Rno-microRNA-30c-5p promotes myocardial ischemia reperfusion injury in rats through activating NF-κB pathway and targeting SIRT1

Rno-microRNA-30c-5p promotes myocardial ischemia reperfusion injury in rats through activating NF-κB pathway and targeting SIRT1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Rno-microRNA-30c-5p promotes myocardial ischemia reperfusion injury in rats through activating NF-κB pathway and targeting SIRT1 Jianfeng Chen, Mingming Zhang, Shouyan Zhang, Junlong Wu, Shufeng Xue This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.15405/v5 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 May, 2020 Read the published version in BMC Cardiovascular Disorders → Version 5 posted 4 You are reading this latest preprint version Show more versions Abstract Background: This study aimed to investigate the regulatory effect of rno-microRNA-30c-5p (rno-miR-30c-5p) on myocardial ischemia reperfusion (IR) injury in rats and the underlying molecular mechanisms. Methods: A rat model of myocardial IR injury was established. The infarct size was detected by 2,3,5-triphenyltetrazolium chloride staining. The pathologic changes of myocardial tissues were detected by hematoxylin-eosin staining. The apoptosis of myocardial cells was measured by TUNEL staining and flow cytometry. The mRNA expression of rno-miR-30c-5p and Sirtuin 1 (SIRT1) was detected by quantitative real-time PCR. The levels of IL-1β, IL-6 and TNF-α were detected by enzyme linked immunosorbent assay. The protein expression of Bax, Bcl-2, caspase-3, p-IκBα, IκBα, p-NF-κB p65, NF-κB p65 and SIRT1 was detected by Western blot. The interaction between rno-miR-30c-5p and SIRT1 was predicted by TargetScan, and further identified by dual luciferase reporter gene and RNA immunoprecipitation assay. Results: The myocardial IR injury model was successfully established in rats. IR induced the myocardial injury in rats and increased the expression of rno-miR-30c-5p. Overexpression of rno-miR-30c-5p enhanced the inflammation, promoted the apoptosis, and activated NF-κB pathway in IR myocardial cells. SIRT1 was the target gene of rno-miR-30c-5p. Silencing of SIRT1 reversed the effects of rno-miR-30c-5p inhibitor on the apoptosis and NF-κB pathway in IR myocardial cells. Conclusions: Rno-miR-30c-5p promoted the myocardial IR injury in rats through activating NF-κB pathway and down-regulating SIRT1. Cardiac & Cardiovascular Systems myocardial ischemia reperfusion injury rno-miR-30c-5p inflammation apoptosis SIRT1 NF-κB pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Ischemic heart disease is a series of diseases characterized by myocardial ischemia, such as angina pectoris and myocardial infarction 1 . Recently, reperfusion of the ischemic myocardium is one of the most common therapeutic strategies for ischemic heart diseases 2 . Although restoring blood flow in time can relieve myocardial infarction to a great extent, the prognosis of patients remains poor due to the ischemia reperfusion (IR) injury on myocardium 3 . Therefore, it is urgent to find out novel therapeutic methods and targets for myocardial IR injury. The genome-wide investigations of genetic variants, epigenetic modifications, and gene expression profiles optimize the search for novel diagnostic or therapeutic targets for IR injury in the post-genomic era 4 . MicroRNAs (miRNAs) are a class of small endogenous noncoding RNAs with 19-25 nucleotides in length, which modulate gene expression at the post-transcriptional level 5 , 6 . A systematic comparison of IR injury-induced miRNA expression changes in rats identifies several potential cardioprotective miRNA targets (protectomiRs), including Rno-miR-125b*, -139-3p, -320, -532-3p, and -188 7 . By using bioinformatics methods based on topological or network dynamical approaches, the mRNA targets of protectomiRs can be predicated. Nevertheless, all unbiased omics approaches and their bioinformatic evaluation need to be verified by rigorous experimental validation at the transcript and protein levels 8 . Recently, studies have indicated that miRNAs play important regulatory roles in myocardial IR injury 9 . Yuan et al. 10 have proved that the inhibition of rno-miR-181b-5p protects cardiomyocytes against I/R injury through targeting AKT3 and PI3KR3. Zhao et al. 11 have reported that mmu-miR-374a protects against myocardial IR injury in mice via targeting MAPK6 pathway. Song et al. 12 have indicated that rno-miR-30b overexpression has anti-apoptotic effect on cardiomyocytes at early phase of myocardial IR injury in a rat model. MiR-30c-5p is another subtype of miR-30 that also involved in the process of IR injury. Zhou et al. 13 have proved that rno-miR-30c-5p is a potential diagnostic marker for I/R-induced kidney injury in rats. Li et al. 14 have shown that hydrogen sulfide protects spinal cord and induces autophagy in a rat model of spinal cord IR injury via regulating rno-miR-30c-5p. However, the regulatory effect and mechanism of rno-miR-30c-5p on myocardial IR injury remain unclear. Nuclear factor κB (NF-κB) is involved in the regulation of multiple biological functions including innate immunity, inflammation, cell proliferation and apoptosis 15 , 16 . Accumulating researches have revealed that myocardial IR injury is associated with the activation of NF-κB 17 . In addition, emerging evidence has indicated that miRNAs play vital roles in myocardial IR injury by regulating NF-κB pathway. For instance, mmu-miR-146a overexpression reduces myocardial IR injury via inhibiting the activation of NF-κB pathway 18 . However, whether the regulatory effect of rno-miR-30c-5p on myocardial IR injury is involved in NF-κB pathway is unknown. In this study, we explored the regulatory effect of rno-miR-30c-5p on myocardial IR injury in rats, as well as the underlying molecular mechanisms. Our results indicated that rno-miR-30c-5p promoted the myocardial IR injury in rats through activating NF-κB pathway and down-regulating SIRT1. Our findings may provide a new theoretical foundation for the treatment of myocardial IR injury in clinical practice. Methods Animals Male Sprague-Dawley (SD) rats (weighting 180-200 g) were provided by Peking University Laboratory Animal Center. All rats were kept at 22-24°C and 55-60% humidity on a 12 h light-dark cycle with free access to water and food. At the end of the study, all rats (220-270g) were anesthetized by an intraperitoneal injection of 50 mg/kg pentobarbital sodium, and then sacrificed by cervical dislocation. All animal experiments were conducted strictly in accordance with the National Institutes of Health guide for the care and use of Laboratory animals. Establishment of the myocardial IR model in rats Rats weighing 200-240g were used to establish the IR model. Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). The left anterior descending coronary artery (LAD) was ligated using 6-0 silk suture slipknot for 30 min, and then reperfused for 2 h. Myocardial ischemia was confirmed by the appearance of regional epicardial cyanosis over the myocardial surface and by arrhythmia. Successful reperfusion was confirmed by the disappearance of epicardial cyanosis and the production of epicardial hyperemia and arrhythmia (IR group). Rats undergoing thoracotomy without LAD ligation were considered as the Sham group. Hemodynamic examination One week after modeling, the hemodynamic parameters including left ventricular ejection fraction (LVEF), left ventricular systolic pressure (LVSP), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic pressure (LVEDP), the maximum up rate of left ventricular pressure (+dP/dt max ), and the maximum down rate of left ventricular pressure (-dP/dt max ) were measured using a Vevo770 scanner (VisualSonics, Toronto, Canada). Infarct size measurement The infarct size was detected using 2,3,5-triphenyltetrazolium chloride (TTC) (Sangon, Shanghai, China) staining. Briefly, the ventricle was sliced into pieces with equal thickness. The slices were then incubated in 2% TTC for 15 min in the dark and fixed in 10% formaldehyde for 10 min. The infarct area was measured by an image analyzer. The infarct size was calculated as the ratio of the infarct area and total area (%). Hematoxylin-Eosin (HE) staining The ventricle was fixed in 4% formaldehyde overnight at 4°C. Followed by dehydration, vitrification, and paraffin-embedding, the tissue samples were cut into 5 μm-thick slices. The sections were then deparaffined in xylene, rehydrated in gradient ethanol, and stained with hematoxylin for 4 min and Eosin for 2 min. The histopathological changes were observed under a light microscope (400 ×). Terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining Cell apoptosis was detected using a TUNEL kit (Beyotime, Shanghai, China). Briefly, the paraffin-embedded tissue sections were deparaffined in xylene, and rehydrated in gradient ethanol. The sections were then incubated with DNase-free Proteinase K for 20 min, with 3% hydrogen peroxide (in PBS) for 10 min, and with TUNEL mix for 60 min. After 30 min of incubation with Streptavidin-HRP, the apoptotic cells were visualized using diaminobenzidine, and re-stained with hematoxylin. The apoptotic cells were counted under a light microscope (400 ×) at five randomly selected fields. Isolation of IR myocardial cells The myocardial tissues at the ischemic site were collected and homogenated. The tissue homogenate was digested with collagenase IV (0.45 mg/ml) containing 0.1% trypsin and 15 μg/ml DNase I. After centrifugation, the residue (myocardial cells) was collected. Myocardial cells were cultured in RPMI 1640 medium (Gibco, USA) containing 15% FBS, and maintained in an incubator at 37°C with 5% CO 2 . Cell transfection and grouping The rno-miR-30c-5p mimics, rno-miR-30c-5p inhibitor, SIRT1 siRNA1-3 and the negative controls (mimics NC, inhibitor NC and si-NC) were purchased from Genepharma (Shanghai, China). IR myocardial cells were seeded into 24-well plates (1 × 10 5 /well), and cultured until 80% confluence. Cells were then transfected with the above agents using Lipofectamine 3000. IR myocardial cells were randomly divided into 9 groups: IR (no treatment), inhibitor NC, rno-miR-30c-5p inhibitor, mimics NC, rno-miR-30c-5p mimics, si-NC + inhibitor NC, siRNA2 + inhibitor NC, siRNA2 + rno-miR-30c-5p inhibitor, and si-NC + rno-miR-30c-5p inhibitor group. After 48 h of transfection, cells were used for subsequent experiments. Flow cytometry Myocardial cells were washed with PBS twice and then stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) for 15 min in the dark. The apoptosis was detected by a flow cytometer (Beckman Coulter, USA). Enzyme linked immunosorbent assay (ELISA) The myocardial cells and tissues were homogenated and maintained on ice. The levels of inflammatory factors including TNF-α, IL-1β and IL-6 were detected using specific ELISA kits (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s instructions. Quantitative real-time PCR Total RNA was extracted from myocardial cells and tissues using TRIZOL (Invitrogen, USA). Total RNA was then reverse-transcribed into cDNA using a Reverse Transcription Kit (Thermo Fisher Scientific, USA). qRT-PCR was performed on a PCR instrument (Bio-Rad, USA) using SYBR Green Mixture (Roche, Switzerland). Primers were shown as follows: rno-miR-30c-5p F: 5′-GGGGTGTAAACATCCTACAC-3′, R: 5′-GTGGAGTCGGCAATTGCACT-3′; U6 F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′, R: 5′-CGCTTCAC GAATTTG CGTGTCAT-3′; SIRT1 F: 5′-AAGGAGCAGATTAGTAAGC-3′, R: 5′-TAGAGGATAAGGCGTCAT-3′; GAPDH F: 5′-GACGGCCGCATCTTCTTGT-3′, R: 5′-CACACCGACCTTCACCATTTT-3′. GAPDH and U6 with stable expression were used as internal controls of SIRT1 and rno-miR-30c-5p, respectively. Western blot Total protein was extracted from myocardial cells and tissues using RIPA lysis buffer (Beyotime, Shanghai, China). The protein samples (50 μg) were separated by 10% SDS-PAGE and then transferred onto polyvinylidenedifluoride membrane. After blocked with 5% skim milk in TBST for 2 h, the membrane was incubated with specific primary antibody (anti-Bax, 1:1000, 14796; anti-Bcl-2, 1:1000, 4228s; anti-IκBα, 1:500, #4814; anti-p-IκBα, 1:500, #2859; anti-SIRT1, 1:1000, #2310, Cell signal, USA; anti-NF-κB p65, 1:1000, SAB4502610; anti-p-NF-κB p65, 1:1000, SAB4301496, Sigma Aldrich, USA; anti-caspase-3, 1:1000, sc-271759; anti-β-actin, 1:1000, sc-517582, Santa Cruz, USA) overnight at 4°C. After washed with TBST for three times, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibody for 2 h at 25°C. The protein bands were visualized using a HRP kit and quantified by an ECL system (Thermo Fisher Scientific, USA). TargetScan prediction The targets of rno-miR-30c-5p were predicted using TargetScan 7.1 (http://www.targetscan.org/vert_71/). A total of 1249 transcripts containing 1835 sites were predicted (Table S1). A target gene SIRT1 ( ENST00000212015.6 ) was selected due to its important role in myocardial IR injury (Table S2). Dual luciferase reporter gene (DLR) assay DLR assay was used to identify the targeting relationship between SIRT1 and rno-miR-30c-5p. The fragment of SIRT1, containing the binding site was amplified and cloned into pmirGLO luciferase vector (Promega, USA) to construct wild pmirGLO-WT-SIRT1-3ʹ-UTR (SIRT1-Wt) and mutant pmirGLO-MUT-SIRT1-3ʹ-UTR (SIRT1-Mt). Myocardial cells were co-transfected with SIRT1-Wt/Mt and rno-miR-30c-5p mimics/mimics NC using Lipofectamine 3000. Myocardial cells were randomly divided into 4 groups: SIRT1-Mt + rno-miR-30c-5p mimics, SIRT1-Mt + mimics NC, SIRT1-Wt + rno-miR-30c-5p mimics, and SIRT1-Wt + mimics NC group. After 48 h of transfection, the luciferase activity was measured using a dual luciferase kit (Promega). RNA immunoprecipitation (RIP) assay RIP assay was performed using a Magna RIP Kit (Millipore, USA). Briefly, myocardial cells were lysed in lysis buffer. The cell lysate was then incubated with anti-Ago2 or IgG-coated beads at 4°C for 2 h. After washed with PBS, the RNA-protein-beads complexes were isolated using Trizol. The expression of rno-miR-30c-5p and SIRT1 was measured by qRT-PCR. Statistical analysis Three independent repetitions were conducted for each sample. Data were expressed as mean ± standard deviation (SD), and analyzed using SPSS 22.0 Statistical Software (Chicago, IL). Differences among multi-groups were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Differences between two groups were analyzed by Student’s t test. The level of statistical significance was set at p < 0.05. Results IR induces myocardial injury in rats As shown in Figure 1A, the levels of LVEF, LVSP, +dP/dt max and -dP/dt max were significantly lower, and the levels of LVEDV, LVESV and LVEDP were significantly higher in the IR group than those in the Sham group (P < 0.05). The infarct size was significantly higher in the IR group than that in the Sham group (P < 0.05) (Figure 1B). HE staining showed that the myocardial fibers in the Sham group were orderly arranged without inflammatory cell infiltration. Disorganized myocardial fibers accompanied with obvious inflammatory cell infiltration were observed in the IR group (Figure 1C). The levels of IL-6, IL-1β and TNF-α in the Sham group were significantly higher than those in the IR group (P < 0.05) (Figure 1D). In addition, TUNEL assay showed that IR significantly promoted the apoptosis of myocardial cells (P < 0.05) (Figure 1E). The protein expression of Bax, caspase-3, and p-NF-κB p65/NF-κB p65 was significantly increased, and the protein expression of Bcl-2 and p-IκBα/IκBα was significantly decreased in the IR group compared with that in the Sham group (P < 0.05) (Figure 1F and H). Note worthily, the expression of rno-miR-30c-5p was significantly higher in the IR group than that in the Sham group (P < 0.05) (Figure 1G). All these results suggested that IR could induce the myocardial injury in rats. Rno-miR-30c-5p enhances the inflammation, promotes the apoptosis, and activated NF-κB pathway in IR myocardial cells As shown in Figure 2A, the expression of rno-miR-30c-5p in IR myocardial cells was significantly decreased in the rno-miR-30c-5p inhibitor group, and increased in the rno-miR-30c-5p mimics group compared with the IR group (P < 0.05). The expression of rno-miR-30c-5p was not significantly influenced by the transfection of either inhibitor NC or mimics NC (Figure 2A). The levels of IL-6, IL-1β and TNF-α were significantly decreased in the rno-miR-30c-5p inhibitor group, and were significantly increased in the rno-miR-30c-5p mimics group compared with those in the IR group (P < 0.05) (Figure 2B). The apoptotic index was significantly lower in the ron-miR-30c-5p inhibitor group and was significantly higher in the rno-miR-30c-5p mimics group than that in the IR group (P < 0.05) (Figure 2C). In addition, the transfection of rno-miR-30c-5p inhibitor significantly decreased the protein expression of Bax, caspase-3 and p-NF-κB p65/NF-κB p65, and increased the protein expression of Bcl-2 and p-IκBα/IκBα in IR myocardial cells. The effect of rno-miR-30c-5p mimics on the expression of the above proteins was opposite to that of rno-miR-30c-5p inhibitor (P < 0.05) (Figure 2D and E). These results indicated that rno-miR-30c-5p might enhance the inflammation, promote the apoptosis and activate NF-κB pathway in IR myocardial cells. SIRT1 is the target gene of rno-miR-30c-5p As shown in Figure 3A, the expression of SIRT1 in the IR group was significantly lower than that in the Sham group (P < 0.05). The expression of rno-miR-30c-5p was negatively correlated with the expression of SIRT1 (P < 0.05) (Figure 3B). The transfection of rno-miR-30c-5p inhibitor and rno-miR-30c-5p mimics significantly increased and decreased the expression of SIRT1 in IR myocardial cells at the mRNA and protein level, respectively (P < 0.05) (Figure 3C). A binding site at 3'-UTR of SIRT1 was predicted on rno-miR-30c-5p by TargetScan (Figure 3D). DLR assay showed that the luciferase activity was significantly reduced in the SIRT1-Wt + rno-miR-30c-5p mimics group compared with that in the SIRT1-Wt + NC-mimics group (P < 0.05) (Figure 3E). RIP assay further indicated the expression of SIRT1 and rno-miR-30c-5p was significantly decreased in the Anti-IgG group compared with that in the Input group (P < 0.05) (Figure 3E). All these results suggested that SIRT1 was the target gene of rno-miR-30c-5p. Silencing of SIRT1 reversed the effects of rno-miR-30c-5p inhibitor on the apoptosis and NF-κB pathway in IR myocardial cells SIRT1 was silenced in IR myocardial cells by the transfection of siRNA1, 2 and 3. As shown in Figure 4A, the protein expression of SIRT1 was significantly decreased by the transfection of siRNA1, 2 or 3 (P < 0.05). siRNA2 with relatively high silence efficiency was used for subsequent experiments. Compared with the si-NC + inhibitor NC group, the apoptotic index was significantly increased in the siRNA2 + inhibitor NC group, and was significantly decreased in the si-NC + rno-miR-30c-5p inhibitor group (P < 0.05). In addition, the protein expression of Bax, caspase-3, and p-NF-κB p65/NF-κB p65 was significantly increased in the siRNA2 + inhibitor NC group, and was significantly decreased in the si-NC + rno-miR-30c-5p inhibitor group compared with the si-NC + inhibitor NC group (P < 0.05). The protein expression of Bcl-2 and p-IκBα/IκBα was opposite to that of Bax in different groups (P < 0.05) (Figure 4B and D). Note worthily, the effects of rno-miR-30c-5p inhibitor on the apoptosis and NF-κB pathway were reversed by the transfection of siRNA2 in IR myocardial cells (P < 0.05) (Figure 4B-D). All these results suggested rno-miR-30c-5p could promote the apoptosis, and activated NF-κB pathway in IR myocardial cells by targeting SIRT1. Discussion Myocardial infarction is one of the most common causes of death worldwide 19 . The therapeutic outcomes of patients receiving reperfusion are greatly limited by the occurrence of myocardial IR injury. It is urgent to explore the potential molecular mechanisms involving myocardial IR injury, and identify novel therapeutic targets. In this study, we demonstrated that rno-miR-30c-5p could promote the myocardial IR injury in rats through activating NF-κB pathway and down-regulating SIRT1. Myocardial IR injury often leads to inflammation, and the inflammatory cascade reaction further induces the apoptosis of myocardial cells 20 , 21 . MiRNAs exert important roles in myocardial I/R injury through regulating inflammation and cell apoptosis 22 , 23 . For example, lentivirus expressing mmu-miR-146a attenuates I/R-induced myocardial apoptosis and inflammatory cytokine production in mice 18 . Intramyocardial injection of mmu-miR322 mimics diminishes cardiac apoptosis and reduces infarct size in IR mice 24 . Overexpression of rno-miR-144 significantly reduces the myocardial injury and apoptosis in IR rats 25 . Mmu-miR-24-3p decreases the infarct area and inhibits cell apoptosis in mice with myocardial IR injury. In this study, we found that the expression of rno-miR-30c-5p was significantly up-regulated in rats with myocardial IR injury. In vitro experiments confirmed that rno-miR-30c-5p enhances the inflammation and promotes the apoptosis of IR myocardial cells. Our findings indicate that rno-miR-30c-5p may enhance the myocardial IR injury via promoting inflammation and cell apoptosis. The promoting role of rno-miR-30c-5p on myocardial IR injury is consistent with that on I/R-induced kidney and spinal cord injury. Zhou et al. 13 have proved that ron-miR-30c-5p is up-regulated in rats with I/R-induced kidney injury. Li et al. 14 have shown that hydrogen sulfide protects spinal cord and induces autophagy in a rat model of spinal cord IR injury via down-regulating rno-miR-30c-5p. Silencing of ron-miR-30c-5p