MiR-138 ameliorates myocardial ischemia/reperfusion injury by targeting intercellular cell adhesion molecule 1

Purpose: To explore the effect of miR-138 on regulating intercellular cell adhesion molecule 1 (ICAM-1) expression in endothelial cells to alleviate cardiac ischemia/reperfusion (I/R) injury and its related mechanisms. Methods: The left anterior descending artery of the heart was occluded for 30 min and then perfused for 2 h to induce a rat model of cardiac I/R injury. H9C2 cells were cultured in an anoxic medium without serum to establish the model of hypoxia/reoxygenation (H/R). Triphenyl tetrazolium chloride (TTC) staining was applied to measure myocardial infarction sizes in rat hearts. The mRNA expression levels of miR-138 and ICAM-1 were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Dual luciferase reporter assay was used to identify the target of miR-138. The agomiR-138 and miR-138 mimics were transfected into H9C2 cells; exogenous ICAM-1 was also administered, and ROS accumulation, cell viability, and apoptosis were measured. Furthermore, the underlying mechanism was investigated. Results: MiR-138 was downregulated both in vitro and in vivo. AgomiR-138 reduced myocardial infarction area, decreased ROS production and suppressed cell apoptosis in a rat model of cardiac I/R injury. On the other hand, miR-138 mimics increased cell viability, enhanced ROS production and induced cell apoptosis in H/R-induced H9C2 cells. Further analysis verified ICAM-1 as a target of miR138. Besides, exogenous ICAM-1 inhibited the protective effect of miR-138 on H/R-induced apoptosis in vitro. Conclusion: MiR-138 may protect against injury of myocardial I/R by targeting ICAM-1. The results also provide insight into miR-138/ICAM-1 axis as new therapeutic targets for myocardial I/R injury.


INTRODUCTION
Ischemic heart diseases (IHD) can promote the development of blood vessel diseases and ultimately result in death [1]. To date, reperfusion of arterial blockages is main treatment method for IHD. However, cardiac ischemia/reperfusion (I/R) injuries were induced by reperfusion(myocardial infarction, myocardial cell death, and microvascular obstruction etc.) [2].
Thus, the underlying mechanisms of cardiac I/Rinduced injury were needed to be further explored.
MicroRNAs, composed of 18 -20 nucleotides, are endogenous, single-stranded RNAs that regulate the expression of gene through targeting the 3'-terminal untranslated region (UTR) of mRNA at the posttranscriptional level [3]. MicroRNAs have been shown to regulate more than 60 % of human genes [4]. Numerous studies have shown that microRNAs regulate many critical pathological and physiological processes [5,6], including cell migration, proliferation, and apoptosis. It has been proved that miR-21, miR-141, and miR-320 act as key regulators of cardiac development and myocardial injury [7]. In particular, miR-138 exhibited protective effects on cerebral I/R injuries in rats [8], while the effect of miR-138 on cardiac I/R injury remains unclear.
MiR-138 was previously found to regulate the growth of pancreatic cancer by targeting FOXC1 [9]. According to a microrna.org prediction, miR-138 may target Intercellular Cell Adhesion Molecule-1 (ICAM-1), a downstream factor regulated by nuclear factor-kappa B (NF-κB). In fibroblasts, macrophages, lymphocytes, and vascular endothelial cells, ICAM-1 was highly expressed [10] and is considered as an important pro-inflammatory factor that mediates the inflammatory response to reperfusion injury [11]. Under normal physiological state, endothelial cells express relatively low levels of ICAM-1, its expression level can be increased upon stimulation of various inflammatory factors, hypertension, and I/R. Extensive studies have shown that reduced ICAM-1 expression acts critical effects on cardiac I/R injuries [11].
This study aimed to clarify the roles of miR-138 in the regulation of ICAM-1activityin endothelial cells during cardiac I/R injury and reveal the underlying mechanisms. Elevated miR-138 levels attenuated myocardial infarction, accumulation of reactive oxygen species (ROS), cell viability, and cell apoptosis by targeting ICAM-1in vivo and in vitro, suggesting that miR-138 might ameliorate the damage of cardiac I/R injury through targeting ICAM-1.

