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microRNA-223-3p attenuates angiotensin II-dependent ROS effect on cell viability by targeting NLRP3 in H9c2 cells

22 Introduction: Recently, enhanced activation of NLRP3 has been reported to be involved in 23 atrial fibrillation (AF). This study aimed to detect the correlation between oxidative stress and 24 NLRP3 and explore the role of miR-223-3p in the injury of ROS induced by Ang II. 25 Material and methods: Serum Ang II levels were examined by ELISA kit. Fibrosis levels of 26 right atrial appendages were determined by Masson’s staining. H9c2 cells tansfected with 27 miR-223-3p mimics were treated with Ang II with or without MCC950 (a potent selective 28 NLRP3 inhibitor). Cell viability was detected by CCK-8 assay. Protein abundance was 29 detected by Western blot. MDA assay and DCFH-DA were used to measured oxidative stress. 30 RT-qPCR was used to assay the expression of miR-223-3p and NLRP3. 31 Results: Totally, 43 patients enrolled in this study, including 20 patients with persistent 32 (chronic) AF (cAF). Comparing with sinus rhythm (SR) group, we found an enhanced 33 activation of NLRP3 inflammasome which were positively correlated with oxidative stress 34 and serum Ang II level in cAF patients. Ang II induced ROS generation and inhibited the 35 H9c2 cell viability. In addition, overexpression of miR-223-3p functioned as MCC950 which 36 inhibited the expression of NLRP3 inflammasome and partly attenuated the effects of ROS 37 induced by Ang II on H9c2 cell viability. Lastly, we used luciferase assay to confirm NLRP3 38 as a direct target gene of miR-223-3p. 39 Conclusion: miR-223-3p has protective effects on oxidative stress induced by Ang II in AF 40 by targeting NLRP3 and could provide a new potential intervention targets for treatment of 41 AF. 42

