下调EZH2表达促进卵巢癌细胞衰老的机制研究

目的:探讨下调 EZH2 表达促进卵巢癌细胞衰老的分子机制。方法:采用real-time PCR、Western blot及免疫组化检测卵巢癌组织和正常组织及4种卵巢癌细胞和人正常卵巢上皮细胞IOSE80中EZH2的表达水平;运用脂质体2000转染法将 EZH2 小干扰RNA转染,或将GSK126作用于卵巢癌细胞和IOSE80细胞,siRNA转染的IOSE80细胞经5 Gy电离辐射照射,以阴性对照siRNA为对照。MTT法、集落形成实验、流式细胞术和SA-β-Gal染色法检测细胞增殖、凋亡率和细胞衰老情况;Western blot检测EZH2、p53、p21、p16、caspase-3、cleaved caspase-3、PARP、cleaved PARP、H3K27me3、H3K27me2和H3K27me1的蛋白水平。结果: EZH2在卵巢癌组织和4种卵巢癌细胞中的表达水平均显著高于正常组织和IOSE80细胞( P <0.01);敲减 EZH2 表达显著抑制卵巢癌细胞的增殖,促进电离辐射诱导的细胞衰老,其作用与GSK126处理的细胞表型相一致。Western blot检测发现敲减 EZH2 表达明显抑制H3K27me3的表达,促进p53、p21和p16的表达( P <0.01),但对细胞凋亡通路关键蛋白的水平无影响。结论: EZH2在卵巢癌组织和卵巢癌细胞中高表达。敲减 EZH2 表达通过降低H3K27me3水平促进卵巢癌细胞衰老,从而抑制细胞增殖。

Knock-down of EZH2 expression promotes senescence of ovarian cancer cells

AIM: To investigate the molecular mechanism of down-regulated EZH2 expression promoting senescence of ovarian cancer cells. METHODS: Real- time PCR, Western blot and immunohistochemistry were used to detect the expression of EZH2 in ovarian cancer tissues, normal tissues, 4 ovarian cancer cell lines and IOSE80 cells. The ovarian cancer cells and IOSE80 cells were transfected with EZH2 siRNA (siEZH2) by Lipofectamine 2000 or treated with GSK126. Transfected IOSE80 cells were treated with ionizing radiation for 72 h, and negative control siRNA served as a control. The cell proliferation, apoptotic rate and senescence were detected by MTT assay, colony formation assay, flow cytometry and SA-β-Gal staining. The protein levels of EZH2, p53, p21, p16, caspase-3, cleaved caspase-3, PARP, cleaved PARP, H3K27me3, H3K27me2 and H3K27me1 were determined by Western blot. RESULTS: The EZH2 expression in the ovarian cancer tissues and ovarian cancer cells was significantly higher than that in the normal tissues and IOSE80 cells, respectively ( P <0.01). siEZH2 significantly inhibited the proliferation of ovarian cancer cells, and promoted ionizing radiation-induced senescence. This effect was consistent with the cell phenotype after GSK126 treatment. Knock-down of EZH2 expression significantly inhibited the expression of H3K27me3, promoted the expression of p53, p21 and p16 ( P <0.01), and had no effect on the protein levels of the key molecules in the apoptotic pathway. CONCLUSION: EZH2 is highly expressed in ovarian cancer tissues and ovarian cancer cells. Knock-down of EZH2 expression promotes the senescence of ovarian cancer cells via decrease in H3K27me3 level, thus inhibiting the proliferation of the cells.