MicroRNA-377 Alleviates Myocardial Injury Induced by Hypoxia/Reoxygenation via Downregulating LILRB2 Expression.

BACKGROUND: miR-377 is closely related to myocardial regeneration. miR-377-adjusted mesenchymal stem cells abducted ischemic cardiac angiogenesis. Nevertheless, there were rarely reports about the impact of miR-377 on myocardial ischemia injury. The purpose of this work is that whether miR-377 can protect against myocardial injury caused by hypoxia/reoxygenation (H/R).
METHODS: Gene expression omnibus database (http://www.ncbi.nlm.nih.gov/geo/; no. GSE53211) was utilized to study the differential expression of miR-377 in patients with an acute ST-segment elevation myocardial infarction and healthy controls. The luciferase activity was determined utilizing the dual-luciferase reporter system. Quantitative real-time polymerase chain reaction and Western blotting were used to measure the messenger RNA and protein level.
RESULTS: Low expression of miR-377 and high expression of leukocyte immunoglobulin-like receptor B2 (LILRB2) were identified in patients with myocardial infarction from analyzing the Gene Expression Omnibus data set. Besides, miR-377 expression was downregulated in cardiomyocyte exposed to H/R. Additionally, overexpression of miR-377 could visibly improve cardiomyocyte injury by regulating cell activity and apoptosis.
CONCLUSIONS: In short, our findings suggested that miR-377/LILRB2 might regard as a hopeful therapeutic target for myocardial ischemic.

Introduction

Cardiovascular disease (CVD) is a heart and blood vessels disorder with a high morbidity and mortality worldwide, especially in the elderly population.[1,2] The evaluation of myocardial ischemia delegates a basis in the approach of CVD.[3] Myocardial ischemia is characterized by short of blood supply to heart.[4] Reconstruction of blood flow in ischemic myocardial tissue has been widely recognized as a feasible therapy for ischemic heart disease.[5] It has indicated that ischemia-induced hypoxia and reoxygenation (H/R) were related to apoptosis, production of reactive oxygen species, inflammation, and so on.[6,7] However, the mechanism of myocardial ischemia remains ill-defined. Therefore, it is important to figure out potential mechanisms of action to develop novel therapeutic regimen for myocardial ischemia injury.

MicroRNA (miRNA), consisting of 19 to 24 nucleotides, is a class of endogenous noncoding RNA molecules. By binding to 3′ untranslated region (3′-UTR), miRNA regulates gene expression at the transcriptional or posttranscriptional level.[8,9] MicroRNAs have been shown to take part in almost all biological processes, including glucose and lipid metabolism, apoptosis, signal transduction, and the like.[10,11] miR-377, as a multifunctional miRNA which targets many genes, is involved in inflammation, oxidative stress, and angiogenesis and closely related to myocardial regeneration.[12,13] Recent evidence has showed that miR-377 was downmodulated in cerebral ischemic injury, indicating its potential participation in ischemia/reperfusion (I/R).[14] Previous work by researchers has presented that depletion of miR-377 alleviated brain inflammation and heightened angiogenesis in reaction to cerebral ischemia.[15] Based on these studies, we deduced that miR-377 might also play a role in myocardial ischemic injury.

The main issues addressed in this article are that whether miR-377 can protect against myocardial injury caused by H/R via forbidding leukocyte immunoglobulin-like receptor B2 (LILRB2) expression. First of all, myocardial H/R injury was established as an in vitro model for myocardial ischemia in human cardiomyocytes. The first section of this article will examine the differential expression of miR-377 and LILRB2 in patients with myocardial ischemia using Gene Expression Omnibus (GEO) database. Section 2 mainly explored the effects of upregulation of miR-377 on cardiomyocyte proliferation and apoptosis. The third section is concerned with the relationship between miR-377 and LILRB2 as well as the functional significance of miR-377/LILRB2 on myocardial H/R injury.

Methods

Differential Expression Analysis of miR-377 and LILRB2

In this article, GEO (http://www.ncbi.nlm.nih.gov/geo/) database (no. GSE53211) was utilized to study the differential expression of miR-377 in patients with an acute ST-segment elevation myocardial infarction (STEMI) and healthy controls. There were 13 samples in this data set, including 9 STEMI and 4 healthy controls. Besides, GEO database (the accessing number was GSE66360) was used to analyze the differential expression of LILRB2 in patients with acute myocardial infarction (n = 49) and healthy cohorts (n = 50). The data set has a total of 99 samples.

