转录因子EB在衰老心肌细胞自噬中的作用机制研究

Study on the mechanism of transcription factor EB in autophagy of aging cardiomyocytes

目的探讨转录因子EB(TFEB)在衰老心肌细胞自噬中的作用机制。方法动物实验:将20只老年Wistar大鼠采取随机数字表法分为假手术组(Sham组)和缺血再灌注组(I/R组)。细胞实验:(1)体外培养大鼠心肌细胞,采用8 g/L D-半乳糖孵育8 d后分为常氧(Normoxia)组和缺氧/复氧(H/R)组。(2)在衰老心肌细胞中分别转染过表达和干扰TFEB腺病毒分为过表达对照(Ad-GFP)组、过表达对照+缺氧/复氧(Ad-GFP+H/R)组、过表达TFEB(AdTFEB)组、过表达TFEB+缺氧/复氧(Ad-TFEB+H/R)组、干扰对照(sh-NC)组、干扰对照+缺氧/复氧(sh-NC+H/R)组、干扰TFEB(sh-TFEB)组、干扰TFEB+缺氧/复氧(sh-TFEB+H/R)组。(3)分别用DNMT1、DNMT3a、DNMT3b的特异性抑制剂DC-05、TFD、NA处理缺氧/复氧后的衰老心肌细胞分为H/R组、DC-05组、TFD组、NA组。(4)在衰老心肌细胞中转染干扰DNMT3b腺病毒分为干扰对照(sh-NC)组、干扰对照缺氧/复氧(sh-NC+H/R)组、干扰DNMT3b(shDNMT3b)组、干扰DNMT3b缺氧/复氧(sh-DNMT3b+H/R)组。实时荧光定量PCR(qPCR)检测TFEB的mRNA表达水平;Western blot检测衰老心肌细胞中自噬相关蛋白TFEB、LC3B和p62的蛋白表达;巢式甲基化特异性PCR(nMSPCR)检测TFEB启动子区的DNA甲基化水平。结果与Sham组或Normoxia组比较,I/R组和H/R组中TFEB的mRNA和蛋白表达水平降低(P<0.01);过表达TFEB后,与Ad-GFP组比较,Ad-TFEB组LC3B-Ⅱ/Ⅰ的蛋白表达水平降低,p62的蛋白表达水平升高(P<0.01),而干扰TFEB后得到相反的结果(P<0.01)。与Sham组或Normoxia组比较,I/R组和H/R组中TFEB启动子区DNA甲基化水平升高(P<0.05)。与H/R组比较,NA组TFEB mRNA和蛋白表达水平升高(P<0.01);干扰DNMT3b后,与sh-NC组比较,sh-DNMT3b组TFEB mRNA和蛋白表达水平升高(P<0.01)。结论DNMT3b通过调控TFEB启动子区DNA甲基化抑制TFEB的表达,进而促进衰老心肌细胞自噬。

Objective To explore the mechanism of transcription factor EB(TFEB)in autophagy of aging cardiomyocytes.Methods Animal experiment:Twenty aged Wistar rats were randomly divided into the sham operation group(Sham group)and the ischemia-reperfusion injury group(I/R group).Cell experiments:(1)Aging cardiomyocytes were cultured in vitro,incubated with 8 g/L D-galactose for 8 days,and then divided into the normoxia group and the hypoxia/reoxygenation group(H/R group).(2)Aging cardiomyocytes transfected by adenovirus overexpressing and interfering with TFEB were divided into the Ad-GFP group,the Ad-GFP+H/R group,the Ad-TFEB group,the Ad-TFEB+H/R group,the sh-NC group,the sh-NC+H/R group,the sh-TFEB group and the sh-TFEB+H/R group.(3)Aging cardiomyocytes treated with specific inhibitors of DNMT1,DNMT3a and DNMT3b after hypoxia/reoxygenation were divided into the H/R group,the DNMT1 specific inhibitor(DC-05)group,the DNMT3a specific inhibitor(TFD)group and the DNMT3b specific inhibitor(NA)group.(4)Aging cardiomyocytes transfected by adenovirus interfering with DNMT3b were divided into the sh-NC group,the sh-NC+H/R group,the sh-DNMT3b group and the sh-DNMT3b+H/R group.Quantitative real-time PCR(qPCR)was used to detect the mRNA level of TFEB,and Western blot assay was used to detect the autophagy related proteins TFEB,LC3B and p62 in aging cardiomyocytes.The DNA methylation levels of TFEB promoter in aging myocardium and cardiomyocytes were detected by nested methylation specific PCR(nMS-PCR).Results Compared with the Sham group or the normoxia group,the mRNA and protein expression of TFEB were decreased in the I/R group and the H/R group(P<0.01).The protein expression of LC3B-Ⅱ/Ⅰwas decreased in aging cardiomyocytes after overexpression of TFEB in the Ad-TFEB group compared with the Ad-GFP group,while the protein expression of p62 was increased(P<0.01).The opposite results were obtained after interfering with TFEB(P<0.01).Compared with the Sham group or the normoxia group,the DNA methylation level of the TFEB promoter was increased in the I/R group and the H/R group(P<0.05).Compared with the H/R group,it was found that the mRNA and protein expression level of TFEB were increased in the NA group(P<0.01).And the TFEB mRNA and protein expression were increased in aging cardiomyocytes after overexpressed interference with DNMT3b(P<0.01).Conclusion DNMT3b inhibits the TFEB expression by regulating DNA methylation of TFEB promoter,thus to promote autophagy of aging cardiomyocytes.