转录因子EB在衰老心肌细胞自噬中的作用机制研究

doi:10.11958/20220920

天津医药 ›› 2023, Vol. 51 ›› Issue (3): 246-251.doi: 10.11958/20220920

转录因子EB在衰老心肌细胞自噬中的作用机制研究

盛思琪¹^,²^,³(), 谢琳¹^,²^,³, 姜怡邓¹^,²^,³, 熊建团¹^,²^,³, 杨安宁¹^,²^,³, 吴凯¹^,²^,³, 杨勇²^,³^,⁴, 杨晓明¹^,²^,^△()

1 宁夏医科大学基础医学院（邮编750004）
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
4 宁夏回族自治区人民医院

收稿日期:2022-06-21 修回日期:2022-08-16 出版日期:2023-03-15 发布日期:2023-03-02
通讯作者: 杨晓明 E-mail:530479964@qq.com;xmyang327@126.com
作者简介:盛思琪（1994），女，硕士在读，主要从事心血管病理生理学方面研究。E-mail：530479964@qq.com
基金资助:
国家自然科学基金重点项目(82160049);国家自然科学基金重点项目(81860044);宁夏回族自治区重点研发计划重点项目(2020BFH02003);宁夏回族自治区重点研发计划重点项目(2021BEG02033);宁夏回族自治区重点研发计划重点项目(2021BEG02028)

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

SHENG Siqi¹^,²^,³(), XIE Lin¹^,²^,³, JIANG Yideng¹^,²^,³, XIONG Jiantuan¹^,²^,³, YANG Anning¹^,²^,³, WU Kai¹^,²^,³, YANG Yong²^,³^,⁴, YANG Xiaoming¹^,²^,^△()

1 School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750004, China
2 NHC Key Laboratory of Metabolic Cardiovascular Diseases Research, Ningxia Medical University
3 Ningxia Key Laboratory of Vascular Injury and Repair Research, Ningxia Medical University
4 Ningxia Hui Autonomous Region People's Hospital

Received:2022-06-21 Revised:2022-08-16 Published:2023-03-15 Online:2023-03-02
Contact: YANG Xiaoming E-mail:530479964@qq.com;xmyang327@126.com

盛思琪, 谢琳, 姜怡邓, 熊建团, 杨安宁, 吴凯, 杨勇, 杨晓明. 转录因子EB在衰老心肌细胞自噬中的作用机制研究[J]. 天津医药, 2023, 51(3): 246-251.

SHENG Siqi, XIE Lin, JIANG Yideng, XIONG Jiantuan, YANG Anning, WU Kai, YANG Yong, YANG Xiaoming. Study on the mechanism of transcription factor EB in autophagy of aging cardiomyocytes[J]. Tianjin Medical Journal, 2023, 51(3): 246-251.

摘要/Abstract

摘要： 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/Ⅰ was 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.

关键词: 肌细胞，心脏, 细胞衰老, 转录因子EB, 细胞自噬, DNA甲基化, DNMT3b

Key words: myocytes, cardiac, cell aging, transcription factor EB, autophagy, DNA methylation, DNMT3b

中图分类号:

R542.2

图/表 10

表1 nMS-PCR引物序列

Tab.1 nMS-PCR primer sequence

基因名称	引物序列（5'→3'）
外引物	上游：TATATTTGGATGGTTTTTTGTGG 下游：TAACCCTAACTAACCCAACATTCAT
甲基化引物	上游：GATAAAAGGGTAAGATTTAGGTCGT 下游：TCCCTCCATATTTACATACTTCGTA
非甲基化引物	上游：ATAAAAGGGTAAGATTTAGGTTGT
非甲基化引物	下游：TCCCTCCATATTTACATACTTCATA

