RNA干扰TRAF4表达下调Notch1信号通路增强肺癌替莫唑胺化疗敏感性

TRAF4 expression by RNA interference enhances chemosensitivity of lungcancer temozolomide chemotherapy by down regulating Notch1 signalingpathway

目的探讨RNA干扰(RNAi)技术沉默肿瘤坏死因子受体相关因子(TRAF) 4表达对肺癌替莫唑胺化疗敏感性的影响。方法 RT-PCR检测SPC-A-1、A549、H322、H1299肺癌细胞相对于人胚肺成纤维细胞MRC5中TRAF4的表达量;TRAF4的siRNA转染和替莫唑胺处理A549细胞,细胞被分为空白对照组、阴性对照组、TRAF4-siRNA组、替莫唑胺组和TRAF4-siRNA+替莫唑胺组,细胞培养48 h,RT-PCR及Western印迹检测各组A549细胞TRAF4的表达; CCK8法及流式细胞术分别检测各组细胞活力(OD值)及凋亡率; Western印迹检测含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-3、Bcl-2相关X蛋白(Bax)及Notch神经同源蛋白(Notch) 1前体信号中Notch1和下游重要靶基因发状分裂相关增强子(Hes) 1的蛋白表达。结果 TRAF4在4个肺癌细胞中的表达水平均显著高于其在MRC5细胞中的表达(P<0. 05); TRAF4-siRNA转染A549细胞后,TRAF4的表达显著低于空白对照组(P<0. 05); TRAF4-siRNA组和替莫唑胺组细胞凋亡率及Caspase-3和Bax的表达均显著高于空白对照组,OD值及Notch1和Hes1表达显著低于空白对照组,TRAF4-siRNA+替莫唑胺组凋亡率及Caspase-3和Bax的表达显著高于TRAF4-siRNA组和替莫唑胺组,OD值及Notch1和Hes1表达显著低于TRAF4-siRNA组和替莫唑胺组(P<0. 05)。结论抑制肺癌细胞TRAF4表达可通过下调Notch1信号通路降低肺癌细胞活力及诱导细胞凋亡,并可增强替莫唑胺化疗敏感性。

Objective To investigate the effect of TRAF4 by RNA interference on chemotherapy sensitivity of lung cancer te-mozolomide by down regulated Notch1 signaling pathway.Methods RT-PCR was used to detect the expressions of TRAF4 in SPC-A-1,A549,H322,H1299 lung cancer cell compared with human embryonic lung fibroblast MRC5;TRAF4-siRNA was transfected cellsand temozolomide treated A549 cells,cells were divided into blank control,negative control,TRAF4-siRNA,temozolomide andTRAF4-siRNA+temozolomide groups,cells were cultured for 48 h,RT-PCR and Western blot were used to detect the expression ofTRAF4;CCK8 assay and flow cytometry were used to detect cell viability and apoptosis rate;the expressions of Bax,Caspase3,Notch1 and Hes1 protein were detected by Western blot. Results The expressions of TRAF4 in four lung cancer cells were significant-ly higher than those in the expression of MRC5( P<0.05);the expression of TRAF4 in TRAF4-siRNA transfected A549 cells was sig-nificantly lower than that in blank control group( P<0.05);the apoptosis rate and the expressions of Caspase3,Bax in RAF4-siRNAand temozolomide groups were significantly higher than those of control group,the OD value and the expressions of Notch1,Hes1 weresignificantly lower than those of control group,the apoptosis rate and the expressions of Caspase3,Bax in TRAF4-siRNA+temozolomidegroup were significantly higher than those of TRAF4-siRNA and temozolomide groups,the OD value and the expressions of Notch1,Hes1 were significantly lower than those of TRAF4-siRNA and temozolomide groups( P<0.05).Conclusions Inhibiting of TRAF4 ex-pression in lung cancer cells could reduce the viability of cancer cells and induce apoptosis by down regulating the Notch1 signalingpathway,and could enhance the chemotherapy sensitivity of temozolomide.