miR-196b调控PI3K/Akt通路对宫颈癌细胞顺铂耐药的影响研究  

Effect of miR-196b on cisplatin resistance of cervical cancer cells by regulating PI3K/Akt pathway

作　　者：张玉清[1] 邢惠海 刘立秋 王敬[4] 徐书方(指导)[1] ZHANG Yu-Qing;XING Hui-Hai;LIU Li-Qiu;WANG Jing;XU Shu-Fang(Shijiazhuang Medical College,Shijiazhuang 050599,China)

机构地区：[1]石家庄医学高等专科学校,石家庄050599 [2]石家庄人民医学高等专科学校,石家庄050000 [3]河北省卫健委综合监督执法局,石家庄050071 [4]河北大学附属医院,保定071000

出　　处：《中国免疫学杂志》2021年第23期2865-2870,共6页Chinese Journal of Immunology

摘　　要：目的:探讨miR-196b对宫颈癌细胞顺铂耐药性及磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(Akt)通路的影响。方法:体外培养人宫颈癌细胞株HeLa、顺铂耐药细胞株HeLa/DDP,检测HeLa、HeLa/DDP对顺铂的耐药性;将HeLa/DDP细胞分为:空白对照(仅用转染试剂处理)+DDP组、阴性对照(转染miR-196b NC)+DDP组、miR-196b inhibitor(转染miR-196b inhibitor)+DDP组,采用实时荧光定量PCR(qRT-PCR)检测各组细胞miR-196b水平,蛋白印迹(WB)法检测Akt、磷酸化蛋白激酶B(p-Akt)、PI3K、磷酸化雷帕霉素靶蛋白(p-mTOR)的表达,采用噻唑蓝(MTT)法检测各组细胞的IC_(50),流式细胞仪检测各组细胞凋亡情况。结果:与HeLa细胞相比,HeLa/DDP细胞miR-196b表达显著升高(P<0.05),随顺铂浓度增加(0.01、0.1、1、5、10、20、40、80、100μmol/L)HeLa/DDP、HeLa细胞活力下降,经计算HeLa对顺铂的IC_(50)为4.86μmol/L,HeLa/DDP对顺铂的IC_(50)为68.13μmol/L,与HeLa相比,HeLa/DDP对顺铂的IC_(50)显著升高(P<0.05);与空白对照+DDP组、阴性对照+DDP组比较,miR-196b inhibitor+DDP组HeLa/DDP细胞中miR-196b表达水平,Akt、p-Akt、PI3K、p-mTOR蛋白水平及顺铂IC_(50)显著下降(P<0.05),细胞凋亡率显著升高(P<0.05)。结论:下调miR-196b表达可能通过下调PI3K/Akt通路相关蛋白表达,降低宫颈癌细胞对顺铂的耐药性。Objective:To investigate the effects of miR-196b on cisplatin resistance and phosphatidylinositol-3 kinase(PI3K)/protein kinase B(Akt)pathway in cervical cancer cells.Methods:Human cervical cancer cell lines HeLa and cisplatin resistant cell lines HeLa/DDP were cultured in vitro,and the resistance of HeLa and HeLa/DDP to cisplatin was detected;HeLa/DDP cells were divided into three groups:blank control(treated with transfection reagent only)+DDP group,negative control(transfected with miR-196b NC)+DDP group,miR-196b inhibitor(transfected with miR-196b inhibitor)+DDP group,the level of miR-196b was detected by real-time fluorescent quantitative PCR(qRT-PCR),the expressions of Akt,phosphorylated protein kinase B(p-Akt),PI3K and phosphorylated mammalian target of rapamycin(p-mTOR)were detected by Western blot,IC_(50) was detected by MTT,and apoptosis was detected by flow cytometry.Results:The expression of miR-196b in HeLa/DDP cells was significantly higher than that in HeLa cells(P<0.05),and with the increase of cisplatin concentration(0.01,0.1,1,5,10,20,40,80,100μmol/L),the viabilities of HeLa/DDP and HeLa cells decreased,the IC_(50) of HeLa to cisplatin and HeLa/DDP to cisplatin were calculated to be 4.86μmol/L and 68.13μmol/L respectively,and compared with HeLa,the IC_(50) of HeLa/DDP to cisplatin was higher(P<0.05);the expression level of miR-196b in HeLa/DDP cells of miR-196b inhibitor+DDP group was higher than that of blank control+DDP group and negative control+DDP group,the Akt,p-Akt,PI3K,p-mTOR protein levels and cisplatin IC_(50) were significantly lower(P<0.05),and the apoptotic rate were significantly higher(P<0.05).Conclusion:The down-regulation of miR-196b may reduce cisplatin resistance of cervical cancer cells by down-regulation of PI3K/Akt pathway related protein expression.

关 键 词：miR-196b 宫颈癌细胞 耐药性 顺铂 机制 

分 类 号：R737.33[医药卫生—肿瘤]