| BackgroundAs the predominant component, cardiac fibroblasts (CFs) account for 70% of all cardiac cells. CFs is the carrier of cardiac structural and physical function in physiological process, such as the development of the heart, cardiac structural composition and cellular signal transduction. Meanwhile, CFs are involved in the pathological process of cardiac remodeling in cardiovascular diseases, including hypertension, diabetic cardiomyopathy, cardiac ischemic/reperfusin injury (IR/I) and heart failure. The excessive proliferation CFs, the balanceless secretion of MMPs (matrix metalloproteinases), plus the over-synthetic ECM (extracellular matrix), are the three major pathological processes, which constitute the pathological basis of myocardial interstitial fibrosis. RAAS (Renin-Angiotensin-aldosterone system) plays a key role in regulating myocardial fibrosis and remodeling. Under the pathological circumstances, take myocardial infarction as an example, by means of autocrine and paracrine, Angiotensin Ⅱ (ANGⅡ) powerfully stimulate collagen I overproduction via TGF-β and MAPKs pathways, resulting in the cardiac fibrosis and myocardial dysfunction. However, the molecular mechanisms underlying cardiac fibrosis are not clear and remain to be elucidated. It is difficult but quite necessary to find a way out of cardiac fibrosis.MicroRNAs (miRNAs, miRs) is a class of endogenous non-coding single and small RNA which is composed with 18～25 bases that negatively regulating the gene expression at post-transcriptional level by degrading target mRNA or translation repression. The main function of miRs is regulating the fundamental process of lives, such as cell growth, differentiation, proliferation, apoptosis and senescence. Recent studies found that the expression of miRa was deregulated in the cardiovascular systems involved in myocardial remodeling, myocardial hypertrophy, myocardial cell apoptosis, heart failure, as well as myocardial fibrosis and other pathological and physiological processes. For example, miRNA-21, miRNA-27 and miRNA-29 are all deeply involved in myocardial fibrosis by regulating the expression and function of their targets. miR-7a/b is an evolutionarily conserved mRNA in manMals, and functions in growth, migration and metastasis in tumor cells and fibrosis in fibroblasts. Among cardiovascular diseases, miR-7 was first reported to be associated with the risk of coronary artery disease, and then miR-7a/b was found deregulated in pediatric heart failure and was capable of protecting cardiomyocytes against in IR/I. In addition, it was found that α2(Ⅰ) collagen is miR-7 target gene in dermal fibroblasts. Reasonably, miR-7a/b maybe involved in regulating collagen I synthesis in CFs.Specific protein 1 (Spl) is a ubiquitously expressed transcription factor, and belongs to the Sp family (Spl-Sp8). Spl is implicated in the regulation the housekeeping genes and actively regulated genes, by its basal promoter binding activity. Growing evidence demonstrated that Spl plays an important regulatory role in the expression of several genes relevant to fibrosis, including TGF-β1 and the downstream targets of TGF-β1, take MMPs as examples. Sp1 is also deeply involved in excessive expression and subsequent deposition of collagen I because Sp1 activates the human collagen I promoter via GC boxes and the TCCTCC motif, and its binding activity tocollagen I promoter increases under fibrotic conditions. Several studies have revealed the capability of ANGⅡ in stimulating Spl activation in adult CFs and in mouse hearts. Therefore, the purpose of this study was to experimentally identify the effect of Sp1 on the anti-fibrotic role of miR-7a/b in neonatal CFs, thereby presenting a viable target for therapeutic intervention of fibrotic cardiovascular diseases.This study includes three parts:Part I The effect and mechanisms of miR-7a/b in Angiotensin Ⅱ-induced collagen I synthesis in cardiac fibroblastPart II The effect and mechanisms of miR-7a/b in regulating the role of Spl in cardiac fibroblastPart Ⅲ The effect of miR-7a/b in post-myocardial infarction fibrosisPart Ⅰ The role of miR-7a/b in ANGiotensin Ⅱ-induced cell proliferation, migration and collagen Ⅰ synthesis in cardiac fibroblastObjective 1. To investigate whether