Construction of Myocardial Ischemia Model and Cell Culture

Human neonatal cardiomyocytes were acquired from ScienCell Research Laboratories. The cells were cultured in Dulbecco’s Modified Eagle’s medium containing 10% fetal bovine serum (FBS) and antibiotics (0.1 mg/mL streptomycin and 100 U/mL penicillin). Later, the cells were placed in the medium without FBS and incubated in an anoxic incubator (95% N2, 5% CO2, 37 °C). After 6 hours, it was converted into a normal condition (95% O2, 5% CO2, 37 °C) for 24 hours to reoxygenation.

Transfection

Si-LILRB2, miR-377 mimic, and their negative controls (NCs) were synthesized by GenePharma. Then they were transfected into cardiomyocyte cells by Lipofectamine 3000 according to the instruction.

Cell Counting Kit-8

After 24-hour transfection, the cells were digested and counted to prepare cell suspension. A 96-well plate was added into 100 µL cell suspension (the concentration was 1 × 103 cells/well). Cells were cultured under standard conditions. When testing the proliferation, 10 µL Cell Counting Kit-8 (CCK8) reagent (Beijing Solarbio Science & Technology, Co, Ltd) was put into each well. The cells were cultured for 1.5 hours at 37 °C in a CO2 incubator, and the cell proliferation was determined every 24 hours. The OD450 was detected utilizing a microplate reader.

Cell Apoptosis Assay

Double staining of Annexin V/fluorescein isothiocyanate (FITC) and propidium iodide (PI) was performed to assess the apoptosis according to the manufacturer’s protocol (BD Biosciences). Specifically, the transfected cells were harvested, washed utilizing 400 µL precooled PBS, and fixed with 70% ethanol at 4 °C overnight. Then, the cells were stained with 5 µL Annexin V/FITC and 10 µL PI and cultivated at room temperature in the dark for 20 minutes. Next, these cells were collected and analyzed by the flow cytometer (version 7.6.3).

Dual Luciferase Vector Construction Experiment

The cells were inoculated into 24-well plates and cultured to 80% confluence. LILRB2-WT and LILRB2-Mut 3′-UTR were cloned into the pmiR-RB-REPORTTM dual fluorescein reporter vector. Cells were cotransfected with pmiR-LILRB2-WT/pmiR-LILRB2-Mut and miR-377 mimic and miR-377 mimic NC. Finally, the luciferase activity was determined utilizing the dual-luciferase reporter system (Promega) following the manufacturer’s instructions.

Quantitative Real-Time Polymerase Chain Reaction

The whole RNA was extracted utilizing TRIzol (Invitrogen). Reverse transcription into complementary DNA was performed utilizing PrimeScript RT Reagent Kit. Real-time polymerase chain reaction (PCR) was carried out using a 7900HT real-time PCR system by SYBR Premix Ex Taq II. Glyceraldehyde 3-phosphate dehydrogenase was utilized as an internal reference for detecting messenger RNA (mRNA). The MiScript SYBR-Green PCR kit (Qiagen) was utilized for real-time PCR detection of miRNA. U6 is used as an internal reference. The 2−ΔΔCt method was applied to calculate the expression values of miR-377 and LILRB2. The primer sequences are illustrated in Table 1.

Table 1.

The Primers Used in qRT-PCR.

Name	Sequences
miR-377 forward	5′-AGGTTGCCCTTGGTGAA-3′
miR-377 reverse	5′-GAACATGTCTGCGTATCTC-3′
U6 forward	5′-AAAGCAAATCATCGGACGACC-3′
U6 reverse	5′-GTACAACACATTGTTTCCTCGGA-3′
LILRB2 forward	5′-GTGTGGTCTTCACCCAGTGATC-3′
LILRB2 reverse	5′-AGCCGACATCAGAGACACACTG-3′
GAPDH forward	5′-TGTGGGCATCAATGGATTTGG-3′
GAPDH reverse	5′-ACACCATGTATTCCGGGTCAAT-3′

Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LILRB2, leukocyte immunoglobulin-like receptor B2; qRT-PCR, quantitative real-time polymerase chain reaction.

