miR-144-3p参与高蛋氨酸饮食诱导Cbs^(+/-)小鼠的肝细胞自噬  

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

作　　者：盛思琪 谢琳[1,2,3] 赵翔宇[1,2,3] 姜怡邓 吴凯[1,2,3] 熊建团[1,2,3] 杨安宁 郝银菊[1,2,3,4] 焦运[2,4] Sheng Siqi;Xie Lin;Zhao Xiangyu;Jiang Yideng;Wu Kai;Xiong Jiantuan;Yang Anning;Hao Yinju;Jiao Yun(School of Basic Medical Sciences,Yinchuan 750004;NHC Key Laboratory of Metabolic Cardiovascular Diseases Research,Yinchuan 750004;Ningxia Key Laboratory of Vascular Injury and Repair Research,Ningxia Medical University,Yinchuan 750004,Ningxia Hui Autonomous Region,China;General Hospital of Ningxia Medical University,Yinchuan 750004,Ningxia Hui Autonomous Region,China)

机构地区：[1]宁夏医科大学基础医学院 [2]宁夏医科大学国家卫生健康委代谢性心血管疾病研究重点实验室 [3]宁夏医科大学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

出　　处：《中国组织工程研究》2024年第8期1289-1294,共6页Chinese Journal of Tissue Engineering Research

基　　金：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。

摘　　要：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(0μmol/L同型半胱氨酸)、同型半胱氨酸组(100μmol/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+100μmol/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+100μmol/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+100μmol/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+100μmol/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�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[0μmol/L homocysteine(Hcy)],Hcy(100μmol/L Hcy),mimic-NC(transfected with mimic-NC),mimic-NC+Hcy(mimic-NC transfecton+100μmol/L Hcy),miR-144-3p mimic(transfected with miR-144-3p mimic),and miR-144-3p mimic+Hcy(miR-144-3p mimic transfection+100μmol/L Hcy),inhibitor-NC(transfected with inhibitor-NC),inhibitor-NC+Hcy(inhibitor-NC transfection+100μmol/L Hcy),miR-144-3p inhibitor(transfected with miR-144-3p inhibitor),and miR-144-3p inhibitor+Hcy(miR-144-3p inhibitor transfection+100μmol/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.RESUL

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 

分 类 号：R459.9[医药卫生—治疗学] R33[医药卫生—临床医学] R97