Improvement of Myocardial Cell Injury by miR-199a-3p/mTOR Axis through Regulating Cell Apoptosis and Autophagy

Background Myocardial ischemia-reperfusion injury (MIRI) is characterized by its high incidence rate and mortality. miR-199a-3p is thought to be strongly linked with the development of some myocardial diseases, but the influence of miR-199a-3p in MIRI remains unclear. Methods AC16 cells were used. The concentrations of mammalian target of rapamycin (mTOR), light chain 3 II/light chain 3 I, and Beclin-1 were detected with western blotting and qRT-PCR. The binding site between mTOR and miR-199a-3p was evaluated via luciferase report assay. Cell apoptosis was evaluated through flow cytometry. Results Knockdown of miR-199a-3p accelerated the myocardial cell injury after L-oxygen treatment. Increased expression of mTOR and suppressed autophagy were observed after knockdown of miR-199a-3p. Knockdown of miR-199a-3p or overexpression of mTOR greatly aggravated cell injury through inhibiting autophagy. Conclusions. This study might be helpful for the therapeutic method of MIRI through by regulating miR-199a-3p/mTOR.


Introduction
Myocardial ischemia-reperfusion injury (MIRI) is characterized by more serious structural injury and metabolic dysfunction than before reperfusion [1], and even irreversible cardiac muscle injury appear in MIRI [2]. MIRI could be found in the pathological process of various heart diseases and operations, including after revascularization of myocardial infarction (MI), after cardiac surgery, or restoring blood supply after heart transplantation [3]. Exploring the potential pathological mechanism is helpful for the prevention and treatment of MIRI.
Mammalian target of rapamycin (mTOR) is an atypical and conserved serine/threonine protein kinase, which mainly acts a role through mTOR complex 1 (mTORC1) [11]. The inhibitor of mTORC1 can protect cardiomyocytes by reducing cell energy consumption and activating autophagy, which is the self-protective mechanism of myocardial ischemia. Autophagy is the initial process of cell decomposition and degradation [12]. It acts a key role in maintaining the most basic energy and nutritional needs of cells. In the state of myocardial ischemia and energy deficiency, mTORC1 could be inhibited [13]. Then, the level of cardiomyocyte autophagy is upregulated, and cardiomyocyte death is reduced [14]. Some studies have proved that the AMPK pathway can directly or indirectly regulate mTORC1 in ischemic state and finally cause the upregulation of autophagy level and protect myocardium. However, if miR-199a-3p could regulate MIRI through targeting mTOR remains unclear.
In this study, the MIRI cell model was established with low oxygen (L-oxygen) condition. mTOR overexpression vector (mTOR OE ) and knockdown and overexpression vectors of miR-199a-3p were constructed. The influence of them on apoptosis and autophagy of cells induced by L-oxygen treatment were measured. This research might unfold the modulating effect of miR-199a-3p in preventing and treating MIRI.

Methods and Materials
2.1. Cell Culture. Cardiomyocytes (AC16, Tongpai biotechnology, Shanghai, China) was chosen in this research for in vitro study. DMEM containing 1% penicillin and 5% FBS (Gibco, US) was applied for cell culture. The incubator with 37°C and with CO 2 condition were used for cell culture. After reaching 70% confluence, cells were used for different experiments.

Statistical Analysis.
The results were shown with mean ± SD. SPSS software was used for data analysis. One-way ANOVA was applied for analyzing differences between different groups. P < 0:05 suggests statistical difference.

