Identification Of Muscle-Related Mirna From Skeletal Muscle In Different Development Stage Goat And Analysis On Their Regulation Roles

Goat is an important agricultural animal for meat production. Knowledge about the molecular developmental mechanisms of skeletal muscle formation is of vital interest. However, over the past few years, studies on skeletal muscle growth and development of goat were limited, and mainly examined the expression and function(s) of single genes or single developmental pathways. Functional studies have demonstrated that micro RNAs(mi RNAs) regulate gene expression at the post-transcriptional level and play an important role in various biological processes. Although studies on mi RNAs expression profiles have been performed in various animals, relatively limited information about goat muscle mi RNAs has been reported. To investigate the mi RNAs involved in regulating different periods of skeletal muscle development, global identification of the known and novel mi RNAs involved in goat skeletal muscle development is essential. In addition, the m RNA profiles were analyzed in this study. Based on these, we briefly analyzed the regulation network of mi RNA-m RNA, and the regulatory mechanism of some muscle-related mi RNA, which will significantly enhance our understanding the effect of mi RNAs on muscle development for goat.The main results are as follow: 1.Identification and profiling of micro RNAs from developing caprine skeletal muscleTwo small RNA libraries from fetal goat and six month-old goat muscle tissue were constructed using Solexa high-throughput technology. 464 known mi RNAs and 83 novel mi RNA candidates were identified. Characterization of the frequency of the position-specific nucleotide composition along the length of mi RNA revealed that nucleotide positions 1 and 9 are significantly enriched for U relative to other bases. Family analysis showed that most of the identified mi RNA families have been shown to be conserved in a variety of species. Furthermore, by comparing the mi RNA profile, 336 differentially expressed mi RNAs were identified. Most of the differentially expressed mi RNAs were higher expressing in fetal goat muscle tissue, suggest that the skeletal muscle mi RNAs composition is dynamically altered during the different development stages. By comparing the novel mi RNA profile, 40 differentially expressed novel mi RNAs were identified, among which 33 were down regulated in juvenile goat muscle tissue. 2.Global transcriptional profiling of longissimus thoracis muscle tissue in fetal and juvenile domestic goat using RNA sequencingWe used comparative transcriptional profiling based on RNA sequencing of longissimus thoracis muscle tissue obtained from fetal and six month-old domestic goats to identify genes that are differentially expressed. Gene annotation revealed that 15,960 and 14,981 genes were expressed in the fetal and six month-old goat libraries, respectively. We detected 6,432 differentially expressed genes, and when considering GO-terms found 34, 27 and 55 terms to be significantly enriched in molecular function, cellular component, and biological process, respectively. Pathway analysis revealed that differentially expressed genes were enriched in 256 KEGG terms, among which 41 were significantly enriched. Further more, large numbers of differentially expressed genes enriched in embryonic myogenesis or cell proliferation and differentiation-related pathways(such as WNT), and most of the genes involved in these pathways were down-regulated in the six month-old goats library. The identified novel transcript units, including both non-coding and coding RNAs, as well as alternative splicing events increase the level of complexity of regulation mechanisms during muscle tissue formation and differentiation. 3.Integration of mi RNA profiling and m RNA profiling analyses reveals network of mi RNA regulationAfter small RNA sequencing and RNA-Seq(Transcriptome) analysis of the paired samples, the integrated analysis of differentially-expressed mi RNAs and their predicted target genes were performed. The result showed that most individual mi RNAs had multiple gene targets and each target was regulated by multiple mi RNAs. According to mi RNA-m RNA-KEGG annotation, 10 pathways with the smallest P-value were screened. It was very interesting that among the 7 networks, mi R-424-5p were very active in that it regulates largest numbers of DE-targets and at the center of the networks. 4.Analysis of expression profiles of muscle-related mi RNAThrough integrated analysis the mi RNA profile and m RNA profile, we obtained the regulatory network for differentially expressed mi RNA and differentially expressed genes. We screened out some muscle-related mi RNAs which in the node of the net, analysed their expression profiles, including some novel mi RNA. Result showed that mi R-129-3p, mi R-424-5p, mi R-432 and mi R-665-3p were higher expressed in muscle tissue compared to other tissues or organs, of which mi R-424-5p and mi R-432 were the highest expressing in muscle tissue on both the two development stages. We detected that Novelmi R-47 is muscle tissue specificity expressed. 5.Genetic variations analysis of muscle-related mi RNAsGenetic variations of 20 above-mentioned muscle-related mi RNAs were detected by PCR-SSCP and DNA sequencing techniques in 270 individuals of three populations(Chinese Haimen goat, Chinese Xuhuai white goat and Boer goat). Result showed that 5 of the 20 detected mi RNAs have SNPs. Of the 5 polymorphic loci in this study, the polymorphic loci of mi R-411c-5p, mi R-424-5p, Novelmi R-47 and mi R-541-3p were located in the precursor sequence, Only mi RNA-767 polymorphic site is located in the pre-mi RNAs. Polymorphic information content distribution was the same in the three species, only different in different locus. Besides mi RNA-411c-3p, the other four sites were moderately polymorphic. 6.Verification of target genes for the mi R-424-5pWe construct the overexpression vector of mi R-424-5p(pc DNA3.1-pri-mi R-424-5p). Then we predict the target genes of the mi R-424-5p using bioinformatics software, namely AKT3, SMAD3, CHEK1, and construct the expression vector of the 3'UTR for the three target genes: wild type(p GL3-control-AKT3/SMAD3/CHEK1) and mutant type(p GL3-control-mut-AKT3/SMAD3/CHEK1). Moreover, the three predicted target genes of mi R-424-5p were verified using dual fluorescence report assay experiment. The results indicated that AKT3 gene is a target of mi R-424-5p. 7.The regulating effect of mi RNA-424-5p to muscle cellIn this study, C2C12 cells were cultured in growth medium(GM) or differential medium(DM) after transfected with pc DNA3.1-pri-mi R-424-5p. Then expression of marker genes of cell proliferation(Ki67) and muscle differentiation(MHC, Mef2 c, Myo D and Myf5) was analyzed by q RCR. The results showed that Myo D,Myf5 and Mef2 c were up-regulated and the difference was significant or extremely significant(P<0.05 or P<0.01). That means mi R-424-5p plays a positive role in the differentiation of C2C12 cells.