may be a potential therapeutic strategy for myocardial IR injury. NF-κB is involved in the regulation of multiple biological processes including innate immunity, inflammation, cell proliferation and apoptosis 15 , 16 . Under normal physiological condition, inactive NF-κB complexes are retained in the cytoplasm by binding to inhibitor of κB (IκB) proteins 26 . The stimuli can promote the phosphorylation and subsequent degradation of IκBα, and subsequently import the active NF-κB into the nucleus 26 . More and more studies have indicated that miRNAs exert vital roles in myocardial IR injury by regulating NF-κB pathway 9 , 18 , 27 . Li et al. 9 have confirmed that ron-miR-340-5p suppresses hypoxia/reoxygenation-induced apoptosis and oxidative stress in myocardial H9C2 cells via regulating Act1/NF-κB signaling. Liu et al. 27 have reported that the inhibition of mmu-miR-27a induces high thoracic epidural block to protect mice against myocardial IR injury via activating NF-κB pathway. In this study, overexpression and silencing of rno-miR-30c-5p significantly activated and blocked NF-κB pathway in IR myocardial cells. We speculate that rno-miR-30c-5p may promote the inflammation and apoptosis of myocardial cells in rats with myocardial IR injury through activating NF-κB pathway. SIRT1 is a member of the sirtuin family that involved in the regulation of cell proliferation, apoptosis and autophagy 28 , 29 . Emerging researches have indicated that SIRT1 is a potential therapeutic target for myocardial IR injury 30 . Yu et al. 31 have indicated that melatonin ameliorates IR-induced oxidative stress and endoplasmic reticulum stress via activating SIRT1 signaling in type 2 diabetic rats. Wang et al. 32 have proved that post-ischemic treatment with lumbrokinase attenuates myocardial IR injury through the activation of Sirt1 signaling. Lin et al. 33 have demonstrated that the activation of SIRT1/Nrf2 signaling induced by Rutin contributes to the reduced oxidative stress and apoptosis of cardiomyocytes in rats with myocardial IR injury. Notably, a recent study showed that rno-miR-34a increases the apoptosis and infarct size and decreases left ventricular function through negatively regulating SIRT1 in rats with myocardial IR injury 34 . In this study, SIRT1 was identified as a target gene of rno-miR-30c-5p. We speculate that the up-regulation of SIRT1 may contribute to the promoting effect of rno-miR-30c-5p on myocardial IR injury. This speculation was further illustrated by that silencing of SIRT1 reversed the effects of rno-miR-30c-5p inhibitor on the apoptosis and NF-κB pathway in IR myocardial cells. Evidence has shown that SIRT1 inhibits the transcription of NF-κB through the deacetylation of NF-κB 35 , 36 . The up-regulation of SIRT1 may relieve myocardial IR injury through blocking NF-κB signaling. This study has some limitations. First, the regulatory role of rno-miR-30c-5p on myocardial IR injury is limited at the cellular level. The therapeutic effect of rno-miR-30c-5p silencing on rats with myocardial IR injury remains to be studied. Second, only rno-miR-30c-5p was studied. More miRNAs involving myocardial IR injury still need to be discovered based on microarray or RNA-seq methodologies. Third, only one target of rno-miR-30c-5p was selected. The discovery of more targets of rno-miR-30c-5p based on omics measurements is needed. Conclusions In conclusion, rno-miR-30c-5p was up-regulated in rats with myocardial IR injury. Rno-miR-30c-5p enhanced the inflammation, promoted the apoptosis, and activated NF-κB pathway in IR myocardial cells through targeting SIRT1. Rno-miR-30c-5p may promote the myocardial IR injury in rats through activating NF-κB pathway and down-regulating SIRT1. Our research discovers a novel regulatory mechanism of rno-miR-30c-5p in myocardial IR injury and points out a novel therapeutic target. Abbreviations left anterior descending (LAD) microRNAs (miRNAs) ischemia reperfusion (IR) Declarations Ethics approval and consent to participate: This study was conducted after obtaining Luoyang Central Hospital Affiliated to Zhengzhou University’s ethical committee approval. Consent for publication: Not applicable. Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. The genes analyzed in the present study are available at https://www.ncbi.nlm.nih.gov/search/ with Gene ID: 100314012 (microRNA-30c-5p, ENSMUSG00000065567; http://asia.ensembl.org/Mus_musculus/Gene/Summary?db=core;g=ENSMUSG00000065567;r=1:23291701-23291784;t=ENSMUST00000083633), and Gene ID: 309757 (Sirtuin 1, ENSMUSG00000020063; http://asia.ensembl.org/Mus_musculus/Gene/Summary?db=core;g=ENSMUSG00000020063;r=10:63319005-63381704). Competing interests: The authors declare that they have no competing interests. Funding: Not applicable. Author Contributions: JFC and SFX: conception, design and analysis of data, performed the data analyses and wrote the manuscript; JFC and MMZ: contributed to the conception of the study and revised the manuscript; JLW: contributed to the conception of the study; SYZ: contributed significantly to analysis and manuscript preparation and revised the manuscript; All authors have read and approved the manuscript. Acknowledgements: Not applicable. 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Toxicol In Vitro. 2012;26:1-6. van Delft MA, Huitema LF, Tas SW. The contribution of NF-kappaB signalling to immune regulation and tolerance. Eur J Clin Invest. 2015;45:529-39. Chandrasekar B, Smith JB, Freeman GL. Ischemia-reperfusion of rat myocardium activates nuclear factor-KappaB and induces neutrophil infiltration via lipopolysaccharide-induced CXC chemokine. Circulation. 2001;103:2296-302. Wang X, Ha T, Liu L, Zou J, Zhang X, Kalbfleisch J, et al. Increased expression of microRNA-146a decreases myocardial ischaemia/reperfusion injury. Cardiovasc Res. 2013;97:432-42. Yu Y, Zhang M, Hu Y, Zhao Y, Teng F, Lv X, et al. Increased Bioavailable Berberine Protects Against Myocardial Ischemia Reperfusion Injury Through Attenuation of NFkappaB and JNK Signaling Pathways. Int Heart J. 2018;59:1378-88. Hausenloy DJ, Yellon DM. Myocardial ischemia-reperfusion injury: a neglected therapeutic target. J Clin Invest. 2013;123:92-100. Wang Z, Yu J, Wu J, Qi F, Wang H, Xu Z. Scutellarin protects cardiomyocyte ischemia-reperfusion injury by reducing apoptosis and oxidative stress. Life Sci. 2016;157:200-7. Mishra PK, Tyagi N, Kumar M, Tyagi SC. MicroRNAs as a therapeutic target for cardiovascular diseases. J Cell Mol Med. 2009;13:778-89. Fan ZX, Yang J. The role of microRNAs in regulating myocardial ischemia reperfusion injury. Saudi Med J. 2015;36:787-93. Chen Z, Su X, Shen Y, Jin Y, Luo T, Kim IM, et al. MiR322 mediates cardioprotection against ischemia/reperfusion injury via FBXW7/notch pathway. J Mol Cell Cardiol. 2019;133:67-74. E L, Jiang H, Lu Z. MicroRNA-144 attenuates cardiac ischemia/reperfusion injury by targeting FOXO1. Exp Ther Med. 2019;17:2152-60. Huang W, Cui X, Chen J, Feng Y, Song E, Li J, et al. Long non-coding RNA NKILA inhibits migration and invasion of tongue squamous cell carcinoma cells via suppressing epithelial-mesenchymal transition. Oncotarget. 2016;7:62520-32. Liu JY, Shang J, Mu XD, Gao ZY. Protective effect of down-regulated microRNA-27a mediating high thoracic epidural block on myocardial ischemia-reperfusion injury in mice through regulating ABCA1 and NF-kappaB signaling pathway. Biomed Pharmacother. 2019;112:108606. Karbasforooshan H, Roohbakhsh A, Karimi G. SIRT1 and microRNAs: The role in breast, lung and prostate cancers. Exp Cell Res. 2018;367:1-6. Poulose N, Raju R. Sirtuin regulation in aging and injury. Biochim Biophys Acta. 2015;1852:2442-55. Pantazi E, Zaouali MA, Bejaoui M, Folch-Puy E, Ben Abdennebi H, Rosello-Catafau J. Role of sirtuins in ischemia-reperfusion injury. World J Gastroenterol. 2013;19:7594-602. Yu L, Liang H, Dong X, Zhao G, Jin Z, Zhai M, et al. Reduced SIRT1 signaling exacerbates myocardial ischemia reperfusion injury in type 2 diabetic rats and the protective effect of melatonin. 2015;59:376–90. Wang YH, Shun-An L, Chao-Hsin H, Hsing-Hui S, Yi-Hung C, T. CJ, et al. Sirt1 Activation by Post-ischemic Treatment With Lumbrokinase Protects Against Myocardial Ischemia-Reperfusion Injury. Frontiers in Pharmacology.9:636-. Qiao L, Xiu-Ying C, Ji Z, Yong-Liang Y, Wen Z, Bo W. Upregulation of SIRT1 contributes to the cardioprotective effect of Rutin against myocardial ischemia-reperfusion injury in rats. Journal of Functional Foods.46:227-36. Fu BC, Lang J-L, Zhang D-Y, Sun L, Chen W, Liu W, et al. Suppression of miR-34a expression in the myocardium protects against ischemia-reperfusion injury via SIRT1 protective pathway. Stem Cells & Development.scd.2017.0062. Haigis MC, Sinclair DA. Mammalian sirtuins: biological insights and disease relevance. Annu Rev Pathol. 2010;5:253-95. Yeung F, Hoberg JE, Ramsey CS, Keller MD, Jones DR, Frye RA, et al. Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase. EMBO J. 2004;23:2369-80. Supplementary Files NC3RsARRIVEGuidelinesChecklistfillable.PDF Cite Share Download PDF Status: Published Journal Publication published 20 May, 2020 Read the published version in BMC Cardiovascular Disorders → Version 5 posted Editorial decision: Accept 08 May, 2020 Editor assigned by journal 08 Apr, 2020 Submission checks completed at journal 07 Apr, 2020 Editor invited by journal 07 Apr, 2020 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6071","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":479794,"identity":"21b9502a-dc8c-4f06-be5f-9f7703b8d784","order_by":1,"name":"Jianfeng Chen","email":"","orcid":"","institution":"Luoyang Central Hospital Affiliated to Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Jianfeng","middleName":"","lastName":"Chen","suffix":""},{"id":479795,"identity":"847c8fea-2bc3-4707-9c39-fe2c5364e30f","order_by":2,"name":"Mingming Zhang","email":"","orcid":"","institution":"Luoyang Central Hospital Affiliated to Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Mingming","middleName":"","lastName":"Zhang","suffix":""},{"id":479796,"identity":"5ede44f2-a192-4054-b14d-97542cdbbe3c","order_by":3,"name":"Shouyan Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIiWNgGAWjYPCCAxDqQ4WNHBt7+wHitTDOOJNmzMdzJoF4Lcy8bYcT50k4GOBVK9+/+NnDL3/uyBncSH72gOfM4fQ2CYYEhh8V23BqYZzxzNxYtu2ZscGNNHMDiYr03DbpxgOMPWdu49TCLHHATFqy4XDihhsJZhIGZ6xz22QOJDAztuHWwiZx/Ju0xB+QlvRvEoltzOlsEgkGeLXw8PeYSX5gA2nJMZM42OacQFCLhARPmTRj22FjyTNvyiQbzqQZtgED+SA+v8j3H98m+ePPYTm+4+nbpP9U2MjLt7cffPCjArcWBokEBmYeEEMgASF4ALd6IOA/wMD4A8oYBaNgFIyCUYAVAAApyWArCY27GQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4483-8131","institution":"Luoyang Central Hospital Affiliated to Zhengzhou University","correspondingAuthor":true,"prefix":"","firstName":"Shouyan","middleName":"","lastName":"Zhang","suffix":""},{"id":479797,"identity":"e78824e3-8ba9-40ce-aff9-c8a6f6759a5b","order_by":4,"name":"Junlong