摘要
目的探讨RNA干扰技术沉默人源磷脂酶Cε(PLCε)基因对肾癌786-0细胞p65核转位的调控,对786-0细胞增殖的影响及其机制。方法用携带PLCεshRNA基因的真核表达质粒pGenesil-PLCε转染786-0细胞,克隆形成实验检测786-0细胞的增殖,RT-PCR检测PLCε、c-Myc和COX-2 mRNA的表达,Western blot检测PLCε、c-Myc、COX-2和p65总蛋白及p65在细胞核的表达,细胞免疫荧光检测p65在细胞核与细胞质的定位。同时应用p65特异的抑制剂BAY11-7082处理786-0细胞,Western blot检测c-Myc和COX-2蛋白的表达。结果转染pGenesil-PLCε质粒的786-0细胞生长明显受抑制;转染pGenesil-PLCε质粒细胞中PLCε、c-Myc和COX-2 mRNA和蛋白表达水平均较对照组pGenesil-NP明显下调(P<0.01),且抑制核中p65蛋白的表达(P<0.01),细胞免疫荧光结果显示转染pGenesil-PLCε质粒组p65在细胞核中的表达较对照组pGenesil-NP明显下降;BAY11-7082抑制c-Myc和COX-2蛋白的表达(P<0.01),且呈时间浓度依赖性。结论沉默PLCε基因后可能是通过抑制p65的核转位及c-Myc、COX-2的表达抑制786-0细胞的增殖。
Objective To determine the effect of silencing phospholipase Ce （PLCe） gene by RNA interference on the nuclear translocation of p65 and cell proliferation in human 786-0 renal cancer cells. Methods A PLCe specific shRNA expression vector pGenesil-PLCe was transfected into 786-0 cells by Lipofectamine 2000. Colony cell survival assay were used to determine effects on cellular proliferation ability after transfection. The expression of PLCe, c-Myc and COX-2 was detected by RT-PCR. The expression of PLCe, c-Myc, COX-2, total p65 and p65 in the nucleus were detected by Western blotting. Immunofluorescence assay was used to detect the location of p65. The expression of c-MyC and COX-2 was also measured by Western blotting after BAY11-7082, an inhibitor of p65, treatment with different doses for different time periods. Results 786-0 cells proliferation was inhibited after PLCe shRNA treatment in vitro. In 786-0 renal cancer cells, the expression of PLCe, transcription factor p65 in the nucleus and its downstream gene c-Myc and COX-2 was decreased using PLCe specific shRNA （P 〈 0.01 ）. Immunofluorescence assay results showed that p65 translocated to the nucleus was suppressed after PLCe shRNA transfection. The expression of c-Myc and COX-2 at protein level were inhibited by BAY11-7082 in a dose- and time-dependent manner （P 〈 0.01 ）. Conclusion Silencing PLCe inhibits 786-0 cell proliferation via inhibiting nuclear translocation of p65 and expression of c-Myc and COX-2.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2014年第19期2010-2015,共6页
Journal of Third Military Medical University
基金
重庆市教委科学技术研究项目(KJ080306)~~