Establishment of a cardiac I/R injury rat model
The Animal Experiment Center of Suzhou Kowloon Hospital provided fifty adults Sprague-Dawley (SD) rats (male, 260-350 g). Rats were housed in standard conditions and provided feed and water weekly. Experiments were followed the guidelines of Animal Use of the National Institutes of Health's Code of Ethics [12] and was approved by the institutional Ethics Committee (Approval no. 2017SME0215). SD rats were randomly grouped(a) Sham group, (b) I/R group, (C) I/R + agomiR-NC group, and (D) I/R + agomiR-138 group (n=10 per group). Except the sham group, rats were housed in standard conditions for 3 days prior to surgical injury by myocardial I/R. Surgical injury was performed as previously reported [13]. Thirty minutes after ischemia, the slip was released, and reperfused the myocardium for 2 h. Finally, collected the blood samples of the sacrificed rats for biochemical analysis, and the peri-infarct zones of the hearts were used for morphological and biochemical tests. The model rats were injected through the tail vein with a agomiR-138 or a scrambled miRNA analog (RiboBio. Co., GuangZhou, China), twice a week, the dose in 20 mL of saline is 20 mg/kg/day.

Measurement of myocardial infarct size
Rat hearts were stored at -18 °C for 15 min, and then five uniform tissue sections were cut parallel to the line below the heart ligature and the coronary sulcus. All sections were weighed and cultured for 15 min in triphenyl tetrazolium chloride (TTC) in the dark at 37 °C for pathological examination. Image-Pro Plus software (Version 4.2, Media Cybernetics, LP, USA) was used to analyze white or pale non-TTC-stained areas and red TTC-stained areas. Finally, the ratio of infarcted myocardium to the entire myocardial tissue section (R) was obtained as in Eq 1. (1) where S and A are the infarct size and total heart area, respectively.

Quantitative RT-PCR analysis
The tissues and cell samples RNAs were extracted by MagBeads Total RNA Extraction Kit (TIANGEN, Beijing, China). Target gene specific primers were shown in Table 1. TB green qPCR master mix (TAKARA BIO INC, Tokyo, Japan) was used for fluorescence quantitative detection according to the manufacturer's instructions. The fluorescence values were expressed by 2 -ΔΔCt and quantified [14].

Determination of creatine kinase (CK) and serum lactate dehydrogenase (LDH) activities
Centrifuged the blood samples for 15 min at 4,500 × g, and serum samples were kept at -80°C. LDH and CK levels were evaluated by a commercial ELISA kit (Thermo Fisher Scientific, USA).

Assessment of ROS levels
Homogenized cells in lysis buffer and at 4°C, centrifuged for 15 min at 12,000 × g, discarded the cell pellets. 2,7-dchlorofluorescein diacetate (DCFH-DA) was used to assess ROS levels by using a ROS assay kit (Abeam, UK).

Apoptosis analysis
Apoptotic cells were formalin-fixed on a 5-mm thick heart tissue section, deparaffined, and treated with proteinase K (10-20 mg/mL) were labeled with TUNEL for 15 min at 25°C. After TUNEL labeling, 0.344-mm 2 sections were viewed at 400× magnification using a light microscope (Olympus, Tokyo, Japan) to quantify apoptotic cells, which were identified as dark green.
In addition, resuspended the cells in Annexin binding buffer (200 μL) with annexin V-FITC (10 μL) and PI (5 μL) and were cultured in the dark for 10 min at 25°C. Flow cytometry (Abcam, USA) was used to analyze the double-stained cells.

Western blot analysis
First, homogenized tissues on ice for 30 min with RIPA buffer (Sigma-Aldrich, USA) and protease inhibitor.
Next, centrifuged the tissue homogenates for 15 min at 12,000 g, and collected the supernatants and stored at -20°C. Separated the proteins in the supernatants by 12% SDS-polyacrylamide gels (PAGE) and transferred onto membranes of PVDF. Nonspecific binding was blocked by Tris-buffered saline (TBS) with 3% BSA (TBS and 0.1% Tween 20) under gentle shaking at 25°C for 1 h.

Statistical analysis
Data was analysed by GraphPad 8.0 and presented as mean ± standard deviation (SD). Variance (ANOVA) was used to analyze differences among the different groups and p < 0.05 was regarded as significant.

