RNAi沉默p53基因对HeLa细胞核转录因子3表达及其对细胞增殖和侵袭能力的影响

Effect of RNAi silenced p53 gene on expression of nuclear transcription factor 3 in cervical cancer HeLa cells

目的:探讨RNAi沉默p53基因对宫颈癌HeLa细胞核转录因子3(STAT3)的表达,及对HeLa细胞增殖和侵袭能力的影响。方法:构建3种靶向p53基因的shRNA1~3序列,并转染HeLa细胞,采用逆转录PCR和Western blot检测细胞p53 mRNA和蛋白的表达,以筛选有效沉默p53的shRNA序列。将筛选的序列转染HeLa细胞后,采用逆转录PCR和Western blot检测STAT3 mRNA和蛋白的表达;采用四甲基噻唑蓝法(MTT)检测细胞增殖;采用Transwell检测细胞侵袭能力。结果:构建的3种p53-shRNA均可沉默宫颈癌HeLa细胞p53的表达,尤以shRNA3的沉默效果最好,转染shRNA3后,HeLa细胞p53 mRNA和蛋白的表达量分别为0.23±0.05和0.41±0.11,显著低于未转染组、空质粒组、p53-shRNA1组和p53-shRNA2组(P均<0.05),故后续试验均以shRNA3转染HeLa细胞。将shRNA3序列转染HeLa细胞沉默p53基因后,p53-shRNA3组STAT3mRNA和蛋白表达量分别为1.09±0.21和1.46±0.25,显著高于未转染组(0.53±0.10和10.74±0.14)及空质粒组(0.55±0.11和0.73±0.15)(P均<0.05)。MTT法分析结果显示,在孵育74、36、48和72h时,p53-shRNA3组HeLa细胞增殖能力均显著大于未转染组和空质粒组(P均<0.05)。Transwell法分析结果显示,转染12 h后p53-shRNA3组穿膜细胞数目(59.36±5.33)显著高于未转染组(15.73±1.29)和空质粒组(16.01±1.34)(P均<0.05)。结论:RNAi沉默p53基因能够上调宫颈癌HeLa细胞STAT3的表达,并促进细胞的增殖及侵袭能力。

OBJECTIVE： To observe the effect of RNAi silenced p53 gene on the expression of nuclear transcription factor STAT3 in cervical cancer HeLa cells,and on their proliferation and invasion. METHODS： 3 kinds of short hairpin RNA （shRNA） which target the p53 gene were constructed and were used for liposome-transfection into HeLa cells. Then,expression of p53 were detected by PCR and Western blot. After silencing the p53 gene,expression of STAT3 was detected by PCR and Western blot. Proliferation and invasion of HeLa cells were detected by the MTT and Transwell assays. RESULTS： The transfection efficiency of p53-shRNA3 group was 85.7%. After the transfection,the expression of p53 mRNA and protein were 0.23±0.05 and 0.41±0.11,respectively,significantly lower than the non-transfection,empty plasmid,p53-shRNA1 and p53-shRNA2 groups （P 〈 0.05）. In addition,expression of STAT3 mRNA and protein of the p53-shRNA3 group were 1.09±0.21 and 1.46±0.25,respectively,significantly higher than non-transfection group and empty plasmid group （P 〈 0.05）. MTT results showed that at 24,36,48 h and 72 h,cell proliferation ability of the p53-shRNA3 group was significantly higher than the non-transfected group and empty plasmid group （P 〈 0.05）. Transwell results showed that the number of cells in the p53-shRNA3 group （0.05±59.36） was significantly higher than the non-transfection group （15.73±1.29） and the empty vector group （16.01±1.34） （P 〈 0.05）. CONCLUSION： Silencing the p53 gene can significantly increase the expression of STAT3,and promote the proliferation and invasive ability of HeLa cells.