Introduction: Recently, enhanced activation of NLRP3 has been reported to be involved in 23 atrial fibrillation (AF). This study aimed to detect the correlation between oxidative stress and 24 NLRP3 and explore the role of miR-223-3p in the injury of ROS induced by Ang II. 25 Material and methods: Serum Ang II levels were examined by ELISA kit. Fibrosis levels of 26 right atrial appendages were determined by Masson's staining. H9c2 cells tansfected with 27 miR-223-3p mimics were treated with Ang II with or without MCC950 (a potent selective 28 NLRP3 inhibitor). Cell viability was detected by CCK-8 assay. Protein abundance was 29 detected by Western blot. MDA assay and DCFH-DA were used to measured oxidative stress. 30 RT-qPCR was used to assay the expression of miR-223-3p and NLRP3. 31 Results: Totally, 43 patients enrolled in this study, including 20 patients with persistent 32 (chronic) AF (cAF). Comparing with sinus rhythm (SR) group, we found an enhanced 33 activation of NLRP3 inflammasome which were positively correlated with oxidative stress 34 and serum Ang II level in cAF patients. Ang II induced ROS generation and inhibited the 35 H9c2 cell viability. In addition, overexpression of miR-223-3p functioned as MCC950 which 36 inhibited the expression of NLRP3 inflammasome and partly attenuated the effects of ROS 37 induced by Ang II on H9c2 cell viability. Lastly, we used luciferase assay to confirm NLRP3 38 as a direct target gene of miR-223-3p. 39 Conclusion: miR-223-3p has protective effects on oxidative stress induced by Ang II in AF 40 by targeting NLRP3 and could provide a new potential intervention targets for treatment of 41 AF. 42 high risk of stroke, heart failure and mortality. 1 More than 30 million people worldwide 48 suffer from AF, with an average incidence of about 3%. 2 A recent survey of current 49 conditions found that there are about 4.87 million patients with AF over the age of 35 in 50 China. 51 At present, the pathogenesis of AF is not completely known. The enhanced activation of 52 nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome 53 and oxidative stress may be two potential mechanisms leading to AF. The NLRP3 54 inflammasome plays an important role in pyroptosis. Pyroptosis is a novel type of caspase 1-55 dependent programmed cell death caused by activated inflammasomes. It has been found to 56 be involved in different cellular processes in various diseases, including renal ischemia 57 reperfusion injury, diabetic nephropathy, nerve injury and atherosclerosis. 3-5 The activated 58 NLRP3 inflammasomes induce pro-caspase-1, which upon maturation subsequently releases 59 inflammatory cytokines leading to pyrotosis. 6 However, there are only a few studies done on 60 the role of NLRP3 in the occurrence and maintenance of AF. In one of our previous studies, 61 we found an increase in the expression of NLRP3 and IL-1β in the peripheral blood 62 mononuclear cells (PBMCs) of AF patients, suggesting that NLRP3 inflammasome signaling 63 had a role in AF. 7 But, the specific molecular mechanisms underlying this process are still 64 not clear. 65 Reactive oxygen species (ROS) that act as second messenger and regulate the activity of 66 various transcription factors are both effectors of oxidative stress and potent triggers of 67 inflammation. Several studies indicated that increased ROS level in myocardial tissue was 68 associated with AF. 8 ROS-mediated activation of inflammatory signaling in cardiac tissues 69 could lead to myocardial fibrosis while activation of transforming growth factor β1(TGF-β1) 70 could cause atrial structural remodeling. Interestingly, ROS was also found to act as a trigger 71 regulate the generation of ROS in cells by activating membrane-bound NAD(P)H oxidase or 73 inflammatory signaling. 9 Taken together, ROS may act as a bridge between NLRP3 74 activation and Renin-angiotensin-aldosterone system (RAAS) activation in AF. Hence, we 75 hypothesized that abnormal secretion of Ang II increased myocardial ROS generation and 76 then activated the NLRP3 inflammasome, which induced cardiac myocytes pyroptosis and 77 activated inflammation in AF. 78 Micro RNAs(miRNAs) are small molecular RNAs composed of 19-25 nucleotides. They 79 regulate target gene expression at the post-transcriptional level by acting on target mRNA and 80 subsequently promoting the degradation of target mRNA or repressing target mRNA 81 translation. 10 The effect of miRNAs in the pathogenesis of cardiac diseases such as 82 arrhythmia, myocardial infarction and heart failure has been shown by many studies. 11-13 But 83 only few studies have focused on the role of miRNAs mediated ROS regulation in AF. One 84 study used right atrial tachypacing and found that overexpression of miR-206 induced ROS 85 generation by negatively regulating SOD1 in the canine model of AF. 14 Another study 86 reported exosomal miR-223 levels being significantly elevated after cardiopulmonary bypass, 87 leading to downregulation of IL-6 and NLRP3 expression in the monocytes. 15 88 However, whether miR-223-3p could regulate Ang II-dependent ROS in AF via NLRP3 89 remains largely unknown. This study aimed to detect the correlation between oxidative stress 90 and NLRP3 and explor the role of miR-223-3p in the injury of ROS induced by Ang II. 91

Tissue samples 93
The right atrial appendage (RAA) tissues were collected from patients with persistent 94 (chronic) AF (cAF) who were undergoing open-heart surgery for coronary bypass grafting or 95 valve replacement. The diagnosis of cAF is in accordance with the latest AHA/ASA 96 guidelines. After tissue harvest, some of the samples were rapidly cryopreserved in liquid with cool PBS and then fixed with 4% paraformaldehyde for fibrosis analysis. All patients 99 included in the study were informed about the project and informed consent were received 100 before the surgery. The experimental protocols were approved by the ethics committee of the 101 first affiliated hospital of Guangxi Medical University. 102

Tissue Fibrosis and Malonadehyde (MDA) assay 103
To measure myocardial fibrosis, the fixed human atrial samples were imbedded in paraffin 104 and serially sectioned into 4um thick slices for Masson's staining. To increase statistical 105 power, three fields of view for each slice were randomly selected and observed under an 106 optical microscope (Olympus, Tokyo, Japan). Image-Pro 6.0 software was used to analyze the 107 results. The MDA levels were detected by using the MDA kits (Nanjing Jiancheng 108 Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. 109

Enzyme-Linked immunosorbent assay (ELISA) 110
The serum Ang II levels were measured by specific ELISA kit (ab10601; Abcam, Cambridge, 111 UK) according to the manufacturer's instructions. 112

