摘要
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饮食对照组(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正常对照组(Control组,0μmol/L Hcy)、Hcy干预组(Hcy组,100μmol/L Hcy)、转染干扰片段对照组(si-NC组,0μmol/L Hcy)、转染LSM4干扰片段组(si-LSM4组,0μmol/L Hcy)、转染DNMT1干扰片段组(si-DNMT1组,0μmol/L Hcy)、Hcy干预下对照干扰组(Hcy+si-NC组,100μmol/L Hcy)、Hcy干预下LSM4干扰组(Hcy+si-LSM4组,100μmol/L Hcy)和Hcy干预下DNMT1干扰组(Hcy+si-DNMT1组,100μmol/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)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;Western blot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Western blot检测Hcy+si-DNMT1组中LSM4蛋白表达。结果与ND组、Control组相比,HMD组、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 hepatocyte apoptosis in mice induced with Hcy.Methods 12 ApoE-/-mice were divided into two groups:normal diet(ND,n=6)and high methionine diet(HMD,n=6)groups.Normal hepatocytes of NCTC1469 were divided into a normal group(control,0μL/L Hcy),Hcy intervention group(Hcy,100μL/L Hcy),NC siRNA-transfected control group(si-NC group,0μmol/L Hcy),LSM4 siRNA-transfected group(si-LSM4 group,0μmol/L Hcy),DNMT1 siRNA-transfected group(si-DNMT1 group,0μmol/L Hcy),NC siRNA-transfected Hcy intervention group(Hcy+si-NC group,100μmol/L Hcy),LSM4 siRNA-transfected Hcy intervention group(Hcy+si-LSM4 group,100μmol/L Hcy),and DNMT1 siRNA-transfected Hcy intervention group(Hcy+si-DNMT1 group,100μmol/L Hcy).Analysis of the expression of LSM4 in various tissues was conducted using the NCBI database.Quantitative real-time PCR(qRT-PCR)and Western blot were used to detect differences in LSM4 protein expression in mouse tissues(HMD and ND)and hepatocytes(control and Hcy).Western blot was used to detect the expression of Bcl2-associated X(Bax)and B-cell lymphoma-2(Bcl-2).The cell apoptosis rate in the Control,Hcy,Hcy+si-NC,and Hcy+si-LSM4 groups were detected by flow cytometry.MethPrimer online software was used to analyze the CpG islands of LSM4 promoter region.The expression of LSM4 in the Hcy+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 ND and Control group(P<0.05).Bax protein expression was significantly higher,but Bcl-2 was significantly lower in Hcy group compared with those of the Control group(P<0.05).The expression of Bax protein was significantly lower,but the level of Bcl-2 was significantly higher in the Hcy+si-LSM4 group compared with those in the Hcy+si-NC group(P<0.05).The cell apoptosis rate in the Hcy group was higher than that in the Control group(P<0.05),while the apoptosis rate in the 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 Hcy+si-DNMT1 group’s expression of LSM4 protein was increased(P<0.05).Conclusions DNMT1 regulates LSM4 hypomethylation to increase its expression,thereby promoting Hcy-induced apoptosis of mouse hepatocytes.
作者
夏童童
马芳
刘虹麟
张正皓
丁寒霜
郝银菊
张慧萍
吴凯
焦运
姜怡邓
李桂忠
XIA Tongtong;MA Fang;LIU Honglin;ZHANG Zhenghao;DING Hanshuang;HAO Yinju;ZHANG Huiping;WU Kai;JIAO Yun;JIANG Yideng;LI Guizhong(NHC Key Laboratory of Metabolic Cardiovascular Diseases Research,Yinchuan 750004,China;School of Basic Medical Sciences,Ningxia Medical University,Yinchuan 750004;School of Laboratory Medicine,Ningxia Medical University,Yinchuan 750004;General Hospital of Ningxia Medical University,Yinchuan 750004;Department of Medical Genetics,Hunan Maternal and Child Health Hospital,Changsha 410008;Department of Infection,General Hospital of Ningxia Medical University,Yinchuan 750004)
出处
《中国比较医学杂志》
CAS
北大核心
2024年第11期34-42,共9页
Chinese Journal of Comparative Medicine
基金
国家自然科学基金(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)。