The Functional Mechanism Of MicroRNA-7? In Protecting Against Cardiomyocyte Injury In Acute Myocardial Infarction

Acute myocardial infarction (MI) is a kind of serious ischemic heart disease, which mainly refers to myocardial necrosis induced by persistent ischemia and hypoxia after acute occlusion of the coronary artery, and has become one leading cause of increasing mortalities among cardiovascular diseases. Ventricular remodeling and heart failure are two main factors to influence the cardiovascular event rate, long term survival and life quality following MI. Emerging evidence has showed that a large quantity of myocardial cell apoptosis can be observed in the infarct and non-infarct area, wherein partial irreversible apoptosis will directly contribute to MI-induced cardiac injuries. After MI, the developing apoptosis of cardiomyocytes, triggered by ischemia stress in the border zone, aggravates the ventricular remodeling of the remaining active myocardium, which further results in heart failure. Thus, the intervention of cardiac myocyte apoptosis has been considered to be one strategy for preventing cardiac remodeling and heart failure progression and improving the prognosis of patients following MI. Further investigation on the mechanism of MI-induced apoptosis and anti-apoptosis pathways in cardiomyocytes might provide newly effective targets for taking intervention measures in clinical applications.MicroRNAs (miRNAs) are endogenous, small non-coding RNAs composed with 18-25 nucleotides that inhibit gene expression at post-transcriptional level by binding the 3'untranslated regions (3'UTRs) of target mRNAs and result in the degradation of target mRNA or translation repression, which participates in various pathophysiological processes of cardiovascular diseases. MiR-7a is a kind of tumor suppressor that inhibits tumor cell proliferation and promotes tumor cell apoptosis in various tumors, and shows evolutionarily conserved in mammals. Previous studies also found that miR-7a is down-regulated in end-stage heart failure or up-regulated in cardiac ischemic/reperfusin injury, showing relation to MI development. However, further studies revealed that miR-7a protected myocardial cells against I/R-induced apoptosis by negatively regulating poly (ADP-ribose) polymerase (PARP), an executioner of apoptosis. Recent study also showed that ectopic expressed miR-7a also provides resistance against unfolded protein response (UPR) mediated apoptosis. These results indicate that miR-7a participates in the process of myocardial apoptosis under different pathological states. However, the upstream regulatory mechanism and downstream pathways related to miR-7a are not fully clarified.Circular RNA (circRNA) is a new type of untranslated RNA induced by special selectivity of shear, with the potential to form a covalently closed continuous loop. Recently, it has been demonstrated that circRNA shows abundantly expression across the eukaryotic tree of life with evolutionary conservation particularly between humans and mice. Similar to linear mRNAs, the well-expressed, stable expression of circRNAs also shows tissue and developmental stage-specific characteristics, indicating several potential biological functions. Currently, one human circRNA, antisense to the cerebellar degeneration-related protein 1 transcript (Cdrlas), also termed as ciRS-7 (circular RNA sponge for miR-7), has been well-studied and identified to be implicated in human diseases. Bioinformatics studies shows that Cdrlas contains more than 60 binding sites or clusters for binding miR-7, and functions as a miR-7 sponge to inhibit the miR-7 activity. In the mouse brain, particularly in the neocortical and hippocampal neurons, Cdrlas and miR-7 were observed to be co-expressed, and Cdrlas binds miR-7 seed sites to regulate the expression of miR-7 and its target genes. There is no related report whether Cdrlas mediating miR-7a participates in myocardial apoptosis following the process of MI. Curcumin is a polyphenol extracted from turmeric, used as a Chinese herbal medicine. In recent years, the application of curcumin in anti-inflammation, anti-oxidation, anti-infection, anti-tumor, anti-atherosclerosis, and coronary artery protection has been increasingly