甲胎蛋白抑制顺铂诱导肝癌细胞凋亡的分子机制  

The molecular mechanism of alpha-fetoprotein inhibiting cisplatin-induced apoptosis of hepatocellular carcinoma cells

作　　者：张超 闫颖[1] 赵海建[1] 李刚 张传宝[1] Zhang Chao;Yan Ying;Zhao Haijian;Li Gang;Zhang Chuanbao(National Center of Clinical Laboratories,Beijing Hospital,National Center of Gerontology,Institute of Geriatric Medicine,Chinese Academy of Medical Sciences,Beijing 100730,China;Department of Biochemistry and Biophysics,Health Science Center,Peking University,Beijing 100191,China)

机构地区：[1]北京医院国家老年医学中心,国家卫生健康委临床检验中心,中国医学科学院老年医学研究院,100730 [2]北京大学医学部基础医学院生物化学与生物物理系,100191

出　　处：《中华消化杂志》2020年第6期400-406,共7页Chinese Journal of Digestion

基　　金：北京医院博士启动基金项目(BJ-2019-148);国家自然科学基金面上项目(81370522);北京市科学技术委员会G20工程支撑保障项目(Z161100001816043)。

摘　　要：目的探讨甲胎蛋白抑制顺铂诱导的肝癌细胞凋亡的分子机制。方法选用HepG2(甲胎蛋白阳性)和QSG-7701(甲胎蛋白阴性)两种常见肝癌细胞系。分别在HepG2细胞中转染甲胎蛋白干扰质粒和在QSG-7701细胞中转染甲胎蛋白过表达质粒,培养12、24、36和48 h后,采用细胞计数试剂盒8(CCK-8)检测细胞增殖情况;在HepG2和QSG-7701细胞中分别干扰和过表达甲胎蛋白24 h后,加入凋亡诱导剂顺铂,采用流式细胞术检测甲胎蛋白对顺铂诱导的肝癌细胞凋亡的影响;采用蛋白质免疫共沉淀技术(CoIP)检测甲胎蛋白与转录因子维甲酸受体(RAR)的相互作用;运用染色质免疫共沉淀技术(ChIP)实验联合蛋白质印迹法验证甲胎蛋白表达水平对DNA损伤诱导转录基因1(DDIT1)表达的影响,以及甲胎蛋白和DDIT1对肝癌细胞凋亡的影响。统计学方法采用t检验。结果 CCK-8结果显示,质粒转染12、24、36和48 h后,过表达甲胎蛋白的QSG-7701细胞相对增殖率分别较对照组升高28.7%±2.7%、49.8%±6.1%、65.8%±3.0%和79.3%±2.0%,而敲减甲胎蛋白后的HepG2细胞相对增殖率分别较对照组降低16.5%±6.1%、28.5%±5.7%、42.5%±1.7%和57.6%±3.8%,差异均有统计学意义(t=3.978、4.357、3.461、3.636、2.858、2.446、3.233、4.492,P均<0.05)。流式细胞术结果显示,QSG-7701细胞过表达甲胎蛋白24和48 h后的细胞凋亡率较单独顺铂处理细胞24和48 h后分别降低46.3%±2.9%和47.7%±7.4%,HepG2细胞敲减甲胎蛋白24和48 h后的细胞凋亡率较单独顺铂处理细胞24和48 h后分别升高86.7%±4.0%和31.6%±10.5%,差异均有统计学意义(t=3.543、3.893、2.336、2.561,P均<0.05)。CoIP实验结果显示,AFP与RAR能够发生相互作用。在HepG2细胞中敲减甲胎蛋白表达后,提取核蛋白发现RAR的入核较对照组增加;而在QSG-7701细胞中过表达甲胎蛋白后,RAR的入核较对照组减少。ChIP实验结果显示,AFP能够调控DDIT1表达。敲减甲胎蛋白的HepG2细胞Objective To investigate the molecular mechanism of alpha-fetoprotein(AFP)inhibiting cisplatin-induced apoptosis of hepatocellular carcinoma cells.Methods HepG2(AFP positive)and QSG-7701(AFP negative),two common hepatocyte carcinoma cell lines were selected.The expression of AFP was knockdown in HepG2 cells with RNA interference method and AFP expression plasmid was transfected in QSG-7701 cells.After the cells were cultured for 12,24,36 and 48 hours,the cell proliferation was detected by cell counting kit-8(CCK-8)assay.After HepG2 and QSG-7701 cell lines were interfered or overexpressed AFP protein for 24 h,respectively,apoptotic inducer cisplatin(CDDP)was added.The effect of AFP on apoptosis induced by CDDP in hepatocellular carcinoma cells was determined by flow cytometry.The interaction between AFP and transcription factor retinoic acid receptor(RAR)was examined by protein coimmun oprecipitation(CoIP)technique.The effect of AFP expression level on the expression of DNA damage inducible transcript 1(DDIT1),and the effects of AFP and DDIT1 on apoptosis of hepatocellular carcinoma cells were tested by chromatin immunoprecipitation(ChIP)assay and Western blotting.T test was performed for statistical analysis.Results The results of CCK-8 test showed that after plasmid transfected for 12,24,36 and 48 hours,the relative proliferation rates of QSG-7701 cells overexpressed AFP increased by 28.7%±2.7%,49.8%±6.1%,65.8%±3.0%and 79.3%±2.0%,respectively;however the relative proliferation rates of HepG2 cells after AFP knockdown decreased by 16.5%±6.1%,28.5%±5.7%,42.5%±1.7%and 57.6%±3.8%,respectively,when compared with the control group,and the differences were statistically significant(t=3.978,4.357,3.461,3.636,2.858,2.446,3.233 and 4.492,all P<0.05).The results of flow cytometry indicated that after AFP overexpression for 24 and 48 hours,the apoptosis rate of QSG-7701 cells decreased by 46.3%±2.9%and 47.7%±7.4%,respectively,compared with cisplatin induced cells;however after AFP knockdown for 24 and 48 hours,the

关 键 词：甲胎蛋白 肝细胞癌 增殖 凋亡 

分 类 号：R735.7[医药卫生—肿瘤]