Wu","email":"","orcid":"","institution":"Luoyang Central Hospital Affiliated to Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Junlong","middleName":"","lastName":"Wu","suffix":""},{"id":479798,"identity":"2bc58266-bdbe-4ebd-92f7-b4eb5fd38545","order_by":5,"name":"Shufeng Xue","email":"","orcid":"","institution":"Luoyang Central Hospital Affiliated to Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Shufeng","middleName":"","lastName":"Xue","suffix":""}],"badges":[],"createdAt":"2019-09-25 15:21:20","currentVersionCode":5,"declarations":"","doi":"10.21203/rs.2.15405/v5","doiUrl":"https://doi.org/10.21203/rs.2.15405/v5","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12872-020-01520-2","type":"published","date":"2020-05-20T20:27:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":899243,"identity":"ff5a267f-1d04-4830-894a-1a80d29cbd38","added_by":"auto","created_at":"2020-04-14 19:37:14","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":528115,"visible":true,"origin":"","legend":"IR induced myocardial injury in rats. (A) The levels of LVEF, LVEDV, LVESV, LVSP, LVEDP, +dP/dtmax and -dP/dtmax (N = 25 each group). (B) Infarct size (N = 10 each group). (C) HE staining of myocardial tissues (× 400, N = 5 each group, representative images were shown). (D) The levels of IL-1β, IL-6 and TNF-α were detected by ELISA (N = 10 each group). (E) The apoptotic cells (%) were measured by TUNEL staining (N = 5 each group, representative images were shown). (F) The expression of Bax, Bcl-2 and caspase-3 was detected by Western blot (N = 5 each group, representative images were shown). (G) The expression of rno-miR-30c-5p was detected by qRT-PCR (N = 5 each group). (H) The expression of p-IκBα, IκBα, p-NF-κB p65 and NF-κB p65 was detected by Western blot (N = 5 each group, representative images were shown). Data were presented as mean ± SD (three independent repetitions for each sample). *P \u003c 0.05, vs. Sham group.","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6071/v5/Fig.1.jpg"},{"id":899246,"identity":"1eaf944d-dd78-4ba4-80a5-75ca4ed4ddd6","added_by":"auto","created_at":"2020-04-14 19:37:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":496556,"visible":true,"origin":"","legend":"Rno-miR-30c-5p enhanced the inflammation, promoted the apoptosis, and activated NF-κB pathway in IR myocardial cells. (A) The mRNA expression of rno-miR-30c-5p was detected by qRT-PCR (N = 3 each group). (B) The levels of IL-1β, IL-6 and TNF-α were detected by ELISA (N = 3 each group). (C) The apoptotic index (%) was detected by Flow cytometry (N = 3 each group). (D) The expression of Bax, Bcl-2 and caspase-3 was detected by Western blot (N = 3 each group). (E) The expression of p-IκBα, IκBα, p-NF-κB p65 and NF-κB p65 was detected by Western blot (N = 3 each group). Data were presented as mean ± SD (three independent repetitions for each sample). *P \u003c 0.05, vs. IR and inhibitor NC group; #P \u003c 0.05, vs. IR and mimics NC group.","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6071/v5/Fig.2.jpg"},{"id":899248,"identity":"377ab4c5-1cca-4920-ba71-0eea6de0c911","added_by":"auto","created_at":"2020-04-14 19:37:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":327898,"visible":true,"origin":"","legend":"SIRT1 was the target gene of rno-miR-30c-5p. (A) The expression of rno-miR-30c-5p was detected by qRT-PCR (N = 5 each group). (B) Correlation analysis between the expression of rno-miR-30c-5p and SIRT1. (C) The mRNA and protein expression of SIRT1 were detected by qRT-PCR and Western blot (N = 3 each group). (D) The binding site of rno-miR-30c-5p at 3'-UTR of SIRT1 was predicted by TargetScan software. (E) The interaction between rno-miR-30c-5p and SIRT1 was analyzed by DLR assay. (F) The interaction between rno-miR-30c-5p and SIRT1 was analyzed by RIP assay. Data were presented as mean ± SD (three independent repetitions for each sample). *P \u003c 0.05, vs. Sham group (A); *P \u003c 0.05, vs. IR and inhibitor NC group, #P \u003c 0.05, vs. IR and mimics NC group (C); *P \u003c 0.05, vs. NC-mimics group (E); *P \u003c 0.05, vs. Anti-IgG group (F).","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6071/v5/Fig.3.jpg"},{"id":899250,"identity":"cebdc8d8-ce42-4619-86f8-07bac0c0588d","added_by":"auto","created_at":"2020-04-14 19:37:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":553408,"visible":true,"origin":"","legend":"Rno-miR-30c-5p promoted the apoptosis, and activated NF-κB pathway in IR myocardial cells by targeting SIRT1. (A) The expression of SIRT1 was detected by Western blot (N = 3 each group). (B) The expression of p-IκBα, IκBα, p-NF-κB p65 and NF-κB p65 was detected by Western blot (N = 3 each group). (C) The apoptotic index (%) was measured by flow cytometry (N = 3 each group). (D) The expression of Bax, Bcl-2 and caspase-3 was detected by Western blot (N = 3 each group). Data were presented as mean ± SD (three independent repetitions for each sample). *P \u003c 0.05, vs. Control and si-NC group (A); *P \u003c 0.05, vs. si-NC + inhibitor NC group, #P \u003c 0.05, vs. siRNA2 + inhibitor NC group (B-D).","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6071/v5/Fig.4.jpg"},{"id":13498605,"identity":"e3bbddb6-40ac-4496-b2d6-858424fea5c5","added_by":"auto","created_at":"2021-09-16 22:58:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":928091,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6071/v5/233be574-6c9c-4d52-9768-b82cd0d1ae90.pdf"},{"id":899245,"identity":"01813387-5102-40a1-aa9c-eac59b157ce7","added_by":"auto","created_at":"2020-04-14 19:37:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1127987,"visible":true,"origin":"","legend":"","description":"","filename":"NC3RsARRIVEGuidelinesChecklistfillable.PDF","url":"https://assets-eu.researchsquare.com/files/rs-6071/v5/NC3Rs ARRIVE Guidelines Checklist (fillable).PDF"}],"financialInterests":"","formattedTitle":"Rno-microRNA-30c-5p promotes myocardial ischemia reperfusion injury in rats through activating NF-κB pathway and targeting SIRT1","fulltext":[{"header":"Background","content":"\u003cp\u003eIschemic heart disease is a series of diseases characterized by myocardial ischemia, such as angina pectoris and myocardial infarction \u003ca href=\"#_ENREF_1\"\u003e\u003csup\u003e1\u003c/sup\u003e\u003c/a\u003e. Recently, reperfusion of the ischemic myocardium is one of the most common therapeutic strategies for ischemic heart diseases \u003ca href=\"#_ENREF_2\"\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/a\u003e. Although restoring blood flow in time can relieve myocardial infarction to a great extent, the prognosis of patients remains poor due to the ischemia reperfusion (IR) injury on myocardium \u003ca href=\"#_ENREF_3\"\u003e\u003csup\u003e3\u003c/sup\u003e\u003c/a\u003e. Therefore, it is urgent to find out novel therapeutic methods and targets for myocardial IR injury.\u003c/p\u003e\n\u003cp\u003eThe genome-wide investigations of genetic variants, epigenetic modifications, and gene expression profiles optimize the search for novel diagnostic or therapeutic targets for IR injury in the post-genomic era \u003ca href=\"#_ENREF_4\"\u003e\u003csup\u003e4\u003c/sup\u003e\u003c/a\u003e. MicroRNAs (miRNAs) are a class of small endogenous noncoding RNAs with 19-25 nucleotides in length, which modulate gene expression at the post-transcriptional level \u003csup\u003e\u003ca href=\"#_ENREF_5\"\u003e5\u003c/a\u003e,\u003ca href=\"#_ENREF_6\"\u003e6\u003c/a\u003e\u003c/sup\u003e. A systematic comparison of IR injury-induced miRNA expression changes in rats identifies several potential cardioprotective miRNA targets (protectomiRs), including Rno-miR-125b*, -139-3p, -320, -532-3p, and -188 \u003ca href=\"#_ENREF_7\"\u003e\u003csup\u003e7\u003c/sup\u003e\u003c/a\u003e. By using bioinformatics methods based on topological or network dynamical approaches, the mRNA targets of protectomiRs can be predicated. Nevertheless, all unbiased omics approaches and their bioinformatic evaluation need to be verified by rigorous experimental validation at the transcript and protein levels \u003ca href=\"#_ENREF_8\"\u003e\u003csup\u003e8\u003c/sup\u003e\u003c/a\u003e.\u003c/p\u003e\n\u003cp\u003eRecently, studies have indicated that miRNAs play important regulatory roles in myocardial IR injury \u003ca href=\"#_ENREF_9\"\u003e\u003csup\u003e9\u003c/sup\u003e\u003c/a\u003e. Yuan et al. \u003ca href=\"#_ENREF_10\"\u003e\u003csup\u003e10\u003c/sup\u003e\u003c/a\u003e have proved that the inhibition of rno-miR-181b-5p protects cardiomyocytes against I/R injury through targeting AKT3 and PI3KR3. Zhao et al. \u003ca href=\"#_ENREF_11\"\u003e\u003csup\u003e11\u003c/sup\u003e\u003c/a\u003e have reported that mmu-miR-374a protects against myocardial IR injury in mice via targeting MAPK6 pathway. Song et al. \u003ca href=\"#_ENREF_12\"\u003e\u003csup\u003e12\u003c/sup\u003e\u003c/a\u003e have indicated that rno-miR-30b overexpression has anti-apoptotic effect on cardiomyocytes at early phase of myocardial IR injury in a rat model. MiR-30c-5p is another subtype of miR-30 that also involved in the process of IR injury. Zhou et al. \u003ca href=\"#_ENREF_13\"\u003e\u003csup\u003e13\u003c/sup\u003e\u003c/a\u003e have proved that rno-miR-30c-5p is a potential diagnostic marker for I/R-induced kidney injury in rats.\u0026nbsp;Li et al. \u003ca href=\"#_ENREF_14\"\u003e\u003csup\u003e14\u003c/sup\u003e\u003c/a\u003e have shown that hydrogen sulfide protects spinal cord and induces autophagy in a rat model of spinal cord IR injury via regulating rno-miR-30c-5p. However, the regulatory effect and mechanism of rno-miR-30c-5p on myocardial IR injury remain unclear.\u003c/p\u003e\n\u003cp\u003eNuclear factor \u0026kappa;B (NF-\u0026kappa;B) is involved in the regulation of multiple biological functions including innate immunity, inflammation, cell proliferation and apoptosis \u003csup\u003e\u003ca href=\"#_ENREF_15\"\u003e15\u003c/a\u003e,\u003ca href=\"#_ENREF_16\"\u003e16\u003c/a\u003e\u003c/sup\u003e. Accumulating researches have revealed that myocardial IR injury is associated with the activation of NF-\u0026kappa;B \u003ca href=\"#_ENREF_17\"\u003e\u003csup\u003e17\u003c/sup\u003e\u003c/a\u003e. In addition, emerging evidence has indicated that miRNAs play vital roles in myocardial IR injury by regulating NF-\u0026kappa;B pathway. For instance, mmu-miR-146a\u0026nbsp;overexpression reduces myocardial IR injury via inhibiting the activation of NF-\u0026kappa;B pathway \u003ca href=\"#_ENREF_18\"\u003e\u003csup\u003e18\u003c/sup\u003e\u003c/a\u003e. However, whether the regulatory effect of rno-miR-30c-5p on myocardial IR injury is involved in NF-\u0026kappa;B pathway is unknown.