studied. In recent years, it also has been found that curcumin plays a role in myocardial cell protection. However, the molecular mechanism remains unclear. In the present study, we confirmed whether miR-7a and apoptosis related protein specific protein 1 (SP1) conferred an involvement in the protective effect of hypoxia-induced myocardial apoptosis using in vitro and in vivo experiments.This study includes four parts:Part I The effect of MI injury or hypoxia stress on the expression of Cdrlas and miR-7a in cardiomyocytesPart II The effect and mechanisms of miR-7a in hypoxia-induced apoptosis of cardiomyocytes by regulating its targets, PARP and SP1Part III The effect and mechanisms of Cdrlas in promoting MI development by inhibiting miR-7a activityPart IV The effect and mechanisms of curcumin in protecting cardiomyocytes from MI-induced injuries by upregulating miR-7aPart I The effect of MI-induced injuries on the expression of Cdrlas and miR-7a in cardiomyocytesObjective1. To investigate whether Cdrlas shows abnormal expression in myocardial tissues after MI.2. To confirm whether the abnormal expression of Cdrlas and miR-7a is synchronous in myocardial tissues after MI.3. To further explore the potential co-expression of Cdrlas and miR-7a in cardiomyocytes under hypoxia treatment.Methods1. Establishment of MI modelAll mice were anesthetized with 0.8% sodium pentobarbital to receive MI introduction or a sham operation under aseptic conditions. MI introduction was performed by permanent ligation of the left anterior descending (LAD) artery. Mice receiving a sham operation were subjected to the same surgical procedures as the experimental ones, but the LAD artery was not ligated.2. Assessments for established MI modelAt 24 h post-ligation, the mice were anesthetized for further determination of myocardial infarct size expressed by a percentage of area at risk (AAR) and also the AAR percentage in the whole left ventricle (LV). Besides, LDH concentration in the serum was measured by using a colorimetric LDH assay kit.3. Cell culturePrimary cardiomyocytes isolated from mouse ventricles or mouse cardiac myocytes (MCMs) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10%FBS and 100?g/ml penicillin/streptomycin, and incubated at 37? under 5% CO2.4. Hypoxia treatmentFor hypoxia treatment, cells were pre-incubated, and then seeded to fresh DMEM medium with incubation in a hypoxic chamber for 3,6, or 12 h.5. Real-time PCR for the detection of Cdrlas expressionThe TRIzol reagent was used to isolate total RNA, including microRNA. For the detection of Cdrlas expression, cDNA synthesis was performed with Oligo (dT), and a SYBR RT-PCR kit was used for mRNA quantification with specific primers (Forward:5'-GTGTCTCCAGTGTATCGGCG-3'; Reverse: 5'-TACTGGCACCACTGGAAACC-3'). All reactions were preceded on the ABI 7500 real-time PCR system by standard protocols. The comparative threshold cycle (Ct) value was calculated and analyzed by using the 2-??CT method, with ?-actin as an internal control.6. Real-time PCR for the detection of miR-7a expressionFor miR-7a expression, microRNA was analyzed by using the TaqMan MicroRNA Reverse Transcription Kit with provided RT-U6 and microRNA-specific stem-loop primers, and the expression levels were determined through TaqMan MicroRNA assays with the TaqMan Universal PCR Master Mix. Results were analyzed by using the 2-??CT method, with U6 as an internal control.Results1. Evaluation of established MI modelTo evaluate the MI development in mice, myocardial infarct size and the extent of AAR were detected accordingly, as well as LDH release, a biochemical marker of myocardial cell necrosis. A significant increase of infarct size, AAR and LDH release were observed in MI mice as compared with the sham control, indicating the successful establishment of a mouse MI model.2. Co-expression of Cdrlas and miR-7a during MI-induced injuryThe expression of Cdrlas and miR-7a in cardiomyocytes of MI mice showed significant increase with a time-dependent manner as compared to that of sham groups, with a respective 2.4-fold and 1.9-fold increase in cardiomyocytes at 24 h after MI surgery.3. Co-upregulation of Cdrlas and miR-7a in hypoxia-treated cardiomyocytesThe co-expression of Cdrlas and