1.国家卫生健康委员会代谢性心血管疾病研究重点实验室;2.宁夏医科大学总医院;3.湖南省妇幼保健院医学遗传科;4.宁夏医科大学基础医学院;5.宁夏医科大学总医院感染科
]国家自然科学(82370293,U21A20343,82060110)；宁夏回族自治区重点研发计划重点项目(2023BEG02074,2022BFH02013,2022BEG02054,2021BEG02028,2020BEG03005)；宁夏医科大学校级科研项目重点项目(XZ2022005)
1.NHC Key Laboratory of Metabolic Cardiovascular Diseases Research;2.General Hospital of Ningxia Medical University;3.Department of Medical Genetics, Hunan Maternal and Child Health Hospital;4.School of Basic Medical Sciences, Ningxia Medical University;5.Department of Infection, General Hospital of Ningxia Medical University
目的 探讨DNA甲基转移酶1（DNA methyltransferase 1, DNMT1）调控LSM4蛋白（sm-like protein-4, LSM4）在同型半胱氨酸（homocysteine, Hcy）诱导小鼠肝细胞凋亡中的作用及机制研究。方法 将ApoE-/-小鼠（12只）均分为2组， 给予普通饲料喂养设为对照组（ND组，n＝6），给予高蛋氨酸饲料喂养设为高蛋氨酸组（HMD组，n＝6）；NCTC1469小鼠正常肝细胞分为对照组（Control组，0 μmol/L Hcy）、Hcy干预组（Hcy组，100 μmol/L Hcy）、转染干扰片段对照组（si-NC组，0 μmol/L Hcy）、转染LSM4干扰片段组（si-LSM4组，0 μmol/L Hcy）、转染DNMT1干扰片段组（si-DNMT1组，0 μmol/L Hcy）、Hcy干预下转染干扰片段对照组（Hcy＋si-NC组，100 μmol/L Hcy）、Hcy干预下转染LSM4干扰片段组（Hcy＋si-LSM4组，100 μmol/L Hcy）和Hcy干预下转染DNMT1干扰片段组（Hcy＋si-DNMT1组，100 μmol/L Hcy）；NCBI数据库分析LSM4在多种组织中表达；实时荧光定量PCR（quantitative real-time-PCR, qRT-PCR）和蛋白质印迹法（western blot,WB）检测小鼠组织（HMD组和ND组）和肝细胞（Control组和Hcy组）LSM4蛋白表达差异；WB检测凋亡指标Bcl-2相关X蛋白（Bcl-2-associated X, Bax）和B淋巴细胞瘤-2蛋白（B-cell lymphoma-2, Bcl-2）表达变化；流式细胞术检测Control组、Hcy组、Hcy＋si-NC组和Hcy＋si-LSM4组细胞凋亡率变化； MethPrimer在线软件分析LSM4启动子区CpG岛；qRT-PCR和WB检测Hcy＋si-DNMT1组中LSM4蛋白表达。结果 与ND/Control组相比，HMD/Hcy组LSM4蛋白表达显著增高（P＜0.05）；与Control组比较，Hcy组中Bax蛋白表达显著上调（P＜0.05），而Bcl-2表达明显降低（P＜0.05）；与Hcy＋si-NC组比较，Hcy＋si-LSM4组中Bax蛋白表达量显著减少（P＜0.05），Bcl-2蛋白表达量明显增多（P＜0.05）；与Control组相比，Hcy组细胞凋亡率显著升高（P＜0.05）；相较Hcy＋si-NC组，Hcy＋si-LSM4组细胞凋亡率下降（P＜0.05）；MethPrimer在线软件分析显示LSM4启动子区GC含量丰富且存在1个CpG岛；与Hcy＋si-NC组相比，Hcy＋si-DNMT1组LSM4蛋白表达增高（P＜0.05）。结论 DNMT1通过调控LSM4低甲基化使其表达升高，从而促进Hcy诱导的小鼠肝细胞凋亡。
Objective To study the effect of DNA methyltransferase 1 (DNMT1) on sm-like protein-4 (LSM4) in the hepatocytes apoptosis of mice induced by Hcy. Methods 12 ApoE-/- mice were divided into two groups: normal diet (ND, n = 6), high methionine diet (HMD, n = 6). Normal hepatocytes of NCTC1469 were divided into normal group (control, 0 μL/L Hcy), Hcy intervented group (Hcy, 100 μL/L Hcy), transfected NC siRNA control group (si-NC group, 0 μmol/L Hcy), transfected LSM4 siRNA group (si-LSM4 group, 0 μmol/L Hcy), transfected DNMT1 siRNA group (si-DNMT1 group, 0 μmol/L Hcy), transfected NC siRNA in the Hcy intervened group (Hcy＋si-NC group, 100 μmol/L Hcy), transfected LSM4 siRNA in the Hcy intervened group (Hcy＋si-LSM4 group, 100 μmol/L Hcy) and transfected siRNA DNMT1 in the Hcy intervened group (Hcy＋si-DNMT1 group, 100 μmol/L Hcy); Analysis of the expression of LSM4 in various tissues by NCBI database; quantitative real-time PCR (qRT-PCR) and Western blot (WB) were used to detect the difference of LSM4 protein expression in mice tissues (HMD and ND) and hepatocytes (control and Hcy). Western blot was used to detect the protein expression of Bcl2-associated X (Bax) and B-cell lymphoma-2 (Bcl-2). The cell apoptosis rate of control, Hcy, Hcy＋si-NC and Hcy＋si-LSM4 were detected by flow cytometry. MethPrimer online software was used to analyze the CpG islands of LSM4 promoter region. The expression of LSM4 in si-DNMT1 group was detected by qRT-PCR and Western blot. Results The expression of LSM4 in HMD/Hcy group was higher than that in the control group (P＜0.05). Bax protein expression in Hcy＋si-LSM4 was significantly higher than that in the control group (P＜0.05), but Bcl-2 was significantly lower (P＜0.05). The expression of Bax in Hcy＋si-LSM4 was significantly lower than that in Hcy＋si-NC group (P＜0.05), and the level of Bcl-2 was significantly increased. The cell apoptosis rate in Hcy group was higher than that in the control group (P＜0.05). The cell apoptosis rate in Hcy＋si-LSM4 group was lower than that in the Hcy＋si-NC group (P＜0.05). MethPrimer database analysis showed that the promoter region of LSM4 was GC rich and there was one CpG island. Compared with the Hcy+si-NC group, the expression of LSM4 protein in the Hcy+si-DNMT1 group was increased (P＜0.05). Conclusions DNMT1 regulates LSM4 hypomethylation to increase its expression, thereby promoting Hcy-induced apoptosis of mouse hepatocytes.
