Inhibition of lncRNA TUG1 upregulates miR-142-3p to ameliorate myocardial injury during ischemia and reperfusion via targeting HMGB1- and Rac1-induced autophagy

https://doi.org/10.1016/j.yjmcc.2019.05.021Get rights and content

Abstract

Background

Long non-coding RNAs (lncRNAs) play a central role in regulating heart diseases. In the present study, we examined the effects of lncRNA taurine up-regulated gene 1 (TUG1) in ischemia/reperfusion (I/R)- or hydrogen peroxide-challenged cardiomyocytes, with specific focus on autophagy-induced cell apoptosis.

Methods

The expressions of miR-142-3p and TUG1 in H2O2-challenged cardiomyocytes and I/R-injured heart tissue were measured by RT-qPCR. Cell death was measured by trypan blue staining assay. Cell apoptosis was determined by Annexin V/PI staining and TUNEL assay. Autophagy was examined by quantifying cells or tissues containing LC3+ autophagic vacuoles by immunofluorescence, or by measuring the expressions of autophagy-related biomarkers by Western blot. The direct interaction between miR-142-3p and TUG1, high mobility group box 1 protein (HMGB1), or Ras-related C3 botulinum toxin substrate 1 (Rac1) was examined using luciferase reporter assay. The significance of miR-142-3p and TUG1 on cell apoptosis or autophagy was examined using both gain-of-function and loss-of-function approaches. The importance of HMGB1 or Rac1 was assessed using siRNA-mediated gene silencing.

Results

miR-142-3p was down-regulated, while TUG1 up-regulated in H2O2-challenged cardiomyocytes in vitro and I/R-injured heart tissues in vivo. Functionally, inhibition of TUG1 and overexpression of miR-142-3p inhibited cell apoptosis and autophagy in cardiomyocytes. The function of TUG1 were achieved by sponging miR-142-3p and releasing the suppression of the putative targets of miR-142-3p, HMGB1 and Rac1. Both HMGB1 and Rac1 essentially mediated cell apoptosis and autophagy induced by TUG1.

Conclusions

TUG1, by targeting miR-142-3p and up-regulating HMGB1 and Rac1, plays a central role in stimulating autophagic cell apoptosis in ischemia/hypoxia-challenged cardiomyocytes. Down-regulating TUG1 or up-regulating miR-142-3p may ameliorate myocardial injury and protect against acute myocardial infarction.

Introduction

Acute myocardial infarction (AMI), also known as heart attack, is caused by insufficient blood flow to heart tissue and characterized by myocardial ischemia and hypoxia. AMI is a leading cause of death worldwide. In China, the AMI-related mortality continues to increase in both rural and urban areas over the past few decades and is 5.6-fold higher in 2014 than in 1987 [1]. Although early mechanic reperfusion is the standard treatment for AMI in clinic, ischemia/reperfusion (I/R) induces myocardial injury and aggravate AMI [2,3]. I/R-induced cell death plays a central role in the pathogenesis of AMI and is achieved through three major mechanisms: apoptosis, necrosis, and autophagy [4].

Unlike apoptosis and necrosis, autophagy is generally considered a pro-survival mechanism, that is, providing amino acids, fatty acids, and/or energy to cells during nutrient deprivation [5]. Autophagy is a multi-step process that starts with Beclin-1/VPS34-mediated phagophore formation, followed by the conjugation of multiple Atg proteins, the conversion of soluble LC3-I to lipid-bound LC3-II, the insertion of lipidated LC3-II into autophagosome aided by p62/SQSTM1 adaptor, the fusion of autophagosome with lysosome, and the protein degradation by lysosomal enzymes [5]. Autophagy is induced in AMI, yet its functional consequence is still under debate, with some studies supporting the protective role [6,7], while others pointing to autophagy-induced cell death [8]. In comparison, less is known on the molecular mechanisms regulating autophagy in I/R- or hypoxia-challenged cardiomyocytes.

Long non-coding RNAs (lncRNAs) are a newly discovered group of RNA molecules containing >200 nucleotides and presenting important regulatory roles in a variety of physiological and pathological processes [9]. LncRNAs control gene expression on multiple levels, from epigenetic, transcriptional, post-transcriptional, to miRNA-mediated mRNA transcription [9]. Recent studies showed that the lncRNA taurine up-regulated gene 1 (TUG1) was up-regulated in response to hypoxia and ischemia [10,11]. Specifically, TUG1 promoted the apoptosis and reduced the viability, migration, and invasion in hypoxia-challenged cardiomyocytes [10]. It is not known, however, whether TUG1 may also regulate cell death by acting on autophagy in cardiomyocytes and if so, what mechanisms are involved.

