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. 2017 Oct;187(10):2288–2299. doi: 10.1016/j.ajpath.2017.06.014

Figure 4.

Figure 4

EZH2 represses miR-34a expression through H3K27 trimethylation in cholangiocarcinoma (CCA) cells. A: Representative immunohistochemistry for EZH2 in human CCA tissue. The brown color indicates positive signals; nuclei were counterstained as blue. The boxed area in the left panel is shown at higher magnification in the right panel. B: Western blot analysis for EZH2 in nonmalignant human cholangiocyte cell (H69) and CCA cells (CCLP1, SG231, HUCCT1, and TFK1). C: The levels of miR-34a in CCA cells with/without GSK126 treatment for 72 hours, as determined by quantitative RT-PCR (RT-qPCR). D: Chromatin immunoprecipitation (ChIP) assay. The chromatin extracted from CCLP1 and SG231 cells treated with or without GSK126 was subjected to immunoprecipitation with H3K27me3 antibody, and the precipitated DNA was subjected to RT-qPCR analysis using two sets of primers to amplify the miR-34a promoter region, as shown in the schematic diagram. Normal mouse IgG was used as the negative control. E: RT-qPCR analysis for miR-34a in SG231 cells transfected with two individual EZH2 shRNA or control vector (pSMP). F: ChIP assay with EZH2 antibody followed by RT-qPCR analysis in SG231 cells with or without GSK126 treatment. Data are expressed as means ± SD (D and F); data are expressed as means ± SEM (C and E). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Original magnification: ×100 (A, left panel); ×200 (A, right panel). TSS, transcription start site.