GADD45A靶向抑制p38 MAPK通路逆转宫颈癌细胞获得性耐药的实验研究

Experimental study on reversal of acquired drug resistance of cervical cancer cells by GADD45A targeting inhibition of p38 MAPK pathway

目的探究生长阻滞和DNA损伤诱导45A(GADD45A)对宫颈癌细胞顺铂(DDP)获得性耐药的影响及可能的分子机制。方法体外培养HeLa细胞,通过DDP诱导建立HeLa耐药细胞HeLa/DDP,实时荧光定量PCR(qPCR)检测GADD45A表达。将HeLa/DDP细胞分为空白对照组(细胞不做任何处理)、阴性对照组(转染阴性对照病毒空载体)、GADD45A转染组(转染GADD45A过表达慢病毒载体)、GADD45A+Anisomycin组(细胞转染GADD45A过表达慢病毒载体后加入JNK/p38激活剂Anisomycin)。CCK-8法和细胞集落形成法检测HeLa/DDP细胞耐药性。彗星实验和流式细胞术检测DDP对各组HeLa/DDP细胞DNA损伤、细胞周期和凋亡的影响。Western blotting法检测各组细胞p38蛋白表达。结果HeLa细胞中美好中GADD45A mRNA表达量低于正常宫颈鳞状上皮细胞H8(0.70±0.09 vs.1.00±0.04,P<0.05)。HeLa/DDP细胞中GADD45A mRNA表达量低于HeLa细胞(0.48±0.05 vs.0.70±0.09,P<0.05)。GADD45A转染组HeLa/DDP细胞对DDP的耐药指数低于空白对照组(23.57±1.26 vs.76.55±5.79),且细胞集落数量减少(119±26 vs.415±33),同时DNA损伤率增加[尾长(75.26±19.83)μm vs.(21.47±6.41)μm],S期细胞比例升高[(34.22±1.49)%vs.(15.40±2.98)%],细胞凋亡率增加[(40.98±4.59)%vs.(5.56±0.77)%],差异具有统计学意义P<0.05)。与空白对照组比较,GADD45A转染组HeLa/DDP细胞GADD45A蛋白表达增强,但p-p38/p38蛋白比值降低(P<0.05)。GADD45A+Anisomycin组上述指标较GADD45A组被逆转。结论在DDP耐药宫颈癌细胞中GADD45A表达普遍下调,上调GADD45A表达可以降低HeLa/DDP细胞活性,增加DDP诱导细胞凋亡和DNA损伤,且与抑制p38磷酸化激活有关。

Objective To investigate the effects of growth arrest and DNA damage-induced 45A(GADD45A)on the acquired drug resistance of cervical cancer cells to cisplatin(DDP)and its possible molecular mechanism.Methods HeLa cells were cultured in vitro and to establish HeLa/DDP resistance cells induced by cisplatin(DDP).The expression of GADD45A was detected by real-time fluorescent quantitative PCR(qPCR).HeLa/DDP cells were divided into blank control group(without any treatment),negative control group(transfected with negative vector),GADD45A group(transfected with GADD45 overexpression vector),GADD45A+Anisomycin group(after transfected with GADD45 overexpression vector,treated with JNK/p38 activator Anisomycin).CCK-8 assay and colony forming assay were used to detect the drug resistance.Comet assay and flow cytometry were used to detect the effects of DDP on DNA damage,cell cycle and apoptosis of HeLa/DDP cells.The expression of p38 protein was detected by western blot.Results The expression level of GADD45A in HeLa cells was lower than that in normal cervical squamous cell H8(0.70±0.09 vs.1.00±0.04,P<0.05).The mRNA expression of GADD45A in HeLa/DDP cells was lower than that in HeLa cells(0.48±0.05 vs.0.70±0.09,P<0.05).The resistance index of HeLa/DDP cells in GADD45A transfected group to DDP was lower than that in blank control group(23.57±1.26 vs.76.55±5.79),and the number of cell colonies decreased(119±26 vs.415±33).At the same time,DNA damage rate increased[tail length(75.26±19.83)μm vs.(21.47±6.41)μm],the proportion of S phase cells increased[(34.22±1.49)%vs.(15.40±2.98)%],The apoptosis rate was increased[(40.98±4.59)%vs.(5.56±0.77)%],and the difference was statistically significant(P<0.05).Compared with blank control group,the expression of GADD45A protein in HeLa/DDP cells of GADD45A transfected group was increased,but the ratio of p-p38/p38 protein was decreased(P<0.05).The above indexes were reversed in the GADD45A+Anisomycin group compared with the GADD45A group.Conclusion The expression of GADD45A was down-regulated in DDP-resistant cervical cancer cells,up-regulated GADD45A could decrease the activity of HeLa/DDP cells,increase the apoptosis and DNA damage induced by DDP,which would be related to the inhibition of p38 phosphorylation.