中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (26): 4221-4225.doi: 10.12307/2024.423

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

METTL3在同型半胱氨酸诱导小鼠胰岛β细胞自噬中的作用

马凌桔1,2,汪乐新2,迟宏扬2,3,张竞文2,彭红建2,4,高春兰2,5,姜怡邓2,黄  晖1,杨  力1,马胜超2,3   

  1. 1宁夏医科大学总医院老年与特需医学科,宁夏回族自治区银川市  750004;2国家卫生健康委员会代谢性心血管疾病研究重点实验室,宁夏回族自治区银川市  750004;3宁夏医科大学检验学院,宁夏回族自治区银川市  750004;4中南大学化学化工学院,湖南省长沙市  410083;5银川市第一人民医院,宁夏回族自治区银川市  750001
  • 收稿日期:2023-06-06 接受日期:2023-07-24 出版日期:2024-09-18 发布日期:2023-10-07
  • 通讯作者: 马胜超,博士,副教授,国家卫生健康委员会代谢性心血管疾病研究重点实验室,宁夏回族自治区银川市 750004;宁夏医科大学检验学院,宁夏回族自治区银川市 750004
  • 作者简介:马凌桔,男,1994年生,宁夏回族自治区平罗县人,回族,2022年宁夏医科大学毕业,硕士,医师,主要从事老年慢性代谢性疾病的研究。
  • 基金资助:
    国家自然科学基金青年项目(81900273),项目负责人:马胜超;国家自然科学基金地区项目(82060139,82270492),项目负责人:马胜超;宁夏自治区自然科学基金优秀青年项目(2023AAC05035),项目负责人:马胜超;宁夏回族自治区重点研发计划项目(2019BEG03006),项目负责人:张竞文;宁夏医科大学校级重点项目(XZ2022004),项目负责人:彭红建

Role of METTL3 in homocysteine-induced autophagy in mouse islet beta cells

Ma Lingju1, 2, Wang Lexin2, Chi Hongyang2, 3, Zhang Jingwen2, Peng Hongjian2, 4, Gao Chunlan2, 5, Jiang Yideng2, Huang Hui1, Yang Li1, Ma Shengchao2, 3   

  1. 1Department of Geriatrics and Special Need Medicine, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 2Key Laboratory of Metabolic Cardiovascular Disease Research of National Health Commission, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 3School of Inspection, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 4College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan Province, China; 5The First People’s Hospital of Yinchuan, Yinchuan 750001, Ningxia Hui Autonomous Region, China
  • Received:2023-06-06 Accepted:2023-07-24 Online:2024-09-18 Published:2023-10-07
  • Contact: Ma Shengchao, MD, Associate professor, Key Laboratory of Metabolic Cardiovascular Disease Research of National Health Commission, Yinchuan 750004, Ningxia Hui Autonomous Region, China; School of Inspection, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • About author:Ma Lingju, Master, Physician, Department of Geriatrics and Special Need Medicine, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; Key Laboratory of Metabolic Cardiovascular Disease Research of National Health Commission, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Supported by:
    the National Natural Science Foundation of China (Youth Project), No. 81900273 (to MSC); National Natural Science Foundation of China (Regional Project), Nos. 82060139 and 82270492 (to MSC); Outstanding Youth Project of Natural Science Foundation of Ningxia Autonomous Region, No. 2023AAC05035 (to MSC); the Key Research and Development Program of Ningxia Hui Autonomous Region, No. 2019BEG03006 (to ZJW); The School-level Key Project of Ningxia Medical University, No. XZ2022004 (to PHJ)

摘要:


文题释义:

自噬:是指以胞质空泡化为特征依赖于溶酶体的一种降解途径,在维持细胞内稳态中发挥重要作用,自噬标志物微管相关蛋白Ⅰ轻链 3 (LC3)、Beclin-1蛋白及p62蛋白反映了自噬流的水平。
N6甲基腺苷甲基转移酶(METTL3) :是m6A甲基转移酶复合体亚复合物MT-ADE含甲基供体S-腺苷甲硫氨酸的结合位点,分子质量70 kD,在转录后水平发挥重要调控作用,包括前体mRNA的剪接、成熟mRNA的输出、mRNA的稳定性调节等。


