miRNA-19b-3p Stimulates Cardiomyocyte Apoptosis Induced by Myocardial Ischemia Reperfusion via Downregulating PTEN

Objective To clarify the function of miRNA-19b-3p in accelerating myocardial ischemia-reperfusion injury- (MIRI-) induced cardiomyocyte apoptosis by downregulating gene of phosphate and tension homology deleted on chromsome ten (PTEN), thus influencing the progression of acute myocardial infarction. Materials and Methods miRNA-19b-3p and PTEN levels in HCM cells undergoing hypoxia/reoxygenation (H/R) were determined. Meanwhile, activities of myocardium injury markers [lactate dehydrogenase (LDH), malondialdehyde; malonic dialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX)] in H/R-induced HCM cells were tested. Through dual-luciferase reporter gene assay, the binding between miRNA-19b-3p and PTEN was verified. Regulatory effects of miRNA-19b-3p and PTEN on apoptotic rate and apoptosis-associated gene expressions (proapoptotic protein Bcl-2 associated X protein (Bax), antiapoptotic protein B-cell lymphoma-2 (Bcl-2), and cytochrome C) in H/R-induced human cardiac myocytes (HCM) cells were examined. Results miRNA-19b-3p was upregulated, while PTEN was downregulated in H/R-induced HCM cells. Knockdown of miRNA-19b-3p decreased activities of LDH, MDA, and GSH-PX, but increased SOD level in H/R-induced HCM cells. The binding between miRNA-19b-3p and PTEN was confirmed. More importantly, knockdown of miRNA-19b-3p reduced apoptotic rate, downregulated proapoptosis gene expressions (Bax and cytochrome C), and upregulated antiapoptosis gene expression (Bcl-2), which were reversed by silence of PTEN. Conclusions miRNA-19b-3p is upregulated in HCM cells undergoing hypoxia and reoxygenation, which accelerates MIRI-induced cardiomyocyte apoptosis through downregulating PTEN.


Introduction
Acute myocardial infarction (AMI) is an important cause of death and disability worldwide [1][2][3]. Timely myocardial reperfusion is the most effective intervention for alleviating ischemiainduced myocardium injury. However, reperfusion itself induces myocardial cell death, that is, myocardial ischemiareperfusion injury (MIRI) [4][5][6]. Apoptosis is a process of programmed cell death influencing MIRI and cardiomyocyte loss during cardiac remodeling at post-AMI [7]. A growing number of evidences have suggested that cardiomyocyte apoptosis occurs primarily in the surviving myocardium following ischemia [8]. It is necessary to uncover the pathogenic mechanism of MIRI and develop effective therapeutic targets for clinical treatment of AMI.
miRNAs are a class of noncoding DNAs expressed in eukaryotic cells, ranging in length from 20 to 25 nucleotides [9][10][11]. Mature miRNAs are processed by primary transcripts through various nucleases, which are then assembled into an RNA-induced silencing complex (RISC). Subsequently, RISC binds 3′UTR of target mRNAs through complementary base pairing, thus degrading mRNAs or inhibiting their translation [12,13]. miRNAs are extensively distributed in different types of cells and human diseases, such as ischemic cardiomyopathy [14], cardiac remodeling [15], heart failure [16], and arrhythmia [17]. In recent years, critical functions of miRNAs in MIRI have been identified [18][19][20]. These miRNAs could be utilized as therapeutic targets for clinical treatment of AMI.
miRNA-19b-3p is a member of the miR-17-92 cluster located on the human chromatin 13. Biological functions of miRNA-19b-3p have been discovered in multiple types of tumors [21][22][23][24][25]. In a recent study, exosomal miRNA-19b-3p of tubular epithelial cells promotes M1 macrophage activation in kidney injury [26]. Further, circulating miR-19a-3p and miR-19b-3p characterize the human aging process and their isomiRs associate with healthy status at extreme ages [27]. However, the role of miR-19b-3p in AMI was unknown. In this paper, regulatory effects of miRNA-19b-3p on AMI-induced cardiomyocyte apoptosis were determined.        2.6. Cell Counting Kit-8 (CCK-8). Cells were inoculated in a 96-well plate at 80% confluence. Viability was determined at the appointed time points using CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan). Absorbance at 450 nm was recorded for plotting the viability curve.  The Student t-test was applied for analyzing differences between the two groups:p < 0:05 was considered statistically significant.

miRNA-19b-3p Was Upregulated in H/R-Induced Cells.
H/R was conducted in HCM cells to mimic the in vitro environment of MIRI. Compared with HCM cells under normoxic conditions, H/R induction markedly upregulated miRNA-19b-3p in HCM cells (Figure 1(a)). Subsequently, transfection of miRNA-19b-3p inhibitor markedly downregulated miRNA-19b-3p level in H/R-induced HCM cells, presenting an effective transfection efficacy (Figure 1(b)). Myocardial injury markers were determined here. As the data revealed, knockdown of miRNA-19b-3p decreased activities of LDH, MDA, and GSH-PX, but increased SOD level in H/R-induced HCM cells (Figures 1(c)-1(f)). It is demonstrated that miRNA-19b-3p was involved in MIRI.