图1 TFEB在衰老心肌组织和心肌细胞中的表达情况 A：TFEB在大鼠衰老心肌组织中的蛋白表达情况；B：TFEB在衰老心肌细胞中的蛋白表达情况。

Fig.1 Expression of TFEB in aging myocardial tissue and cardiomyocytes

图2 在衰老心肌细胞中过表达和干扰TFEB后检测其表达 A：转染Ad-TFEB后衰老心肌细胞中TFEB的蛋白表达情况；B：转染sh-TFEB后衰老心肌细胞中TFEB的蛋白表达情况。

Fig.2 Detection of TFEB expression after overexpression and interference of TFEB in aging cardiomyocytes

图3 过表达和沉默TFEB对衰老心肌细胞自噬的影响 A：转染Ad-TFEB；B：转染sh-TFEB；1：Ad-GFP组；2：Ad-GFP+H/R组；3：Ad-TFEB组；4：Ad-TFEB+H/R组；5：sh-NC组；6：sh-NC+H/R组；7：sh-TFEB组；8：sh-TFEB+H/R组。

Fig.3 The effect of TFEB on autophagy in aging cardiomyocytes

表2 各组LC3B-Ⅱ/Ⅰ和p62蛋白表达水平比较 (n=3,$\bar{x}±s$)

Tab.2 Comparison of the protein levels of LC3B-Ⅱ/Ⅰ and p62 between the groups

组别	LC3B-Ⅱ/Ⅰ	p62
Ad-GFP组	2.149±0.145	0.421±0.062
Ad-GFP+H/R组	4.135±0.246	0.114±0.021
Ad-TFEB组	1.144±0.112^a	0.963±0.077^a
Ad-TFEB+H/R组	1.880±0.104^b	0.261±0.040^b
F	186.600^＊＊	138.500^＊＊
sh-NC组	0.975±0.233	0.741±0.034
sh-NC+H/R组	1.165±0.005	0.348±0.064
sh-TFEB组	2.222±0.140^c	0.307±0.074^c
sh-TFEB+H/R组	4.248±0.259^d	0.169±0.027^d
F	191.900^＊＊	63.040^＊＊

图4 衰老心肌组织和心肌细胞中TFEB启动子区的DNA甲基化水平 A：TFEB的CPG岛；B：衰老心肌组织中TFEB启动子区的DNA甲基化水平；C：衰老心肌细胞中TFEB启动子区的DNA甲基化水平。

Fig.4 DNA methylation levels of TFEB promoter region in aging myocardial tissue and cardiomyocytes

图5 DNA甲基化转移酶抑制剂对衰老心肌细胞中TFEB表达的影响

Fig.5 Effects of DNA methyltransferase inhibitors on the expression of TFEB in aging cardiomyocytes

表3 DNA甲基化转移酶抑制剂对衰老心肌细胞中TFEB表达的影响 (n=3,$\bar{x}±s$)

Tab.3 Effects of DNA methyltransferase inhibitors on the expression of TFEB in aging cardiomyocytes

组别	TFEB
组别	mRNA	蛋白
H/R组	1.111±0.145	0.443±0.041
DC-05组	1.031±0.125	0.466±0.018
TFD组	1.150±0.216	0.478±0.045
NA组	2.386±0.132^a	0.929±0.139^a
F	49.590^＊＊	28.020^＊＊

Fig.6 DNMT3b对衰老心肌细胞中TFEB表达的影响 1：sh-NC组；2：sh-NC+H/R组；3：sh-DNMT3b组；4：sh-DNMT3b+H/R组。

Fig.6 The effect of DNMT3b on the expression of TFEB in aging cardiomyocytes

表4 Effects of DNMT3b on TFEB expression in aging cardiomyocytes

Tab.4 DNMT3b对衰老心肌细胞中TFEB表达的影响 (n=3,$\bar{x}±s$)

组别	TFEB
组别	mRNA	蛋白
sh-NC组	0.391±0.021	0.244±0.014
sh-NC+H/R组	0.306±0.055	0.081±0.005
sh-DNMT3b组	1.182±0.061^a	0.548±0.044^a
sh-DNMT3b+H/R组	0.595±0.031^b	0.218±0.065^b
F	229.500^＊＊	72.900^＊＊

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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

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