miR-7a was also confirmed in both primary cardiomyocytes from normal CL57B6 mice and MCM cells with a time dependent manner after hypoxia treatment for simulating MI injury in vitro.Conclusion1. Cdrlas expression was upregulated in myocardial tissues after MI, in line with miR-7a upregulation.2. Hypoxia could induce the co-upregulation of Cdrlas and miR-7a in mouse cardiomyocytes.Part ? The effect and mechanisms of miR-7a in hypoxia-induced apoptosis of cardiomyocytes by regulating its targets, PARP and SP1Objective1. To validate whether SP1 is one miR-7a targets.2. To investigate the functional mechanism of PARP and SP1 in regulating miR-7a-mediated pathways during hypoxia-induced apoptosis of cardiomyocytes.Methods1. Cell cultureMouse cardiac myocytes (MCM) cell line were cultured in DMEM, containing 10% FBS and 100?g/ml penicillin or streptomycin, at 37? under 5% CO2. For hypoxia treatment, cells were incubated in a hypoxic chamber for 12 h.2. Cell transfection and groupsMCM cells were pre-cultured overnight until 50% confluence and then transfected with plasmids or microRNAs by Lipofectamine 2000. At 6h post transfection, the supernatant of cell cultures was removed and fresh medium was added. After incubation of an additional 48 h, cells were collected for further experiments. MCM cells were randomly divided into four groups to receive the transfection of miR-7a mimic or scrambled miRNA (negative control), and then the co-transfection of pcDNA-PARP-SP1 or pcDNA (empty control) for functional studies.3. Real-time PCR for the detection of PARP and SP1 mRNATRIzol reagent was used to isolate total RNA, and cDNA was synthesized for further real-time PCR analysis with PARP and SP1 specific primers. Results were analyzed by using the 2"AACT method, with ?-actin as an internal control.4. Western blot analysis for PARP and SP1Whole lysates were prepared from cultured cardiomyocytes, and protein concentration was determined by the BCA method. Samples with equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes for the detection of PARP, SP1 and GAPDH or P-actin levels after being incubated with specific antibodies. Immunoreactive bands were visualized on the ImageQuant LAS 4000 System by using an enhanced chemiluminescence kit.5. Luciferase assayThe 3'untranslated region (UTR) sequence of SP1 or PARP containing the predicted mmu-miR-7a binding sites was sub cloned into the pmirGLO vector, resulting in the wild-type luciferase reporter constructs, pmirGLO-SP1-3'UTR-WT and pmirGLO-PARP-3'UTR-WT, or the mutant types, pmirGLO-SP1-3'UTR-Mut and pmirGLO-PARP-3'UTR-Mut, with the corresponding mutant seed sequence. These constructs were co-transfected into cells with miR-7a mimics, and luciferase activity was measured with the Dual-Luciferase Reporter Assay System, with Renilla luciferase activity as an internal control.6. Caspase-3 activity analysisCaspase-3 activity was measured by a Colorimetric Assay Kit. Absorbance at 405 nm was measured on a microplate reader, and results were normalized to those from control cells.7. Annexin V/propidium iodide (PI) stainingAfter the indicated treatment, cells were collected and assayed by using an FITC-labeled Annexin V (Annexin V-FITC) apoptosis detection kit. The percentage of apoptotic cells was quantified by flow cytometry.Results1. MiR-7a inhibited the activity of PARP or SP13'UTRMiR-7a strongly inhibited luciferase activity of the wild-type luciferase reporter constructs, pmirGLO-SP1 3'UTR-WT or pmirGLO-PARP-3'UTR-WT, but showed little influence on the mutant types, pmirGLO-SP1-3'UTR-Mut or pmirGLO-PARP-3'UTR-Mut.2. MiR-7a targeted the expression of PARP and SP1The expression of PARP and SP1 at both mRNA and protein level were detected to be inhibited significantly in miR-7a overexpressing cells. Taken together, SP1 was validated as one miR-7a target, in line with PARP.3. miR-7a protected MCM cells from hypoxia-induced apoptosis by targeting PARP and SP1Hypoxia treatment induced significant activation of caspase-3 with increased apoptotic cells, while the overexpression of miR-7a could rescue these apoptosis phenotypes induced by hypoxia. Further results showed that hypoxia-induced apoptosis could be recovered by the co-overexpression of PARP and SP1, through the