Western Blotting

After 48-hour transfection, cells were harvested and lysed utilizing radioimmunoprecipitation assay buffer (Beyotime) on the basis of the protocol of manufacturer and centrifuged at 12,000 rpm for 20 minutes at 4 °C. Then, bicinchoninic acid assay kit was used to measure the protein concentration. Next, protein samples were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Subsequently, these membranes were blocked with 5% skimmed milk for 1 hour and probed with primary antibodies at 4 °C overnight. After being washed with tris buffered saline containing Tween, the second antibody was used to incubate the membranes at room temperature for 1 hour. Enhanced chemiluminescence kit was used to visualize the protein bands. Glyceraldehyde 3-phosphate dehydrogenase was applied as the internal control. All antibodies were purchased from Santa Cruz Biotechnology.

Statistical Analysis

The experimental data were analyzed by using SPSS 22.0 and GraphPad Prism 6.0 statistical analysis software. Student t test was applied for comparison between 2 groups. The mean comparison among multiple samples was carried out by 1-way analysis of variance and post hoc Dunnett (multiple groups and 1 group) and Bonferroni (comparison among more than 2 groups) tests.

Results

miR-377 Expression Is Downregulated and LILRB2 Expression Is Upregulated in Myocardial infarction

Based on GSE53211 (P = .0485; Figure 1A) data obtained from the GEO database, the expression level of miR-377 was obviously lower in the STEMI group (n = 9) than that in the healthy control group (n = 4). The expression level of LILRB2 was significantly higher in the myocardial infarction group (n = 49) than that in the control group (n = 50) based on the data obtained from the GEO database (no. GSE66360; P < .0001; Figure 1B).

Figure 1.

The expression value of miR-377 and leukocyte immunoglobulin-like receptor B2 (LILRB2) in patients with myocardial ischemia utilizing Gene Expression Omnibus (GEO) database. A, The expression of miR-377 was downmodulated in ST-segment elevation myocardial infarction (STEMI) patients. B, The expression of LILRB2 was highly expressed in STEMI patients.

Upregulation of miR-377 Increased Myocardial Cell Viability Impaired by H/R

In order to investigate the effect of miR-377 on H/R model, miR-377 mimics and its control were transfected into myocardial cells to achieve high expression of miR-377. As shown in Figure 2A, miR-377 expression was declined in the H/R group than in the sham group. Compared with the control group, miR-377 mimic treatment caused obviously enhanced miR-377 expression (Figure 2B). Besides, compared to the miR-377 mimic NC group, miR-377 mimic was significantly increased. These results indicated that miR-377 was successfully upregulated in the H/R group. To examine the effect of miR-377 upregulation on cardiomyocyte viability, we performed CCK-8 assay. As illustrated in Figure 2C, compared to the sham group, the cell viability was significantly decreased in the H/R group. Besides, compared with the miR-377 mimic NC group, the miR-377 mimic group had an increased proliferation rate at 72 hours (Figure 2C). Above all, these data demonstrated that the upregulation of miR-377 could accelerate myocardial cell viability.

Figure 2.

Downregulated expression of miR-377 in hypoxia/reoxygenation (H/R) model and upregulated miR-377 heightened cardiomyocyte activity. A, By quantitative real-time polymerase chain reaction (qRT-PCR), the expression level of miR-377 in myocardial cells was detected after H/R treatment. B, The expression level of miR-377 in myocardial cells was detected after transfection of miR-377 mimic. C, Cell viability was tested by Cell Counting Kit-8 (CCK8) assay. **P < .01 versus sham group, ## P < .01 versus H/R + negative control (NC) group. H/R, hypoxia/reoxygenation.

