| Background:Cardiac fibrosis,associated with adverse prognosis in cardiovascular disease,is one of the most common pathophysiological processes induced by acute or chronic stimuli such as myocardial infarction and hypertension.Following the mechanical stimulation,pressure/volume overload,humoral and other pathological factors,cardiac fibroblasts proliferate and trans-differentiate to myofibroblasts,resulting in excessive secretion of extracellular matrix(ECM),collagen scar formation,decreased cardiac compliance and cardiac remodeling,and ultimately heart failure.Currently,efficacious treatments for cardiac fibroblast activation and fibrosis are lacking.Therefore,discovering key molecules for cardiac fibrosis and elucidating the mechanism is of great value for cardiovascular treatment.mRNAs,non-coding RNAs and proteins play an essential role in the initiation and development of cardiac fibrosis.Besides,there is a complex interaction between these molecules.Long non-coding RNA(lncRNA)is a non-coding RNA class with a length of more than 200 nucleotides.Although lncRNA is considered a potential therapeutic target for cardiovascular diseases,some inherent difficulties,such as its conservatism,secondary structure effect,and cell type-specific expression profile,contribute to only a few lncRNAs with definite biological functions related to diseases.Thus,it is worthwhile to further explore the lncRNAs related to cardiac fibrosis and gain insights into their molecular basis.The advent of high-throughput technologies,such as microarray and second-generation sequencing,has made it possible to discover more known and unknown molecules,including mRNA and non-coding RNA.Therefore,whole transcriptome sequencing contributes to revealing mRNA and non-coding RNA expression and finding molecules playing critical roles in diseases from the aspects of molecular interaction and genotype-phenotype association.Furthermore,using bioinformatics is helpful to find the key molecules in the process of cardiac fibrosis.Weighted Gene Co-expression Network Analysis(WGCNA)is a systems biology method originally conceived for describing correlation patterns among genes across microarrays data.Now WGCNA has been successfully applied to cancer-related studies.Therefore,the association analysis between clinical traits and gene expression patterns is helpful to mine key lncRNAs in many gene expression changes.Gene-set enrichment analysis(GSEA)is a gene enrichment method,which can correlate lncRNA with mRNA expression regardless of specific thresholds,making the most of the information of RNA-seq to find the potential biological function of lncRNA.Therefore,in this study,we first established a mouse model of cardiac fibrosis and performed RNA-seq.Then,we screened some key lncRNAs from transcriptome data by bioinformatics method.Finally,we carried out animal and cell experiments to verify the selected lncRNAs.Objective:(1)Establishing a mouse model of cardiac fibrosis induced by thoracic aortic constriction(TAC)and observing the dynamic changes of cardiac anatomy and function;obtaining cardiac transcriptome profiling of TAC mice heart at different stages using the next-generation sequencing.(2)Screening critical lncRNAs associated with cardiac fibrosis using bioinformatics analysis based on RNA sequencing.Validation of the lncRNAs we mined in vitro and in vivo.(3)Exploration whether lncRNA Gm29233 regulates cardiac fibroblast activation through miR-221/222-TGFBR1 axis in vitro.(4)Exploration of the role of lncRNA Gm29233 on cardiac fibrosis in vivo.Materials and Methods:1.Establishment of a mouse cardiac fibrosis model and RNA-seq.(1)Thoracic aortic constriction(TAC)operation was performed on mice to establishthe mouse model of cardiac fibrosis.After TAC,the heart structure and function changes were observed by echocardiography,and the liver and kidney functions were detected by an automatic biochemical analyzer.Hematoxylin-eosin(HE)staining and Masson staining were used to observe the cardiac hypertrophy and collagen deposition in myocardial tissues.Quantitative PCR and Western-blot were carried out to determine the expression of the markers of cardiac fibrosis in cardiac tissues.The above indexes were used to evaluate whether the mouse cardiac fibrosis model induced by pressure overload was successfully constructed.