Effects of agomiR-138 on myocardial infarction area, ROS production and cell apoptosis
The I/R group showed an obvious increase (p < 0.001) in myocardial infarction area as compare to that in the sham group. However, I/R + agomiR-138 group exhibited a significant decrease in myocardial infarction area, compared to that in I/R + agomiR-NC group (Figure 1 A, p < 0.001). Downregulation of miR-138 was observed in I/R group compared to sham group. Moreover, miR-138 level was markedly increased in the IR + agomiR-138 group as compared to that in I/R + agomiR-NC group (p < 0.001; Figure 1 B). The serum LDH and CK activities were significantly elevated during I/R injury (p < 0.001, Figure 1 C). However, agomiR-138 treatment reduced the activities of LDH (p < 0.01) and CK (p < 0.001) after I/R as compare to that of the I/R + agomiR-NC group. ROS was accumulated in I/R rats as compared to that of the sham group (Figure 1 D). In the I/R + agomiR-138 group, ROS production was markedly decreased as compare to that of the I/R + agomiR-NC group. In addition, TUNEL assay showed that the apoptotic cell number was markedly higher in the I/R group compared to that in sham group, whereas agomiR-138 suppressed cell apoptosis upon I/R treatment (Figure 1 E).

Effects of niR-138 mimics on cell viability, ROS production and cell apoptosis on H/Rinduced H9C2 cells
MiR-138 was significantly downregulated in H/R cells (p < 0.001), whereas miR-138 were upregulated in the H/R + miR-138 mimic group (p < 0.001) as compare to that in the H/R + NCmimic group (Figure 2 A). MTT analysis showed that, as compare to the control group, the cell viability was markedly decreased in the H/R group (p < 0.01). The higher cell viability was found in the miR-138 mimic+ H/R group than NC-mimic + H/R group (Figure 2 B, p < 0.01). Serum LDH and CK activities were obviously improved in H/R-induced H9C2 cells (p < 0.01), while miR-138 mimic reduced LDH and CK activities under H/R treatment as compare to that of the H/R + NC-mimic group (Figure 2 C, p < 0.05;). ROS levels in H9C2 cells were markedly enhanced in the H/R group, whereas miR-138 mimics under H/R condition led to markedly decreased ROS production (Figure 2 D). Besides, H/R treatment significantly induced cell apoptosis as shown by flow cytometry (p < 0.001). However, miR-138 mimics markedly inhibited H/R-induced cell apoptosis (Figure 2 E, p < 0.01). In addition, reduced Bcl-2 expression and elevated expression levels of cleaved caspase-9, Bax and cleaved caspase-3 (all p < 0.001) were found in H9C2 cells induced by H/R .However, these effects (p < 0.001) were ameliorated by miR-138 mimics as compared to the NC-mimic + H/R group (Figure 2 F).

ICAM-1 was targeted by miR-138
Microrna.org predicted that ICAM-1) was a putative target of miR-138. The MUT and WT 3′-UTRs of ICAM-1 containing the miR-138 binding site were cloned into the reporter system (Figure  3 A). The luciferase activity was inhibited after co-transfection of miR-138 mimic and 3'-UTR of WT-ICAM-1 (Figure 3 A, p < 0.001), while had no significant effects on that of MUT-ICAM-1 3'-UTR. Moreover, the ICAM-1 mRNA and protein expressions in H/R cells were significantly reduced by miR-138 mimic (p < 0.001 and p < 0.01), whereas ICAM-1 mRNA and protein expressions (p < 0.01) were significantly improved in miR-138 inhibitor + H/R group (Figure 3 B and C). Moreover, ICAM-1 protein expression was increased in I/R rats as compared to sham group (p< 0.001), but was decreased by agomiR-138 as compared to I/R + agomiR-NC group (Figure 3 D, p < 0.01).