Cell culture 113
We purchased H9c2 cell line from Chinese Academy of Sciences (Shanghai, China). After 114 H9c2 cells were resuscitated, the cells were cultured in DMEM (10569010; Gibco, 115 Gaithersburg, USA) with proper volume of Penicillin-Streptomycin mix (15140148; Gibco, 116 Gaithersburg, USA). The incubator envionment was adjusted to 37℃ and 5% CO2. Then cells 117 were seeded in appropriate plates for the following experiments. 118

Cell counting kit-8 (CCK-8) assay 119
We used CCK-8 assay (CK04; Dojindo, Kumamoto, Japan) to assess cell viability. Briefly, 120 H9c2 cells were seeded in a 96-well plate and either incubated in different concentration of 121 P r e p r i n t for different lengths of time (from 0 h to 48 h). MCC950 is a potent specific inhibitor of the 123 NLRP3 inflammasome and it blocks the release of IL-1β induced by NLRP3 activators such 124 as lipopolysaccharide (LPS) and inflammatory factors. In the inhibitor group, MCC950 (50 125 μM) (HY12815; MedChem Express, Monmouth, USA) was added in culture medium before 126 stimulation with Ang II. After adding 10ul of CCK-8 reagent into each well of the 96-well 127 plate, the plate was incubated at 37℃ for 2 h. Absorbance at 450nm was then measured by 128 Varioskan Lux system (Thermo Fisher Scientific, Waltham, USA). The viability of the treated 129 cells was measured according to the manufacturer's instructions. Experiments were 130 performed at least in triplicates. 131

Transfection 132
After culturing for 24h, all the cells were observed and evaluated under microscope. Cells at 133 70%-80% confluency in 6-cell-culture dish were transfected with miR-223-3p mimics and 134 miR-223-3p mimics negative control (NC) by Lipofectamine 3000 Reagent (Invitrogen, 135 Carlsbad, USA). The sequences of the miR-223-3p mimics and NC used in transfection 136 experiment are listed in Table 1. Both miR-223-3p mimics and miR-223-3p mimics NC were 137 constructed by GenePharma company (Shanghai, China). The transfection efficiency was 138 determined by RT-qPCR. The transfected cells were cultured in serum-free medium for 139 another 24 h and then treated with Ang II and MCC950 for further experiments. 140

Real-time Quantification PCR (RT-qPCR) analysis 141
Total RNA from the RAA tissues or harvested cells were extracted by Trizol (9108; Takara, 142 Tokyo, Japan). cDNA was synthesized by using tailing reaction kit (B532451; Sangon 143 Biotech, Shanghai, China) for miRNA or using reverse transcription kit (RR047A; Takara, 144 Tokyo, Japan) for mRNA. Subsequently, the cDNA was submitted for RT-qPCR analysis on 145 SetpOne system (Thermo Fisher Scientific, Waltham, USA), and quantified using the 2 -△△ CT 146 sequences for RT-qPCR are listed in Table 1. 148

Western Blot analysis 149
The RAA tissues or harvested cells were lysed by RIPA lysis buffer (P0013B; Beyotime,  Image-Pro 6.0 software was used to measure the average fluorescence intensity. 169

miR-223-3p target gene prediction 170
In order to find potential binding sites, we searched TargetScan (http://www.targetscan.org/) 171 prediction, the sequence of potential target genes with high score were analysed by the 173 National Biotechnology Information Center BLAST program. 174

Statisical Analysis 183
In our article, the categorical data were present as frequency and the continuous data were 184 expressed as mean ± standard deviation (SD) or mean. The SPSS version 20 software was 185 used for the statistical analysis. For the categorical data, the differences between groups were 186 compared by chi-square test. For continus data, the difference in means between two groups 187 with normally distributed data was compared using the two-tailed Students's t-test. In cases 188 when experimentally designed groups were more than two, one-way ANOVA and Holm-189 sidak's test were used for comparison. If the data were not normally distributed, difference 190 between groups was tested by Mann-Whitney and Kruskal-Wallis test. Difference was 191 considered statistically significant if P-value was less than 0.05. Analyzed data were plotted 192 as histograms using GraphPad Prism version 5 software.  Table 2.  (Figure 2 D). 213

Upregulation of serum Ang II level in patients with cAF 214
To investigate the serum Ang II level in patients with SR or cAF, the serum levels of Ang II 215 were examined using a specific ELISA kit. As shown in Figure 2 E

miR-223-3p expression level upregulated in patients with cAF 219
fold higher in patients with cAF than that in the SR group (Figure 2 F). 221