co-transfection of pcDNA-PARP-SP 1 into miR-7a overexpressing cells.Conclusion1. MiR-7a targeted the expression of PARP and SP1 by binding 3'UTR.2. MiR-7a showed cardioprotective effects under hypoxia-induced apoptosis by inhibiting PARP and SP1.Part ? The effect and mechanisms of Cdrlas in promoting MI development by inhibiting miR-7a activityObjective1. To investigate the potential regulation of Cdrlas on cardiomyocyte apoptosis.2. To investigate whether Cdrlas functioned as a miR-7a inhibitor in regulating cardiomyocyte apoptosis.3. To confirm the mechanism of Cdrlas/miR-7a pathway in MI development.Methods1. Cell cultureMouse cardiac myocytes (MCM) cell line were cultured in DMEM containing 10% FBS and 100?g/ml penicillin/streptomycin at 37? under 5% CO2.2. Cell transfectionMCM cells were randomly divided into four groups to receive the transfection of pcDNA-Cdrlas or pcDNA (empty control), and then the co-transfection of miR-7a mimic or scrambled miRNA (negative control) by using Lipofectamine 2000 (Invitrogen). At 24 h post-transfection, cells were collected for quantitative Real-time PCR for the detection of Cdrlas or miR-7a expression.3. Real-time PCR for the detection of PARP and SP1 mRNATRIzol reagent was used to isolate total RNA, and cDNA was synthesized for further real-time PCR analysis with PARP and SP1 specific primers. Results were analyzed by using the 2-??CT method, with ?-actin as an internal control.PARP and SP14. Western blot analysis for PARP and SP1Whole lysates were prepared, separated by SDS-PAGE and then transferred to PVDF membranes for the detection of PARP and SP1 after being incubated with specific antibodies. Immunoreactive bands were visualized by using an enhanced chemiluminescence kit.5. Caspase-3 activity analysisCaspase-3 activity was measured by a Colorimetric Assay Kit. Absorbance at 405 nm was measured on a microplate reader, and results were normalized to those from control cells.6. Annexin V/PI stainingAfter the indicated treatment, cells were collected and assayed by using an FITC-labeled Annexin V (Annexin V-FITC) apoptosis detection kit. The percentage of apoptotic cells was quantified by flow cytometry.7. In vivo delivery of plasmids or miRNAsPlasmids, pre-treated with Lipofectamine 2000, or miRNAs contained lentivirus were delivered by intramyocardial injection into four sites of the left ventricular free wall with an insulin syringe. After injection, mice were allowed to recover for three days, and then sham or MI surgery was performed. At 24 h post-ligation, the mice were anesthetized for the determination of myocardial infarct size, the AAR percentage in the whole left ventricle (LV) and also LDH concentration in the serum.8. ImmunohistochemistryPartial mouse hearts, removed at 24 h after the ligation, were fixed, embedded in paraffin and routinely processed for frozen sectioning. Then, immunohistochemical staining was performed for the detection of SP1 and PARP expression in the myocardium sections. Images of sections were photographed and analyzed, while the mean optical density of SP1 or PARP was determined by calculation of the ratio of integrated optical density in the area of interest.Results1. The expression of PARP and SP1 were upregulated by CdrlasAfter the transfection of pcDNA-Cdrlas, Cdrlas expression was confirmed to be increased by around 3.3-fold as compared to the control, while the expression levels of endogenous miR-7a showed no obvious changes. In contrast, however, the expression of PARP and SP1, two miR-7a target genes, was strongly increased by the overexpression of Cdrlas.2. miR-7a protected cardiomyocyte from Cdrlas-induced apoptosisCdrlas significantly increased the caspase-3 activity and apoptotic cells, while co-overexpression of miR-7a reversed Cdr las-induced phenotypes, resulting in reduced caspase-3 activity and apoptotic cells. These results implied that the Cdr1as overexpression might regulate cardiomyocyte apoptosis by reducing miR-7a activity.3. Cdrlas aggravated MI injuries with upregulated PARP and SP1, which could be rescued by the co-overexpression of miR-7aMI injuries, including enlarged infarct size, increased AAR and elevated LDH release, were all strongly aggravated by Cdrlas overexpression through the