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In this study, we explored the regulatory effect of rno-miR-30c-5p on myocardial IR injury in rats, as well as the underlying molecular mechanisms. Our results indicated that rno-miR-30c-5p promoted the myocardial IR injury in rats through activating NF-\u0026kappa;B pathway and down-regulating SIRT1. Our findings may provide a new theoretical foundation for the treatment of myocardial IR injury in clinical practice.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimals\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale Sprague-Dawley (SD) rats (weighting 180-200 g) were provided by Peking University Laboratory Animal Center. All rats were kept at 22-24\u0026deg;C and 55-60% humidity on a 12 h light-dark cycle with free access to water and food. At the end of the study, all rats (220-270g) were anesthetized by an intraperitoneal injection of 50 mg/kg pentobarbital sodium, and then sacrificed by cervical dislocation. All animal experiments were conducted strictly in accordance with the National Institutes of Health guide for the care and use of Laboratory animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEstablishment of the myocardial IR model\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e in rats\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats weighing 200-240g were used to establish the IR model. Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). The left anterior descending coronary artery (LAD) was ligated using 6-0 silk suture slipknot for 30 min, and then reperfused for 2 h. Myocardial ischemia was confirmed by the appearance of regional epicardial cyanosis over the myocardial surface and by arrhythmia. Successful reperfusion was confirmed by the disappearance of epicardial cyanosis and the production of epicardial hyperemia and arrhythmia (IR group). Rats undergoing thoracotomy without LAD ligation were considered as the Sham group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHemodynamic examination \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne week after modeling, the hemodynamic parameters including left ventricular ejection fraction (LVEF), left ventricular systolic pressure (LVSP), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic pressure (LVEDP), the maximum up rate of left ventricular pressure (+dP/dt\u003csub\u003emax\u003c/sub\u003e), and the maximum down rate of left ventricular pressure (-dP/dt\u003csub\u003emax\u003c/sub\u003e) were measured using a Vevo770\u0026nbsp;scanner (VisualSonics, Toronto, Canada).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInfarct size measurement\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe infarct size was detected using 2,3,5-triphenyltetrazolium chloride (TTC) (Sangon, Shanghai, China) staining. Briefly, the ventricle was sliced into pieces with equal thickness. The slices were then incubated in 2% TTC for 15 min in the dark and fixed in 10% formaldehyde for 10 min. The infarct area was measured by an image analyzer. The infarct size was calculated as the ratio of the infarct area and total area (%).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHematoxylin-Eosin (HE) staining\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ventricle was fixed in 4% formaldehyde overnight at 4\u0026deg;C. Followed by dehydration, vitrification, and paraffin-embedding, the tissue samples were cut into 5 \u0026mu;m-thick slices. The sections were then deparaffined in xylene, rehydrated in gradient ethanol, and stained with hematoxylin for 4 min and Eosin for 2 min. The histopathological changes were observed under a light microscope (400 \u0026times;).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTerminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell apoptosis was detected using a TUNEL kit (Beyotime, Shanghai, China). Briefly, the paraffin-embedded tissue sections were deparaffined in xylene, and rehydrated in gradient ethanol. The sections were then incubated with DNase-free Proteinase K for 20 min, with 3% hydrogen peroxide (in PBS) for 10 min, and with TUNEL mix for 60 min. After 30 min of incubation with Streptavidin-HRP, the apoptotic cells were visualized using diaminobenzidine, and re-stained with hematoxylin. The apoptotic cells were counted under a light microscope (400 \u0026times;) at five randomly selected fields.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIsolation of IR myocardial cells\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe myocardial tissues at the ischemic site were collected and homogenated. The tissue homogenate was digested with collagenase IV (0.45 mg/ml) containing 0.1% trypsin and 15 \u0026mu;g/ml DNase I. After centrifugation, the residue (myocardial cells) was collected. Myocardial cells were cultured in RPMI 1640 medium (Gibco, USA) containing 15% FBS, and maintained in an incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell transfection and grouping\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rno-miR-30c-5p mimics, rno-miR-30c-5p inhibitor, SIRT1 siRNA1-3 and the negative controls (mimics NC, inhibitor NC and si-NC) were purchased from Genepharma (Shanghai, China). IR myocardial cells were seeded into 24-well plates (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e/well), and cultured until 80% confluence. Cells were then transfected with the above agents using Lipofectamine 3000. IR myocardial cells were randomly divided into 9 groups: IR (no treatment), inhibitor NC, rno-miR-30c-5p inhibitor, mimics NC, rno-miR-30c-5p mimics, si-NC + inhibitor NC, siRNA2 + inhibitor NC, siRNA2 + rno-miR-30c-5p inhibitor, and si-NC + rno-miR-30c-5p inhibitor group. After 48 h of transfection, cells were used for subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFlow cytometry\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMyocardial cells were washed with PBS twice and then stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) for 15 min in the dark. The apoptosis was detected by a flow cytometer (Beckman Coulter, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEnzyme linked immunosorbent assay (ELISA)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe myocardial cells and tissues were homogenated and maintained on ice. The levels of inflammatory factors including TNF-\u0026alpha;, IL-1\u0026beta; and IL-6 were detected using specific ELISA kits (Thermo Fisher Scientific, USA) in accordance with the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eQuantitative real-time PCR\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from myocardial cells and tissues using TRIZOL (Invitrogen, USA). Total RNA was then reverse-transcribed into cDNA using a Reverse Transcription Kit (Thermo Fisher Scientific, USA). qRT-PCR was performed on a PCR instrument (Bio-Rad, USA) using SYBR Green Mixture (Roche, Switzerland). Primers were shown as follows: rno-miR-30c-5p F: 5\u0026prime;-GGGGTGTAAACATCCTACAC-3\u0026prime;, R: 5\u0026prime;-GTGGAGTCGGCAATTGCACT-3\u0026prime;; U6 F: 5\u0026prime;-GCTTCGGCAGCACATATACTAAAAT-3\u0026prime;, R: 5\u0026prime;-CGCTTCAC GAATTTG CGTGTCAT-3\u0026prime;; SIRT1 F: 5\u0026prime;-AAGGAGCAGATTAGTAAGC-3\u0026prime;, R: 5\u0026prime;-TAGAGGATAAGGCGTCAT-3\u0026prime;; GAPDH F: 5\u0026prime;-GACGGCCGCATCTTCTTGT-3\u0026prime;, R: 5\u0026prime;-CACACCGACCTTCACCATTTT-3\u0026prime;. GAPDH and U6 with stable expression were used as internal controls of SIRT1 and rno-miR-30c-5p, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWestern blot\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal protein was extracted from myocardial cells and tissues using RIPA lysis buffer (Beyotime, Shanghai, China). The protein samples (50 \u0026mu;g) were separated by 10% SDS-PAGE and then transferred onto polyvinylidenedifluoride membrane. After blocked with 5% skim milk in TBST for 2 h, the membrane was incubated with specific primary antibody (anti-Bax, 1:1000, 14796; anti-Bcl-2, 1:1000, 4228s; anti-I\u0026kappa;B\u0026alpha;, 1:500, #4814; anti-p-I\u0026kappa;B\u0026alpha;, 1:500, #2859; anti-SIRT1, 1:1000, #2310, Cell signal, USA; anti-NF-\u0026kappa;B p65, 1:1000, SAB4502610; anti-p-NF-\u0026kappa;B p65, 1:1000, SAB4301496, Sigma Aldrich, USA; anti-caspase-3, 1:1000, sc-271759; anti-\u0026beta;-actin, 1:1000, sc-517582, Santa Cruz, USA) overnight at 4\u0026deg;C. After washed with TBST for three times, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibody for 2 h at 25\u0026deg;C. The protein bands were visualized using a HRP kit and quantified by an ECL system (Thermo Fisher Scientific, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTargetScan prediction\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe targets of rno-miR-30c-5p were predicted using TargetScan 7.1 (http://www.targetscan.org/vert_71/). A total of 1249\u0026nbsp;transcripts containing 1835 sites were predicted (Table S1). A target gene SIRT1 (\u003ca href=\"http://www.ensembl.org/Homo_sapiens/Transcript/Summary?db=core;t=ENST00000212015.6\"\u003eENST00000212015.6\u003c/a\u003e) was selected due to its important role in myocardial IR injury (Table S2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDual luciferase reporter gene (DLR) assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDLR assay was used to identify the targeting relationship between SIRT1 and rno-miR-30c-5p. The fragment of SIRT1, containing the binding site was amplified and cloned into pmirGLO luciferase vector (Promega, USA) to construct wild pmirGLO-WT-SIRT1-3ʹ-UTR (SIRT1-Wt) and mutant pmirGLO-MUT-SIRT1-3ʹ-UTR (SIRT1-Mt). Myocardial cells were co-transfected with SIRT1-Wt/Mt and rno-miR-30c-5p mimics/mimics NC using Lipofectamine 3000. Myocardial cells were randomly divided into 4 groups: SIRT1-Mt + rno-miR-30c-5p mimics, SIRT1-Mt + mimics NC, SIRT1-Wt + rno-miR-30c-5p mimics, and SIRT1-Wt + mimics NC group. After 48 h of transfection, the luciferase activity was measured using a dual luciferase kit (Promega).