SIRT4通过抑制GLS2表达增强肝细胞癌对索拉菲尼敏感性的研究

刘晓萌; 刘文慧; 高珊; 李梦宇; 马晓青; 王洪权; 唐博

doi:10.12354/j.issn.1000-8179.2023.20230501

SIRT4通过抑制GLS2表达增强肝细胞癌对索拉菲尼敏感性的研究

SIRT4 enhances hepatocellular carcinoma sensitivity to sorafenib via inhibiting GLS2 expression

摘要

摘要:
目的探索Sirtuin4（SIRT4）调控肿瘤谷氨酰胺代谢的机制，明确SIRT4对肝癌细胞干性及索拉菲尼治疗敏感性的影响。
方法通过实时定量 PCR（real-time quantitative reverse transcription PCR，qRT-PCR）和免疫组织化学染色检测SIRT4在肝癌中的mRNA和蛋白表达水平。免疫印迹及细胞成球实验检测肿瘤细胞干性，细胞计数实验（CCK-8）和平板克隆实验检测肝癌细胞索拉菲尼治疗敏感性。免疫共沉淀检测SIRT4对蛋白的去乙酰化作用。
结果在肝癌的临床样本组织和细胞系中，SIRT4的表达显著低于癌旁组织和正常肝细胞。在体外实验中，过表达SIRT4能够明显降低肝癌细胞的成球数量（P<0.01）和干性标志物表达，并使索拉菲尼治疗的肝癌细胞克隆数和OD₄₅₀值进一步下降（均P<0.01）。机制研究表明，SIRT4通过去乙酰化降低谷氨酰胺合成酶2（glutamine synthase 2，GLS2）的表达，进而抑制肝癌细胞中谷氨酰胺的分解代谢，从而调节肝癌细胞的干性和对索拉菲尼的敏感性。
结论 SIRT4在肝癌组织中表达显著降低，能够通过降低谷氨酰胺分解代谢，进而抑制肝癌细胞的干性并增强其对索拉菲尼的敏感性。

Abstract:
Objective To explore the mechanism by which Sirtuin4 (SIRT4) regulates tumor glutamine metabolism, and to clarify the effect of SIRT4 on hepatocellular carcinoma (HCC) cell stemness and sensitivity to sorafenib therapy.
Methods SIRT4 mRNA and protein expression levels in HCC were detected using real-time quantitative reverse transcription PCR (qRT-PCR) and immunohistochemistry, respectively. Tumor cell stemness was assessed by Western blot and sphere formation. HCC cell sensitivity to sorafenib was quantitated using cell counting Kit-8 (CCK-8) and plate colony formation. Deacetylation of glutamine synthase 2 (GLS2) protein by SIRT4 was detected via co-immunoprecipitation.
Results SIRT4 expression was significantly lower in clinical samples and human HCC cell lines than in para-cancerous tissues and normal human hepatocytes. In vitro, SIRT4 overexpression not only reduced the spheroid formation number (P<0.01) and stemness marker expression of HCC cells, but also decreased the number of colony formation and OD₄₅₀(P<0.01) in sorafenib-treated HCC cells. A mechanistic study revealed that SIRT4 reduced GLS2 expression through deacetylation, in turn inhibiting glutamine catabolism in HCC cells, thereby modulating their stemness and sensitivity to sorafenib.
Conclusions SIRT4 expression was significantly reduced in HCC, inhibiting HCC cell stemness and enhancing HCC cellsensitivity to sorafenib by decreasing glutamine catabolism.

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