Fu Junhao, Yu Min, Xu Wenxia, Yu Shian
Central Laboratory, Affiliated Jinhua Hospital, Zhejiang University School of Medicine, Jinhua, Zhejiang Province, China.
Department of Hepatobiliary and Pancreatic Surgery, Affiliated Jinhua Hospital, Zhejiang University School of Medicine, Jinhua, Zhejiang Province, China.
Cell J. 2023 Feb 1;25(2):118-125. doi: 10.22074/cellj.2022.557564.1077.
Chemotherapeutic drug resistance is the main obstacle that affects the efficacy of current therapies of hepatocellular carcinoma (HCC), which needs to be addressed urgently. High expression of histone methyltransferase G9a was reported to play a pivotal role in the progression of HCC. Regulatory mechanism of aberrant activation of G9a in HCC and the association with subsequent cisplatin (DDP) resistance still remains ambiguous. This study strived to investigate mechanism of G9a overexpression and its impact on cisplatin resistance in HCC cells.
In this experimental study, we investigated effects of different concentrations of cisplatin in combination with BIX-01294 or PR-619 on viability and apoptosis of HuH7 and SNU387 cells via CCK-8 kit and flow cytometric analysis, respectively. Colony formation capacity was applied to evaluate effect of cisplatin with or without BIX-01294 on cell proliferation, and western blotting was used to verify expression level of the related proteins. Global mRNA expression profile analysis was adopted to identify differentially expressed genes associated with overexpression of G9a.
We observed that overexpression of G9a admittedly promoted cisplatin resistance in HCC cells. Global mRNA expression profile analysis after G9a inhibition showed that DNA repair and cell cycle progression were downregulated. Moreover, we identified that deubiquitination enzymes (DUBs) stabilized high expression of G9a in HCC through deubiquitination. Additionally, cisplatin could significantly inhibit proliferation of DUBs-deficient HCC cells, while promoting their apoptosis.
Collectively, our data indicated that DUBs stabilize G9a through deubiquitination, thereby participating in the cisplatin resistance of HCC cells. The elucidation of this mechanism contributes to propose a potential alternative intervention strategy for the treatment of HCC patients harboring high G9a levels.
化疗耐药是影响当前肝细胞癌（HCC）治疗疗效的主要障碍，亟待解决。据报道，组蛋白甲基转移酶G9a的高表达在HCC进展中起关键作用。HCC中G9a异常激活的调控机制及其与随后顺铂（DDP）耐药的关联仍不明确。本研究旨在探讨G9a过表达的机制及其对HCC细胞顺铂耐药的影响。
在本实验研究中，我们分别通过CCK-8试剂盒和流式细胞术分析，研究了不同浓度顺铂联合BIX-01294或PR-619对HuH7和SNU387细胞活力和凋亡的影响。采用集落形成能力评估顺铂联合或不联合BIX-01294对细胞增殖的影响，并用蛋白质印迹法验证相关蛋白的表达水平。采用全基因组mRNA表达谱分析来鉴定与G9a过表达相关的差异表达基因。
我们观察到G9a过表达确实促进了HCC细胞的顺铂耐药。G9a抑制后的全基因组mRNA表达谱分析表明，DNA修复和细胞周期进程下调。此外，我们发现去泛素化酶（DUBs）通过去泛素化稳定了HCC中G9a的高表达。此外，顺铂可显著抑制DUBs缺陷型HCC细胞的增殖，同时促进其凋亡。
总体而言，我们的数据表明DUBs通过去泛素化稳定G9a，从而参与HCC细胞的顺铂耐药。对这一机制的阐明有助于提出一种潜在的替代干预策略，用于治疗G9a水平高的HCC患者。