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRNA immunoprecipitation (RIP) assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRIP assay was performed using a Magna RIP Kit (Millipore, USA). Briefly, myocardial cells were lysed in lysis buffer. The cell lysate was then incubated with anti-Ago2 or IgG-coated beads at 4\u0026deg;C for 2 h. After washed with PBS, the RNA-protein-beads complexes were isolated using Trizol. The expression of rno-miR-30c-5p and SIRT1 was measured by qRT-PCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree\u0026nbsp;independent\u0026nbsp;repetitions were conducted for each sample. Data were expressed as mean \u0026plusmn; standard deviation (SD), and analyzed using SPSS 22.0 Statistical Software (Chicago, IL). Differences among multi-groups were analyzed by one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. Differences between two groups were analyzed by Student\u0026rsquo;s t test. The level of statistical significance was set at p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIR induces myocardial injury in rats\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 1A, the levels of LVEF, LVSP, +dP/dt\u003csub\u003emax\u003c/sub\u003e and -dP/dt\u003csub\u003emax\u003c/sub\u003e were significantly lower, and the levels of LVEDV, LVESV and LVEDP were significantly higher in the IR group than those in the Sham group (P \u0026lt; 0.05). The infarct size was significantly higher in the IR group than that in the Sham group (P \u0026lt; 0.05) (Figure 1B). HE staining showed that the myocardial fibers in the Sham group were orderly arranged without inflammatory cell infiltration. Disorganized myocardial fibers accompanied with obvious inflammatory cell infiltration were observed in the IR group (Figure 1C). The levels of IL-6, IL-1\u0026beta; and TNF-\u0026alpha; in the Sham group were significantly higher than those in the IR group (P \u0026lt; 0.05) (Figure 1D). In addition, TUNEL assay showed that IR significantly promoted the apoptosis of myocardial cells (P \u0026lt; 0.05) (Figure 1E). The protein expression of Bax, caspase-3, and p-NF-\u0026kappa;B p65/NF-\u0026kappa;B p65 was significantly increased, and the protein expression of Bcl-2 and p-I\u0026kappa;B\u0026alpha;/I\u0026kappa;B\u0026alpha; was significantly decreased in the IR group compared with that in the Sham group (P \u0026lt; 0.05) (Figure 1F and H). Note worthily, the expression of rno-miR-30c-5p was significantly higher in the IR group than that in the Sham group (P \u0026lt; 0.05) (Figure 1G). All these results suggested that IR could induce the myocardial injury in rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRno-miR-30c-5p enhances the inflammation, promotes the apoptosis, and activated NF-\u0026kappa;B pathway in IR myocardial cells\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 2A, the expression of rno-miR-30c-5p in IR myocardial cells was significantly decreased in the rno-miR-30c-5p inhibitor group, and increased in the rno-miR-30c-5p mimics group compared with the IR group (P \u0026lt; 0.05). The expression of rno-miR-30c-5p was not significantly influenced by the transfection of either inhibitor NC or mimics NC (Figure 2A). The levels of IL-6, IL-1\u0026beta; and TNF-\u0026alpha; were significantly decreased in the rno-miR-30c-5p inhibitor group, and were significantly increased in the rno-miR-30c-5p mimics group compared with those in the IR group (P \u0026lt; 0.05) (Figure 2B). The apoptotic index was significantly lower in the ron-miR-30c-5p inhibitor group and was significantly higher in the rno-miR-30c-5p mimics group than that in the IR group (P \u0026lt; 0.05) (Figure 2C). In addition, the transfection of rno-miR-30c-5p inhibitor significantly decreased the protein expression of Bax, caspase-3 and p-NF-\u0026kappa;B p65/NF-\u0026kappa;B p65, and increased the protein expression of Bcl-2 and p-I\u0026kappa;B\u0026alpha;/I\u0026kappa;B\u0026alpha; in IR myocardial cells. The effect of rno-miR-30c-5p mimics on the expression of the above proteins was opposite to that of rno-miR-30c-5p inhibitor (P \u0026lt; 0.05) (Figure 2D and E). These results indicated that rno-miR-30c-5p might enhance the inflammation, promote the apoptosis and activate NF-\u0026kappa;B pathway in IR myocardial cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSIRT1 is the target gene of rno-miR-30c-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 3A, the expression of SIRT1 in the IR group was significantly lower than that in the Sham group (P \u0026lt; 0.05). The expression of rno-miR-30c-5p was negatively correlated with the expression of SIRT1 (P \u0026lt; 0.05) (Figure 3B). The transfection of rno-miR-30c-5p inhibitor and rno-miR-30c-5p mimics significantly increased and decreased the expression of SIRT1 in IR myocardial cells at the mRNA and protein level, respectively (P \u0026lt; 0.05) (Figure 3C). A binding site at 3'-UTR of SIRT1 was predicted on rno-miR-30c-5p by TargetScan (Figure 3D). DLR assay showed that the luciferase activity was significantly reduced in the SIRT1-Wt + rno-miR-30c-5p mimics group compared with that in the SIRT1-Wt + NC-mimics group (P \u0026lt; 0.05) (Figure 3E). RIP assay further indicated the expression of SIRT1 and rno-miR-30c-5p was significantly decreased in the Anti-IgG group compared with that in the Input group (P \u0026lt; 0.05) (Figure 3E). All these results suggested that SIRT1 was the target gene of rno-miR-30c-5p.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSilencing of SIRT1 reversed the effects of rno-miR-30c-5p inhibitor on the apoptosis and NF-\u0026kappa;B pathway in IR myocardial cells\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSIRT1 was silenced in IR myocardial cells by the transfection of siRNA1, 2 and 3. As shown in Figure 4A, the protein expression of SIRT1 was significantly decreased by the transfection of siRNA1, 2 or 3 (P \u0026lt; 0.05). siRNA2 with relatively high silence efficiency was used for subsequent experiments. Compared with the si-NC + inhibitor NC group, the apoptotic index was significantly increased in the siRNA2 + inhibitor NC group, and was significantly decreased in the si-NC + rno-miR-30c-5p inhibitor group (P \u0026lt; 0.05). In addition, the protein expression of Bax, caspase-3, and p-NF-\u0026kappa;B p65/NF-\u0026kappa;B p65 was significantly increased in the siRNA2 + inhibitor NC group, and was significantly decreased in the si-NC + rno-miR-30c-5p inhibitor group compared with the si-NC + inhibitor NC group (P \u0026lt; 0.05). The protein expression of Bcl-2 and p-I\u0026kappa;B\u0026alpha;/I\u0026kappa;B\u0026alpha; was opposite to that of Bax in different groups (P \u0026lt; 0.05) (Figure 4B and D). Note worthily, the effects of rno-miR-30c-5p inhibitor on the apoptosis and NF-\u0026kappa;B pathway were reversed by the transfection of siRNA2 in IR myocardial cells (P \u0026lt; 0.05) (Figure 4B-D). All these results suggested rno-miR-30c-5p could promote the apoptosis, and activated NF-\u0026kappa;B pathway in IR myocardial cells by targeting SIRT1.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMyocardial infarction is one of the most common causes of death worldwide \u003ca href=\"#_ENREF_19\"\u003e\u003csup\u003e19\u003c/sup\u003e\u003c/a\u003e.\u003c/p\u003e\n\u003cp\u003eThe therapeutic outcomes of patients receiving reperfusion are greatly limited by the occurrence of myocardial IR injury. It is urgent to explore the potential molecular mechanisms involving myocardial IR injury, and identify novel therapeutic targets. In this study, we demonstrated that rno-miR-30c-5p could promote the myocardial IR injury in rats through activating NF-\u0026kappa;B pathway and down-regulating SIRT1.\u003c/p\u003e\n\u003cp\u003eMyocardial IR injury often leads to inflammation, and the inflammatory cascade reaction further induces the apoptosis of myocardial cells \u003csup\u003e\u003ca href=\"#_ENREF_20\"\u003e20\u003c/a\u003e,\u003ca href=\"#_ENREF_21\"\u003e21\u003c/a\u003e\u003c/sup\u003e. MiRNAs exert important roles in myocardial I/R injury through regulating inflammation and cell apoptosis \u003csup\u003e\u003ca href=\"#_ENREF_22\"\u003e22\u003c/a\u003e,\u003ca href=\"#_ENREF_23\"\u003e23\u003c/a\u003e\u003c/sup\u003e. For example, lentivirus expressing mmu-miR-146a attenuates I/R-induced myocardial apoptosis and inflammatory cytokine production in mice \u003ca href=\"#_ENREF_18\"\u003e\u003csup\u003e18\u003c/sup\u003e\u003c/a\u003e. Intramyocardial injection of mmu-miR322 mimics diminishes cardiac apoptosis and reduces infarct size in IR mice \u003ca href=\"#_ENREF_24\"\u003e\u003csup\u003e24\u003c/sup\u003e\u003c/a\u003e. Overexpression of rno-miR-144 significantly reduces the myocardial\u0026nbsp;injury\u0026nbsp;and apoptosis in IR rats \u003ca href=\"#_ENREF_25\"\u003e\u003csup\u003e25\u003c/sup\u003e\u003c/a\u003e. Mmu-miR-24-3p decreases the infarct area and inhibits cell apoptosis in mice with myocardial IR injury. In this study, we found that the expression of rno-miR-30c-5p was significantly up-regulated in rats with myocardial IR injury. In vitro experiments confirmed that rno-miR-30c-5p enhances the inflammation and promotes the apoptosis of IR myocardial cells. Our findings indicate that rno-miR-30c-5p may enhance the myocardial\u0026nbsp;IR injury via promoting inflammation and cell apoptosis. The promoting role of rno-miR-30c-5p on myocardial\u0026nbsp;IR injury is consistent with that on I/R-induced kidney and spinal cord injury. Zhou et al. \u003ca href=\"#_ENREF_13\"\u003e\u003csup\u003e13\u003c/sup\u003e\u003c/a\u003e have proved that ron-miR-30c-5p is up-regulated in rats with I/R-induced kidney injury.\u0026nbsp;Li et al. \u003ca href=\"#_ENREF_14\"\u003e\u003csup\u003e14\u003c/sup\u003e\u003c/a\u003e have shown that hydrogen sulfide protects spinal cord and induces autophagy in a rat model of spinal cord IR injury via down-regulating rno-miR-30c-5p. Silencing of ron-miR-30c-5p may be a potential therapeutic strategy for myocardial\u0026nbsp;IR injury.\u003c/p\u003e\n\u003cp\u003eNF-\u0026kappa;B is involved in the regulation of multiple biological processes including innate immunity, inflammation, cell proliferation and apoptosis \u003csup\u003e\u003ca href=\"#_ENREF_15\"\u003e15\u003c/a\u003e,\u003ca href=\"#_ENREF_16\"\u003e16\u003c/a\u003e\u003c/sup\u003e. Under normal physiological condition, inactive\u0026nbsp;NF-\u0026kappa;B complexes are retained in the\u0026nbsp;cytoplasm\u0026nbsp;by binding to inhibitor of \u0026kappa;B (I\u0026kappa;B) proteins \u003ca href=\"#_ENREF_26\"\u003e\u003csup\u003e26\u003c/sup\u003e\u003c/a\u003e. The stimuli can promote the phosphorylation and subsequent degradation of I\u0026kappa;B\u0026alpha;, and subsequently import the active NF-\u0026kappa;B into the nucleus \u003ca href=\"#_ENREF_26\"\u003e\u003csup\u003e26\u003c/sup\u003e\u003c/a\u003e. More and more studies have indicated that miRNAs exert vital roles in myocardial IR injury by regulating NF-\u0026kappa;B pathway \u003csup\u003e\u003ca href=\"#_ENREF_9\"\u003e9\u003c/a\u003e,\u003ca href=\"#_ENREF_18\"\u003e18\u003c/a\u003e,\u003ca href=\"#_ENREF_27\"\u003e27\u003c/a\u003e\u003c/sup\u003e. Li et al. \u003ca href=\"#_ENREF_9\"\u003e\u003csup\u003e9\u003c/sup\u003e\u003c/a\u003e have confirmed that ron-miR-340-5p suppresses hypoxia/reoxygenation-induced apoptosis and oxidative stress in myocardial H9C2 cells via regulating Act1/NF-\u0026kappa;B signaling. Liu et al. \u003ca href=\"#_ENREF_27\"\u003e\u003csup\u003e27\u003c/sup\u003e\u003c/a\u003e have reported that the inhibition of mmu-miR-27a induces high thoracic epidural block to protect mice against myocardial IR injury via activating NF-\u0026kappa;B pathway. In this study, overexpression and silencing of rno-miR-30c-5p significantly activated and blocked NF-\u0026kappa;B pathway in IR myocardial cells. We speculate that rno-miR-30c-5p may promote the inflammation and apoptosis of myocardial cells in rats with myocardial IR injury through activating NF-\u0026kappa;B pathway.