ANGⅡ could effect the expression of collagen Ⅰ and miR-7a/b.2. To study the effect of miR-7a/b in the regulation of collagen Ⅰ synthesis in ANG Ⅱ-treated CFs.3. To investigate the signal pathways involved in the role of miR-7a/b played in ANG Ⅱ-treated CFs.Methods1. Cell cultureThe extraction of primary CFs:obtain the heart of 2-3-day old neonatal Wistar rat, shred to 1nM3, and then digested with collagenase. After differential adhesion, the cells were put in incubator at 37℃ and CFs at passage 2 or 3 was used. The special marker of CFs, vemintin was used for the identification of CFs.2. miR-7a/b overexpression/inhibitionTransfection of miR-7a/b mimics:For miR-7a/b over-expression, miR-7a/b mimics were transfected into CFs with Lipofectamine2000 according to the manufactures’instruction. For miR-7a/b inhibition, miR-7a/b inhibitors were used. InMunofluorescent staining and qRT-PCR were used to qualify the transfection efficiency of miR-7a/b mimics.3. InMunofluorescent staining and confocal microscopyInMunofluorescence analysis was used to identify CFs by staing of vemintin, α-actin and CD31.4. Western blottingProteins were extracted from CFs. The protein expression of collagen Ⅰ、 MMP-2、MMP-9、TGF-β、ERK、p-ERK、JNK、p-JNK、p38 and p-p38 was analyzed in our experiment.5. Real-time quantitative PCRFor collagen Ⅰ mRNA quantification, CFs was harvested total RNA was isolated using TRIzol reagent, and cDNA synthesis was performed with oligo(dT) primers. Quantitative RT-PCR (qRT-PCR) analysis was performed using a SYBR RT-PCR kit. miR-7a/b levels were measured using Taqman MicroRNA assays and Taqman Universal PCR Master Mix.6. Assessment of cell proliferation and migration assayCFs proliferation assays were determined by MTT assay. CFs migration assays were performed in Transwell chambers.7. Gelatin zymographyThe activity of MMP-2 and MMP-9 in CFs was determined by zymography.8. Luciferase assayspRL-cmv vectors containing control siRNA or miR-7a/b mimics were co-transfected with wild type collagen Ⅰ vector (with a 3’UTR segment harboring the putative miR-7a/b binding sequence) or mutant collagen Ⅰ vector (with a 3’ UTR segment harboring the putative miR-7a/b binding mutant sites). The CFs was harvested and lysed 24 h after transfection, and Renilla luciferase activities were measured consecutively using dual-luciferase assays, according to the manufacturer’s instructions. Luciferase assays9. Statistic analysisAll statistical analyses involved use of SPSS 18.0. Data are reported as mean ± standard deviation. P<0.05 was considered statistically significant.Results1. Identification of CFsOver 95% of the cultured cells was vimentin positive, and a-actin and CD31 negative.2. Enhanced activation of collagen Ⅰ in ANGⅡ-treated CFsANG Ⅱ (100 nM) effectively activated the expression of collagen Ⅰ in a time-dependent manner. The dose response of ANG Ⅱ was measured in the CFs at 24 h and expression of collagen Ⅰ increased gradually in a dose-dependent manner.3.100 nM ANGⅡ down-regulated miR-7a/b in CFsWe quantified miR-7a/b levels in 100 nM ANG Ⅱ-treated CFs using qRT-PCR. Compared with the levels in normal untreated CFs, miR-7a/b was down-regulated in CFs treated with ANG Ⅱ.4. miR-7a/b repressed ANG Ⅱ-induced collagen Ⅰ in CFsmiR-7a/b effectively repressed the elevated protein expression of collagen Ⅰ induced by ANG Ⅱ.5. Collagen Ⅰ is a target of miR-7amiR-7a/b overexpression suppressed the protein expression of collagen Ⅰ in untreated CFs, while miR-7a/b inhibition further stimulated collagen Ⅰ protein level. miR-7a, but not miR-7b could effluence collagen Ⅰ mRNA expression. Luciferase assays to confirmed miR-7a directly targeted collagen Ⅰ6. miR-7a/b regulated MMP-2 and MMP-9miR-7a/b dramatically reduced the ANG Ⅱ-induced protein expression of MMP-2 and MMP-9, as well as the activity of MMP-2.7. miR-7a/b inhibited ANG Ⅱ-induced CF proliferation and migrationCFs displayed rapid growth following ANG Ⅱ stimulation, which was slowed by the miR-7a/b mimics. Meanwhile, the results of the Transwell assay demonstrated reduced migration by CFs treated with miR-7a/b mimics compared with CFs treated with control siRNA.8. miR-7a/b reduced ANG Ⅱ-induced TGF-β and MAPK activationTGF-β was activated by ANG Ⅱ but was suppressed by miR-7a/b mimics. Although