| Development of skeletal muscle is a complex process:first, proliferating myoblasts get into differentiation under regulation of MRFs; then myotubes which possess several nucleus are formed by integration of several myoblasts; finally all the nucleus disperse to membrane and forms myofibers. More and more researches suggest that miRNAs play a very important role in the regulation of muscle development during various periods, however, most researches mainly concentrate in muscle-specific miRNAs, such as miR-1, miR-133 and miR-206. In order to discover more miRNAs involved in the regulation of muscle development, solexa sequencing technology was performed to identify miRNAs existing in skeletal muscle, and this was helpful for disclosure of miRNAs’biological functions.1. Identification of miRNAs in porcine skeletal musclePhysiological state together with genes participate in the regulation of muscle development are various during different periods, which suggests that species and abundance of functional miRNAs might also possess diversity in these periods. To obtain as much as possible a wide range of coverage, more time points spanned different growth periods were selected, including pregnant in 33,40,45,50,55,60,65,70,75,80,85,90, 95,100,105 dpc, postnatal 0,10 days and adult, to make a RNA pool and solexa sequencing were performed. Finally,23813176 total reads were obtained and 19937559 were high quality. After getting rid of reads without 3’primer or insert tag, reads with polyA or 5’primer contaminants and reads shorter than 18nt,19854980 clean reads of total sRNAs were obtained. Sequences of all sRNAs mainly ranged from 21-23 nt in length with a distribution peak at 22 nt.164783 unique sRNAs and 15182311 total sRNAs could match to porcine genome, which accounted for 39.69% and 76.47% respectively compared to total reads number. To make each unique sRNA mapped to only one annotation, we follow the following priority rule:rRNAetc (in which GenBank> Rfam)> known miRNA> repeat> exon> intron. The sRNAs which had no matched to any known sequences were used to predict novel miRNAs. Finally,14034350 total reads and 3407 unique miRNAs standing for 172 matured miRNAs were obtained. BLASTN searches against porcine genome revealed that 78 sRNAs were considered as predict novel miRNAs in our solexa results. The seed region was crucial for recognition of miRNAs to their target genes, and 42 miRNAs in our solexa sequencing possessed mutations in seed region.QPCR results of partial high abundance miRNAs in different organs suggested: ssc-let-7a and ssc-miR-21 expressed in a comparative level in different organs, which recommended that this kind of miRNAs might participated in widely regulations in whole body; ssc-miR-206 only expressed in skeletal muscle; ssc-miR-1 expressed both in skeletal muscle and heart; ssc-miR-378 expressed much higher in skeletal muscle and heart than other organs, suggesting that might have an important biological function in muscle; ssc-miR-10b expressed high in skeletal muscle, while it mainly expressed in kidney; ssc-miR-181a expressed high in skeletal muscle, spleen, kidney, small intestine and colon; ssc-miR-30d expressed high in skeletal muscle and kidney; ssc-miR-127 expressed high in skeletal muscle and spleen; ssc-miR-143 expressed mainly in colon; ssc-miR-148a expressed in liver and colon; and ssc-miR-30a expressed extremely high in kidney; ssc-miR-199a expressed high in longissimus muscle and low in biceps femoris muscle, this might attribute to different containing of MyHC II in different type of skeletal muscle.Longissimus muscle of adult pig, fetuses in the 33,65,90 dpc and piglets in the postnatal 0,10,100,180 days were isolated and qPCR was performed to identify the expression pattern of miR-378 in the process of myogenesis. As the results showed, the expression of ssc-miR-378 increased during the gestation, and got to maximum in postnatal 0 day, then declined gradually. It could be speculated that miR-378 might play an important role in promoting formation of muscle fibers.2. Biological functions of miR-378 in skeletal muscle developmentThe qPCR result in mouse showed that mmu-miR-378 was mainly espressed in skeletal muscle, which was consistent with the expression pattern in pig, suggesting that the function of miR-378 in regulation of skeletal muscle development might be conservative between pig and mouse. Expression of miR-378 increased during the differentiation of C2C12, got a maximum at second day of differentiation, and then maintained at a relative stable level, which suggested that miR-378 might involve in skeletal muscle differentiation. To probe the change of genes and pathway signaling downstream of miR-378, microarray was used to detect down-regulated genes in C2C12 after over-expression of miR-378. GO and pathway analysis revealed that all the 2-folds down-regulated gene involved in the biological process of proliferation, differentiation, apoptosis and transport of ion, and referred JAK-STAT, ErbB, MAPK pathway signaling. GRB2 both existed in down-regulated genes from microarray and results of target prediction. Luciferase reporter system verified the regulation of miR-378 to GRB23’UTR and found out accurate binding site using the method of mutation. MiR-378 might repress MAPK pathway in an indirect manner and promote differentiation of C2C12 by repressing of GRB2 which fulfilled a role of activator up-stream of MAPK signaling. |
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