ICAM-1 suppressed the protection of miR-138 in H9C2 cells induced by H/R
To investigate whether exogenous ICAM-1 affected cell viability in H9C2 cells induced by H/R, MTT analysis exhibited that the cell viability was markedly reduced in themiR-138 mimic + ICAM-1+ H/R group, compared to miR-138 mimic + H/R group (Figure 4 A, p < 0.05). In addition, serum levels of LDH and CK were significantly upregulated (p < 0.05, p < 0.01) with miR-138 mimic + ICAM-1 treatment upon H/R conditions, compared to miR-138 mimic + H/R group (Figure 4 B). In addition, the ROS levels were markedly improved in the miR-138 mimic + ICAM-1+ H/R group as compared to the miR-138 mimic + H/R group (Figure 4

DISCUSSION
The underlying mechanisms of cardiac I/R injury are not fully understood due to a variety of factors, including mitochondrial dysfunction, onset of cardiomyocyte apoptosis, and mitochondrial defects [16]. Understanding the complexity of pathological reperfusion injury and its underlying mechanisms might provide insights into developing promising therapeutic strategies targeting cardiac I/R injury. In this study, a decreased expression of miR-138 was observed in both in vivo and in vitro. AgomiR-138 reduced myocardial infarction area, decreased ROS production and suppressed cell apoptosis in a rat model of cardiac I/R injury.
On the other hand, miR-138 mimics increased cell viability, enhanced ROS production and induced cell apoptosis in H/R-induced H9C2 cells. Further analysis verified ICAM-1 as a target ofmiR-138.
Besides, exogenous ICAM-1 abolished the protection of miR-138 in H9C2 cells that induced by H/R. These data demonstrated that miR-138 expression might be involved in the I/R-induced injury of cardiomyocyte through ICAM-1. MiR-106b-5p, miR-424, and miR-99a play critical roles in cardiac I/R injury [17]. In particular, miR-138is reported to be a cardiacenriched miRNA, [18]. In the present experiments, a decreased expression ofmiR-138 was observed in both I/R rats and H/R cells.
Lactate dehydrogenase (LDH) and CK are important serum enzyme biomarkers of myocardial infarction and are released from damaged cells [19]. Energy produced by healthy mitochondria functions in the form of ATP, while damaged mitochondria result in ROS accumulation [20]. Reduction of damaged mitochondria protects against cell death and maintains normal mitochondrial function. Apoptosis exerts a critical effect on cardiac I/R injury by activating apoptosis-associated proteins [21]. Results from our study indicated that, in the rat I/R injury model, downregulation of miR-138 occurred together with an increased myocardial infarction area, accumulation of LDH and CK expression, elevated ROS levels, and apoptotic rates. Moreover, upon H/R stimulation, cell viability, LDH and CK expression, formation of ROS, and cell apoptosis showed similar trends to those of in vivo experiments, which demonstrated upregulated cleaved caspase-9, Bax and cleaved caspase-3activitiesand decreased Bcl-2 activity. Importantly, miR-138 overexpression promoted these changes in cellular activity and ROS accumulation in I/R rats and partially attenuated these events in H9C2 cells upon H/R stimulation.
The biological functions of most miRNAs are exerted through binding to their target molecules, but it is unclear whether miR-138 affects I/Rinduced injury [22]. Therefore, bioinformatics website (www.microrna.org/were) was used to screen the potential targets of miR-138, and its downstream target (ICAM-1) may participate in I/R-induced injury. ICAM-1 is widely expressed in fibroblasts, macrophages, lymphocytes, and vascular endothelial cells, among other cell types [10]. For example, ICAM-1 is expressed in several human colonic epithelial cell lines and is upregulated in these cells upon invasive bacterial infection [23].
The present study showed that ICAM-1 expression was improved in I/R injury-induced rats. In addition, ICAM-1 was identified as a target of miR-138, which was evaluated by dual luciferase assays. MiR-138 can also negatively regulate ICAM-1 expression. Functional analysis further indicated that exogenous ICAM-1 significantly decreased H9C2 cell survival rates under H/R conditions and increased LDH and CK activities, ROS levels and induced cell apoptosis, as indicated by increased cleaved caspase-3, Bcl-2, cleaved caspase-9 and decreased Bax expression.

CONCLUSION
This study demonstrates that miR-138 mediates cardioprotection against myocardial I/R injury by targeting ICAM-1. It also throws some light on miR-138/ICAM-1 axis as a new therapeutic target for heart disease.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
We declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors.

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