TRMT61A在肝癌细胞中的功能及其机制研究

doi:10.3969/j.issn.1674-8115.2022.06.008

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (6): 742-750.doi: 10.3969/j.issn.1674-8115.2022.06.008

• 论著 · 基础研究 • 上一篇

TRMT61A在肝癌细胞中的功能及其机制研究

胡哲轩¹(), 张欣¹, 沃璐璐¹, 李静池¹, 王娇¹, 周佽想¹, 赵倩¹^,²()

^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

收稿日期:2022-03-16 接受日期:2022-05-25 出版日期:2022-06-28 发布日期:2022-08-19
通讯作者: 赵倩 E-mail:okaydokay@yeah.net;qzhao@shsmu.edu.cn
作者简介:胡哲轩（1996—），男，硕士生；电子信箱：okaydokay@yeah.net。
基金资助:
国家自然科学基金(81772831)

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

HU Zhexuan¹(), ZHANG Xin¹, WO Lulu¹, LI Jingchi¹, WANG Jiao¹, ZHOU Cixiang¹, ZHAO Qian¹^,²()

^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

Received:2022-03-16 Accepted:2022-05-25 Online:2022-06-28 Published:2022-08-19
Contact: ZHAO Qian E-mail:okaydokay@yeah.net;qzhao@shsmu.edu.cn
Supported by:
National Natural Science Foundation of China(81772831)

摘要/Abstract

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

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

中图分类号:

R735.7

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.

HU Zhexuan, ZHANG Xin, WO Lulu, LI Jingchi, WANG Jiao, ZHOU Cixiang, ZHAO Qian. Study on the function of TRMT61A in liver cancer cell and its mechanism[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(6): 742-750.

图/表 6

图1 TRMT61A 在肝癌组织样本中的表达及其与预后的关系Note：A. Expression of TRMT61A in tumor and normal tissues of hepatocellular carcinoma patients from TCGA database. B. The overall survival curves of hepatocellular carcinoma patients with low or high TRMT61A expression. ①P=0.000, compared with the normal tissues. TPM—transcripts per kilobase million.

Fig 1 Expression of TRMT61A in hepatocellular carcinoma samples and its correlation with prognosis

图2 稳定敲低 TRMT61A 肝癌细胞系Huh7和HepG2的构建和验证Note：A/B.TRMT61A expression in Huh7 cells (A) and HepG2 cells (B) detected by Western blotting. C/D. Relative levels of TRMT61A protein in Huh7 cells (C) and HepG2 cells (D). E/F. m1A modification in Huh7 cells (E) and HepG2 cells (F). ①P=0.000, compared with the NC group.

Fig 2 Establishment and confirmation of stably-TRMT61A-knockdown Huh7 and HepG2 cells

图3 敲低 TRMT61A 对肝癌细胞增殖功能的影响Note：A/B.Growth curves of Huh7 cells (A) and HepG2 (B) cells after TRMT61A knockdown. C-F.Colony formation (C/D) and colony numbers (E/F) of Huh7 cells and HepG2 cells after TRMT61A knockdown. ①P=0.000, ②P=0.001, compared with the NC group.

Fig 3 Effects of TRMT61A knockdown on proliferation of Huh7 and HepG2 cells

图4 敲低 TRMT61A 对肝癌细胞周期的影响Note：A/B. Cell percentage of Huh7 cells in different cell cycle phases after TRMT61A knockdown. C/D. Cell percentage of HepG2 cells in different cell cycle phases after TRMT61A knockdown. ①P=0.014, ②P=0.024, ③P=0.005, compared with the NC group.

Fig 4 Effects of TRMT61A knockdown on cell cycle of Huh7 and HepG2 cells

图5 敲低 TRMT61A 对肝癌细胞调控细胞周期相关蛋白表达的影响Note：A/B.Expression of cell cycle-associated protein in Huh7 (A) and HepG2 cells (B) after TRMT61A knockdown detected by Western blotting. C-D.Relative levels of cell cycle-associated protein in Huh7 (C) and HepG2 cells (D) after TRMT61A knockdown. ①P=0.000, ②P=0.001, ③P=0.004, compared with the NC group.

Fig 5 Effects of TRMT61A knockdown on cell cycle-related protein levels in Huh7 and HepG2 cells

图6 敲低 TRMT61A 对 Huh7和HepG2细胞凋亡的影响Note：A/B. Cell apoptosis rate of Huh7 cells detected by Annexin V/PI staining. C/D. Cell apoptosis rate of HepG2 cells detected by Annexin V/PI staining. E/F. Cleaved caspase3 in Huh7 (E) and HepG2 cells (F) after TRMT61A knockdown detected by Western blotting. G.Relative levels of cleaved caspase3 in Huh7 and HepG2 cells after TRMT61A knockdown. ①P=0.000, ②P=0.018, ③P=0.002, compared with the NC group.

Fig 6 Effects of TRMT61A knockdown on cell apoptosis in Huh7 cells and HepG2 cells

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TRMT61A在肝癌细胞中的功能及其机制研究

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

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