miR-223-3p suppresses inflammation to protect cardiomyocytes by targeting NLRP3 in acute myocardial infarction patients

Acute myocardial infarction (AMI) is an ischemic heart disease that is caused by acute and persistent ischemia/hypoxia of the coronary arteries. When myocardial infarction occurred, the cardiomyocytes caused irreversible damage and necrosis due to hypoxia and decreased supply of ATP (Boersma et al., 2003; Reed et al., 2017). The necrotic cells activate d the autoimmune system and released a severe inflammatory response after releasing their contents. On the one hand, the release of inflammatory mediators initiated the repair of damaged tissues by the body (Michaels et al, 2000; Maier et al., 2005). On the other hand, inflammatory cytokines could induce cardiomyocyte apoptosis, and cardiomyocyte apoptosis further promoted the increase of inflammatory cytokines, which was one of the main causes of AMI progression (Pop et al., 2014; Westman et al., 2016). Therefore, anti-inflammatory treatment was an important part of the treatment of patients with AMI (Ong et al., 2018).


Introduction
Acute myocardial infarction (AMI) is an ischemic heart disease that is caused by acute and persistent ischemia/hypoxia of the coronary arteries. When myocardial infarction occurred, the cardiomyocytes caused irreversible damage and necrosis due to hypoxia and decreased supply of ATP (Boersma et al., 2003;Reed et al., 2017). The necrotic cells activate d the autoimmune system and released a severe inflammatory response after releasing their contents. On the one hand, the release of inflammatory mediators initiated the repair of damaged tissues by the body (Michaels et al, 2000;Maier et al., 2005). On the other hand, inflammatory cytokines could induce cardiomyocyte apoptosis, and cardiomyocyte apoptosis further promoted the increase of inflammatory cytokines, which was one of the main causes of AMI progression (Pop et al., 2014;Westman et al., 2016). Therefore, anti-inflammatory treatment was an important part of the treatment of patients with AMI (Ong et al., 2018).
microRNA is a small non-coding RNA that plays an important role in regulating the transcription of genes. Previous studies had found that miRNAs participated in cardiomy--ocyte development, proliferation, apoptosis and other processes (Bang et al., 2014;Lu et al., 2010), and could be used as a new biomarker, playing an important role in the early diagnosis, prognosis (Wang et al., 2010;Yang et al., 2017), and even treatment of acute myocardial infarction. More and more research have showed that miRNAs were involved in the regulation of inflammatory response in AMI patients (Hristov & Weber, 2015;Yang et al., 2015a). In animal experiments, miR-223-3p had been found as a novel MicroRNA Regulator of expression of voltage-gated K+ channel Kv4.2 in acute myocardial infarction (Liu et al., 2016). And lots of previous researches had shown that miR-223-3p could inhibit inflammation in patients with various diseases by targeting inhibition of NLRP3 expression, such as acute lung injury (Feng et al., 2017), intestinal inflammation (Neudecker et al., 2017) and others (Haneklaus et al., 2012;Bauernfeind et al., 2012).
However, the regulatory mechanism of miR-223-3p on circulating inflammation in patients with AMI is unknown. In this study, we detected the expression of miR-223-3p by Real-time quantitative PCR, and found that miR-223-3p was not only up-regulated in the circulating of AMI patients, but also was associated with myocardial injury and inflammation in AMI patients. In vitro experiments shown that miR-223-3p suppressed inflammation to protect cardiomyocytes by targeting NLRP3 in peripheral blood mononuclear cells. All in all, this paper provide a new idea for AMI anti-inflammatory treatment.

Patients and blood sample
53 cases AMI people which were diagnosed according to WHO standards for AMI (Mendis et al., 2011) and 53 cases UA patients which were diagnosed according to ACC/AHA 2007 guidelines (Anderson et al., 2007) were randomly included in this study, and their blood samples were collected within 12 hours after the onset of chest pain. At the same time, there were 53 healthy people who were accepted a medical examination. In addition, 10 cases healthy volunteers donated their blood for this study.
The present study was performed with the approval of the Ethics Committee of the Changzhou No.2 People' s Hospital, Affiliated Nanjing Medical University. All aspects of the study complied with the Declaration of Helsink. (Cook et al., 2003) In addition, all participants signed the informed consent.

Cell apoptosis assay
24 hours after inducing by different dose of IL-1β (CYT-708, AmyJet Scientific, WuHan, China), H9C2 cells were collected, and the Annexin V FITC/PI kit (Invitrogen, USA) was used for flow cytometry to detect apoptosis. Beckman CytoFLEX Flow cytometry (BECKMAN, USA) was used to analysis the apoptosis of cells.

Statistical analysis
Data was presented in (mean±standard deviation) and analyzed by SPSS 25.0. Student's t-test or chi-square test was used to compare differences between two groups. One-way ANOVA with Duncan's post-hoc test was used for comparing multiple groups. The correlation between serum level of miR-223-3p and inflammatory factors / myocardial enzyme in patients with AMI was analyzed by Pearson's correlation coefficient. Logistic regression models were constructed to determine the odds ratio (OR) and 95% confidence interval (CI) for putative risk factors associated with AMI. P < 0.05 was considered to indicate a statistically significant difference.

miR-223-3p was up-regulated in the circulating of AMI patients
The expression of miR-223-3p in the serum or PBMC of healthy people (n = 53), UA patients (n=53) and AMI patients (n = 53) were measured by RT-qPCR, and results showed that the serum or PBMC level of miR-223-3p in the AMI patients was significantly higher than that in UA group (P < 0.001), and that in control group was the lowest of three group (Figure 1).

miR-223-3p was associated with the occurrence of AMI
Univariate analysis showed that (Table 1) there were no significant differences in gender, age, smoking, hypertension, TC, TG, HDL-C and LDL-C between three group (P > 0.05). However, the serum CK-MB, cTnI, AST, LDH, TNF-α, IL-6, IL-1β and IL-8 in AMI patients was significantly higher than that in health people and UA patients (P < 0.001).

