Involvement of miR-144-3p in Cbs+/-mouse hepatocyte autophagy induced by high-methionine diet
- VernacularTitle:miR-144-3p参与高蛋氨酸饮食诱导Cbs+/-小鼠的肝细胞自噬
- Author:
Siqi SHENG
1
,
2
,
3
;
Lin XIE
;
Xiangyu ZHAO
;
Yideng JIANG
;
Kai WU
;
Jiantuan XIONG
;
Anning YANG
;
Yinju HAO
;
Yun JIAO
Author Information
- Keywords: microRNA; miR-144-3p; homocysteine; autophagy; liver injury; high-methionine diet
- From: Chinese Journal of Tissue Engineering Research 2024;28(8):1289-1294
- CountryChina
- Language:Chinese
- 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.
