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MicroRNA-384-5p/Beclin-1 As Potential Indicators For Epigallocatechin Gallate Against Cardiomyocytes Ischemia Reperfusion Injury By Inhibiting Autophagy Via PI3K/Akt Pathway

Authors Zhang C, Liang R, Gan X, Yang X, Chen L, Jian J

Received 11 June 2019

Accepted for publication 2 October 2019

Published 17 October 2019 Volume 2019:13 Pages 3607—3623


Checked for plagiarism Yes

Review by Single-blind

Peer reviewer comments 2

Editor who approved publication: Professor Manfred Ogris

Chan Zhang,1 Ronggan Liang,2 Xiaowen Gan,2 Xiufang Yang,2 LingLin Chen,2 Jie Jian2

1Department of Pharmacology, Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China; 2Department of Pharmacology, Guilin Medical University, Guilin, Guangxi, People’s Republic of China

Correspondence: Jie Jian
Department of Pharmacology, Guilin Medical University, 109, North 2nd Huancheng Road, Guilin, Guangxi 541004, People’s Republic of China
Tel +86 183 7833 0768

Background/Aims: Epigallocatechin gallate (EGCG) has established protective actions against myocardial ischemia/reperfusion (I/R) injury by regulating autophagy. However, little is known about the mechanisms of EGCG in posttranscriptional regulation in the process of cardioprotection. Here we studied whether microRNAs play a role in EGCG-induced cardioprotection.
Methods: The myocardial I/R injury in vitro and in vivo model were made, with or without EGCG pretreatment. The upregulation and silencing of microRNA-384-5p (miR-384) and Beclin-1 in H9c2 cell lines were established. Rats were transfected with miR-384 specific shRNA. Dual-luciferase reporter gene assay was conducted to verify the relationship between miR-384 and Beclin-1. TTC staining was performed to analyze the area of myocardial infarct size. Cell viability was monitored by cell counting kit-8 (CCK-8). The release of cardiac troponin-I (cTnI) was examined by ELISA. The levels of autophagy-related genes or proteins expression were evaluated by qRT-PCR or Western blotting. Autophagosomes of myocardial cells were detected by transmission electron microscopy and laser scanning confocal microscope.
Results: I/R increased both autophagosomes and autolysosomes, thereby increasing autophagic flux both in vitro and in vivo. Pretreatment with EGCG attenuated I/R-induced autophagic flux expression, accompanied by an increase in cell viability and a decrease in the size of myocardial infarction. MiR-384 expression was down-regulated in H9c2 cell lines when subjected to I/R, while this suppression could be reversed by EGCG pretreatment. The dual-luciferase assay verified that Beclin-1 was a target of miR-384. Both overexpression of miR-384 and knocking down of Beclin-1 significantly inhibited I/R-induced autophagy, accompanied by the activation of PI3K/Akt pathway, thus enhanced the protective effect of EGCG. However, these functions were abrogated by the PI3K inhibitor, LY294002.
Conclusion: We confirmed that EGCG has a protective role in microRNA-384-mediated autophagy by targeting Beclin-1 via activating the PI3K/Akt signaling pathway. Our results unveiled a novel role of EGCG in myocardial protection, involving posttranscriptional regulation with miRNA-384.

Keywords: epigallocatechin gallate, ischemia/reperfusion, autophagy, microRNA-384-5p, Beclin-1, PI3K/Akt pathway

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