Cardiomyocytes apoptosis by IL-1β in a dose-dependent
We examined the effects of different concentrations of IL-1β on apoptosis of H2C9 cells, and found that the apoptotic rate of H2C9 cells increased with increasing TNF-α concentration ( Figure 5).

Discussion
In this paper, we found that the circulating miR-223-3p in AMI patients was significantly higher than that in UA patients and healthy people. Moreover, univariate and logistic regression analysis showed the circulating miR-223-3p was a protective factor in the occurrence of AMI. Although there were no clinical data in published studies indicating that miR-223-3p was highly expressed in peripheral blood of patients with AMI, animal studies had shown that (Liu et al, 2016) upregulation of miR-223-3p in AMI repressed the expression of KCND2/Kv4.2 resulting in reduction of Ito density that could cause APD prolongation and promote arrhythmias in AMI.
In addition, we also found that circulating miR-223-3p in AMI patients was negatively correlated with serum myocardial enzyme, such as CK-MB, cTnI, AST and LDH, and was negatively correlated with serum inflammatory cytokines, such as TNF-α, IL-6, IL-1β and IL-8. Myocardial local and systemic inflammatory responses played an important role in the development of ventricular remodeling and heart failure after acute myocardial infarction. In AMI patients, myocardial injury came from two aspects. In addition to myocardial ischemic injury after AMI, immune-mediated inflammatory response also played a role in aggravating myocardial damage and expanding myocardial infarction. Necrotic myocardial tissue after AMI caused complement activation, cytokine release, inflammation, and chemotaxis and infiltration of immune cells, as well as myocardial damage through pathological autoimmune responses (Frangogiannis et al., 2002). Cytokines, such as TNF-α, IL-6, IL-1β and IL-8, were polypeptides secreted by various cells such as lymphocytes, monocytes, macrophages, and vascular endothelial cells. They were released into the blood during the inflammatory reaction, and their    factor / myocardial enzyme. More importantly was miR-223-3p suppressed circulating inflammation to protect cardiomyocytes by targeting NLRP3 in AMI patients.
concentration in the blood reflected the stage of inflammation of the body degree (Hartman et al., 2018).
miR-223-3p was located at the Xq12 position of human chromosome, and previous studies had shown that it could regulate inflammation in human body. Zhuang et al. (2012) found that miR-223-3p could affect obesity-related tissue inflammation by regulating macrophage activation. Neudecker et al. (2017) found that the bone marrow-derived miR-223-3p could regulated the seriousness of the human intestine. Moreover, miR-223-3p also played a role in regulating inflammation in organ damage (Feng et al., 2017;Yang et al., 2015b), infection and cancer (Haneklaus et al., 2013). Combined with our findings above, it suggested that miR-223-3p might also regulate peripheral blood inflammation in patients with AMI. It was well known that miRNAs were non-coding RNAs and must function by regulating target genes. For miR-223-3p, many studies had shown that miR-223-3p regulated inflammation by targeting NLRP3 expression (Bauernfeind et al., 2012). And the luciferase reporter gene system confirmed that miR-223-3p targeted inhibition of NLRP3 expression in THP-1 and human peripheral blood mononuclear cell in this study.
The NLRP3 inflammatory corpuscle was an important component of the pattern recognition receptor family in the innate immune system. Once activated, the NLRP3 inflammatory corpuscle would recruit and interact with the apoptosis-associated speck-like protein containing CARD, thereby recruiting and binding procaspase-1 to activate its own cleavage and activation into caspase-1, maturing the IL-1β precursor (pro-IL-1β) and IL-18 precursor (pro-IL-18) into active pro-inflammatory cytokines (IL-1β and IL-18), ultimately acting as a pro-inflammatory factor (Schroder et al., 2010;Zhou et al., 2011). The NLRP3 inflammatory body recognized different risk signals and induced a sterile inflammatory response, making it to be the best candidate for pattern recognition receptors that function in the AMI process. Mezzaroma et al. found that inhibited the activation of NLRP3 inflammatory bodies by siRNA or drugs could reduce cardiomyocyte apoptosis and ultimately reduce myocardial remodeling after myocardial infarction in permanent myocardial infarction model mice (Mezzaroma et al., 2011). Liu et al. (2014) detected an up-regulation of NLRP3 expression in the ischemic heart, increased caspase-1 activity, and increased IL-1β and IL-18 in the C57BL/6J mouse myocardial ischemia-reperfusion model. And intramyocardial injection NLRP3 siRNA or intraperitoneal injection of NLRP3 inflammatory inhibitor BAY 11-7028 could reduce macrophage and neutrophil infiltration in myocardial ischemia-reperfusion injury, and reduce myocardial cell apoptosis and myocardial infarct size.
In this study, we found miR-223-3p-mimic could reduce the expression of NLRP3 and the secretion of IL-1β human PBMC in vitro. At the same time, IL-1β induced the apoptosis of cardiomyocytes in a dose-dependent in vitro. IL-1β was an important cytokine that played a pro-inflammatory role in the body and was also one of the two cytokines regulated by NLRP3 (Mulay et al., 2013;Mishra et al., 2013).

Conclusion
Circulating miR-223-3p was highly expressed in AMI patients and was negatively correlated with serum inflammatory