: 10.22074/cellj.2021.7010

Fig.4.

Fig.4

pcDNA-PVT1 vector for overexpression (PVT1) regulated expression of yes-associated protein 1 (YAP1) through enhancer of zeste homolog 2 (EZH2) -mediated microRNA-497(miR-497) promoter methylation. A. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis for the expression levels of EZH2, miR-497 and YAP1 in A549 and H1299 cells after EZH2 knockdown, treated with si-EZH2 or si-NC. B. Western blot analysis for the protein levels of EZH2 and YAP1 in A549 and H1299 cells after EZH2 knockdown, treated with EZH2 siRNA for knockdown (si-EZH2) or si-NC. C. ChIP–qPCR of EZH2 occupancy and H3K27me3 binding in the miR-497 promoter in H1299 cells with or without PVT1 overexpression. a, b and c represented respectively three pairs of amplification primers for miR-497 promoter; and d and e represented respectively two pairs of amplification primers for U6 promoter as internal references. D. MSP analysis for the methylation level of miR-497 promoter in A549 and H1299 cells treated with si-EZH2 or 5-Aza-dC. E. qRT-PCR analysis for the expression levels of miR-497 and YAP1 in A549 and H1299 cells treated with 5-Aza-dC or combined with PVT1 overexpression. F. Western blot analysis for the protein levels of LAST2 and YAP1 phosphorylation in A549 and H1299 cells treated with 5-Aza-dC or combined with PVT1 overexpression. Data are mean ± SD of three independent experiments, *; P<0.05, **; P<0.01 compared between siRNA negative control (si-NC) and si-EZH2 groups or compared between groups as shown with a horizontal line.5-aza2-deoxycytidine;A549 and H1299; Two of human lung carcinoma cell lines, ChIP; Chromatin immunoprecipitation, MSP; Methylation-specific polymerase chain reaction, LAST2; Large tumor suppressor kinase 2 protein, and SD; Standard deviation.