Chinese Journal of Tissue Engineering Research ›› 2024, Vol. 28 ›› Issue (8): 1289-1294.doi: 10.12307/2024.201

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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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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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