(2)RNA-seq: 2 μg total RNA of the left ventricle was extracted to construct a chain-specific library for Illumina sequencing.Then quality control,quantitative and differential analysis,and functional annotation using transcriptome sequencing data were carried out.2.Screening the key lncRNAs in the process of cardiac fibrosis using bioinformatics.(1)WGCNA: Based on the expression of lncRNAs,quality control,co-expression network construction,module clustering,module and trait association,and core genes screening were carried out.(2)GSEA: using the R language,single-gene GSEA was performed to analyze the function of lncRNA.First,the correlation coefficient between lncRNA and mRNA was calculated and ranked,and then the enrichment score and permutation test were conducted to determine whether the pathway was enriched or not.3.Expression and function of lncRNA Gm29233.(1)In the mouse model of cardiac fibrosis induced by TAC,quantitative PCR was used to detect the expression changes of lncRNA Gm29233 and other lncRNAs screened by bioinformatics.(2)Determining the expression of lncRNAs in activated cardiac fibroblasts:mouse neonatal cardiac fibroblasts were isolated and treated with transforming growth factor-β1(TGF-β1,10 ng/m L)for 24 h to induce fibroblast activation in vitro.The expression of fibrotic markers,such as CTGF,POSTN and α-SMA,were detected by RT-q PCR and Western-blot.The fluorescence intensity of α-SMA was observed by confocal laser scanning microscopy.The cell proliferation rate was measured by the EdU incorporation assay.The cell migration rate was measured by scratch assay,and the collagen secretion ability was measured by collagen gel contraction assay.These detections aim to identify the activation phenotype of cardiac fibroblasts.After cardiac fibroblasts were induced to myofibroblasts phenotype,the expression of lncRNAs in activated cardiac fibroblasts was detected by RT-q PCR.(3)Subcellular localization: nucleus and cytoplasm separation and RNA-FISH(RNA-fluorescence in situ hybridization)were used to identify lncRNA expression in cytoplasm or nucleus.(4)To determine the role of lncRNA Gm29233 in the activation of cardiac fibroblasts: the expression of Gm29233 in cardiac fibroblasts was silenced with specific siRNA(100 n M),or Gm29233 overexpressed with adenoviral vector(200MOI),followed by TGF-β1 stimulation of cardiac fibroblasts for 24 h.The effects of knockdown or overexpression of lncRNA Gm29233 on fibrosis markers and activated phenotypes(fluorescence intensity of α-SMA,EDU,scratch test,collagen secretion ability)were observed.4.To clarify the regulatory relationship between lncRNA Gm29233 and miR-221/222-TGFBR1.(1)Bioinformatics analysis: Rnahybrid and miRanda software were used to predict the targeted miRNA of lncRNA Gm29233 and TGFBR1.(2)Dual-Luciferase Reporter Assay: A wild-type dual-luciferase reporter vector containing lncRNA Gm29233 and TGFBR1 sequences and a mutant-type vector containing the mutation site were constructed.Additionally,the miR-221/222 sensor,a plasmid with complementary sequences with miR-221/222,was also constructed.Next,the plasmid DNA and miR-221/222 mimics were transfected into HEK 293 T cells.Firefly luciferase activity was analyzed by Dual-Glo Luciferase Assay System(Promega)and was normalized to the Renilla luciferase activity.(3)To determine the role of miR-221/222 in the activated cardiac fibroblasts:specific miR-221/222 mimics were transfected in cardiac fibroblasts to overexpress miR-221/222 and specific miR-221/222 antagomirs to inhibit the expression of miR-221/222.The changes in the expression of fibrosis markers were detected by Western-blot to observe the role of miR-221/222 on the activated cardiac fibroblasts.