Upregulation of miR-377 Inhibits Cardiomyocyte Apoptosis Seduced by H/R

To detect the effect of miR-377 on cardiomyocyte apoptosis, we performed flow cytometry. Compared with the sham group, the apoptosis rate was significantly enhanced in the H/R groups (Figure 3A and B). Compared with the H/R + miR-377 mimic NC group, the apoptosis rate was significantly weakened in the H/R + miR-377 mimic group (Figure 3A and B). Simultaneously, the expression of Bcl2 (an antiapoptotic protein) was reduced and the expression of Bax (a proapoptotic protein) was augmented in the H/R groups compared to the sham group (Figure 3C and D). Besides, compared to the miR-377 mimic NC group, miR-377 mimic enhanced Bcl2 expression and lessened Bax expression in the H/R groups (Figure 3C and D). These outcomes indicated that upregulation of miR-377 restrained cell apoptosis exposed to H/R, suggesting that miR-377 could improve cardiomyocyte cell injury.

Figure 3.

miR-377 upregulation restrained myocardial cell apoptosis. A and B, By flow cytometry, apoptotic ratio of myocardial cell was tested. C and D, By Western blot, the levels of apoptotic proteins were detected. **P < .01 versus sham group, ## P < .01 versus hypoxia/reoxygenation (H/R) + negative control (NC) group.

-377 Could Bind to LILRB2

Leukocyte immunoglobulin-like receptor B2 was forecasted as a target of miR-377 by utilizing TargetScan, miRanda, miRWalk, and miRDB software. The latent binding sites between m Figure 4. The relationship of miR-377 and LILRB2. (a) The 3’-UTR of LILRB2 containing mutant (MUT) or wide type (WT) binding sites between LILRB2 and miR-377. (b) The relative luciferase activity of LILRB2-WT/LILRB2-MUT in miR-377 mimic/mimic NC groups. n=5, **P < 0.01 vs. control group.

Figure 4.

The relationship of miR-377 and LILRB2. A, The 3′ untranslated region (3′-UTR) of leukocyte immunoglobulin-like receptor B2 (LILRB2) containing mutant (MUT) or wide-type (WT) binding sites between LILRB2 and miR-377. B, The relative luciferase activity of LILRB2-WT/LILRB2-MUT in miR-377 mimic/mimic negative control (NC) groups. n = 5, **P < .01 versus control group.

miRiR-377 and LILRB2 are displayed in Figure 4A. Through luciferase reporter assay, the luciferase activity of LILRB2-WT was significantly suppressed in the miR-377 mimic group compared to the miR-377 mimic NC group, while there was no significant difference between the miR-377 mimic and miR-377 mimic NC groups (Figure 4B). These results suggested that LILRB2 is a target of miR-377 and is adjusted by miR-377.

Leukocyte Immunoglobulin-Like Receptor B2 Silence Heightens the Ameliorated Impact of miR-377 on H/R Myocardial Injury

To further verify whether miR-377 manifested its function via targeting LILRB2, we designed cotransfection of LILRB2 knockdown combined with miR-377 mimic. It was found that the overexpression of miR-377 could impair the expression of LILRB2 both on the mRNA and protein level (Figure 5A-C). The CCK8 results indicated that LILRB2 silence accelerated the elevated role of miR-377 mimic on the proliferation of cardiomyocyte in H/R (Figure 5D). Flow cytometry results revealed that LILRB2 silence stressed the prohibitive influence of miR-377 mimic on the apoptosis of cardiomyocyte treated with H/R (Figure 5E and F). Together, these results provide important insights into that LILRB2 mediates the impacts of miR-377 on cell proliferation and apoptosis in myocardial H/R injury.

Figure 5.

Leukocyte immunoglobulin-like receptor B2 (LILRB2) silence stressed the repressive functions of miR-377 on hypoxia/reoxygenation (H/R) myocardial injury. A, Leukocyte immunoglobulin-like receptor B2 rescuing the influence of miR-377 on myocardial H/R injury was tested by quantitative real-time polymerase chain reaction (qRT-PCR). B, Leukocyte immunoglobulin-like receptor B2 rescuing the impact of miR-377 on myocardial H/R injury was tested by Western blot. C, Quantification of (B). D, Leukocyte immunoglobulin-like receptor B2 can rescue the influence of miR-377 on the viability of myocardial cells. E, Leukocyte immunoglobulin-like receptor B2 can rescue the impact of miR-377 on the apoptosis ability of myocardial cells. F, quantification of (E).

