Transcriptome Profile Analysis Of Porcine Muscle Fiber By High-throughput Sequencing And Research On The Biological Roles Of MiR-423-5p

The analysis of transcriptome contributes to more precisely identification of gene structure and function, and RNA-seq has been developed to utilize depth transcriptome analysis technology, which significantly promoted the understanding of the transcriptome.However, the molecular mechanism of porcine muscle fiber type remains to be determined. In this study, we apply RNA-seq for analyzing the wide range of transcriptional changes in porcine muscle tissues. In addition, we performed the advanced knowledge of the expression profile, GO terms, Pathway analysis, interaction network, novel transcripts, refinement of gene structure, alternative splicing events and SNP analysis.Through analyzing different types of muscle transcriptome library, we found that the expression pattern of gene SUFU differed in different types of muscles, but the specific mechanism in the muscle development is still not very clear. In order to analyze the role of SUFU during muscle development, we analyze SUFU expression pattern in different tissues and its role in muscle cell proliferation and differentiation by RNA interference technology.Bioinformatics analysis has found that the miR-423-5p binding sites is presented in3’UTR region of the SUFU gene. As the target mi RNA of SUFU, the mechanism of mi R-423-5 p in muscle differentiation remains to be elucidated. In this study, We first detected the expression profile of different tissue and C2C12 cells. Then we analyzed the role of miR-423-5p in myoblast proliferation and differentiation, to be fully understand the regulation mechanism of muscle development.There are major results based on investigation above, as follows:1. RNA-seq analysis yielded 89,658,562 total reads for EDL muscle and 46,723,568 total reads for Sol muscle. Expressed short reads were mapped to 32,432 reference genes(16,415/16,017, EDL/Sol) of the Sus scrofa genome.2. The expression pattern revealed that up to 2,152 differentially expressed genes(DEGs)between two types of muscle. Overall, we identified 1,534 up-regulated and 618down-regulated genes. 1,422, 1,481, and 1,526 genes were mapped to one or multiple GOterms for biological process, molecular function and cellular component.3. Specific enrichment of DEGs was observed in 241 signaling pathways using the KEGG pathway database, in addition, 230 and 216 genes were affected by the up- and down-regulated. We investigated the transcription levels of 57 genes related to muscle characteristics, and there was no significant discrepancy for 22 genes between EDL and Sol libraries. We next studied potential protein-protein interactions of 18 muscle-related DEGs,and found most of the targeted protein has different expression level between two types of muscle. In EDL sample, 3,271 genes were refined in the 5’-end region, and 1,599 genes had an extension at the 3’-end. In Sol sample, 2,814 genes were detected to be extended at the5’-end, and 1,385 genes had at least an extension at the 3’-end. 10,962 and 9,686 novel transcripts were identified in EDL and Sol muscles. RNA-seq has found that 9,334 and 8,585 alternative splicing events were observed in EDL and Sol libraries, as well as 58,362 and58,359 potential SNPs.4. SUFU was identified as the differential expressed transcripts based on RNA-seq data,and the research has found that SUFU is widely expressed in different tissues of mice; SUFU showed the expression trend of first rise after falling during the proliferation and differentiation of C2C12 cells; Interference of SUFU could partially inhibit the proliferation and differentiation of C2C12 cells.5. mi R-423-5p, as the potential target of SUFU, was wide expressed in different tissues of mice; During the proliferation of C2C12 cells, the expression trend of miR-423-5p indicated to be early rising to peak, then gradually decreasing to smooth expression level;Over-expression of miR-423-5p could inhibit the proliferation of C2C12 cells as well as cell division level.6. During the differentiation of C2C12 cells, The expression pattern of miR-423-5p is gradually increased, reaching its peak in post differentiation, and then gradually decline;Over-expression of miR-423-5p could inhibit the differentiation of C2C12 cells and the formation of myotube.In all, our transcriptome sequencing analysis has obtained high quality data, and also identified and analyzed the function of gene SUFU and its targeted miR-423-5p, which provides the deep understanding of muscle development and muscle fiber type.