MicroRNA 142-3p (miR-142-3p) is a miRNA molecule reported to be targeted by TUG1 [12] and closely associated with cardiovascular diseases [[13], [14], [15], [16]]. A recent study showed that miR-142-3p protects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis and fibrosis by targeting high mobility group box 1 protein (HMGB1) [16]. While characterizing the interaction between TUG1 and miR-142-3p and screening for potential miR-142-3p targets using bioinformatic tools, we found that TUG1 bound to miR-142-3p at a potential site where the latter interacted with HMGB1 and Ras-related C3 botulinum toxin substrate 1 (Rac1). This finding, combined with the important roles of HMGB1 and Rac1 in regulating autophagy in heart [17,18], prompted us to hypothesize that TUG1, by sponging miR-142-3p, up-regulates HMGB1 and Rac1, stimulates autophagic cell apoptosis in cardiomyocytes in response to I/R or hypoxia. To test this hypothesis, we established an in vivo I/R model and an in vitro model where cultured primary cardiomyocytes were treated with hydrogen peroxide (H2O2) to mimic hypoxia-induced injury, and assessed the functional roles of TUG1 and miR-142-3p, with specific focus on autophagy, using both gain-of-function and loss-of-function strategies. We also examined the mechanistic involvement of HMGB1 and Rac1 in TUG1/miR-142-3p-regulated autophagy of cardiomyocytes. Findings from this study supported our hypothesis and revealed that TUG1 was up-regulated in I/R- or hypoxia-challenged cardiomyocytes and stimulated autophagic cell apoptosis. Therefore, targeting TUG1 in cardiomyocytes may benefit AMI treatment in clinic.

Section snippets

Cells and reagents

All animal-related protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Guangxi Medical University (Guangxi, China). The primary neonatal cardiomyocytes were isolated from C57BL/6 mice of 1–3 days (Shanghai SLAC Experiment Animal Co., Shanghai, China) using Pierce Primary Cardiomyocyte Isolation Kit (ThermoFisher Scientific, Waltman, MA, USA) according the manufacturer's instructions and cultured in DMEM medium containing

MiR-142-3p was down-regulated in H2O2-challenged cardiomyocytes in vitro and I/R-injured heart tissue in vivo, essentially inhibited both apoptosis and autophagy, and thus protected against cell death

Ischemia and hypoxia are characteristic changes in cardiomyocytes during AMI. To assess the biological significance of miR-142-3p in hypoxia- or ischemia-challenged cardiomyocytes, we treated primary cardiomyocytes with H2O2 in vitro and established an I/R model in vivo. First, by monitoring the expressions of miR-142-3p by RT-qPCR, we found that miR-142-3p level decreased in a time-dependent pattern in primary cardiomyocytes challenged with H2O2 (Fig. 1A) and in hearts from mouse exposed to

Discussion

In present study, we presented our novel findings that lncRNA TUG1 was up-regulated in I/R-stressed heart tissues in vivo and H2O2-challenged cardiomyocytes in vitro; by sponging miR-142-3p, TUG1 elevated the expressions of HMGB1 and Rac1, both molecules essentially contributing to autophagic cell apoptosis. These findings suggest that autophagy may contribute to the apoptosis of ischemia/hypoxia-challenged cardiomyocytes, and targeting TUG1 or up-regulating miR-142-3p may ameliorate I/R- or

Conflict of interests

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by National Natural Science Foundation of China (No. 81600283) and Guangxi Natural Science Foundation (No. 2017GXNSFAA198069).

References (49)

  • R.S. Whelan et al.

    Cell death in the pathogenesis of heart disease: mechanisms and significance

    Annu. Rev. Physiol.

    (2010)
  • D. Glick et al.

    Autophagy: cellular and molecular mechanisms

    J. Pathol.

    (2010)
  • H. Kanamori et al.

    Autophagy limits acute myocardial infarction induced by permanent coronary artery occlusion

    Am. J. Physiol. Heart Circ. Physiol.

    (2011)
  • J. McCormick et al.

    STAT1 deficiency in the heart protects against myocardial infarction by enhancing autophagy

    J. Cell. Mol. Med.

    (2012)
  • K. Wang et al.

    APF lncRNA regulates autophagy and myocardial infarction by targeting miR-188-3p

    Nat. Commun.

    (2015)
  • J.T. Kung et al.

    Long noncoding RNAs: past, present, and future

    Genetics

    (2013)
  • Z. Wu et al.

    LncRNA TUG1 serves an important role in hypoxia-induced myocardial cell injury by regulating the miR1455pBinp3 axis

    Mol. Med. Rep.

    (2018)
  • Q. Liu et al.

    Downregulation of long noncoding RNA TUG1 inhibits proliferation and induces apoptosis through the TUG1/miR-142/ZEB2 axis in bladder cancer cells

    Onco Targets Ther.

    (2017)
  • J. Liu et al.

    MiR-142-3p attenuates the migration of CD4(+) T cells through regulating actin cytoskeleton via RAC1 and ROCK2 in arteriosclerosis obliterans

    PLoS One

    (2014)
  • N. Nair et al.

    Circulating miRNA as novel markers for diastolic dysfunction

    Mol. Cell. Biochem.

    (2013)
  • K.L. Ellis et al.

    Circulating microRNAs as candidate markers to distinguish heart failure in breathless patients

    Eur. J. Heart Fail.

    (2013)
  • Y. Wang et al.

    MicroRNA-142-3p inhibits hypoxia/reoxygenationinduced apoptosis and fibrosis of cardiomyocytes by targeting high mobility group box 1

    Int. J. Mol. Med.

    (2016)
  • E. Foglio et al.

    HMGB1 inhibits apoptosis following MI and induces autophagy via mTORC1 inhibition

    J. Cell. Physiol.

    (2017)
  • Y.M. Wei et al.

    Enhancement of autophagy by simvastatin through inhibition of Rac1-mTOR signaling pathway in coronary arterial myocytes

    Cell. Physiol. Biochem.

    (2013)
  • Cited by (114)

    View all citing articles on Scopus
    1

    These are co-first authors.

    View full text