摘要
目的探讨枸橼相互作用的丝氨酸/苏氨酸激酶(CIT)对阿霉素(ADM)耐药肝癌细胞耐药性的调控作用及其机制。方法用脂质体将乱序无意义阴性序列(si-NC)、CIT小干扰RNA(si-CIT)转染至耐药细胞;Realtime PCR和Western blotting检测CIT基因、连锁凋亡抑制蛋白(XIAP)、B细胞淋巴瘤蛋白2(Bcl-2)、Bcl-2相关X基因(Bax)、细胞色素C(Cyt C)、磷酸酶基因诱导的假定激酶1(PINK1)、常染色体隐性遗传性青少年帕金森综合征致病基因(Parkin)和自噬微管相关蛋白轻链3抗体Ⅰ/Ⅱ(LC3-Ⅰ/Ⅱ)的表达;细胞计数试剂盒-8(CCK-8)、流式细胞术和荧光探针(JC-10)染色法检测细胞的抑制率、凋亡和线粒体膜电位。结果耐药细胞中CIT基因表达高于正常细胞,敲减CIT可增强其对ADM的敏感性。敲减CIT促进耐药细胞凋亡和线粒体自噬能力,抑制细胞线粒体膜电位,并上调细胞Bax、PINK1、Parkin和LC3-Ⅰ/Ⅱ及胞质中Cyt C,下调细胞中Bcl-2和线粒体中Cyt C。结论CIT基因可通过线粒体途径的凋亡和自噬调节阿霉素耐药肝癌细胞的耐药性。
Objective To investigate the regulatory effect of citron rho-interacting serine/threonine kinase(CIT on drug resistance of adriamycin(ADM)resistant hepatocellular carcinoma cells and its mechanism.Methods Random nonsense negative sequence(si-NC),CIT small interfering RNA(si-CIT)trasfected into SK-Hep1/ADM cells with liposomes.Real-time PCR,Western blotting were used to detect the expression of CIT,X linked inhibitor of apoptosis protein(XIAP),B-cell lymphoma protein 2(Bcl-2),Bcl-2 related X gene(Bax),cytochrome C(Cyt C),phosphatase gene induction putative kinase 1(PINK1),autosomal recessive adolescent Parkinson’s disease pathogenic gene(Parkin)and autophagy microtubule-associated protein light chain 3 antibodyⅠ/Ⅱ(LC3-Ⅰ/Ⅱ);Cell counting kit 8(CCK-8),flow cytometry,and fluorescence probe(JC-10)staining were used to detect cell inhibition rate,apoptosis and mitochondrial membrane potential.Results The expression level of CIT in drug-resistant cells was significantly higher than that in normal cells.Knocking down CIT significantly increased the sensitivity to ADM.Knocking down CIT promoted drug-resistant cell apoptosis and mitochondrial autophagy,down-regulated cell mitochondrial membrane potential,and up-regulated Bax,Cyt C,and PINK1,Parkin,LC3-Ⅰ/Ⅱ,down-regulate XIAP,Bcl-2,Cyt C.Conclusion CIT gene could regulate the drug resistance of adriamycin-resistant HCC cells through mitochondrial pathway apoptosis and autophagy.
作者
郭丽
赵虹
张俊涛
刘志贞
弓韬
于保锋
GUO Li;ZHAO Hong;ZHANG Jun-tao;LIU Zhi-zhen;GONG Tao;YU Bao-feng(Department of Basic Medicine,Linfen Vocational and Technical College,Shanxi Linfen 041000,China;Department of Biochemistry and Molecular Biology,Shanxi Medical University,Taiyuan 030001,China)
出处
《解剖学报》
CAS
CSCD
北大核心
2020年第5期772-777,共6页
Acta Anatomica Sinica
基金
山西省重点研发计划项目(201703D421023)
山西省青年科技研究基金(201801D221069)。
关键词
枸椽相互作用的丝氨酸/苏氨酸激酶
阿霉素耐药肝癌细胞
线粒体
自噬
免疫印迹法
Citron rho-interacting serine/threonine kinase
Adriamycin-resistant hepatic carcinoma cells
Mitochondria
Autophagy
Western blotting