Knockdown of miR-199a-3p Accelerated the Myocardial
Cell Injury after L-Oxygen Treatment. The myocardial cell injury model was established through L-oxygen treatment, and the influence of knocking down miR-199a-3p on cell viability and apoptosis of cardiomyocyte was evaluated. Loxygen treatment significantly inhibited cell proliferation and promoted apoptosis (Figures 1(a)-1(c)). In addition, supplementary treatment with knocking down miR-199a-3p remarkably aggravated the effects of L-oxygen. Significant decrease of cell viability and increased of cell apoptosis were found in the group L-oxygen+ miR-199a-3p inhibitor (Figures 1(a)-1(c)).

miR-199a-3p
Inhibitor Increased the Level of mTOR and Suppressed Autophagy. The expression of phosphorylated mTOR (p-mTOR) was increased, but light chain 3 II/light chain 3 I (LC3 II/I) and Beclin-1 were suppressed in the group L-oxygen compared to group N-oxygen (Figures 2(a)-2(c)). In addition, knockdown of miR-199a-3p remarkably promoted p-mTOR expression but inhibited Beclin-1 and LC3 levels on the condition of both L-oxygen and N-oxygen (Figures 2(a)-2(c)).

Overexpression of mTOR or miR-199a-3p Inhibitor
Remarkably Aggravated Cell Injury. The binding site was identified successfully with luciferase report assay (Figures 3(a) and 3(b)). Overexpression vector of mTOR was successfully constructed. Overexpression of mTOR  (Figures 4(a)-4(c)) compared with group L-oxygen+vector. Meanwhile, supplementary treatment with knocking down miR-199a-3p remarkably strengthened the function of mTOR OE . Remarkable decrease of cell viability and increased of apoptosis were observed in the group L-oxy-gen+ mTOR OE +miR-199a-3p inhibitor in comparison with group L-oxygen+mTOR OE (Figures 4(a)-4(c)).

Overexpression of mTOR Significantly Inhibited
Autophagy. The influence of mTOR OE and knockdown of miR-199a-3p on autophagy-linked proteins were also investigated. The protein and mRNA levels of p-mTOR were increased, but Beclin-1 and LC3 II/I were suppressed after overexpression of mTOR compared with group L-oxygen (Figures 5(a) and 5(c)). In addition, of miR-199a-3p inhibitor significantly promoted p-mTOR level but suppressed

Discussion
MIRI is characterized by high incidence rate and mortality in the cardiovascular diseases [3]. Although, new treatment methods (thrombolysis, percutaneous angioplasty, percutaneous coronary intervention, and cardiac bypass) have been greatly improved. The reperfusion injury still cannot be completely solved [15]. MIRI process might be involved in inflammatory factor infiltration, calcium overload, intracellular pH change, and aerobic free radical injury [16,17]. Unfolding the potential pathological mechanism of MIRI might be helpful to develop new treatment for MIRI.
In the process of autophagy, the transformation of LC3-II is an important step of autophagosomes [18]. In this present study, overexpression of mTOR or knockdown of miR-199a-3p could significantly inhibit autophagy process by suppressing Beclin-1 and LC3II/I ratio (Figures 5(a)-5(c)). These data indicated that regulation of mTOR or miR-199a-3p might be the potential function targets treating MIRI. miRNAs can regulate most biological processes, including autophagy and apoptosis. miRNAs could regulate the courses of some cardiovascular diseases [9,19]. Previous research indicated that miR-199a-3p modulated myocardial hypertrophy, but the regulatory function of miR-199a-3p in MIRI has not been fully unfold. In this study, the inhibition of autophagy by knocking down miR-199a-3p was demonstrated (Figures 5(a)-5(c)).
In the normal growth state of cells with sufficient growth factors and ATP [20], mTOR could downregulate autophagy signal transduction pathway and maintain a low basic level of autophagy [21]. However, in the state of starvation, autophagy is activated by PI3K-AMPK/mTOR axis. The influence of the PI3K-AMPK/mTOR signaling pathway on MIRI is important and complicated and needs to be further explored.

Conclusions
In this study, we demonstrated that overexpression of mTOR or miR-199a-3p inhibitor greatly inhibited autophagy but accelerated apoptosis. The modulating effects of miR-199a-3p/mTOR axis in MIRI was identified.

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