背景:高同型半胱氨酸(homocysteine,Hcy)血症与胰岛β细胞功能密切相关,但其具体分子机制尚不完全明确。

目的:探讨METTL3在Hcy诱导小鼠胰岛β细胞自噬中的作用。
方法:取第3,4代小鼠胰岛β细胞进行实验。①细胞模型建立和分组:对照组细胞不加入Hcy,Hcy组细胞加入浓度为100 µmol/L Hcy干预48 h;②按LipofectamineTM 2000说明书将过表达质粒Ad-METTL3及si-METTL3转染小鼠胰岛β细胞,设计3种不同干扰片段,PCR验证、筛选出干扰效率最好的干扰片段;③转染后实验分组:对照组、Hcy组、Ad-NC(阴性对照)+Hcy组、Ad-METTL3+Hcy组、si-NC(阴性对照)+Hcy组和si-METTL3+Hcy组;④采用qRT-PCR及Western blot检测细胞中METTL3及细胞自噬相关蛋白LC3Ⅱ/Ⅰ、p62的表达;ELISA法测定胰岛素水平来评价胰岛β细胞胰岛素分泌能力;分别过表达和干扰METTL3后检测细胞自噬相关蛋白及胰岛素水平。

结果与结论:①与对照组相比,Hcy组自噬相关蛋白LC3Ⅱ/Ⅰ的表达水平升高(P < 0.05),而p62表达明显降低( P < 0.05),胰岛素分泌能力明显下降(P < 0.05);②与对照组相比,Hcy组中METTL3蛋白及mRNA水平表达均降低(P < 0.05);③胰岛β细胞中沉默METTL3后,Hcy进一步上调了细胞中LC3Ⅱ/Ⅰ的表达(P < 0.05),而p62表达显著下降(P < 0.05),细胞中胰岛素水平增加(P < 0.05);过表达METTL3后,Hcy则使LC3Ⅱ/Ⅰ表达显著降低且p62 表达则增高( P < 0.05);④结论:METTL3参与了Hcy诱导的胰岛β细胞自噬调控,对胰岛素的分泌发挥着调控作用。

https://orcid.org/0009-0003-5188-3181(马凌桔)

中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程

关键词: N6-甲基腺苷甲基转移酶3, 胰岛β细胞, 同型半胱氨酸, 细胞自噬, LC3Ⅱ/Ⅰ

Abstract: BACKGROUND: Hyperhomocysteinemia is closely related to the function of islet β cells, but its specific molecular mechanism is not fully understood.
OBJECTIVE: To investigate the role of N6 methyltransferase-like 3 (METTL3) in homocysteine (Hcy)-induced autophagy of mouse islet β cells. 
METHODS: The 3rd and 4th generation mouse islet β cells were taken for the experiment. (1) Cell modeling and grouping: cells in control group were not treated with Hcy, while those in homocysteine group were treated with 100 µmol/L Hcy for 48 hours. (2) The mouse islet β-cells were transfected with the plasmids overexpressing Ad-METTL3 and si-METTL3 according to the instructions of LipofectamineTM 2000. Three different interfering fragments were designed, and the one with the best interfering efficiency was verified and screened by PCR. (3) After transfection, the cells were divided into control group, Hcy group, Ad-NC (negative control)+Hcy group, Ad-METTL3+Hcy group, si-NC (negative control)+Hcy group and si-METTL3+Hcy group. (4) qRT-PCR and western blot were used to detect the expression levels of METTL3 and autophagy-related proteins LC3II/I and p62 in cells. Insulin level was determined by ELISA to evaluate insulin secretion capacity of islet cells. Autophagy-related proteins and insulin level were detected after overexpression and interference with METTL3. 
RESULTS AND CONCLUSION: Compared with the control group, the expression level of LC3II/I was increased (P < 0.05), the expression of p62 was significantly reduced (P < 0.05), and the insulin secretion capacity was significantly decreased (P < 0.05) in the Hcy group. Compared with the control group, the protein and mRNA levels of METTL3 were reduced in the Hcy group (P < 0.05). After METTL3 silencing in islet β cells, Hcy further upregulated the expression of LC3II/I (P < 0.05), significantly dowregulated the expression of p62 (P < 0.05), and increased the insulin level (P < 0.05). After overexpression of METTL3, Hcy significantly decreased the LC3II/I expression and increased the p62 expression in islet β cells (P < 0.05).  To conclude, METTL3 is involved in the Hcy-induced autophagy regulation of islet β cells and plays a role in the regulation of insulin secretion.

Key words: methyltransferase-like 3, islet β cell, homocysteine, autophagy, LC3II/I

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