中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (8): 1289-1294.doi: 10.12307/2024.201

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

miR-144-3p参与高蛋氨酸饮食诱导Cbs+/-小鼠的肝细胞自噬

盛思琪1，2，3，谢  琳1，2，3，赵翔宇1，2，3，姜怡邓1，2，3，吴  凯1，2，3，熊建团1，2，3，杨安宁1，2，3，郝银菊1，2，3，4，焦  运2，4   

  1. 宁夏医科大学，1基础医学院，2国家卫生健康委代谢性心血管疾病研究重点实验室，3宁夏血管损伤与修复研究重点实验室，宁夏回族自治区银川市  750004；4宁夏医科大学总医院，宁夏回族自治区银川市  750004
  • 收稿日期:2022-11-15 接受日期:2022-12-26 出版日期:2024-03-18 发布日期:2023-07-19
  • 通讯作者: 焦运，主任医师，宁夏医科大学总医院，宁夏回族自治区银川市 750004；国家卫生健康委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
  • 作者简介:盛思琪，女，1994年生，宁夏回族自治区石嘴山市人，汉族，宁夏医科大学在读硕士，主要从事同型半胱氨酸对细胞损伤方向的研究。
  • 基金资助:
    国家自然科学基金重点项目(82060110)，项目负责人：焦运；宁夏回族自治区重点研发计划重点项目(2021BEG02033)，项目负责人：熊建团；宁夏回族自治区重点研发计划重点项目(2020BFH02003)，项目负责人：杨安宁；宁夏回族自治区重点研发计划重点项目(2022BFH02013)，项目负责人：郝银菊；中国医学科学院中央级公益性科研院所基本科研业务费项目(2019PT330002)，项目负责人：姜怡邓

Involvement of miR-144-3p in Cbs+/- mouse hepatocyte autophagy induced by high-methionine diet

Sheng Siqi1, 2, 3, Xie Lin1, 2, 3, Zhao Xiangyu1, 2, 3, Jiang Yideng1, 2, 3, Wu Kai1, 2, 3, Xiong Jiantuan1, 2, 3, Yang Anning1, 2, 3, Hao Yinju1, 2, 3, 4, Jiao Yun2, 4   

  1. 1School of Basic Medical Sciences, 2NHC Key Laboratory of Metabolic Cardiovascular Diseases Research, Yinchuan 750004, 3Ningxia Key Laboratory of Vascular Injury and Repair Research, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 4General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Received:2022-11-15 Accepted:2022-12-26 Online:2024-03-18 Published:2023-07-19
  • Contact: Jiao Yun, Chief physician, NHC Key Laboratory of Metabolic Cardiovascular Diseases Research, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • About author:Sheng Siqi, Master candidate, School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; NHC Key Laboratory of Metabolic Cardiovascular Diseases Research, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; Ningxia Key Laboratory of Vascular Injury and Repair Research, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Supported by:
    the National Natural Science Foundation of China, No. 82060110 (to JY); Ningxia Hui Autonomous Region Key R&D Program, No. 2021BEG02033 (to XJT); Ningxia Hui Autonomous Region Key R&D Program, No. 2020BFH02003 (to YAN); Ningxia Hui Autonomous Region Key R&D Program, No. 2022BFH02013 (to HYJ); Basic Scientific Research Expense Project of the Central Public Welfare Research Institute of the Chinese Academy of Medical Sciences, No. 2019PT330002 (to JYD)

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摘要：


文题释义：

自噬(autophagy)：是真核生物细胞内溶酶体降解的途径，通过溶酶体或胞液进行营养物质缺乏即“饥饿”状态下的营养动员，从而清除受损蛋白质，调节细胞的新陈代谢和内环境稳态。细胞自噬活性可通过LC3B、p62等自噬关键蛋白的表达水平来评估。
微小RNA(miRNA)：是一类长度为18-22个核苷酸且高度保守的非编码RNA，可将RNA诱导的沉默复合物结合到特定的序列位置上，在转录后水平沉默特定的靶基因，从而调节靶蛋白的生成，参与多种细胞生物学功能的调控。


背景：高蛋氨酸饮食可导致Cbs+/-小鼠发生肝损伤，高同型半胱氨酸血症与肝脂肪变性、自身免疫性肝炎、酒精性脂肪肝等多种肝脏相关疾病的发生和进展有关。微小RNA (miRNAs)参与细胞存活、分化和细胞自噬等各种细胞过程，具有重要意义。

目的：探讨miR-144-3p在高蛋氨酸饮食诱导Cbs+/-小鼠肝细胞自噬中的关键作用。
方法：①选取4周龄体质量相近的雄性胱硫醚β-合成酶基因正常(Cbs+/+)小鼠和单基因敲除(Cbs+/-)小鼠各10只，均饲以高蛋氨酸饮食，12周后处死，留取肝脏组织。②体外培养人源肝细胞(HL-7702)，分为对照组(0 μmol/L同型半胱氨酸)、同型半胱氨酸组(100 μmol/L同型半胱氨酸)、mimic-NC组(转染mimic-NC)、mimic-NC+同型半胱氨酸组(转染mimic-NC+100 μmol/L同型半胱氨酸)、miR-144-3p mimic组(转染miR-144-3p mimic)、miR-144-3p mimic+同型半胱氨酸组(转染miR-144-3p mimic+100 μmol/L同型半胱氨酸)、inhibitor-NC组(转染inhibitor-NC)、inhibitor-NC+同型半胱氨酸组(转染inhibitor-NC+100 μmol/L同型半胱氨酸)、miR-144-3p inhibitor组(转染miR-144-3p inhibitor)、miR-144-3p inhibitor+同型半胱氨酸组(转染miR-144-3p inhibitor+100 μmol/L同型半胱氨酸)。采用荧光定量PCR检测肝组织和肝细胞中miR-144-3p的表达水平；转染miR-144-3p模拟物或抑制剂后，采用荧光定量PCR和Western blot分别检测miR-144-3p的转染效率及其对LC3B和p62蛋白表达的影响；酶联免疫法检测肝细胞上清液中丙氨酸氨基转移酶和门冬氨酸氨基转移酶的表达情况；Pearson相关性分析肝细胞miR-144-3p表达与肝细胞上清液中丙氨酸氨基转移酶和门冬氨酸氨基转移酶含量的相关性。