(4)To determine whether lncRNA Gm29233 regulates cardiac fibroblasts activation through the miR-221/222-TGFBR1 axis: Quantitative PCR or Western-blot were used to detect the fibrosis markers and TGF-β/Smad signaling pathway in the activated cardiac fibroblasts.The α-SMA fluorescence intensity,EDU,scratch test,and collagen secretion experiment were performed to evaluate cardiac fibroblasts’ activation.To observe the effect of overexpression or knockdown of lncRNA Gm29233 on activated cardiac fibroblasts,miR-221/222antagomirs(inhibitors of miR-221/222)treatment after knockdown of lncRNA Gm29233 with siRNA,or miR-221/222 mimics(agonists of miR-221/222)or Si TGFBR1(knockdown of TGFBR1 expression)treatment after overexpression of lncRNA Gm29233 with adenovirus were carried out.The changes in the activation of cardiac fibroblasts were observed.5.To investigate the effect of specific knockdown of lncRNA Gm29233 in activated fibroblasts on cardiac fibrosis.Mice were treated 7 days after TAC with either shRNA contained periostin promoter-driven,specifically knockdown Gm29233 in activated cardiac fibroblasts or shNc by administering adeno-associated virus via tail vein.Cardiac function and fibrosis were assessed by echocardiography,HE staining and Masson staining four weeks later.Quantitative PCR and Western-blot were used to evaluate the expression of fibrosis markers.6.Conservation analysis of lncRNA Gm29233,and expression and functional analysis of its homologous sequences AC037198.2.(1)Human lncRNA with the conserved position of lncRNA Gm29233 was found in UCSC Genome Browser and Ensemble Genome Browser.After the homologous lncRNA AC037198.2 was found,sequence alignment software MAFFT was used to compare the sequence conservation.(2)PCA,lncRNA co-expression analysis and functional annotation were performed using GSE152250 in the GEO database.Results:1.Evaluation of mouse cardiac fibrosis model and RNA-sequence.(1)Compared with the Sham group,cardiac fibrosis was observed at 4 and 8weeks after TAC,characterized by impaired cardiac function and increased collagen deposition,accompanied by liver function injury and slight deterioration of renal function.(2)Transcriptome RNA-sequence: library data were qualified.Samples could be clustered by the group.Compared with the Sham group,there were 2053up-regulated and 1344 down-regulated genes in the TAC＿2W group and 1028up-regulated and 517 down-regulated genes in the TAC＿4W group.Differential genes in TAC＿2W vs.Sham and TAC＿4W vs.Sham were enriched in several pathways related to fibrosis formation(extracellular matrix formation).2.LncRNA Gm29233 is a crucial molecule in the process of cardiac fibrosis.(1)WGCNA analysis: Through gene clustering,it was found that the cyan module was significantly positively correlated with sample stage and heart/body weight ratio and negatively with ejection fraction.By comparing module member(MM)and module significance(MS)in the cyan module,we select five lncRNA associated with cardiac fibrosis: Gm13054,Gm9913,Gm29233,D030025P21 Rik and Dnm3 os.(2)Compared with the Sham group,the expression of lncRNA Gm13054,Gm9913,Gm29233,D030025P21 Rik,Dnm3os were significantly increased in the TAC group.The expression of Gm29233,D030025P21 Rik and Dnm3 os were upregulated in the activated cardiac fibroblasts induced by TGF-β1 compared with the control group,and the expression of Gm29233 and Dnm3 os showed a continuous rise trend consistently over TGF-β1 stimulation time.(3)Single-gene GSEA analysis of lncRNA Gm29233 revealed significant activation of some signaling pathways related to cardiac fibrosis,including epithelial-mesenchymal transformation,TGF-β,extracellular matrix connectivity,cell cycle and inflammatory response.Based on the most significant contribution to the enrichment of the gene set,Gm29233 was significantly correlated with some fibrosis markers and transforming growth factor-beta receptor I(TGFBR1).3.LncRNA Gm29233 mainly localizes in the cytoplasm.In vitro,lncRNA Gm29233 regulates the activation of cardiac fibroblasts.(1)Nucleo-cytoplasmic separation and RNA-FISH experiment revealed that lncRNA Gm29233 was mainly localized in the cytoplasm.(2)In vitro,the specific knockdown of lncRNA Gm29233 by siRNA reduced TGF-β1-induced activation of cardiac fibroblasts and attenuated their proliferation,migration and collagen secretion ability.(3)In vitro,adenovirus-specific overexpression of lncRNA Gm29233 promoted TGF-β1-induced activation of cardiac fibroblasts and enhanced their proliferation,migration and collagen contraction.4.LncRNA Gm29233 regulates cardiac fibroblast activation through the miR-221/222-TGFBR1 axis.