摘要
目的:探究美洲大蠊多肽PAE_(2)基于腺苷-磷酸活化蛋白激酶(AMPK)调节自噬对人肝癌耐药细胞株BEL-7402/5-FU多药耐药性的影响。方法:以人肝癌细胞株BEL-7402及人肝癌耐药细胞株BEL-7402/5-FU为研究对象,采用噻唑蓝(MTT)比色法测定AICAR的作用时间及浓度;酶联免疫吸附试验(ELISA)测定细胞中AMPK含量;实时聚合酶链式反应(real-time PCR)检测细胞中AMPK mRNA的表达水平;real-time PCR检测细胞中自噬相关蛋白5(ATG5)、自噬相关蛋白7(ATG7)、磷酸肌醇-3-激酶3(PIK3C3)、Beclin 1、微管相关蛋白1亲链3-Ⅱ(LC3-Ⅱ)、P62、Zeste同源物增强子2(EZH2)等mRNA的表达水平;激光共聚焦显微镜观察细胞中LC3-Ⅱ的表达;免疫细胞化学法(ICC)检测细胞中P62蛋白的表达。结果:美洲大蠊多肽PAE_(2)能够降低AMPK含量(P<0.05),也能降低AMPK mRNA的表达水平,显著降低自噬相关因子ATG5 mRNA、ATG7 mRNA、PIK3C3mRNA、Beclin 1 mRNA、LC3-ⅡmRNA的表达水平(P<0.05),明显提高P62 mRNA及EZH2 mRNA的表达水平(P<0.05),增加P62蛋白的表达(P<0.05)。结论:美洲大蠊多肽PAE_(2)能够通过下调AMPK的表达进而抑制自噬来逆转BEL-7402/5-FU细胞的多药耐药性。
Objective:To investigate the effects of the Periplaneta americana polypeptide PAE_(2) on the multi-drug resistance of BEL-7402/5-FU cells based on adenosine monophosphate-activited protein kinase(AMPK)regulation of autophagy.Methods:Human hepatocellular carcinoma sensitive cell line BEL-7402 and human hepatocellular carcinoma drug-resistant cell line BEL-7402/5-FU were used as the research objects.The action time and concentration of AICAR were determined by methyl thiazolyl tetrazolium(MTT)colorimetry assay.AMPK content in cells was determined by enzyme linked immunosorbent assay(ELISA).Real-time polymerase chain reaction(real-time PCR)was used to detect AMPK mRNA expression level in cells.Real-time PCR was used to detect the mRNA expression levels of autophagy related protein 5(ATG5),autophagy related protein 7(ATG7),phosphoinositide-3-kinase class 3(PIK3C3),Beclin 1,microtubuler-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ),P62 and enhancer of Zeste homolog 2(EZH2).The expression of LC3-Ⅱ was observed by laser scanning confocal microscope.The expression of P62 protein was detected by immunocytochemistry(ICC).Results:Periplaneta americana polypeptide PAE_(2) can reduce the expression of AMPK and its mRNA,greatly reduce the expression of related autophagy proteins ATG5 mRNA,ATG7 mRNA,PIK3C3 mRNA,Beclin 1 mRNA,and LC3-域mRNA(P<0.05),and significantly increase the expression of P62 mRNA和EZH2 mRNA(P<0.05),increase the expression of P62 protein(P<0.05).Conclusion:Periplaneta americana polypeptide PAE_(2) can reverse the multi-drug resistance of BEL-7402/5-FU cells by inhibiting AMPK so as to inhibit autophagy.
作者
欧玲
李彩琳
李银蕊
周杰
单婉歆
吴定宇
彭芳
Ou Ling;Li Cailin;Li Yinrui;Zhou Jie;Shan Wanxin;Wu Dingyu;Peng Fang(College of Pharmacy,Dali University,Dali,Yunnan 671000,China;Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D,Dali,Yunnan 671000,China;The Asset and Laboratory Management Division,Dali University,Dali,Yunnan 671000,China)
出处
《大理大学学报》
2022年第4期1-7,F0003,共8页
Journal of Dali University
基金
国家自然科学基金项目(81560600)
云南省地方本科高校(部分)基础研究联合专项资金项目(2017FH001-010)。
关键词
美洲大蠊多肽
腺苷-磷酸活化蛋白激酶
自噬
多药耐药性
Periplaneta americana polypeptide
adenosine monophosphate-activited protein kinase
autophagy
multi-drug resistance
