摘要
目的:观察RNAi沉默膀胱癌BIU-87细胞磷脂酶C-ε(Phospholipase C epsilon,PLCε)表达后对藏花素(Crocin)抑制人膀胱癌BIU-87细胞增殖作用的影响。方法:将携有siRNA基因的真核表达质粒PGenesil-PLCε转染BIU-87细胞后,分为5组:未转染组、pGenesil-NP阴性质粒组、pGenesil-PLCε质粒组、Crocin组、pGenesil-PLCε联合Crocin组。MTT法观察不同处理组对BIU-87细胞的生长抑制作用。RT-PCR法检测转染后对PLCεmRNA表达的影响及各处理组PCNA(Proliferating cell nuclear antigen)、CyclinD1 mRNA表达。Western-blot检测PCNA、CyclinD1蛋白表达。结果:RNAi可抑制BIU-87细胞78.06%的PLCεmRNA表达。pGenesil-PLCε联合Crocin组可增强Crocin对BIU-87细胞的生长抑制作用,48h抑制率达45.30%,且与pGenesil-PLCε质粒组和Crocin组相比较有显著性差异(P<0.01)。RT-PCR和Western-blot结果显示与RNAi沉默PLCε组比较,RNAi PLCε联合Crocin组明显下调了PCNA、Cyclin D1基因和蛋白的表达(P<0.05);较单独Crocin组比较,RNAi PLCε联合Crocin组显著降低了PCNA基因和蛋白的表达(P<0.05),降低了Cyclin D1蛋白的表达(P<0.05),Cyclin D1 mRNA表达变化两组间无显著性差异(P>0.05)。结论:沉默PLCε基因可增强Crocin对BIU-87细胞的抑制作用,其机制与进一步下调PCNA、CyclinD1的表达有关。
Objective：To investigate the effects of Crocin on proliferation regulation after silencing PLC ε gene expression by RNAi interference in human bladder cancer cells BIU-87. Methods ：BIU-87 cells were transfected with plasmid pGenesil-PLC ε.The experiment was designed as five groups to compared with each other,they were non-transfected cells as control group,pGenesil-NP negative plasmid transfected group,pGenesil-PLC ε plasmid transfected group, Crocin group, pGenesil-PLC ε plasmid transfected together with Crocin group. The influence on proliferation of every group was determined by methyl thiazolyl tetrazolium（MTY）. The expression of PLC ε mRNA of BIU-87 cells after transfection with the plasmid pGenesil-PLC ε was observed by RT-PCR. RT-PCR and Western-blot were used to detect cyclin D1 and PCNA expression. Results：78.06% mRMA of PLC ε was reduced clearly after transfected with plasmid pGenesil-PLC ε in BIU-87 cells. pGenesil-PLC ε plasmid transfected together with croin group could enhance the inhibitive effect of Crocin on proliferation up to 45.30% at 48h,which was strikingly compared with the pGenesil-PLC ε plasmid transfected group and the Crocin group （P〈O.O1）. Both RT-PCR and western-blot indicated that the expression of PCNA,CyclinD1 both in mRNA and protein were markedly decreased in the RNAi together with Crocin group compared with the RNAi group（P〈O.05）. Compared with the Crocin group,The expression of PCNA both in mRNA and protein were reduced remarkably（P〈O.O5）,the expression of CyclinD1 protein was decreased clearly（P〈O.O5）in the RNAi together with Crocin group, but the expression of CyclinD1 mRNA had no significant difference between the two groups（P〉O.05）. Conclution：RNAi PLC ε together with Crocin can enhance the inhibit effect of Crocin,they can further reduce PCNA,CyclinD1 expressionthe was the mechanism.
出处
《重庆医科大学学报》
CAS
CSCD
2008年第5期536-540,共5页
Journal of Chongqing Medical University
基金
重庆市科委自然科学基金(NO CSTC2005 BB5309)
