Screening And Identification Of MiRNA Related To Muscle Development In Dazu Black Goats

Goat is one of the important domestic animals and its genetic basis related to main economic traits has become the focus of goat breeding research.Dazu Black goat,as a representative local breed of mutton sheep in southwest China,plays an important role in local animal husbandry industry.The growth rate of livestock and poultry is closely related to muscle development,which is regulated and affected by a series of complex and precise multi-level factors.In particular,Micro RNA(miRNA)has been widely confirmed to be involved in the genetic mechanism of animal muscle development,but the mechanism of miRNA action in goat muscle development is rarely reported.In order to further identify key candidate miRNA related to skeletal muscle development of Dazu Black goats and reveal their regulatory mechanisms,RNA-Seq was used to sequencing miRNA transcriptomics from longissimus dorsi muscle of Dazu Black goats at different developmental stages(embryonic 75 days(ET)and postnatal 1 day(DC)).Candidate miRNA that may be related to muscle growth were screened and obtained,and the function of chi-miR-27b-3p in skeletal muscle satellite cells was preliminarily verified.Therefore,this study not only provides data support for molecular selection of meat goat breeding in the future,but also provides theoretical basis for revealing the genetic basis related to muscle growth and development of goats.The main results obtained in this study are as follows:1.Quality control of original data.A total of 218,161,800 clean tags were obtained by transcriptome sequencing of the longissimus dorsi sequencing libraries of 12 Dazu Black goats.In addition,the sequence length of 4.45%-45.57% clean tags is distributed between20-24 nt,accounting for the largest 22nt(45.57%),followed by 23nt(20.15%).2.Identification of differentially expressed miRNA(DE＿miRNA)between groups and functional annotation of target genes.A total of 1514 miRNA were identified in this study,of which 546 were miRNA with significantly different expression between groups(P <0.05).Compared with DC group,321 miRNA were up-regulated and 225 were down-regulated in ET group.A total of 7162 target genes were predicted from 546DE＿miRNA.The GO analysis results of target gene function annotation showed that 4933,4673 and 5482 target genes were significantly enriched in 10454 biological process entries,1244 cellular component entries and 2415 molecular functions items,respectively.It includes a large number of items related to muscle development such as skeletal system development,osteoblast development,skeletal muscle tissue development and so on.Secondly,KEGG enrichment analysis showed that a total of 3121 target genes were enriched into 345 KEGG pathways,including signaling pathways that have been clearly related to muscle development,such as PI3K/Akt and Wnt.3.RT-q PCR verification of differentially expressed miRNA.Twenty-two miRNA(chi-miR-27b-3p,chi-miR-133a-5p,chi-miR-154a-3p,etc.)with significantly differentially expressed miRNA transcriptomic sequencing results were randomly selected for RT-q PCR verification,and their relative expression levels were 100% consistent with the sequencing results.The results of miRNA transcriptome sequencing and data analysis in this study are reliable.4.Biological effects of chi-miR-27b-3p on skeletal muscle satellite cells(SMSC)of goats.In this study,the leg muscle of Dazu Black goat at 1 day after birth was used for primary goat SMSC culture,and the culture results showed that the primary cells were in line with the morphology of skeletal muscle satellite cells.After 48 hours of culture,the isolated and purified skeletal muscle satellite cells were identified by immunofluorescence.The expression of Pax7,the marker gene of skeletal muscle satellite cells,was positive,indicating that the primary SMSC was successfully isolated and cultured.chi-miR-27b-3p mimics/mimic NC/inhibitor/inhibitor NC were transfected into goat SMSC,and their effects on the proliferation of goat SMSC were detected.The results showed that over-expression of chi-miR-27b-3p inhibited the proliferation of goat SMSC.Interference with chi-miR-27b-3p promotes the proliferation of SMSC in goats.The expression level of MDFI after over-expression of chi-miR-27b-3p was detected by RT-q PCR,and the results showed that over-expression of chi-miR-27b-3p significantly inhibited the expression of MDFI gene(P < 0.05).The targeting relationship between chi-miR-27b-3p and MDFI gene was verified by dual luciferase reporting system.Therefore,it is further inferred that chi-miR-27b-3p is involved in regulating the proliferation of SMSC in goats through targeting MDFI.In this study,a large number of miRNAs that may be related to goat muscle development were screened by miRNA transcriptome sequencing,the effect of chi-miR-27b-3p on the proliferation of goat skeletal muscle satellite cells was clarified,and the targeting relationship between chi-miR-27b-3p and MDFI was further revealed.Therefore,the results of this study not only contribute to the understanding of the mechanism of miRNA action in skeletal muscle development of goats,but also provide valuable genetic data for molecular breeding of Dazu Black goats in the future.