The Role And Underlying Mechanism Of Long Non-coding RNA FIRL In Cardiac Fibrosis

Aims:Cardiac fibrosis is the main characteristic of heart failure induced by various heart disease,which would increase the stiffness of heart chamber wall,reduce cardiac compliance,and ultimately promotes cardiac dysfunction.However,the underline mechanism of cardiac fibrosis is not very clear.Long noncoding RNAs(lnc RNAs)play an important regulatory role in the development of cardiovascular system and a variety of diseases.Previously,we profiled the expression of lnc RNAs by microarray in the failing hearts of patients with dilated cardiomyopathy(DCM),among which RP11-544 D21.2,we named as FIRL,was up-regulated most significantly.We aimed to elucidate the function and mechanism of FIRL in cardiac fibrosis,which might provide novel therapeutic targets in the future.Methods: Quantitative real-time polymerase chain reaction(q PCR)and in situ hybridization assays(FISH)were used to detect the location and expression of FIRL in human hearts.CCK8 assay and Ed U assay were used to determine the proliferation of cardiac fibroblasts.The level of supernatant IL-6 secreted by cardiac fibroblasts was measured by ELISA assay.The expression of fibrosis related markers ACTA2,POSTN and FN1 was shown by immunofluorescence staining(IF).RNA-pull down,mass spectrometry,site-specific mutation,dual luciferase report assay,Chromatin immunoprecipitation(Ch IP)and RNA immunoprecipitation(RIP)were used to explore the underlying molecular mechanism.Transverse aortic constriction(TAC)was used to establish the animal model of heart failure.Results: FIRL was mainly localized in nucleus of cardiac fibroblast and robustly increased in the failing hearts of patients with DCM as well as TAC treated mice.In vivo,Gapme R mediated knockdown of FIRL before or after TAC could attenuate TAC-induced cardiac fibrosis and improve cardiac dysfunction.Furthermore,cardiac fibroblasts specifically knockout of FIRL by conditional knockout mice revealed that knockout of FIRL could mitigate TAC-induced cardiomyocyte hypertrophy and cardiac fibrosis,ameliorating adverse cardiac remodeling.In vitro,FIRL could increase the proliferation of cardiac fibroblasts,promote the differentiation of cardiac fibroblasts into myofibroblasts,and upregulate the expressions of fibrosis related markers ACTA2,POSTN and FN1.Mechanistically,FIRL could recruit glycolytic enzyme ENO1 increasing the transcription and secretion of IL-6.Moreover,neutralization antibody of IL-6 could block the effect of FIRL on fibroblast proliferation in vitro.Gapme R-FIRL combined with IL-6 neutralization antibody could not further improve cardiac function and alleviate cardiac fibrosis,indicating that the pro-cardiac fibrosis effect of FIRL was IL-6 dependent.Besides,when cardiomyocytes were cultured with supernatant isolated from FIRL-si RNA pretreated cardiac fibroblasts,cardiomyocytes area and hypertrophy markers were dramatically decreased.These results showed that knockdown of FIRL in fibroblasts could indirectly diminish cardiomyocytes hypertrophy.Conclusion:Cardiac fibroblast localized lnc RNA-FIRL was significantly up-regulated during heart failure.Mechanistically,FIRL recruited glycolytic enzyme ENO1 to enhance IL-6 transcription.Furthermore,FIRL knockdown of fibroblasts directly prevented proliferation of fibroblasts and fibrosis whereas indirectly attenuated myocytes hypertrophy through IL-6 mediated myocytes-fibroblast crosstalk.