MiR-599 Protects Cardiomyocytes against Oxidative Stress-Induced Pyroptosis

Oxidative stress is a crucial factor and key promoter of a variety of cardiovascular diseases associated with cardiomyocyte injury. Emerging literatures suggest that pyroptosis plays a key role in cardiac damages. However, whether pyroptosis contributes to cardiomyocyte injury under oxidative stress and the underlying molecular mechanisms are totally unclear. This study was designed to investigate the potential role of pyroptosis in H2O2-induced cardiomyocyte injury and to elucidate the potential mechanisms. Primary cardiomyocytes from neonatal Wistar rats were utilized. These myocytes were treated with different concentrations of H2O2 (25, 50, and 100 μM) for 24 h to induce oxidative injury. Our results indicated that mRNA and protein levels of ASC were remarkably upregulated and caspase-1 was activated. Moreover, the expressions of inflammatory factors IL-1β and IL-18 were also increased. Luciferase assay showed that miR-599 inhibited ASC expression through complementary binding with its 3′UTR. MiR-599 expression was substantially reduced in H2O2-treated cardiomyocytes. Upregulation of miR-599 inhibited cardiomyocyte pyroptosis under oxidative stress, and opposite results were found by decreasing the expression of miR-599. Consistently, miR-599 overexpression ameliorated cardiomyocyte injury caused by H2O2. Therefore, miR-599 could be a promising therapeutic approach for the management of cardiac injury under oxidative condition.


Introduction
Cardiovascular disease is a pressing worldwide public health problem and the main cause of death and birth defects all over the world [1]. Oxidative stress is involved in the initiation and progression of numerous cardiovascular diseases including cardiac hypertrophy, heart failure, hypertension, and atherosclerosis [2,3]. Furthermore, considerable research has been conducted to explore antioxidants that can reduce oxidative stress in order to ameliorate cardiovascular diseases [4,5]. However, the underlying mechanisms of pathophysiological elevated ROS in the cardiovascular system are still not completely revealed [6].
Pyroptosis, a proinflammatory programmed cell death quite different from apoptosis and necrosis, leads to cytokine release that activates proinflammatory immune cell mediators [7]. Inflammasomes are multimeric protein complexes that assemble in the cytosol after sensing pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Nucleotide-binding oligomerization domain-like receptor family 3 (NLRP3) is a cytosolic receptor activated by PAMPs and DAMPs [8]. The N-terminal pyrin domain (PYD) of NLRP3 serves as a scaffold to nucleate apoptosis-associated speck-like protein containing a CARD (ASC) [9]. Then, pro-caspase-1 is recruited to form an inflammasome. Pro-caspase-1 induces autoproteolytic cleavage into activated cleaved caspase-1 (C-caspase-1). Active caspase-1 cleaves pro-IL-1β and pro-IL-18 into biologically activated IL-1β and IL-18, respectively [10,11]. Several studies have provided evidence that pyroptosis is involved in the inflammation process of cardiovascular diseases [12][13][14]. However, the role of pyroptosis in cardiomyocytes under oxidative stress is totally unclear. Additionally, the potential relationship and pathological mechanisms between oxidative stress and pyroptosis in cardiomyocytes remain to be investigated.
It has been well recognized that microRNAs (miRNAs) play central roles in regulating some key protein-coding genes related to cardiovascular diseases [15]. In this study, we used TargetScan and http://microRNA.org/ to predict the putative complementary miRNAs of ASC. Among them, miR-599 is the most reducing miRNA after H 2 O 2 treatment. MiR-599 has been identified as an important tumor suppressor gene in multiple studies [16]. However, its role in cardiovascular diseases is not clear.
Taken together, in this study, we investigated the roles of miR-599 and pyroptosis in cardiomyocytes under oxidative stress. Furthermore, the interaction between miR-599 and ASC in the regulation of cardiomyocyte oxidative stress and the possible mechanism was also revealed.