\u003c/p\u003e\n\u003cp\u003eSIRT1 is a member of the sirtuin family that involved in the regulation of cell proliferation, apoptosis and autophagy \u003csup\u003e\u003ca href=\"#_ENREF_28\"\u003e28\u003c/a\u003e,\u003ca href=\"#_ENREF_29\"\u003e29\u003c/a\u003e\u003c/sup\u003e. Emerging researches have indicated that SIRT1 is a potential therapeutic target for myocardial IR injury \u003ca href=\"#_ENREF_30\"\u003e\u003csup\u003e30\u003c/sup\u003e\u003c/a\u003e. Yu et al. \u003ca href=\"#_ENREF_31\"\u003e\u003csup\u003e31\u003c/sup\u003e\u003c/a\u003e have indicated that melatonin ameliorates IR-induced oxidative stress and endoplasmic reticulum stress via activating SIRT1 signaling in type 2 diabetic rats. Wang et al. \u003ca href=\"#_ENREF_32\"\u003e\u003csup\u003e32\u003c/sup\u003e\u003c/a\u003e have proved that post-ischemic treatment with lumbrokinase attenuates myocardial IR injury through the activation of Sirt1 signaling. Lin et al. \u003ca href=\"#_ENREF_33\"\u003e\u003csup\u003e33\u003c/sup\u003e\u003c/a\u003e have demonstrated that the activation of SIRT1/Nrf2 signaling induced by Rutin contributes to the reduced oxidative stress and apoptosis of cardiomyocytes in rats with myocardial IR injury. Notably, a recent study showed that rno-miR-34a increases the apoptosis and infarct size and decreases left ventricular function through negatively regulating SIRT1 in rats with myocardial IR injury \u003ca href=\"#_ENREF_34\"\u003e\u003csup\u003e34\u003c/sup\u003e\u003c/a\u003e. In this study, SIRT1 was identified as a target gene of rno-miR-30c-5p. We speculate that the up-regulation of SIRT1 may contribute to the promoting effect of rno-miR-30c-5p on myocardial\u0026nbsp;IR injury. This speculation was further illustrated by that silencing of SIRT1 reversed the effects of rno-miR-30c-5p inhibitor on the apoptosis and NF-\u0026kappa;B pathway in IR myocardial cells. Evidence has shown that SIRT1 inhibits the transcription of NF-\u0026kappa;B through the deacetylation of NF-\u0026kappa;B \u003csup\u003e\u003ca href=\"#_ENREF_35\"\u003e35\u003c/a\u003e,\u003ca href=\"#_ENREF_36\"\u003e36\u003c/a\u003e\u003c/sup\u003e. The up-regulation of SIRT1 may relieve myocardial IR injury through blocking NF-\u0026kappa;B signaling.\u003c/p\u003e\n\u003cp\u003eThis study has some limitations. First, the regulatory role of rno-miR-30c-5p on myocardial\u0026nbsp;IR injury is limited at the cellular level. The therapeutic effect of rno-miR-30c-5p silencing on rats with myocardial\u0026nbsp;IR injury remains to be studied. Second, only rno-miR-30c-5p was studied. More miRNAs involving myocardial\u0026nbsp;IR injury still need to be discovered based on microarray or RNA-seq methodologies. Third, only one target of rno-miR-30c-5p was selected. The discovery of more targets of rno-miR-30c-5p based on omics measurements is needed.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, rno-miR-30c-5p was up-regulated in rats with myocardial IR injury. Rno-miR-30c-5p enhanced the inflammation, promoted the apoptosis, and activated NF-\u0026kappa;B pathway in IR myocardial cells through targeting SIRT1. Rno-miR-30c-5p may promote the myocardial IR injury in rats through activating NF-\u0026kappa;B pathway and down-regulating SIRT1. Our research discovers a novel regulatory mechanism of rno-miR-30c-5p in myocardial IR injury and points out a novel therapeutic target.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eleft anterior descending (LAD)\u003c/p\u003e\n\u003cp\u003emicroRNAs (miRNAs)\u003c/p\u003e\n\u003cp\u003eischemia reperfusion (IR)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate: This study was conducted after obtaining Luoyang Central Hospital Affiliated to Zhengzhou University\u0026rsquo;s ethical committee approval.\u003c/p\u003e\n\u003cp\u003eConsent for publication: Not applicable.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials:\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eThe genes analyzed in the present study are available at https://www.ncbi.nlm.nih.gov/search/ with Gene ID: 100314012 (microRNA-30c-5p, ENSMUSG00000065567; http://asia.ensembl.org/Mus_musculus/Gene/Summary?db=core;g=ENSMUSG00000065567;r=1:23291701-23291784;t=ENSMUST00000083633), and Gene ID: 309757 (Sirtuin 1, ENSMUSG00000020063; http://asia.ensembl.org/Mus_musculus/Gene/Summary?db=core;g=ENSMUSG00000020063;r=10:63319005-63381704).\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding: Not applicable.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions: JFC and SFX: conception, design and analysis of data, performed the data analyses and wrote the manuscript;\u003c/p\u003e\n\u003cp\u003eJFC and MMZ: contributed to the conception of the study and revised the manuscript;\u003c/p\u003e\n\u003cp\u003eJLW: contributed to the conception of the study;\u003c/p\u003e\n\u003cp\u003eSYZ: contributed significantly to analysis and manuscript preparation and revised the manuscript;\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgements: Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWong ND. 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Reduced SIRT1 signaling exacerbates myocardial ischemia reperfusion injury in type 2 diabetic rats and the protective effect of melatonin. 2015;59:376\u0026ndash;90.\u003c/li\u003e\n\u003cli\u003eWang YH, Shun-An L, Chao-Hsin H, Hsing-Hui S, Yi-Hung C, T. CJ, et al. Sirt1 Activation by Post-ischemic Treatment With Lumbrokinase Protects Against Myocardial Ischemia-Reperfusion Injury. Frontiers in Pharmacology.9:636-.\u003c/li\u003e\n\u003cli\u003eQiao L, Xiu-Ying C, Ji Z, Yong-Liang Y, Wen Z, Bo W. Upregulation of SIRT1 contributes to the cardioprotective effect of Rutin against myocardial ischemia-reperfusion injury in rats. Journal of Functional Foods.46:227-36.\u003c/li\u003e\n\u003cli\u003eFu BC, Lang J-L, Zhang D-Y, Sun L, Chen W, Liu W, et al. Suppression of miR-34a expression in the myocardium protects against ischemia-reperfusion injury via SIRT1 protective pathway. Stem Cells \u0026amp; Development.scd.2017.0062.\u003c/li\u003e\n\u003cli\u003eHaigis MC, Sinclair DA. Mammalian sirtuins: biological insights and disease relevance. Annu Rev Pathol. 2010;5:253-95.\u003c/li\u003e\n\u003cli\u003eYeung F, Hoberg JE, Ramsey CS, Keller MD, Jones DR, Frye RA, et al. Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase. EMBO J. 2004;23:2369-80.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cardiovascular-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcar","sideBox":"Learn more about [BMC Cardiovascular Disorders](http://bmccardiovascdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcar/default.aspx","title":"BMC Cardiovascular Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"myocardial ischemia reperfusion injury, rno-miR-30c-5p, inflammation, apoptosis, SIRT1, NF-κB pathway","lastPublishedDoi":"10.21203/rs.2.15405/v5","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.15405/v5","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e This study aimed to investigate the regulatory effect of rno-microRNA-30c-5p (rno-miR-30c-5p) on myocardial ischemia reperfusion (IR) injury in rats and the underlying molecular mechanisms.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e A rat model of myocardial IR injury was established. The infarct size was detected by 2,3,5-triphenyltetrazolium chloride staining. The pathologic changes of myocardial tissues were detected by hematoxylin-eosin staining. The apoptosis of myocardial cells was measured by TUNEL staining and flow cytometry. The mRNA expression of rno-miR-30c-5p and Sirtuin 1 (SIRT1) was detected by quantitative real-time PCR. The levels of IL-1β, IL-6 and TNF-α were detected by enzyme linked immunosorbent assay. The protein expression of Bax, Bcl-2, caspase-3, p-IκBα, IκBα, p-NF-κB p65, NF-κB p65 and SIRT1 was detected by Western blot. The interaction between rno-miR-30c-5p and SIRT1 was predicted by TargetScan, and further identified by dual luciferase reporter gene and RNA immunoprecipitation assay.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The myocardial IR injury model was successfully established in rats. IR induced the myocardial injury in rats and increased the expression of rno-miR-30c-5p. Overexpression of rno-miR-30c-5p enhanced the inflammation, promoted the apoptosis, and activated NF-κB pathway in IR myocardial cells. SIRT1 was the target gene of rno-miR-30c-5p. Silencing of SIRT1 reversed the effects of rno-miR-30c-5p inhibitor on the apoptosis and NF-κB pathway in IR myocardial cells.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Rno-miR-30c-5p promoted the myocardial IR injury in rats through activating NF-κB pathway and down-regulating SIRT1.\u003c/p\u003e","manuscriptTitle":"Rno-microRNA-30c-5p promotes myocardial ischemia reperfusion injury in rats through activating NF-κB pathway and targeting SIRT1","msid":"","msnumber":"","nonDraftVersions":[{"code":5,"date":"2020-04-14 19:36:42","doi":"10.21203/rs.2.15405/v5","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2020-05-08T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-04-08T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-04-07T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-04-07T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cardiovascular-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcar","sideBox":"Learn more about [BMC Cardiovascular Disorders](http://bmccardiovascdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcar/default.aspx","title":"BMC Cardiovascular Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":4,"date":"2020-03-14 19:59:29","doi":"10.21203/rs.2.15405/v4","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2020-04-01T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-03-08T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-03-07T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-03-07T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cardiovascular-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcar","sideBox":"Learn more about [BMC Cardiovascular Disorders](http://bmccardiovascdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcar/default.aspx","title":"BMC Cardiovascular Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":3,"date":"2020-02-28 17:25:48","doi":"10.21203/rs.2.15405/v3","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2020-03-03T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-03-02T12:00:00+00:00","index":1,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-03-02T12:00:00+00:00","index":1,"fulltext":"Recommendation: Accept without revision\nForm responses:\n---\n\nComments to Author:\n---\nThe authors have answered adequately both of my remaining questions. I have no further comments.