transfection of pcDNA-Cdrlas via intracardial injection, while the expression of PARP and SP1 was upregulated consistently in heart tissues. Moreover, Cdrlas-induced effects could all be reversed by the overexpression of miR-7a through the co-transfection of lentivirus with miR-7a mimic.Conclusion1. MiR-7a protected cardiomyocyte from Cdr las-induced apoptosis.2. Cdrlas functioned as a miR-7a inhibitor in promoting MI development, involving the upregulated expression of PARP and SP1.Part IV Curcumin protects cardiac myocyte against hypoxia-induced apoptosis through upregulating miR-7a expressionObjective1. To confirm the role of curcumin in cardioprotective effects following MI.2. To investigate the effects of curcumin on miR-7a and SP1 expression of myocardial cell.3. To explore the potential mechanism of miR-7 in the protective effect of curcumin against MI injury.Methods1. Curcumin administration and MI mouse model establishment:Mice received 50 mg/kg/d curcumin by intragastric administration for one week and underwent ligation of left anterior descending (LAD) coronary artery occlusion for the establishment of MI model. Twenty-four hours after operation, the infarct size, AAR and serum LDH content were detected.2. Cell cultureMCM were cultured in DMEM containing 10% FBS and 100?g/ml penicillin/streptomycin at 37? under 5% CO2. Curcumin (10 ?M) was used to pre-incubate with MCM for 2h before the treatment of hypoxia. The equal volume of DMSO, a solvent of curcumin, acted as a control.3 Cell transfection and treatmentsThe MCMs were transfected with miR-7a inhibitor or scrambled siRNA (NC) using Lipofectamine2000 reagent for inhibit the expression of miR-7a. MSCs were transfected with pcDNA-SP1 or pcDNA for overexpression of SP1. Forty-eight hours after transfection, MCMs were pre-incubated with curcumin for 2h and then cultured at the atmosphere with 0% oxygen for another 24h.4. Real-time PCR for the detection of miR-7a expressionTotal RNA was abstracted from tissues and cells using TRIzol and then reverse-transcribed into complementary DNA. The relative expression of miR-7a was quantified by real-time PCR, with U6 acting as reference gene.5. Real-time PCR for the detection of SP1 mRNA expressionTotal RNA was abstracted from tissues and cells using TRIzol and then reverse-transcribed into complementary DNA. The relative expression of SP1 was quantified by real-time PCR, with p-actin acting as reference gene.6. Western blot analysis for SP1 protein levelProtein sample was isolated with RIPA lysis buffer from tissue and cells. The SP1 protein was detected using anti-SP1 antibody.7. Caspase-3 activityCaspase-3 activity of MCM was determined using Caspase-3 Activity Assay Kit.8. Apoptosis assayTUNEL assay was used to evaluate the MSC apoptosis.Results1. The effects of curcumin on myocardial damage and the expression of miR-7a and SP1Curcumin treatment significantly decreased the infarct size, area at risk and LDH release, and markedly reduced the myocardial damage following MI. Curcumin also up-regulated miR-7a and down-regulated SP1 expression.2. The effect of curcumin on the apoptosis of MCM exposed to hypoxia MCM was pre-incubated with curcumin prior to the treatment of hypoxia. The results showed that the increase of caspase-3 activity and TUNEL positive cell percentage was reversed by curcumin treatment. In MCM exposed to hypoxia, miR-7a level was significantly reduced while SP1 expression in mRNA and protein levels was elevated. The pre-treatment of curcumin also reversed the decrease of miR-7a expression and the increase of SP1 expression3. The role of miR-7a and SP1 in the effects of curcumin on hypoxia induced MCM apoptosisIn MCM exposed to hypoxia, the decreased levels of caspase-3 activity and TUNEL positive cells induced by curcumin was also reversed by miR-7a inhibitor or SP1 overexpression.4. Curcumin played an anti-apoptosis effect through mediating SP1 in MCM The decrease of SP1 expression induced by curcumin was inhibited by miR-7a inhibitor.Conclusion1. MiR-7 is involved in the regulation of SP1 expression in MCM exposed to hypoxia.2. Curcumin modulated SP1 expression through miR-7a in hypoxia induced MCM and protect myocardial cell against damage and apoptosis following MI and hypoxia.