结果与结论：①与Cbs+/+组比较，Cbs+/-组小鼠肝组织和同型半胱氨酸组肝细胞中miR-144-3p的表达水平降低(P < 0.01)；②转染miR-144-3p模拟物后，与mimic-NC比较，miR-144-3p mimic组中LC3B-Ⅱ/Ⅰ蛋白的表达水平降低，p62蛋白的表达水平升高(P < 0.01)；转染miR-144-3p inhibitor后，得到相反的结果(P < 0.01)；③与mimic-NC组相比，miR-144-3p mimic组肝细胞上清液中丙氨酸氨基转移酶和门冬氨酸氨基转移酶的含量降低(P < 0.01)；与inhibitor-NC组比较，得到相反的结果(P < 0.01)；④肝细胞中miR-144-3p的表达与肝细胞上清液中丙氨酸氨基转移酶(P < 0.01，r=-0.887 6)和门冬氨酸氨基转移酶(P < 0.01，r=-0.829 9)的含量呈负相关；⑤结果表明，同型半胱氨酸通过抑制miR-144-3p的表达促进肝细胞的自噬，进而加重肝损伤。

https://orcid.org/0000-0003-3270-0637(盛思琪)

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: 微小RNA, miR-144-3p, 同型半胱氨酸, 细胞自噬, 肝损伤, 高蛋氨酸饮食

Abstract: BACKGROUND: High-methionine diet can cause liver injury in Cbs+/- mice, and hyperhomocystinemia is related to the occurrence and progression of various liver-related diseases, such as hepatic steatosis, autoimmune hepatitis, and alcoholic fatty liver disease. MicroRNAs (miRNAs) are involved in various cellular processes including cell survival, differentiation and autophagy, which are of great significance. 
OBJECTIVE: To investigate the critical role of miR-144-3p on Cbs+/- mouse hepatocyte autophagy induced by high methionine die.
METHODS: (1) Ten male cystathione-β-synthase normal (Cbs+/+) mice and another 10 male mice with single gene knockout (Cbs+/-) of similar body mass, 4 weeks of age, were fed a high-methionine diet and executed after 12 weeks to take liver tissue. (2) Human hepatocytes (HL-7702) were cultured in vitro and divided into control [0 μmol/L homocysteine (Hcy)], Hcy (100 μmol/L Hcy), mimic-NC (transfected with mimic-NC), mimic-NC + Hcy (mimic-NC transfecton+100 μmol/L Hcy), miR-144-3p mimic (transfected with miR-144-3p mimic), and miR-144-3p mimic + Hcy (miR-144-3p mimic transfection+100 μ mol/L Hcy), inhibitor-NC (transfected with inhibitor-NC), inhibitor-NC + Hcy (inhibitor-NC transfection + 100 μmol/L Hcy), miR-144-3p inhibitor (transfected with miR-144-3p inhibitor), and miR-144-3p inhibitor + Hcy (miR-144-3p inhibitor transfection + 100 μmol/L Hcy). Quantitative real-time PCR was used to detect the expression of miR-144-3p in liver tissue and hepatocytes. After transfection of miR-144-3p mimic or inhibitor, quantitative real-time PCR and western blot were used to detect the transfection efficiency of miR-144-3p and its effect on the expression of autophagy-related proteins LC3B and p62. The levels of alanine transferase and aspartate aminotransferase in hepatocyte supernatants were determined by enzyme linked immunosorbent assay. The correlation between the expression of miR-144-3 in hepatocyte and the levels of alanine transferase and aspartate aminotransferase in hepatocyte supernatants were analyzed by Pearson correlation analysis.
RESULTS AND CONCLUSION: Compared with the Cbs+/+ group and control group, the expression of miR-144-3p in the liver tissue of the Cbs+/- group and in hepatocytes of the Hcy group was decreased (P < 0.01). The expression of LC3B-II/I was decreased in hepatocyte after transfection of miR-144-3p mimic, while the protein expression of p62 was increased (P < 0.01). The opposite results were obtained after transfection of miR-144-3p inhibitor (P < 0.01). Compared with the mimic-NC group, the levels of alanine transferase and aspartate aminotransferase were decreased in the miR-144-3p mimic group (P < 0.01), while the opposite results were obtained in the inhibitor-NC group (P < 0.01). The expression of miR-144-3p in hepatocytes was negatively correlated with the levels of alanine transferase (P < 0.01, r=-0.887 6) and aspartate aminotransferase (P < 0.01, r=-0.829 9) in the supernatant of hepatocytes. To conclude, Hcy promotes hepatocyte autophagy by inhibiting the expression of miR-144-3p, which subsequently aggravates liver injury.

Key words: microRNA, miR-144-3p, homocysteine, autophagy, liver injury, high-methionine diet

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