\n* Are the methods appropriate and well described?: **Yes**\n* Does the work include the necessary controls?: **Yes**\n* Are the conclusions drawn adequately supported by the data shown?: **Yes**\n* Are you able to assess any statistics in the manuscript or would you recommend an additional statistical review?: **Not relevant to this manuscript**\n* Quality of written English: **Acceptable**\n* Declaration of competing interests: **I declare that I have no competing interests**\n* I agree to the open peer review policy of the journal. I understand that my name will be included on my report to the authors and, if the manuscript is accepted for publication, my named report including any attachments I upload will be posted on the website along with the authors' responses. I agree for my report to be made available under an Open Access Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0/). I understand that any comments which I do not wish to be included in my named report can be included as confidential comments to the editors, which will not be published.: ** I agree to the open peer review policy of the journal**\n"},{"type":"reviewersInvited","content":"","date":"2020-03-01T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-02-27T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-02-26T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-02-26T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cardiovascular-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcar","sideBox":"Learn more about [BMC Cardiovascular Disorders](http://bmccardiovascdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcar/default.aspx","title":"BMC Cardiovascular Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":2,"date":"2020-01-24 14:57:00","doi":"10.21203/rs.2.15405/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2020-02-14T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-02-07T12:00:00+00:00","index":1,"fulltext":"Recommendation: Accept without revision\nForm responses:\n---\n* Are the methods appropriate and well described?: **Yes**\n* Does the work include the necessary controls?: **Yes**\n* Are the conclusions drawn adequately supported by the data shown?: **Yes**\n* Are you able to assess any statistics in the manuscript or would you recommend an additional statistical review?: **I am able to assess the statistics**\n* Quality of written English: **Needs some language corrections before being published**\n* Declaration of competing interests: **I declare that I have no competing interests.**\n\nComments to Author:\n---\n"},{"type":"editorInvitedReview","content":"","date":"2020-02-07T12:00:00+00:00","index":2,"fulltext":"Recommendation: Major revisions required\nForm responses:\n---\n* Are the methods appropriate and well described?: **Yes**\n* Does the work include the necessary controls?: **Yes**\n* Are the conclusions drawn adequately supported by the data shown?: **Yes**\n* Are you able to assess any statistics in the manuscript or would you recommend an additional statistical review?: **I recommend additional statistical review**\n* Quality of written English: **Needs some language corrections before being published**\n* Declaration of competing interests: **I declare that I have no competing interests**\n\nComments to Author:\n---\nMultiple questions were addressed by the authors, and also the language usage was corrected, however there are at least two important points where the manuscript should be improved.\n\n1. It is still necessary to discuss the current recommendations (as suggested in my original comments) to investigate the role of microRNAs and transcirptomics changes in cardiovascular diseases. These recommendations are based on an emerging consensus promoting an unbiased approach supported by global omics measurements and bioinformatics analysis which could help to avoid arriving at results that could not be translated into the clinical practice as it happened in the field in the last 30 years. Therefore, in the introduction it is recommended to add a section detailing the current unbiased trends and to provide arguments in favor of the selected biased approach despite the consensus on the usage of unbiased approaches.\n\n2. The results of TargetScan search is still missing from the manuscript. Based on the unbiased methodology it would be really interesting to see all the predicted targets in the order of their Context++ Scores, and also the rank of SIRT1 in this list. Thus, I recommend to include the top TargetScan predictions as a Table in the manuscript and the full list of predicted targets as a supplementary table."},{"type":"reviewerAgreed","content":"","date":"2020-01-24T12:00:00+00:00","index":1,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-01-24T12:00:00+00:00","index":2,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-01-23T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-01-22T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-01-21T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-01-21T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cardiovascular-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcar","sideBox":"Learn more about [BMC Cardiovascular Disorders](http://bmccardiovascdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcar/default.aspx","title":"BMC Cardiovascular Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2019-10-01 00:31:47","doi":"10.21203/rs.2.15405/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2019-12-22T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2019-12-02T12:00:00+00:00","index":2,"fulltext":"Recommendation: Major revisions required\nForm responses:\n---\n* Are the methods appropriate and well described?: **Yes**\n* Does the work include the necessary controls?: **Yes**\n* Are the conclusions drawn adequately supported by the data shown?: **No**\n* Are you able to assess any statistics in the manuscript or would you recommend an additional statistical review?: **I recommend additional statistical review**\n* Quality of written English: **Not suitable for publication unless extensively edited**\n* Declaration of competing interests: **I declare that I have no competing interests**\n\nComments to Author:\n---\n1. There are serious grammatical errors in the manuscripts, extensive language revision is required.\n2. It is not clear from the text why the authors chose to investigate the role of miR-30c. It should be discussed in more detail, what are the supporting evidences behind this choice.\n3. To avoid biased selection of pathways to study in such a cardiovascular disease model, accoding to current recommendations (like in Perrino C et al. 2017 Cardiovasc Res and Schulz R et al. 2017 Basic Res Cardiol.) it would be necessary to support the choice of the studied microRNA by global transcriptomic measurements. Thus it would be recommended to perform microRNA profiling by microarray or RNA-seq methodologies. This way it can be shown if miR-30c is really a key player in this condition.\n4. To name microRNAs correctly the new microRNA nomenclature available on miRBase should be used to avoid inconsistencies. Authors thus should confirm, that microRNA they investigated is rno-miR-30c-5p or not. (There are also other rat microRNAs with similar names like rno-miR-30c-1-3p etc.)\n5. In the methods section it is stated that TargetScan was used to predict targets. The detailed results of the target prediction should be also included in the manuscript. The post-transcriptional regulatory network of the underlying microRNAs (preferably assessed by one of the omics measurements instead of PCR of only one biasedly selected microRNA) could reveal some other important targets in addition to the selected ones.\n\n\n"},{"type":"reviewerAgreed","content":"","date":"2019-11-18T12:00:00+00:00","index":2,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2019-11-08T12:00:00+00:00","index":1,"fulltext":"Recommendation: Accept after discretionary revisions\nForm responses:\n---\n* Are the methods appropriate and well described?: **Yes**\n* Does the work include the necessary controls?: **Yes**\n* Are the conclusions drawn adequately supported by the data shown?: **Yes**\n* Are you able to assess any statistics in the manuscript or would you recommend an additional statistical review?: **I am able to assess the statistics**\n* Quality of written English: **Acceptable**\n* Declaration of competing interests: **I declare that I have no competing interests.'**\n\nComments to Author:\n---\nIn an article BCAR-D-19-00838 authors identified a SIRT1 as a target of miR-30c, which is believed to promote myocardial ischemia in reperfusion injury. Huge amount of work has been done; however, there are certain suggestions that would improve the manuscript.\nOn myocardial OI\"\n- Abstract\no Abbreviation TTC should be avoided or supported by full name in the abstract.\no Real-time or quantitative real-time PCR (qPCR is appropriate).\n- Background\no Last paragraph: \"we explored the function of miR-146a on myocardial IR injury.\" Was truly miR-146a?\n- Methods\no What was GAPDH expression across tested samples; since it is recognized as one of the poorest reference genes.?\n- Discussion\no Too much about other genes (third paragraph of the discussion).\no A little more should be stressed on discussion of analysed miR-30c and other genes in the last paragraph.\n"},{"type":"reviewerAgreed","content":"","date":"2019-10-11T12:00:00+00:00","index":1,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2019-10-09T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2019-09-24T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2019-09-24T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2019-09-23T12:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2019-09-20T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cardiovascular-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcar","sideBox":"Learn more about [BMC Cardiovascular Disorders](http://bmccardiovascdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcar/default.aspx","title":"BMC Cardiovascular Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e422d553-db2d-4034-a2ca-1a709a7494bb","owner":[],"postedDate":"April 14th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":83426,"name":"Cardiac \u0026 Cardiovascular Systems"}],"tags":[],"updatedAt":"2021-08-09T17:59:44+00:00","versionOfRecord":{"articleIdentity":"rs-6071","link":"https://doi.org/10.1186/s12872-020-01520-2","journal":{"identity":"bmc-cardiovascular-disorders","isVorOnly":false,"title":"BMC Cardiovascular Disorders"},"publishedOn":"2020-05-20 20:27:38","publishedOnDateReadable":"May 20th, 2020"},"versionCreatedAt":"2020-04-14 19:36:42","video":"","vorDoi":"10.1186/s12872-020-01520-2","vorDoiUrl":"https://doi.org/10.1186/s12872-020-01520-2","workflowStages":[]},"version":"v5","identity":"rs-6071","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-6071","version":["v5"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}