Cell viability decreaed notably after exposure to 1uM Ang II for 24h 222
To identify the effect of Ang II on cell viability, the H9c2 cells were stimulated with different concentration, the lowest cell viability was detected after Ang II exposure for 24 h (Figure 3  228   B). So, we used 1 μM Ang II for 24 h as an intervention condition for subsequent experiments. 229

ROS level induced by Ang II in H9c2 cells 231
To further explore the regulatory relationship between miR-223-3p and NLRP3, miR-223-3p 232 mimics and miR-223-3p NC were transfected into H9c2 cells before intervention with Ang II. 233 RT-qPCR results confirmed elevated levels of miR-223-3p in the mimic group (Figure 4 A). 234 The protein expression of NLRP3 decreased in the mimic group (Figure 4 B). After 235 intervention with Ang II, compared to the Ang II group, the protein expression of NLRP3, 236 ASC, Caspase-1p20 and IL-1β were markedly lower in the mimic group and Ang 237 II+MCC950 group (P＜0.05) (Figure 4 C). Results of ROS test showed Ang II treatment 238 promoted ROS generation in H9c2 cells. Meanwhile, the ROS level of mimic group was 239 lower compared with the Ang II group, while the same result was observed in Ang II + 240 MCC950 group (P＜0.05) (Figure 4 D). Intriguingly, cell viability was negatively correlated 241 with ROS level in each group (Figure 4 E). 242

4.Discussion 254
At present, several achievements have been made in AF Catheter ablation treatment, 16 255 however, the risk of AF continues to affect people's lives and increases with age. Increasing 256 evidence supports that the oxidative stress and enhanced activation of NLRP3 inflammasome 257 participate in the pathophysiological processes of AF. To our knowledge, the activation of 258 NLRP3 inflammasome regulated by miRNAs in AF is not completely known. At present 259 study, we described the effect of miR-223-3p on regulation of Ang II/ROS/NLRP3 axis in AF 260 by targeting NLRP3. The schematic overview is shown in Figure 6. 261 Ang II is well known as an upstream regulatory factor that induces fibrosis in different organs. 262 It is generally thought the abnormal secretion of Ang II directly mediates myocardial fibrosis 263 by activating fibroblasts via AT1 targeting. Consistent with our study, Qiu et al 17 reported 264 significantly elevated serum Ang II level in a chronic kidney disease rat model whose AF 265 inducibility was significantly induced by transesophageal atrial burst pacing. 266 It is increasingly accepted that Ang II can induce cardiac inflammatory response in atrial 267 remodeling due to the notable expression of inflammatory genes at the early stage of Ang II 268 infusion. 18 Furthermore, Epelman et al 19 observed the recruitment of CCR2+ monocytes intervention in the heart, however, this phenomenon could be inhibited by the CCR2 receptor. Ang II via NADPH pathway. 29 At present, it has been found that CaMKII has four major 289 isoforms (including α, β, γ and δ), while CaMKIIδ is mainly expresses isoform in mammalian 290 heart. Recently, the non-ischemic stresses including Ang II was found to act as an activator of 291 selective inhibitor of NLRP3, can block NLRP3 activation. As discussed earlier, there is a 340 mutual regulating cycle between ROS production and CaMKII activation. After the cells were 341 treated with MCC950, the effect of Ang II on enhanced NLRP3 activation was blocked, so 342 the NLRP3 expression decreased comparing Ang II group. The release of inflammatory 343 cytokines decreased as well. As a result, the activation of CaMKII decreased due to 344 mitochondrial damage decrease, which resulted in the decrease of ROS production. Above 345 results indicated that miR-223-3p might function like MCC950 and negatively regulate 346 NLRP3 inflammasome-mediated ROS generation, suggesting that the upregulated miR-223-347 3p play a protective role in AF. 348 By using bioinformatics methods, NLRP3 was found to be a potential target gene of miR-349 223-3p. There was one predicted conserved binding site of miR-223-3p and NLRP3. 223-3p and NLRP3. Furthermore, using a dual-luciferase reporter assay, we found that miR-352 223-3p could directly bind the 3′-UTR of NLRP3, thus confirming that NLRP3 is a direct
Powered by TCPDF (www.tcpdf.org) P r e p r i n t Figure 6. The schematic overview of current study.