上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2022 , Vol. 42 >Issue 6: 742 - 750

DOI: https://doi.org/10.3969/j.issn.1674-8115.2022.06.008

论著 · 基础研究

TRMT61A在肝癌细胞中的功能及其机制研究

  • 胡哲轩 ,
  • 张欣 ,
  • 沃璐璐 ,
  • 李静池 ,
  • 王娇 ,
  • 周佽想 ,
  • 赵倩
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  • 1.上海交通大学医学院病理生理学系，细胞分化与凋亡教育部重点实验室，上海 200025
    2.中国医学科学院应激与肿瘤创新单元（2019RU043），上海 200025
胡哲轩（1996—），男，硕士生；电子信箱：okaydokay@yeah.net。
赵 倩，电子信箱：qzhao@shsmu.edu.cn。

收稿日期: 2022-03-16

  录用日期: 2022-05-25

  网络出版日期: 2022-08-19

基金资助

国家自然科学基金(81772831)

收起

Study on the function of TRMT61A in liver cancer cell and its mechanism

  • Zhexuan HU ,
  • Xin ZHANG ,
  • Lulu WO ,
  • Jingchi LI ,
  • Jiao WANG ,
  • Cixiang ZHOU ,
  • Qian ZHAO
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  • 1.Department of Pathophysiology, Key Laboratory for Cell Differentiation and Apoptosis Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
    2.Research Units of Stress and Tumor(2019RU043), Chinese Academy of Medical Sciences, Shanghai 200025, China
ZHAO Qian, E-mail: qzhao@shsmu.edu.cn.

Received date: 2022-03-16

  Accepted date: 2022-05-25

  Online published: 2022-08-19

Supported by

National Natural Science Foundation of China(81772831)

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摘要

目的·探究N1-甲基腺苷（N1-methyladenosine，m1A）甲基转移酶催化亚基（tRNA methyltransferase 61 Homolog A，TRMT61A）对肝癌细胞功能的影响及其潜在机制。方法·通过生物信息学软件分析TCGA数据库中TRMT61A在肝细胞肝癌患者样本中的表达情况并绘制相关生存曲线。通过CRISPR-Cas9技术构建稳定敲低TRMT61A表达的肝癌细胞系Huh7细胞和HepG2细胞，通过蛋白质印记法（Western blotting）检测对照组和敲低组细胞TRMT61A蛋白水平；通过斑点印迹实验检测2组细胞总RNA的m1A修饰水平。利用CCK-8法和平板克隆形成实验检测2组细胞增殖能力。使用碘化丙啶（propidium iodide，PI）染色和流式细胞术检测细胞周期；通过Western blotting检测细胞周期相关蛋白表达水平。使用Annexin V/PI细胞凋亡检测试剂盒检测细胞凋亡情况；通过Western blotting检测细胞凋亡相关蛋白表达水平。结果·经生物信息学软件分析，TCGA数据库中TRMT61A在肝癌患者癌组织中高表达，通过绘制生存曲线发现TRMT61A与肝癌患者的预后呈负相关。在肝癌细胞系Huh7和HepG2中稳定敲低TRMT61A后，细胞中TRMT61A蛋白水平下降，总RNA的m1A修饰水平下降。CCK-8实验结果显示在Huh7和HepG2细胞中稳定敲低TRMT61A后细胞增殖明显受到抑制；平板克隆实验结果显示在Huh7和HepG2细胞中稳定敲低TRMT61A后，克隆形成数目显著降低。TRMT61A敲低的Huh7和HepG2细胞出现G0/G1期细胞周期阻滞，细胞中P21蛋白水平显著上升，cyclin D1蛋白水平下降。此外，细胞凋亡检测结果显示敲低TRMT61A后Huh7和HepG2细胞凋亡率上升，而细胞剪切活化caspase3蛋白水平也明显上升。结论·敲低TRMT61A可抑制肝癌细胞增殖，并诱导其发生凋亡。

关键词： N1-甲基腺苷甲基化修饰; TRMT61A; 肝细胞肝癌; 细胞增殖; 细胞凋亡

本文引用格式

胡哲轩 , 张欣 , 沃璐璐 , 李静池 , 王娇 , 周佽想 , 赵倩 . TRMT61A在肝癌细胞中的功能及其机制研究[J]. 上海交通大学学报（医学版）, 2022 , 42(6) : 742 -750 . DOI: 10.3969/j.issn.1674-8115.2022.06.008

Abstract

Objective

·To investigate the effect of tRNA methyltransferase 61 homolog A (TRMT61A) on liver cancer cell function and its mechanism.

Methods

·The expression of TRMT61A in tumor and paired peri-tumor tissues of hepatocellular carcinoma patients was analyzed from TCGA database and the survival curves were plotted by using bioinformatics software. Stably-TRMT61A-knockdown Huh7 cells and HepG2 cells were established by using CRISPR-Cas9 system. TRMT61A protein levels in negative control group and knockdown group were detected by Western blotting. M1A methylation levels in the two groups were detected by Dot blot assay. The cell proliferation of the two groups was investigated through CCK-8 assay and colony formation assay. Flow cytometry was used to analyze cell cycle after propidium iodide (PI) staining. Cell cycle-related protein level was detected by Western blotting. Cell apoptosis was detected by using Annexin V/PI kit. Cell apoptosis-related protein level was detected by Western blotting.

Results

·TRMT61A was highly expressed in hepatocellular carcinoma samples from TCGA database through bioinformatic analysis. Survival curves showed that TRMT61A was negatively correlated with patient prognosis. TRMT61A protein level and m1A modification level were lower in TRMT61A-knockdown liver cancer cells. CCK-8 assay showed that the proliferation ability was aberrantly inhibited in Huh7 and HepG2 cells after TRMT61A knockdown. Colony formation assay showed that the number of colonies was reduced in Huh7 and HepG2 cells after TRMT61A knockdown. Mechanism investigation showed that TRMT61A-knockdown Huh7 and HepG2 cells both displayed cell cycle arrest on G0/G1 phase with elevated P21 protein level and decreased cyclin D1 protein level. Cell apoptosis rates were higher in Huh7 and HepG2 cells after TRMT61A knockdown with elevated cleaved-caspase3 protein level.

Conclusion

·TRMT61A knockdown inhibits the proliferation of liver cancer cells and induces cell apoptosis.

Key words： N1-methyladenosine (m1A) methylation; TRMT61A; hepatocellular carcinoma; cell proliferation; cell apoptosis

参考文献

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