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目的通过RNA干扰技术抑制卵巢癌SKOV3细胞分泌型clusterin(sCLU)基因的表达,研究其对卵巢癌细胞增殖、凋亡及侵袭能力的影响。方法通过脂质体介导靶向sCLU基因的siRNA转染卵巢癌SKOV3细胞,实验分为4组:实验组(sCLU-siRNA+脂质体)、阴性对照组Ⅰ(negative control siRNA+脂质体)、阴性对照组Ⅱ(sCLU-siRNA)、空白对照组(等体积的完全培养液)。RT-PCR和蛋白质印迹分析法检测sCLU表达;利用MTT法、Transwell小室体外侵袭实验及流式细胞仪AnnexinⅤ/PI法检测评价sCLU基因抑制后对卵巢癌细胞增殖、侵袭及凋亡的影响。结果靶向sCLU基因的siRNA明显、特异性地抑制了sCLU mRNA和蛋白的表达水平;MTT实验结果显示实验组细胞增殖能力较各对照组明显降低,差异有统计学意义(P<0.05)。流式细胞仪AnnexinⅤ/PI法检测结果显示实验组细胞凋亡率较各对照组明显升高,达到(15.84±1.53)%,较空白对照组升高了约9%,差异有统计学意义(P<0.01)。侵袭实验结果提示实验组细胞侵袭能力受到明显抑制,实验组穿膜细胞数量为(26.52±6.22)个/视野,较各对照组明显降低,差异有统计学意义(P<0.01)。结论 sCLU基因沉默明显抑制了卵巢癌SKOV3细胞的增殖、侵袭能力,并增加了其凋亡率,sCLU基因有望成为卵巢癌治疗的新靶点。
Objective To investigate the effect of RNA interference on the expression of secreted clusterin (sCLU) gene in ovarian cancer cell line SKOV3 and its effect on the proliferation, apoptosis and invasion of ovarian cancer cells. Methods Liposome-mediated siRNA targeting sCLU gene was transfected into SKOV3 ovarian cancer cells. The experiment was divided into four groups: experimental group (sCLU-siRNA + liposome), negative control siRNA group (liposome) Negative control group Ⅱ (sCLU-siRNA), blank control group (equal volume of complete medium). The expression of sCLU was detected by RT-PCR and Western blotting. The effects of sCLU gene inhibition on the proliferation, invasion and apoptosis of ovarian cancer cells were evaluated by MTT assay, Transwell chamber invasion assay and flow cytometry Annexin V / PI assay. Results siRNA targeting sCLU significantly and specifically inhibited the expression of sCLU mRNA and protein. The results of MTT assay showed that the proliferation of cells in experimental group was significantly lower than that in control group (P <0.05). Flow cytometry Annexin Ⅴ / PI assay results showed that the apoptosis rate of the experimental group was significantly higher than that of the control group, reaching (15.84 ± 1.53)%, compared with the blank control group increased by about 9%, the difference was statistically significant ( P <0.01). The results of invasion assay indicated that the invasion ability of cells in experimental group was significantly inhibited. The number of transmembrane cells in experimental group was (26.52 ± 6.22) / field, which was significantly lower than that in control group (P <0.01). Conclusion sCLU silencing significantly inhibits the proliferation and invasion of ovarian cancer SKOV3 cells and increases the apoptosis rate. The sCLU gene is expected to be a new therapeutic target for ovarian cancer.