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目的:构建膜-细胞骨架联接蛋白Ezrin基因特异性短发卡RAN表达载体(small hairpin RNA,shRNA),探讨其对肾癌细胞株786-0细胞凋亡、增殖的影响。方法:以Ezrin为靶基因,以Pgenesil-1质粒为载体,设计和构建重组体,设计2条发夹式RNA(shRNA),合成后克隆入载体Pgenesil-1,扩增并中量提取质粒,应用脂质体Lipofectamine 2000转染进786-0细胞,重组质粒转染786-0肾癌细胞株,用运实时荧光定量PCR进行筛选鉴定,筛选出抑制率较高的重组质粒载体shRNA-Ezrin1,用shRNA-Ezrin1转染786-0细胞,采用MTT、流式细胞仪、电镜检测,观察RNA干扰Ezrin后肾癌细胞株786-0细胞增殖能力的改变。结果:shRNA干扰后786-0细胞增殖活性减弱,G0/G1时段明显延长(P<0.01),PI缩短(P<0.01),细胞凋亡率增加(P<0.01)。结论:Ezrin与肾癌细胞凋亡、增殖有关,有望成为肾癌基因治疗的一个新靶点。
OBJECTIVE: To construct small hairpin RNA (shRNA) targeting Ezrin gene of membrane-cytoskeleton connexin, and to investigate its effect on the apoptosis and proliferation of human renal cell carcinoma cell line 786-0. Methods: Ezrin was used as the target gene. Pgenesil-1 plasmid was used as a vector to design and construct a recombinant plasmid. Two hairpin RNAs (shRNAs) were designed and cloned into vector Pgenesil-1. The plasmids were amplified, 786-0 cells were transfected with the recombinant plasmid Lipofectamine 2000. The recombinant plasmids were transfected into 786-0 human renal carcinoma cell lines and were screened by real-time fluorescence quantitative PCR. The recombinant plasmid vector shRNA-Ezrin1 with high inhibitory rate was selected, 786-0 cells were transfected with shRNA-Ezrin1. The proliferation of 786-0 cells was detected by MTT, flow cytometry and electron microscopy. Results: After 786-0 shRNA was knocked down, the proliferation of 786-0 cells was weakened, the G0 / G1 phase was significantly prolonged (P <0.01), the PI was shortened (P <0.01) and the apoptosis rate was increased (P <0.01). Conclusion: Ezrin is related to apoptosis and proliferation of renal cell carcinoma, which may be a new target for gene therapy of renal cell carcinoma.