Regulation And Mechanism Of MiRNA-206 On Proliferation And Differentiation Of Goat Skeletal Muscle Satellite Cells

The goat industry is an important part of China’s animal husbandry.With the improvement of people’s living standards,the demand for lamb is increasing.Therefore,it has become a hot topic to study the regulation and mechanism of goat muscle growth.MicroRNA is a class of non-coding small molecule RNA with regulatory effect that widely exists in eukaryotes.Many studies have shown that miRNA can affect the expression of target genes in mRNA and protein levels,and miRNA is involved in cell proliferation,differentiation and apoptosis.In particular,a large number of studies have confirmed that miRNA play an important role in the regulation of muscle development in animals by regulating the metabolism of skeletal muscle satellite cells(SMSC).Based on the published results of genome-wide miRNA differential expression of dorsal longest muscle tissue at different developmental stages of fetal development in Jianzhou Daer goats,we chose chi-miR-206 as candidate miRNAs that showed significant differences(P <0.01),and used SMSC of Dazu Black goats as experimental materials.We studied the regulatory effect of miR-206 on the proliferation and differentiation of SMSC in vitro by overexpression technology,and further chose and verify the possible target genes of this miRNA and their interactions.These studies provide a basis data for the study of the molecular genetic mechanism of goat muscle development.The following results were obtained:1.Expression profile and cellular localization of miR-206 in different tissues of Dazu Black goat.Expressions of miR-206 in four stages(fetus,day 1,3 months,and 18 months)of Dazu Black goats were detected by fluorescence quantitative PCR.The results showed that miR-206 was specifically expressed in the skeletal muscle(longissimus dorsi,leg muscles,pectoral muscle,and gluteal muscle)from all the three stages,while it was hardly expressed in other tissues.The results confirmed the chi-miR-206 specific expression in muscle tissue,and the expression level is related to the degree of muscle development.The results of fluorescence in situ hybridization indicated that chi-miR-206 was abundantly expressed in the cytoplasm,and low expressed in the nucleus.2.Study on the regulation of miR-206 on the proliferation and differentiation of goat SMSC.Firstly,qPCR was used to detect the expression of Pax7 and MyoD.Pax7 and MyoD were verified as marker genes for SMSC proliferation.Simultaneously it also indicated that the goat skeletal muscle in vitro culture system was successfully established in this study.Secondly,it was found that miR-206 had no significant effect on the proliferation of SMSC through the overexpression and inhibition methods.Thirdly,myo-differentiation culture system IV was used to cultured cells in vitro,then extracted RNA at 1d,2d,3d,and 4d to detect the expression of MyoG and MyHC.The results proved again that MyoG and MyHC can be marker genes for SMSC proliferation.Further,it was found that overexpression of mi R-206 promoted the differentiation of goat SMSC,and inhibition of miR-206 reduced the differentiation of goat SMSC.3.Preliminary study on the regulatory mechanism of miR-206 on SMSC differentiation.TargetScan software was used to predict the target genes of miR-206.Through comparison analysis,HDAC4 was selected as the target gene of miR-206 and verified.MiR-206 overexpression and inhibitor were transfected into goat SMSC,respectively,and Real time-PCR and Western blotting were used to detect the expression of HDAC4 at different levels.The results showed that over-expression of miR-206 significantly inhibited the expression of HDAC4 at the transcription and protein levels(P<0.05),while inhibiting the expression of miR-206 significantly increased the expression of HDAC4 protein.Thus,it was verified that the HDAC4 gene is a target gene of goat miR-206.At the same time,the RNAs were extracted in SMSC with miR-206 mimics and inhibitor transfected,and 11 differentially expressed genes(ANKRD1、TIMP3、CTHRC1、TGFB3、ELN、C1QTNF3、LIPG、PTBP2、FZD4 and new genes LOC102184087 and LOC102176691)were screened by high-throughput sequencing.The results provide scientific reference for further studying the molecular genetic mechanism of miR-206 in goat muscle development.Generally speaking,the results indicate that miR-206,which is specifically expressed in goat skeletal muscle tissue,may regulate the differentiation of satellite cells by regulating on target gene HDAC4,and affect the growth and development of goat skeletal muscle.