Cell
Culture. Primary cardiomyocytes were taken from 1to 3-day-old neonatal Wistar rats using collagenase, as described previously [17,18]. Neonatal rats were sterilized with 75% ethanol, then sacrificed by decapitation. Hearts were isolated and digested by collagenase. Dispersed cells were suspended in total DMEM culture medium and centrifuged. Pooled cells were plated into culture flasks. Bromodeoxyuridine was added into the medium to deplete nonmyocytes. Cardiomyocytes were incubated at 37°C with 5% CO 2 . The cells were treated with different concentrations of H 2 O 2 (25, 50, and 100 μM) for 24 h.
2.4. Cell Viability Assay. 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was performed to evaluate cell viability of different groups. We followed the methods of Zhang et al. 2017 [17,18]. Briefly, cells were seeded in 96-well plates followed by gene transfection or H 2 O 2 treatment for 24 h. 20 μL MTT solution was added into cell culture medium for 4 h. Then, 150 μL DMSO was added to dissolve the formazan crystals. Absorbance was measured at 570 nm on a plate reader. 2.6. Western Blotting. The protein isolation was performed as previously described [17]. Protein samples of 100 μg/well were loaded in a 10% SDS polyacrylamide gel and transferred onto a nitrocellulose filter membrane, then blocked by 5% nonfat milk dissolved in PBS for 2 h. The membrane was probed with primary antibodies against ASC (Abcam), caspase-1 (Abcam), and C-caspase-1 (Abcam). GAPDH (Zhongshanjinqiao, Inc., Beijing, China) was used as an internal control. Western blot bands were quantified by the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA) by measuring band intensity (Area × OD).

Bioinformatic
Methods for Targetscan and microrna.org. http://www.targetscan.org/ and http://www.microrna.org/ were used to predict the complementary binding miRNAs with ASC 3 ′ UTR. After getting the 3 ′ UTR region of the ASC in NCBI, targetscan and http://microrna.org/ were used to predict potential miRNAs that may be able to bind to 3 ′ UTR of ASC (>5mer).
2.9. Dual-Luciferase Gene Reporter Assay. For the dualluciferase gene reporter assay between ASC and miR-599, the full length of wild-type ASC was amplified by PCR, and then, the PCR products were subcloned into psiCHECK-2 luciferase reporter vector (Luc-ASC-WT; Promega, WI, USA). Luc-miR599 mutant and Luc-miR599 WT was also constructed.         (Figures 1(b) and 1(c)). Moreover, procaspase-1 was recruited by ASC to form inflammasome, which was responsible for pyroptosis initiation. Our results revealed that cleaved caspase-1 was also increased by H 2 O 2 incubation (Figures 1(d) and 1(e)). These results indicated an induction of cardiomyocyte pyroptosis by H 2 O 2 . Furthermore, pyroptosis of cardiomyocytes was substantially enhanced by H 2 O 2 along with consistently observed increasing in IL-1β and IL-18 (Figures 2(a)-2(d)).

MiR-599 Is Downregulated in Cardiomyocytes under
Oxidative Stress. We further investigated the possible involvement of miRNAs in H 2 O 2 -induced cardiomyocyte pyroptosis. As ASC was remarkably increased in cardiomyocytes treated by H 2 O 2 , http://MicroRNA.org/ and Targetscan databases were used to predict the complementary binding miRNAs with ASC 3 ′ UTR. We found that miR-122, miR-  BioMed Research International 383, and miR-599 were the potential upstream regulator of ASC as predicted by http://MicroRNA.org/ (Figure 3(a)). However, there was no conserved predicted miRNA in Targetscan database (Figure 3(b)). In 100 μM of H 2 O 2 -treated cardiomyocytes, expression levels of three miRNAs were tested by qRT-PCR. The results suggested that miR-599 demonstrated the most pronounced downregulation in the oxidative condition (Figure 3(c)), and H 2 O 2 treatment decreased miR-599 expression in a dose-dependent manner (Figure 3(d)). And we noticed that inhibition of miR-599 alone did not induce the pyroptosis of cardiomyocytes (Supplemental Fig. 1A, B).

MiR-599
Directly Targets ASC. We next performed a series of functional studies to determine the link between miR-599 and ASC. Computational analysis predicted a conserved binding site for miR-599 in the 3 ′ -UTR of ASC gene (PYCARD) (Figure 4(a)). To verify that miR-599 directly targets ASC, we prepared luciferase reporter carrying the PYCARD 3 ′ -UTR (Figure 4(b)). The sequence of the PYCARD 3′UTR mutation was available in supplemental Fig 1D. Cotransfection of miR-599 with the luciferase reporter vector into HEK293 cells caused a sharp decrease in luciferase activity compared with transfection of the luciferase vector alone. The miR-599-induced depression of luciferase activity was rescued by an antisense inhibitor oligonucleotide (miR-599 inhibitor) used to knockdown miR-599 (Figure 4(c)). However, miR-599 failed to affect the luciferase activity elicited by the construct carrying the mutant ASC 3 ′ -UTR fragment (Figure 4(d)). The data indicated that miR-599 inhibited PYCARD translation through complementary binding to its 3 ′ UTR.

