山萘酚逆转肝癌耐药细胞Bel-7402/5-Fu的作用机制研究  

Study on the effect and mechanism of kaempferol in reversing drug-resistantBel-7402/5-Fu cells

作　　者：梁大敏[1] 杨正久[1] 张子萍 钱静[1] 毛朝坤 LIANG Damin;YANG Zhengjiu;ZHANG Ziping;QIAN Jing;MAO Chaokun(Department of Medical Technology,Zunyi Medical and Pharmaceutical College,Zunyi 563003,China)

机构地区：[1]遵义医药高等专科学校医学技术系,563003

出　　处：《天津医药》2024年第9期900-906,共7页Tianjin Medical Journal

基　　金：遵义市科技与大数据局基金资助项目[遵市科合HZ字(2022)151号,遵市科合社字(2018)41号]。

摘　　要：目的探讨山萘酚(KAE)对肝癌耐药细胞Bel-7402/5-Fu功能的影响。方法采用KAE处理Bel-7402/5-Fu细胞,将细胞分为对照组和药物组(0.064、0.320、1.600、8、40、200μmol/L KAE);根据干扰DNA依赖性激酶催化亚基(DNA-PKcs)、加入蛋白酶体抑制剂MG132或加入自噬抑制剂CQ,将细胞分为si-NC组和DNA-PKcs干扰(siRNA-1664、siRNA-2142、siRNA-3785)组,对照组、KAE组和KAE+si-DNA-PKcs组,对照组、KAE+DMSO组、KAE+MG132组和KAE+CQ组。采用CCK-8检测细胞增殖,实时荧光定量PCR(RT-qPCR)和Western blot检测组蛋白H_(2)AX磷酸化(γ-H_(2)AX)、DNA-PKcs、DNA双链断裂修复/V(D)J重组蛋白(Artemis)和药泵基因(P-gp)mRNA和蛋白的表达,流式细胞术检测细胞周期和细胞凋亡,蛋白稳定性实验检测DNA-PKcs蛋白稳定性,免疫共沉淀检测DNA-PKcs蛋白泛素化。结果与对照组相比,8μmol/L KAE处理细胞24 h可抑制约50%的细胞增殖能力,选择此时间和浓度进行后续研究。与对照组相比,KAE组γ-H_(2)AX mRNA和蛋白表达水平升高,DNA-PKcs、Artemis和P-gp mRNA和蛋白表达水平降低(P<0.05);与对照组相比,KAE促进Bel-7402/5-Fu细胞周期阻滞于G2/M期,增加细胞凋亡;与si-NC组相比,siRNA-1664能显著下调DNA-PKcs mRNA和蛋白表达水平(P<0.05);与KAE组相比,KAE+si-DNAPKcs组进一步促进了KAE对细胞的效应;与对照组相比,KAE+DMSO组DNA-PKcs蛋白表达水平降低(P<0.05);与KAE+DMSO组相比,KAE+MG132组DNA-PKcs蛋白表达水平升高(P<0.05),而KAE+CQ组DNA-PKcs蛋白表达水平无明显变化(P>0.05);与对照组相比,KAE+DMSO组促进DNA-PKcs蛋白的泛素化,而KAE+MG132组则可抑制其泛素化(P<0.05)。结论KAE能够诱导肝癌耐药细胞Bel-7402/5-Fu的细胞凋亡和细胞周期阻滞。Objective To investigate the effect of kaempferol(KAE)on the function of drug-resistant Bel-7402/5-Fu cells.Methods Bel-7402/5-Fu cells were treated with KAE,and cells were divided into the control group and the drug group(0.064,0.320,1.600,8,40,200μmol/L KAE).Cells were divided into the si-NC group and the DNA-PKcs interference group,or the control group,the KAE group,the KAE+si-DNA-PKcs group or the KAE+DMSO group,the KAE+MG132 group and the KAE+CQ group based on interfering DNA dependent kinase catalytic subunits(DNA-PKcs)or addition of proteasome inhibitor MG132 or autophagy inhibitor CQ.Cell proliferation was detected using CCK-8.The expression level of histone H_(2)AX phosphorylation(γ-H_(2)AX),DNA-PKcs,DNA double strand break repair/V(D)J recombinant protein(Artemis)and drug pump gene(P-gp)were analyzed using real-time fluorescence quantitative PCR(RT-qPCR)and Western blot assay.Cell cycle and apoptosis were detected by flow cytometry.The stability of DNA-PKcs proteins was analyzed by protein stability experiments.Ubiquitination of DNA-PKcs protein was evaluated by immunoprecipitation assay.Results Compared to the control group,treating cells with 8μmol/L KAE for 24 h inhibited about 50%of cell proliferation ability.Therefore,this time and concentration were chosen for subsequent research.Compared to the control group,the expression level ofγ-H_(2)AX mRNA and protein significantly increased,while expression levels of DNA-PKcs,Artemis and P-gp mRNA and proteins significantly decreased in the KAE group(P<0.05).Compared to the control group,KAE promoted cell cycle arrest in the G2/M phase of Bel-7402/5-Fu cells and increased cell apoptosis.Compared to the si-NC group,siRNA-1664 significantly downregulated the mRNA and protein expression levels of DNAPKcs(P<0.05).Compared with the KAE group,the effect of KAE was further promoted in the KAE+si-DNA-PKcs group of Bel-7402/5-Fu cells.Compared with the control group,the protein expression level of DNA-PKcs decreased in the KAE+DMSO group(P<0.05).Compared with t

关 键 词：癌 肝细胞 山萘酚 细胞凋亡 细胞周期 Bel-7402/5-Fu DNA-PKCS 

分 类 号：R735.7[医药卫生—肿瘤]