摘要
目的观察c-Myc蛋白在宫颈癌HeLa细胞中的表达,探讨胡桃醌通过影响c-Myc蛋白泛素化降解进而影响HeLa细胞的增殖与凋亡。方法培养人宫颈癌HeLa细胞,分为对照组:正常培养;10、20、50μmol/L胡桃醌组:培养液中分别加入对应浓度的胡桃醌。以Westernblot方法分析胡桃醌处理后c-Myc蛋白的表达。HeLa细胞分为对照组和20μmol/L胡桃醌组,在培养0、2、4、8 h分别用Westernblot检测c-Myc蛋白表达的变化;放线菌酮(CHX)法检测c-Myc蛋白半衰期的变化;CoIP方法检测c-Myc蛋白降解影响。HeLa细胞分为空白对照组:正常培养;胡桃醌组:20μmol/L胡桃醌培养液培养;0.5、1.0、2.0μmol/LMG132组:分别与0、1.0、2.0μmol/L蛋白酶体抑制剂MG132加20μmol/L胡桃醌培养,检测c-Myc蛋白表达的水平。将si c-Myc转染入HeLa细胞后,将细胞分siNC组:空载体组;si-NC;胡桃醌组:空载体加20μmol/L胡桃醌处理;si-cMyc组:转染Myc RNA;sicMyc20μmol/L胡桃醌组:转染Myc RNA加20μmol/L胡桃醌处理。MTT及流式细胞术检测20μmol/L胡桃醌处理后对敲除及未敲除c-Myc基因的细胞增殖及凋亡的影响。结果与对照组相比,不同浓度胡桃醌可下调c-Myc蛋白表达且曾时间及剂量依赖性(P<0.05;P<0.01);胡桃醌可缩短c-Myc蛋白的半衰期,加入不同浓度的MG132后,即可明显上调c-Myc蛋白水平(P<0.05;P<0.01)。未用胡桃醌组相比,胡桃醌可明显增加c-Myc蛋白的泛素降解水平。未敲除c-Myc组相比,敲除组在胡桃醌处理后吸光度值明显增加(P<0.05)和早期凋亡率明显下降(P<0.05)。结论胡桃醌通过影响c-Myc蛋白的泛素化降解过程,进而抑制HeLa细胞增殖并促进其凋亡的发生。
Objective To observe the expression of c-Myc protein in cervical cancer HeLa cells and explore the effect of juglone on the proliferation and apoptosis of HeLa cells by affecting c-Myc ubiquitination.Methods HeLa cells treated with different concentrations(0,10,20,or 50μmol/L)of juglone or with 20μmol/L juglone for different time lengths were examined for expression of c-Myc protein with Western blotting.The half-life of c-Myc protein was determined using cycloheximide(CHX)and c-Myc protein degradation was detected using coimmunoprecipitation.We also assessed the effects of 20μmol/L juglone combined with 0,1.0 or 2.0μmol/L MG132(a proteasome inhibitor)on c-Myc expression.The effects of 20μmol/L juglone on the proliferation and apoptosis of HeLa cells with RNA interference-mediated knockdown of c-Myc were evaluated with MTT assay and flow cytometry.Results Treatment with juglone significantly lowered c-Myc protein expression in HeLa cells in a concentration-and time-dependent manner(P<0.05).Juglone obviously shortened the half-life of c-Myc protein,and the addition of MG132 significantly up-regulated the expression level of c-Myc protein(P<0.05).Juglone treatment also promoted ubiquitination of c-Myc protein in HeLa cells.Compared with the cells transfected with a negative control construct,the cells transfected with si-c-Myc showed significantly decreased proliferation inhibition and a lowered cell rate with early apoptosis after juglone treatment(P<0.05).Conclusion Juglone inhibits proliferation and promotes apoptosis of HeLa cells by affecting the ubiquitination of c-Myc protein.
作者
赵行宇
杨昆
宋梓桐
何涵
张巍
ZHAO Xingyu;YANG Kun;SONG Zitong;HE Han;ZHANG Wei(Department of Biochemistry,Jilin Medical College,Jilin 132013,China;Department of Biochemistry,School of Basic Medicine,Yanbian University,Yanbian 133000,China)
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2022年第7期1026-1031,共6页
Journal of Southern Medical University
基金
吉林省科技厅科技发展项目(20190701062GH)
吉林省教育厅“十三五”科学技术研究项目(JJKH20200453KJ,JJKH20220464KJ)。
