外源性小分子双链RNA激活肾癌细胞野生型P53蛋白表达作用的研究

目的 通过转染外源性低分子双链RNA （dsRNA） dsP53~285至人肾癌细胞系ACHN和786-O，观察其激活肾癌细胞中抑癌蛋白野生型P53的表达作用。方法 根据对肾癌细胞的不同处理分为3组，即空白对照（mock）组、阴性对照（转染dsControl）组和实验（dsP53-285）组。荧光实时定量聚合酶链反应（QPCR）和免疫印迹法（Western blot）分别检测P53 mRNA、P21 mRNA和相应蛋白的表达水平。MTT法和集落形成实验检测各组细胞活力及增殖能力。结果 QPCR检测显示dsP53-285能明显促进两种肾癌细胞中P53 mRNA和P21 mRNA水平的升高；与阴性对照组dsControl组相比，dsP53-285分别上调ACHN和786-O细胞中P53 mRNA的表达2.84倍（P<0.01）和3.20倍（P<0.01），dsP53-285分别上调ACHN和786-O细胞中P21 mRNA的表达2.33倍（P<0.01）和2.59倍（P<0.01）；mock组与dsControl组相比，两种细胞系中P53 mRNA和P21 mRNA的表达差异均没有统计学意义（P>0.05）。Western blot法检测显示在两种细胞系中，P53蛋白、P21蛋白表达水平的上调与相应的P53 mRNA、P21 mRNA水平的上调一致。MTT检测结果显示，与dsControl组相比，转染dsP53-285后，ACHN和786-O细胞活力显著降低（P<0.01），mock组与dsControl组无明显差异（P>0.05）。集落形成实验显示，dsP53-285组的集落数数量显著较少（P<0.05），mock组与dsControl组无明显差异（P>0.05）。结论 外源性dsP53-285能显著激活肾癌细胞中P53基因的表达，激活的P53蛋白为野生型而非突变型，并可以显著抑制肾癌细胞的生长。

Study on Exogenous Small Double-stranded RNA's Activating Effects on Wild-type P53 Protein Expression in Renal Cell Carcinoma

Objective To investigate the activating effects of dsP53-285 on wild-type P53 expression in human renal carcinoma cells, dsP53-285 was transfected into human renal carcinoma cell lines ACHN and 786-O.Methods According to the different treatment, renal carcinoma cells were randomly divided into blank control group (mock), negative control group (transfected with dsControl) and experimental group (dsP53-285). The expression of P53 mRNA, P21 mRNA and corresponding protein were detected by real-time quantitative polymerase chain reaction (QPCR) and Western blot. MTT assay and colony formation assay were used to detect the viability and proliferation ability of each group.Results dsP53-285 significantly enhanced the expression of P53 mRNA and P21 mRNA in the two renal cell lines. Compared with the negative control group, dsP53-285 increased the expression of P53 mRNA to 2.84 -fold (P<0.01) in ACHN and 3.20 -fold (P<0.01) in 786-O cells, dsP53-285 up-regulated the expression of P21 mRNA to 2.33 -fold (P<0.01) in ACHN and 2.59 -fold (P<0.01) in 786-O cells. Compared with mock, there was no significant difference in the expression of P53 mRNA and P21 mRNA in both cell lines transfected with dsControl (P>0.05). Western blot analysis showed that the up-regulation of P53 protein and P21 protein expression was consistent with the increase of P53 mRNA and P21 mRNA in the two cell lines. MTT assay showed that the viability of ACHN and 786-O decreased significantly (P<0.01) compared with dsControl group, and there was no significant difference between mock group and dsControl group (P>0.05). Colony formation experiments showed that the number of colonies in the dsP53-285 group was significantly lower (P<0.05), and there was no significant difference between the mock group and the dsControl group (P>0.05).Conclusion Exogenous dsP53-285 can significantly activate the expression of P53 gene in renal cell carcinoma. The activated P53 protein is wild type rather than mutant, and can significantly inhibit the growth of renal cell carcinoma.