Figure 5. LILRB2 silence stressed the repressive functions of miR-377 on H/R myocardial injury. (a) LILRB2 rescuing the influence of miR-377 on myocardial H/R injury was tested by qRT-PCR. (b) LILRB2 can rescue the impact of miR-377 on myocardial H/R injury was tested by western blot. (c) quantification of (b). (d) LILRB2 can rescue the influence of miR-377 on the viability of myocardial cells. (e) LILRB2 can rescue the impact of miR-377 on the apoptosis ability of myocardial cells. (f) quantification of (e).

Discussion

In our study, we revealed that miR-377 receded H/R exposed to cardiomyocyte impairment by strengthening cell viability and inhibited cell apoptosis. Besides, it was demonstrated that upregulation of miR-377 decreased LILRB2 by direct targeting of the 3′-UTR of LILRB2 mRNA. Our results also demonstrated that miR-377/LILRB2 potentially behaved as a significant regulator to modulate myocardial H/R injury through detecting LILRB2 silence accelerated the ameliorative effect of miR-377 on H/R-induced myocardial damage. Briefly, miR-377/LILRB2 pair modulation maybe contributes to unveil novel mechanism of action and is also proposed to function as a significant therapeutic target for myocardial ischemia.

Several reports have announced that miRNAs have taken part in many biological processes, including glucose and lipid metabolism, cell proliferation, cell survival, cell apoptosis, signal transduction, and so on.[16-18] In many models of myocardial ischemic injury, miRNA expression alters across different species from miRNAs, indicating that miRNAs play a key role in myocardial ischemic injury.[19,20] Prior study has noted the overexpression of miR-126 relieved acute myocardial ischemia injury.[21] According to Lu et al, miRNA 144 weakened cardiac I/R injury through targeting FOXO1.[22] As mentioned in the article, miR-377-modulated mesenchymal stem cells abducted ischemic cardiac angiogenesis.[23] It has revealed that transplantation of miR-377 knockout human CD34+ cells in ischemic myocardium accelerated the ability of angiogenic and weakened cardiac fibrosis.[24] Comparison of the findings with those of above studies, our results also confirmed that miR-377 weakened cardiomyocyte H/R injury by enhancing cell viability and repressing cell apoptosis via targeting LILRB2.

By binding to 3′-UTR, miRNA modulates several targeting genes.[25] In this article, among the candidate genes, predicting through multiple software and analyzing the expression trends of LILRB2 in patients, LILRB2 was selected as a potential target for miR-377. Leukocyte immunoglobulin-like receptor B2, as a protein coding gene and located at a gene cluster of chromosomal region 19q13.4, is one member of the leukocyte immunoglobulin-like receptor family.[26] The function of human leukocyte antigen (HLA)-G is directed to immunosuppression and induction of tolerance.[27] Through direct binding to the inhibitory receptor LILRB2, HLA-G mediates short-term inhibition of NK cells to produce T regulatory type 1 cells.[28,29] The expression of HLA-G was controlled by a G/C single-nucleotide polymorphism because the presence of G may increase the affinity of mRNA for miR-148a, miR-148b, and miR-152.[30] As mentioned in the literature, the mRNA levels of LILRB2 was obviously upregulated in acute myocardial infarction patients when compared with the stable angina patients.[31] However, there were few articles concerning the role of LILRB2 on myocardial ischemia. In our study, we indicated that LILRB2 may act a significant role in myocardial ischemia. Besides, LILRB2 was upregulated in H/R model, whereas it was reversed by miR-377 mimic. Furthermore, LILRB2 silence stressed the inhibitory influence of miR-377 mimic on the proliferation and apoptosis of cardiomyocyte treated with H/R.

In summary, one of the more significant findings to emerge from this study is that boosting of miR-377 and deletion of LILRB2 might reduce H/R injury. Besides, the second major finding was that LILRB2 could serve as a target of miR-377. This study has also identified that a low expression of miR-377 and a high expression of LILRB2 were associated with bad result in patients with myocardial infarction via utilizing GEO online analysis tool GEO2 R. Our study sheds new light on a potential regulation mechanism for H/R injury, and the overexpression of miR-377 might take on a cardioprotective role against myocardial ischemia.