南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (7): 1026-1031.doi: 10.12122/j.issn.1673-4254.2022.07.09

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胡桃醌通过促进c-Myc泛素化降解抑制宫颈癌细胞的生长和促进凋亡

赵行宇，杨 昆，宋梓桐，何 涵，张 巍   

  1. 吉林医药学院生化教研室，吉林 吉林 132013；延边大学基础医学院生化教研室，吉林 延边 133000
  • 出版日期:2022-07-20 发布日期:2022-07-15

Juglone induces proliferation inhibition and apoptosis of cervical cancer cells via promoting c-Myc ubiquitination

ZHAO Xingyu, YANG Kun, SONG Zitong, HE Han, ZHANG Wei   

  1. Department of Biochemistry, Jilin Medical College, Jilin 132013, China; Department of Biochemistry, School of Basic Medicine, Yanbian University, Yanbian 133000, China
  • Online:2022-07-20 Published:2022-07-15

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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、50 μmol/L胡桃醌组：培养液中分别加入对应浓度的胡桃醌。以Western blot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20 μmol/L胡桃醌组，在培养0、2、4、8 h分别用Western blot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：20 μmol/L胡桃醌培养液培养；0.5、1.0、2.0 μmol/L MG132组：分别与0、1.0、2.0 μmol/L蛋白酶体抑制剂MG132加20 μmol/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20 μmol/L胡桃醌处理；si-cMyc组：转染Myc RNA；si-cMyc20 μmol/L胡桃醌组：转染Myc RNA加20 μmol/L胡桃醌处理。MTT及流式细胞术检测20 μmol/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。

关键词: 胡桃醌；宫颈癌；HeLa；c-Myc；泛素化

Abstract: 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 50 μmol/L) of juglone or with 20 μmol/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 20 μmol/L juglone combined with 0, 1.0 or 2.0 μmol/L MG132 (a proteasome inhibitor) on c-Myc expression. The effects of 20 μmol/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

Key words: juglone; cervical cancer; HeLa cells; c-Myc; ubiquitination