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. 2022 Jul 20;42(7):1026-1031.
doi: 10.12122/j.issn.1673-4254.2022.07.09.

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

[Article in Chinese]
X Zhao  1 , K Yang  1 , Z Song  1 , H He  2 , W Zhang  1
Affiliations

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

[Article in Chinese]
X Zhao et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

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.

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cMyc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；sicMyc20 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。

Keywords: HeLa cells; c-Myc; cervical cancer; juglone; ubiquitination.

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Figures

图 1
图 1
胡桃醌对HeLa细胞c-Myc蛋白表达的影响 Effect of juglone on expression of c-Myc protein in HeLa cells. A: Western blots of c-Myc protein in HeLa cells treated with different concentrations of juglone. B: Western blots of c-Myc in HeLa cells treated with 20 μmol/L juglone for different time lengths. C: Quantitative analysis of c-Myc protein expression. **P < 0.01 vs untreated (0 μmol/LJuglone) group.
图 2
图 2
胡桃醌对c-Myc蛋白半衰期的影响 Effect of juglone on half-life of c-Myc protein. A, B: Effect of juglone on expression of c-Myc protein at different time points. C: Quantitative analysis of protein expression. *P < 0.05, **P < 0.01 vs 0 h group; #P < 0.05, ##P < 0.01 vs CHX+20 μmol/L group.
图 3
图 3
蛋白酶体抑制剂对胡桃醌处理后c-Myc蛋白的影响 Effects of proteasome inhibitor on c-Myc protein expression in HeLa cells after juglone treatment. A: Western blots of c-Myc protein expressions in different groups. B: Quantitative analysis of the protein expression. *P < 0.05 vs jugloen single treated group; #P < 0.05, ##P < 0.01 vs untreated group.
图 4
图 4
胡桃醌促进c-Myc蛋白泛素化降解 Ubiquitination of c-Myc protein in juglone-treated HeLa cells.
图 5
图 5
胡桃醌对敲及不敲除c-Myc基因HeLa细胞后增殖及凋亡的影响 Effects of juglone on proliferation and apoptosis of HeLa cells with and without c-Myc gene knockout. A, B: estern blotting after c-Myc gene knockout and quantitative analysis of the protein expression. C, D: Effects of juglone on proliferation and apoptosis of cells with and without c-Myc gene knockout. E: Comparison of early apoptosis rate. *P < 0.05; **P < 0.01 vs siNAgroup; #P < 0.05 vs siNAplus jugone group.
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