表皮生长因子受体shRNA联合西罗莫司对Colo-16细胞体外增殖、凋亡的研究  

In vitro effect of a short hairpin RNA targeting epidermal growth factor receptor combined with sirolimus on proliferation and apoptosis of the human cutaneous squamous cell carcinoma cell line Colo-16

作　　者：王慧 刘董 田芳 魏志平 Wang Hui;Liu Dong;Tian Fang;Wei Zhiping(Department of Dermatology,Qingdao Chengyang People's Hospital,Qingdao 266109,Shandong,China;Department of Pharmacy,Qingdao Chengyang People's Hospital,Qingdao 266109,Shandong,China;Outpatient Department,Qingdao Chengyang People's Hospital,Qingdao 266109,Shandong,China;Department of Dermatology,The Affiliated Hospital of Xuzhou Medical University,Xuzhou 221002,Jiangsu,China)

机构地区：[1]青岛市城阳区人民医院皮肤科,青岛266109 [2]青岛市城阳区人民医院药剂科,青岛266109 [3]青岛市城阳区人民医院门诊部,青岛266109 [4]徐州医科大学附属医院皮肤科,徐州221002

出　　处：《中华皮肤科杂志》2022年第2期135-141,共7页Chinese Journal of Dermatology

摘　　要：目的探讨表皮生长因子受体(EGFR)shRNA联合西罗莫司对人皮肤鳞状细胞癌Colo-16细胞增殖、凋亡的影响及机制。方法Colo-16细胞分为5组:正常细胞组(常规培养+磷酸盐缓冲液)阴性对照组(转染shRNA-NC质粒+磷酸盐缓冲液)、西罗莫司组(常规培养+西罗莫司)、EGFR shRNA组(转染EGFR shRNA质粒)、联合组(转染EGFR shRNA质粒±西罗莫司)。采用MTT法检测各组24~96h各时间点细胞增殖活性,流式细胞仪分析处理48h后各组细胞凋亡情况。RT-PCR检测Bcl-2、Bax mRNA的表达,Western印迹法检测凋亡相关蛋白cleaved caspase3、cleaved caspase9、Bcl-2、Bax、细胞增殖相关蛋白p-mTOR、p-AKT、p-P70S6K及细胞周期蛋白D1(cyclin D1)的表达。多组间比较采用单因素方差分析,组间两两多重比较采用SNKq检验。结果MTT检测显示,24~96 h西罗莫司组ECFR shRNA组及联合组Colo-16细胞增殖活性均显著低于正常细胞组(均P<0.05),且联合组细胞增殖活性显著低于西罗莫司组和EGFRshRNA组(均P<0.001),正常细胞组与阴性对照组各时间点细胞增殖活性差异均无统计学意义(均P>0.05)。流式细胞实验结果显示,西罗莫司组、EGFR shRNA组和联合组细胞凋亡率分别为9.52%±0.25%、12.65%±0.23%、19.81%±0.31%,均显著高于正常细胞组(3.33%±0.18%,q值分别为60.07、78.08、122.81,均P<0.001)和阴性对照组(3.42%±0.19%,q值分别为59.90、77.91、122.64,均P<0.001),且联合组凋亡率最高。RT-PCR和Western印迹法显示,与正常细胞组相比,西罗莫司组、EGFR shRNA组和联合组Bcl-2 mRNA及cyclin D1、p-AKT、p-mTOR、p-P70S6K、Bcl-2蛋白表达均显著降低(均P<0.05),Bax mRNA及cleaved caspase3、cleaved caspase9、Bax蛋白表达均显著增加(均P<0.05),其中联合组Bcl-2 mRNA及cyclin D1、p-AKT、p-mTOR、p-P70S6K、Bcl-2蛋白表达显著低于西罗莫司组及EGFR shRNA组(均P<0.05),而Bax mRNA及cleaved caspase3、cleaved caspase9、Bax蛋白表达均显著高于西罗莫司组及EGFR sObjective To investigate the effect of a short hairpin RNA(shRNA)targeting epidermal growth factor receptor(EGFR)combined with sirolimus on proliferation and apoptosis of the human cutaneous squamous cell carcinoma cell line Colo-16,and to explore underlying mechanisms.Methods Cultured Colo-16 cells were divided into 5 groups:normal cell group receiving conventional culture and treatment with phosphate-buffered saline(PBS),negative control group transfected with a shRNA-NC-expressing plasmid and treated with PBS,sirolimus group receiving conventional culture and sirolimus treatment,EGFR shRNA group transfected with an EGFR shRNA-expressing plasmid and treated with PBS,and combined group transfected with an EGFR shRNA-expressing plasmid and treated with sirolimus.Methyl thiazol tetrazolium(MTT)assay was performed to evaluate cellular proliferative activity in the above groups from 24 to 96 hours,and flow cytometry to detect cell apoptosis after 48-hour treatment.Semiquantitative RT-PCR was conducted to determine the mRNA expression of Bcl-2 and Bax,and Western blot analysis to determine the expression of apoptosis-related proteins cleaved caspase-3,cleaved caspase-9,Bcl-2,Bax,cell proliferation-related proteins phosphorylated mammalian target of rapamycin(p-mTOR),phosphorylated protein kinase B(p-AKT),phosphorylated 70-kDa ribosomal protein S6 kinase(p-P70S6k),and cyclin Dl.Comparisons among groups were carried out by using one-way analysis of variance,and multiple comparisons between 2 groups by using Student-Newman-Keuls q test.Results MTT assay showed that the proliferative activity of Colo-16 cells was significantly lower in the sirolimus group,EGFR shRNA group and combined group during 24-96 hours than in the normal cell group(all P<0.05),and higher in the combined group than in the sirolimus group and EGFR shRNA group at 24-96 hours(all P<0.001),and there was no significant difference in the cellular proliferative activity at any time points between the normal cell group and negative control group(all P>0.05

关 键 词：癌 鳞状细胞 受体 表皮生长因子 RNA干扰 西罗莫司 细胞增殖 细胞凋亡 

分 类 号：R739.5[医药卫生—肿瘤]