Knockdown of miRNA-19b-3p Alleviated Cardiomyocyte
Apoptosis. In H/R-induced HCM cells, transfection of miRNA-19b-3p inhibitor accelerated cell viability (Figure 2(a)). Nevertheless, apoptotic rate decreased after knockdown of miRNA-19b-3p in H/R-induced HCM cells (Figure 2(b)). Expression levels of apoptosis-associated genes, Bax, Bcl-2, and cytochrome C were determined. Both mRNA and protein levels of Bax and cytochrome C were downregulated, and Bcl-2 was upregulated in H/R-induced HCM cells transfected with miRNA-19b-3p inhibitor (Figures 2(c) and 2(d)).  To further uncover the mechanism of miRNA-19b-3p in influencing MIRI, we found potential binding sequences in the promoter regions of miRNA-19b-3p and PTEN as predicted in Targetscan (Figure 3(a)). Dual-luciferase reporter gene assay demonstrated that the overexpression of miRNA-19b-3p quenched luciferase activity in wild-type PTEN vector, while it did not affect mutant-type PTEN vector (Figure 3(b)). In addition, PTEN level was markedly upregulated in H/R-induced HCM cells transfected with miRNA-19b-3p inhibitor (Figure 3(c)). It is concluded that PTEN was the direct target of miRNA-19b-3p and negatively regulated by it.

Discussion
Currently, thrombolysis, bypass surgery, and other interventions have been applied for reperfusion of blood flow and protection of ischemic myocardium [28].

Disease Markers
Nevertheless, the sudden reperfusion of blood flow would result in secondary cardiovascular injury, that is, MIRI. MIRI results in cardiomyocyte apoptosis and necrosis, and even cardiac arrest [29]. Cell apoptosis is a vital event during the prognosis of MI. Inhibition of cardiomyocyte apoptosis and reduction of infarcted myocardium area could effectively alleviate the prognosis of AMI [30,31].
Accumulating evidences have uncovered the role of miRNAs in regulating reperfusion in ischemic myocardium. In this paper, miRNA-19b-3p was upregulated in HCM cells under H/R precondition. Silence of miRNA-19b-3p markedly reduced activities of LDH, MDA, and GSH-PX and elevated SOD level in H/R-induced HCM cells. In addition, knockdown of miRNA-19b-3p resulted in viability elevation and apoptosis suppression in HCM cells. Our findings demonstrated the involvement of miRNA-19b-3p in MIRIinduced pathological changes. Furthermore, apoptosisrelated genes were determined in H/R-induced HCM cells. Previous studies proposed that Bcl-2/Bax ratio is the key indicator reflecting the apoptotic level [32,33]. Bcl-2 protein locates in the outer mitochondrial membrane, exerting an antiapoptosis function. Under normal circumstance, Bax is expressed in the cytoplasm. Once AMI occurs, apoptosisrelated signaling triggers the translocation of cytoplasmic Bax into mitochondria, thus initiating the endogenous apoptosis. Here, silence of miRNA-19b-3p downregulated mRNA and protein levels of Bax and cytochrome C, and upregulated Bcl-2.
PTEN is a lipoprotein phosphatase that negatively regulates the PI3K/Akt pathway through PIP3 dephosphorylation and Akt translocation on the cell membrane [34,35]. A recent study illustrated the crucial role of PTEN in mitochondrial-dependent apoptosis [36].
PTEN is considered to be an important pathway involved in MI. Previous studies have demonstrated that the upregulated miR-21 during MI affects collagen production by interfering with VEGF-mediated PTEN pathway [37]. In previous studies, miRNAs were reported to regulating target genes by binding to the 3 ′ UTR area as a sponge thus to inhibit the translation of mRNA [38,39]. In this paper, PTEN was confirmed to be the direct target of miRNA-19b-3p and negatively regulated by it. Besides, PTEN was lowly expressed in H/R-induced HCM cells. To elucidate the involvement of PTEN in HCM cell behaviors influenced by miRNA-19b-3p, gain-of-function experiments were conducted. Notably, knockdown of PTEN reversed regulatory effects of miRNA-19b-3p on apoptotic rate and apoptosis-associated gene expressions in H/R-induced HCM cells. As a result, PTEN was responsible for miRNA-19b-3p to influence MIRIinduced cardiomyocyte apoptosis.

Data Availability
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.