miR-7a/b did not affect total ERK, JNK or p38 levels, miR-7a/b mimics effectively inhibited the ANG Ⅱ-stimulated phosphorylation of ERK, JNK and p38.Conclusion1. ANG Ⅱ up-regulated collagen Ⅰ, and down-regulated miR-7a/b in CFs.2. miR-7a/b overexpression repressed the expression of collagen Ⅰ, TGF-β, MMP-2, MMP-9, p-ERK, p-JNK and p38, as well as inhibited the proliferation and migration in CFs.3. miR-7a directly targets collagen Ⅰ, and the regulation of miR-7b on collagen I maybe indirect.Part II The effect and mechanisms of miR-7a/b in regulating the role of Spl in cardiac fibroblastObjective1. To investigate the function of Sp 1 that performed in ANGⅡ-treated CFs.2. To investigate whether miR-7a/b is involved in the function of Sp1.Methods1. Inhibition of Sp 1 binding activityThe methods of extraction and culture of primary CFs was performed as Part Ⅰ. Different concentration of mithramycin, an effective inhibitor of Spl DNA binding activity, was used 1 h before treatment of ANG Ⅱ.2. InMunofluorescent staining and confocal microscopyInMunofluorescence analysis was used to evaluate the disposition of Sp1 and collagen Ⅰ in CFs.3. Western blottingProteins were extracted CFs. The protein expression of Sp1、collagen Ⅰ、MMP-2、 MMP-9、TGF-β、ERK、p-ERK、JNK、p-JNK、p38 and p-p38 was analyzed in our experiment.4. Real-time quantitative PCRReal-time quantitative PCR was used for the analysis of Sp1 and miR-7a/b.5. Assessment of cell proliferation and migration assayCFs proliferation assays were determined by MTT assay. CFs migration assays were performed using Transwell chambers.6. Gelatin zymographyThe activity of MMP-2 and MMP-9 in CFs was determined by zymography.7. Statistic analysisAll statistical analyses involved use of SPSS 18.0. Data are reported as mean± standard deviation. P<0.05 was considered statistically significant.Results1. miR-7a/b regulated the protein expression of SplOverexpression miR-7a/b suppressed the protein expression of Sp1 in untreated CFs, while miR-7a/b inhibition stimulated Spl protein level. Neither miR-7a nor miR-7b could regulate Sp1 mRNA expression. ANG Ⅱ (100 nM) effectively activated the expression of Sp1, ANG Ⅱ (100 nM) effectively activated the expression of Sp1. InMunofluorescent staining confirmed that Sp1 located in the nucleus and collagen Ⅰ located in the cytoplasm.2. Inhibition of Spl repressed collagen Ⅰ synthesis in CFsCompared to the control group (100 nM ANG Ⅱ-treated only), pretreatment of mithramycin effectively inhibited collagen I synthesis with the maximum effect at 100 nM. Transfection of miR-7a/b inhibitors partially neutralized the effect of 100 nM mithramycin. Inhibition of Spl repressed collagen I synthesis.3. Inhibition of Spl reduced MMP-2 expression/activity and MMP-9 expression in CFsCompared to the control group (100 nM ANG Ⅱ-treated only), pretreatment of 100 nM mithramycin down-regulated MMP-2 and MMP-9 expression, as well as the activity of MMP-2. Compared to the negative control group (transfection of scrambled siRNA+pretreatment of 100 nM mithramycin), knockdown of miR-7a/b up-regulated the expression MMP-2 and MMP-9, as well as the activity of MMP-2.4. Inhibition of Spl inhibited the proliferation and migrationCompared to the control group (100 nM ANG Ⅱ-treated only), pretreatment of 100 nM mithramycin displayed reduced growth and migration, while knockdown of miR-7a/b up-regulated the mithramycin-repressed growth and migration.5. Inhibiton of Spl repressed TGF-β expression and ERK activationCompared to the control group (100 nM ANG Ⅱ-treated only), pretreatment of 100 nM mithramycin inhibited TGF-β and ERK activation, but not JNK or p38 activation, whereas silence of miR-7a/b by miR-7a/b inhibitors abolished the effect of mithramycin on TGF-β and ERK.Conclusion1. ANG Ⅱ induced Sp1 expression in CFs.2. Inhibition of Sp1 binding activity effectively repressed collagen Ⅰ synthesis, MMP-2 expression/activity, MMP-9 and TGF-β expression, as well as the phosphyration of ERK.3. Spl partially mediated the anti-fibrotic role of miR-7a/b in CFs.Part Ⅲ The effect of miR-7a/b in post-myocardial infarction fibrosisObjective1. To investigate whether MI caused alternation in the expression of miR-7a/b.2. To investigate the role of miR-7a/b plaed in post-myocardial