Fibroblast-derived Exosome MiRNA-133 Obviously Promotes Cardiomyocyte-like Differentiation

BackgroundExosomes are nano-scale biofilm structures that contain proteins,microRNAs,cytokine receptors,and other biologically active substances.MiRNAs can regulate gene expression at the transcription level to regulate cell proliferation and apoptosis.Exosomal-derived miRNAs are currently found to have biological characteristics,participate in the physiological and pathological processes of many diseases,and are expected to become molecular diagnostic markers of diseases.So far whether miRNA-133 in exosomes can promote myocardial differentiation has not been reported.ObjectiveIn order to investigate the role of exosome miRNA-133 secreted by cardiac fibroblasts(CFs)in promoting cardiomyocyte differentiation.Methods1.Neonatal rat cardiac fibroblasts were cultured in vitro.2.The cultured CFs were divided into three groups:(1)induction group(2)miRNA-133 high expression group(3)miRNA-133 inhibition group.3.miRNA-133 was transfected into CFs with lentivirus as a vector;CFs were transfected with miRNA-133 inhibitor,and the markers of cardiomyocyte-like cardiac muscle were detected by Western Blotting,immunofluorescence staining and real-time qPCR at 3d,8d and 14 d respectively.Expression of cardiac troponin T(cTNT)and cardiac actin(?-actin),real-time qPCR was used to detect the expression of miRNA-133 in fibroblast exosomes.Results1.Immunofluorescence staining showed that CFs expressed vimentin and DDR.2.Exosomes secreted by CFs were observed by transmission electron microscopy as small vesicles of 30-100 nm,and Western blotting was used to detect exosome-specific protein CD63 and CD9 expression.3.Compared with the induction group,the expression levels of cTNT,?-actin and exosome miRNA-133 secreted into the supernatant of miRNA-133 high expression group increased gradually at different time points,and reached the highest level at 14 d.The cTNT,?-actin and exosome miRNA-133 in miRNA-133 inhibition group was the lowest.ConclusionsThe exosome miRNA-133,an exosome derived from cardiac fibroblasts,can obviously promote the differentiation of fibroblasts into cardiomyocytes.