MiR-599 Prevents Cardiomyocyte Pyroptosis under
Oxidative Stress by Targeting ASC. Next, we wondered if increasing miR-599 expression would have prevented cardiomyocyte pyroptosis. SATB2 and TGFB2 are well-known targets for miR-599; the results shown that miR-599 was transfected effectively (Supplemental Fig. 1C). We found that transient upregulation of miR-599 inhibited ASC expression

Discussion
Oxidative stress-induced cardiac injury is the primary cause of cardiovascular diseases [20][21][22]. Studies on the underlying mechanisms are very crucial to develop therapeutic strategies and prevent premature cardiac cell loss in patients. Pyroptosis is a highly inflammatory form of programmed cell death and is triggered by ASC-inflammasome formation [11,23].
In recent years, a tremendous amount of effort has been devoted to discover the mechanisms of pyroptosis in many diseases and to determine the genes and pathways involved in this process [24]. Published evidence indicated that serelaxin attenuated myocardial I/R injury and the subsequent caspase-1 activation via eNOS-dependent mechanism [25]. Huang et al. found that H 2 S suppressed HG-induced cardiomyocyte inflammation and apoptosis by inhibiting the TLR4/NF-κB pathway and its downstream NLRP3 inflammasome activation [26]. Studies from other group also revealed DM-induced arrhythmias could be successfully treated by inhibiting the IL-1β axis with either IL-1 receptor antagonist or by inhibiting the NLRP3 inflammasome [27,28]. In fact, different concentrations of H 2 O 2 have different effects on cells. More than 200 μM of hydrogen peroxide induces apoptosis in endothelial cells, smooth muscle cells, and epithelial cells [29] [30,31]. Tong et al. reported that low level of H 2 O 2 significantly promoted endothelial cell proliferation, migration, and tube formation; the mechanism is related to Nox-derived ROS [32]. And we found that there are no studies confirming that low concentrations of H 2 O 2 promote the proliferation of smooth muscle cells and epithelial cells.
Reactive oxygen species (ROS) serves as important inflammation activating signals. Recently, several studies have revealed that ROS induces NLRP3 inflammasomedependent pyroptosis in human keratinocyte HaCaT cells, intestinal epithelial cells, and astroglial cells [24,33,34]. Mitogen-activated protein kinases (MAPK) and Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signaling pathways were involved in this process [35]. However, whether pyroptosis inflammasome could be induced by oxidative stress in cardiomyocytes is still unknown. Our study is the first study that demonstrated H 2 O 2 stress brought out pyroptosis in cardiomyocytes as a concentration-dependent manner. ASC mRNA and protein expressions were upregulated, and activated caspase-1 and increased IL-1β and IL-18 levels were also observed. We further elucidated the molecular mechanisms involved in these processes.
In just over two decades since the discovery of the first microRNA (miRNA), the field of miRNA biology has expanded considerably. Insights into the roles of miRNAs in development and disease have made miRNAs attractive tools and targets for novel therapeutic approaches [15]. The regulatory link between oxidative stress and pyroptosis in cardiomyocytes remained to be discovered. Whether miR-NAs responsible for this setting remains unclear, although some microRNAs have been identified that are regulated by oxidative stress that modulate cardiovascular physiopathology [36]. Here, we firstly identified a directly inhibitory miRNA of ASC, miR-599, using luciferase reporter assay. miR-599 was a well-characterized tumor-suppressor that regulated tumor cell proliferation, migration, and invasion. Periostin, SATB2, TGFB2, and MYC have been proved to be its targets to data [37][38][39]. Its role in cardiovascular diseases still remains unclear. Our study showed that miR-599 was downregulated in primary cardiomyocytes after 50 and 100 μM of H 2 O 2 treatment for 24 h. Moreover, gain-offunction as well as loss-of-function experiments further confirmed the antipyroptotic effects of miR-599. Meanwhile, PI/Hoechst staining and cell viability analysis suggested that overexpression of miR-599 ameliorated cardiomyocyte injury caused by H 2 O 2 treatment, whereas miR-599 inhibition contributed to oxidative-induced cardiomyocyte injury.

Conclusions
The present study provided three novel findings: (1) the significant contribution of pyroptosis to oxidative stressinduced cardiomyocytes injury, (2) the antipyroptotic property of miR-599 by directly targeting ASC, and (3) the cardiac protective effects of miR-599 under oxidative stress, while the AMO-599 aggravated cardiac damages. These findings will help to gain knowledge about the molecular mechanisms of cardiomyocyte injury during oxidative stress and show that miRNA-based approaches may contribute to the development of more effective antioxidant therapies.

Data Availability
The original data of our experiment has been send separately. The result of immunofluorescence is edited as PDF version, and Western blot is edited as DOC version. Especially, all the experimental data could be open by GraphPad Prism 5 application.