infarction fibrosis.Methods1. Establishment of MI model1.5% isoflurane was used for anesthesia, and the mice then underwent LAD occlusion surgery.2. Transfection of lentivirusAfter LAD ligation,3 injections of total 20 ul GFP-labeled lentiviral vectors were injected around the ligated spot. The vectors separately carried nonsense siRNA (GFP-NC), miR-7a mimics (GFP-7a), miR-7b mimics (GFP-7b), miR-7a inhibitors (GFP-anti-7a) or miR-7b inhibitors (GFP-anti-7b) to overexpress miR-7a/b using mimics (synthetic RNA oligonucleotides mimicking mature miRs) or to silence miR-7a/b using inhibitors (chemically modified antisense oligonucleotides). Finally, we closed the chests and fed the mice regularly under standard temperature and humidity conditions in the IVC center of the Animal Care Center of the Key Laboratory of Cardiovascular Remodeling and Function Research at Shandong University.3. Cardiac function measurementTransthoracic echocardiography was performed using a high-resolution echocardiography system (Visual Sonics, Toronto, Canada). The derived echocardiography parameters included the diastolic left ventricular internal diameter (LVIDd; in nM) and systolic left ventricular internal diameter (LVIDs; in nM). The left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (FS) were calculated via Visual Sonics measurement software.4. Western blotProtein of cardiac tissues were extracted on ice and the expression of Sp1 and collagen Ⅰ was tested.5. RT-PCRRT-PCR is performed to analyze the level of miR-7a/b after MI.6. Histology and inMunohistochemistryTissue was paraffin-embedded and sectioned for staining with Masson’s trichome and Picrosirius red to examine quantify extracellular matrix (ECM) deposition. InMunohistochemistry was used to determine the levels of Sp1 and collagen Ⅰ.7. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) stainingApoptosis in mice was detected by using Situ Cell Death Detection Kit according to the manufacturer’s instructions. At least five fields positive for apoptotic cells were counted in a blinded selection.8. Statistical analysisData are presented as the mean ± SD and were subjected to Student’s t test or, where applicable, ANOVA for group comparisons using SPSS 18.0 and GraphPad Prism 6. P<0.05 was considered statistically significant.Results1. miR-7a/b expression fluctuated after MImiR-7a/b expression was quantified in the border zone of the myocardium post MI. Compared to that in sham-operated mice, the expression of miR-7a/b fluctuated overtime after MI.2. Transfection efficacyAfter 4 weeks of miR-7a/b overexpression/silencing treatment, the transfection efficiency was assessed and confirmed to be effective.3. miR-7a/b overexpression reduced the expression of SplThe expression of Sp1 was elevated in the border zone of the hearts post MI. Forced miR-7a/b expression dowen-regulated the expression of Sp1. On the other hand, inhibition of miR-7a, but not miR-7b further up-regulated the expression of Spl.4. miR-7a/b overexpression ameliorated fibrosisData obtained by Masson’s trichrome staining of heart sections revealed ECM (extracellular matrix) deposition in the interstitial regions in the border zone of the hearts post MI. Forced miR-7a/b expression diminished the area of collagen deposition, and in parallel, enhanced expression of collagen I provoked by MI were ameliorated in the GFP-7a/b groups. Whereas, knocking down miR-7a/b extended the fibrotic area and up-regulated collagen I expression.5. miR-7a/b overexpression reduced apoptosisCompared with the control group, MI induced apoptosis in the border zone and the restoration of miR-7a/b reduced TUNEL-positive cells, whereas the GFP-anti-7a/b groups processed more apoptotic cells6. miR-7a/b overexpression improved cardiac functionCompared with the sham control group, the GFP-NC group exhibited cardiac dysfunction with decreased LVEF and FS values, and with expanded ventricular diameters and damaged contractile capabilities. miR-7a/b overexpression improved while GFP-anti-7a/b groups further weakened cardiac function.Conclusion1. miR-7a/b expression fluctuated after MI2. miR-7a/b overexpression ameliorated fibrosis, reduced apoptosis and improved cardiac function after MI. |
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