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目的:探讨针对UHRF1基因的小干扰RNA(siRNA)转染人卵巢癌细胞株OVCAR-3后UHRF1表达的变化及细胞的增殖和凋亡,为UHRF1基因作为新的靶点治疗卵巢癌提供依据。方法:用siRNA基因沉默技术,将体外化学合成的针对UHRF1基因的siRNA转染至OVCAR-3细胞(转染组),并以转染阴性对照siRNA序列的细胞作为阴性对照组,未经转染的细胞作为空白组;实时荧光定量PCR和Western blot分别检测转染前后细胞UHRF1 mRNA和蛋白表达水平的变化;CCK-8实验和流式细胞术分别检测沉默UHRF1基因后对细胞增殖和凋亡的影响。结果:实时荧光定量PCR结果显示,siRNA转染后UHRF1基因在卵巢癌细胞株OVCAR-3中表达显著降低(P<0.01)。Western blot结果显示,转染组、阴性对照组和空白组细胞UHRF1蛋白相对表达量分别为0.16±0.04、0.33±0.03、0.35±0.07;转染组细胞UHRF1蛋白表达水平显著低于阴性对照组和空白组(P<0.05),而阴性对照组和空白组之间无显著差异(P>0.05)。CCK-8实验结果显示,转染后48,72,96h,转染组细胞增殖率均显著低于空白组(P<0.01)。流式细胞术结果显示,转染组、阴性对照组细胞凋亡率分别为(15.83±2.22)%、(7.46±2.93)%,差异显著(P<0.01)。结论:UHRF1基因沉默后可显著抑制卵巢癌OVCAR-3细胞UHRF1的表达,并抑制细胞增殖,促进细胞凋亡。
OBJECTIVE: To investigate the changes of UHRF1 expression and proliferation and apoptosis of human ovarian cancer cell line OVCAR-3 transfected with small interfering RNA (siRNA) against UHRF1 gene and to provide evidence for the treatment of ovarian cancer with UHRF1 gene as a new target. METHODS: siRNAs against UHRF1 gene were synthesized and transfected into OVCAR-3 cells (transfected group) by siRNA gene silencing technique. Cells transfected with negative control siRNA sequences were used as negative control group, untransfected . The changes of UHRF1 mRNA and protein expression before and after transfection were detected by real-time fluorescence quantitative PCR and Western blot. The cell proliferation and apoptosis were detected by CCK-8 assay and flow cytometry after silenced UHRF1 gene influences. Results: The real-time PCR results showed that the expression of UHRF1 gene was significantly decreased in OVCAR-3 ovarian cancer cells after siRNA transfection (P <0.01). The results of Western blot showed that the relative expression of UHRF1 in transfection group, negative control group and blank group were 0.16 ± 0.04, 0.33 ± 0.03 and 0.35 ± 0.07, respectively. The expression of UHRF1 in transfected group was significantly lower than that in negative control group (P <0.05), while there was no significant difference between the negative control group and the blank group (P> 0.05). The results of CCK-8 assay showed that at 48, 72 and 96 h after transfection, the cell proliferation rate in transfection group was significantly lower than that in blank group (P <0.01). The results of flow cytometry showed that the apoptotic rates in transfected group and negative control group were (15.83 ± 2.22)% and (7.46 ± 2.93)%, respectively (P <0.01). Conclusion: The silencing of UHRF1 can significantly inhibit the expression of UHRF1 in OVCAR-3 cells and inhibit cell proliferation and apoptosis.