(1)In vitro,the expression of TGFBR1 decreased after the specific knockdown of lncRNA Gm29233 compared with the control group.In contrast,the expression of TGFBR1 increased after the overexpression of lncRNA Gm29233 with adenovirus.(2)RNAhybrid and miRanda software predicted that miR-221 and miR-222 might interact with lncRNA Gm29233 and TGFBR1.(3)Compared with the control group,the expression of miR-221 and miR-222 decreased after TGF-β1 stimulation,respectively.After the specific knockdown of lncRNA Gm29233,the expression of miR-221 and miR-222 were significantly upregulated.(4)After co-transfection of miR-221/222 sensor and wild-type dual-luciferase reporter vector containing Gm29233 and TGFBR1 sequence with specific miR-221/222 mimics,the fluorescence intensity of HEK 293 T cells was significantly decreased.(5)Knockdown of miR-221/222 with antagomirs promoted TGF-β1-induced activation of cardiac fibroblasts,whereas overexpression of miR-221/222 with mimics reduced cardiac fibroblast activation.In the presence of TGF-β1 stimulation,the expression of fibrosis markers was significantly decreased in the overexpression of Gm29233 plus miR-221/222 group compared with the overexpression group.(6)Compared with the mimic NC group,overexpression of miR-221/222 could significantly reduce the protein level of TGFBR1.Compared with the miR-221/222 mimic group,the overexpression of lncRNA Gm29233 plus mimic recovered the TGFBR1 expression decrease.(7)Under TGF-β1 stimulation,the expression of fibrosis markers,the fluorescence intensity of α-SMA,proliferation,migration and collagen contraction of cardiac fibroblasts were significantly mitigated in the overexpression of Gm29233 plus miR-221/222 mimic group and the overexpression of Gm29233 plus knockdown TGFBR1 group,compared with the overexpression group.Additionally,the levels of P-Smad2/T-Smad2/3 and P-Smad3/T-Smad2/3 were also reduced.5.Specific knockdown of lncRNA Gm29233 targeting activated cardiac fibroblasts alleviates TAC-induced cardiac fibrosis in mice.(1)4 weeks after TAC,compared with the Sh-NC group,the H/W ratio,cardiac function and left ventricular diameter of mice in the Sh-Gm29233 treatment group were significantly improved,and the deposition of cardiac collagen fibers was also reduced.(2)4 weeks after TAC,compared with the Sh-NC group,the expression of fibrosis markers was decreased in the Sh-Gm29233 group,and the expression of lncRNA Gm29233 and TGFBR1 in myocardial tissues was decreased,while the expression of miR-221/222 increased.Additionally,the levels of P-Smad2/T-Smad2/3 and P-Smad3/T-Smad2/3 were reduced.6.AC037198.2 is the homologous sequence of lncRNA Gm29233 and AC037198.2 might be involved in regulating cardiac fibrosis.(1)Through genome alignment and conservation analysis,AC037198.2 is the homologous sequence of lncRNA Gm29233 in the human genome.(2)Analysis of GSE152250 showed that,compared with the control group,the expression of AC037198.2 was significantly upregulated in human cardiac fibroblasts after TGF-β1 stimulation,and the upward increase was more significant after 48 hours compared with 24 hours.AC037198.2 was positively correlated with TGFBR1,with a correlation coefficient of 0.96.(3)Through the functional annotation and pathway-gene co-expression network,AC037198.2 was significantly correlated with POSTN,CTGF(CCN2),α-SMA and FBN1,and might be involved in the formation of extracellular structure,extracellular matrix formation,connective tissue development and bone formation pathways.Conclusions:(1)RNA-sequence and bioinformatics analysis reveal that lncRNA Gm29233 involves in the process of cardiac fibrosis induced by pressure overload.(2)Knockdown of lncRNA Gm29233 alleviates cardiac fibroblasts’ activation,while overexpression of lncRNA Gm29233 promotes the further activation of cardiac fibroblasts.Specific knockdown of lncRNA Gm29233 targeting activated cardiac fibroblasts alleviates TAC-induced cardiac fibrosis in mice.(3)LncRNA Gm29233 regulates cardiac fibroblast activation and cardiac fibrosis through the miR-221/222-TGFBR1 axis.(4)AC037198.2 is the homologous sequence of lncRNA Gm29233 in the human genome,which may be involved in the process of cardiac fibrosis.AC037198.2 is expected to become a potential target for the treatment of cardiac fibrosis. |
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