摘要
目的研究抑制p53基因的表达是否影响小双链RNA(dsRNA)分子dsP21-322对p21基因表达的激活效应。方法体外培养人肾癌ACHN细胞(p53野生型)和膀胱癌5637细胞(p53突变型)。采用RT-PCR方法筛选具有抑制p53基因表达的干扰RNA分子dsP53-3。人工合成具有激活效应的dsRNA分子dsP21-322,以及与人体已知RNA序列无同源性的阴性对照小dsRNA分子dsCon。将上述dsRNA分子转染到两种肿瘤细胞中,每种细胞均分为5组,即:1空白组;2阴性对照组;3dsP21-322单转染组;4dsP53单转染组;5dsP21-322与dsP53共转染组。通过荧光染色检测转染效率,采用Real-time PCR和Western-blot技术检测各组细胞中p53和p21转录和翻译水平上的表达差异。结果 1在单转染组中,dsP21-322能够激活ACHN细胞、5637细胞中p21基因表达;2dsP53-3能够抑制ACHN细胞、5637细胞中p53基因表达;3在共转染组中ACHN细胞、5637细胞中p53基因下调,而dsP21-322依然能够激活p21基因表达。结论外源合成的saRNA分子dsP21-322能激活ACHN细胞、5637细胞中p21基因表达,该激活效应不受p53蛋白表达量的影响。
Objective To investigate the effect of down-regulation of expression of p53 gene on saRNA activate p21 gene. Methods Culturey human kidney cancer cell line ACHN（wild-type p53）and human bladder cancer cell line 5637（mutant p53）in vitro.Synthesized the sequence-specific corresponding saRNA fragments of the promoter of p21 gene,sequence-specific corresponding siRNA fragments of mRNA of p53 gene,and negative control small dsRNA dsCon.Co-transfected into human kidney cancer cell line ACHN and human bladder cancer cell line 5637 in vitro,respectively with lipofecta-mine reagent,Real-time PCR and Western-blot were performed to examine the difference expression level of p53 and p21in each group. Results 1p21 gene expression in human kidney cancer cell line ACHN and human bladder cancer cell line 5637 significantly increased.2p53 gene expression in human kidney cancer cell line ACHN and human bladder cancer cell line 5637 significantly declined.3when p53 gene down regulated in human kidney cancer cell line ACHN and human bladder cancer cell line 5637,p21 gene expression significantly increased. Conclusions Synthetic saRNA dsP21-322 can activate both the p21 gene in human kidney cancer cell line ACHN and human bladder cancer cell line 5637,when the p53 gene is down regulated,regardless of the p53 gene is wild-type or mutant,did not affect the dsP21-322 to activate the p21 gene.
出处
《现代泌尿生殖肿瘤杂志》
2015年第6期350-353,共4页
Journal of Contemporary Urologic and Reproductive Oncology
