Identification Of Target Genes Of Microrna-1and Microrna-206in Muscle Development Of PIG

MicroRNAs (miRNAs), widely existed in plants and animals, were a class of evolutionarily conserved endogenous non-coding small RNA molecules, about18-25nucleotides in length. They played an important role in organism growth and development, aging and death process. Previous studies had shown that microRNAs had an important regulatory function in the skeletal muscle growth and development process. In this study, we focused on microRNA-1and microRNA-206which were specific expressed in skeletal muscle as the main object to identify their target genes and explore the function of these two miRNAs in skeletal muscle development. The results of our study are following:1. The target genes of miRNA-1and miRNA-206were predicted by Two bioinformatics software, TargetScan and PicTar. Results showed that there were258target genes could be predicted by both prediction programs. GO and KEGG pathway analysis of the258predicted target genes were performed using the DAVID database. Two targeted genes, Secreted Frizzled-Related Protein1(SFRP1) and Secreted Frizzled-Related Protein2(SFRP2) genes which were related to skeletal muscle development, were selected as the candidate target genes for further research.2. The mRNA sequence of porcine SFRP1gene was assembled and the mRNA3’UTR sequences of SFRP1and SFRP2were amplified. With Psi-Check2as the skeleton vector to construct the Psi-Check2-SFRP1and Psi-Check2-SFRP2vectors, PIEC cells were co-transfected with these two vectors and miRNA-1or miRNA-206mimics, respectively. After transfection for36h, the activity of Dual-Luciferase were detected. The results showed that compared with the negative control group, the Dual-Luciferase activity of the transfected miRNA-1mimics or miRNA-206mimics group were significantly decreased (P<0.01), and indicated that the seed sequence of miRNA-1and miRNA-206might be interaction with the3’UTR binding sites of the SFRP1and of SFRP2genes, and could inhibit the expression of these two genes.3. The bridging PCR method was used to knockout the miRNA-1and miRNA-206binding sites of SFRP13’UTR which was predicted by bioinformatics methods. With Psi-Check2as the skeleton vector to construct the Psi-Check2-SFRP1mutation vector, after had transfected PIEC cells for36h, the activity of Dual-Luciferase were detected. The results showed that compared with the NC control group, the Dual-Luciferase activity of mutation vector had no significantly changed, and indicated that this site might be the target binding site of miRNA-1and miRNA-206.4. Because the expression level of SFRP1gene in PIEC cells was very low, the CDS of porcine SFRP1gene was amplified, and the Psi-Check2-SFRP1CDS-SFRP13’UTR vector was constructed. PIEC cells were co-transfected with this vector and miRNA-1or miRNA-206mimics. After had transfected for36h, the mRNA and protein were extracted to detect the mRNA and protein level of SFRP1. The real-time quantitative PCR (RT-PCR) results showed that compared with the NC group, the SFRP1mRNA expression levels (co-transfected the SFRP1over-expression vector and miR-1/206mimics) had no significant difference (P>0.05). The Western-blot analysis showed that compared with the NC group, SFRP1protein expression levels at the experimental group were significantly decreased. It was indicated that SFRP1was the common target of miRNA-1and miRNA-206and the expression of SFRP1were mainly regulated by these two mircoRNAs at the translate level.5. The spatial-temporal expression profiles of SFRP1and SFRP2genes were carried out using real-time PCR. The spatial expression levels of9different tissues in adult Tongcheng pigs and the temporal expression levels in developmental skeletal muscle at Tongcheng (27time points) and Landrace pigs (23time points) were detected. The results showed that the SFRP1gene were highly expressed in the small intestine, stomach, kidney and lung, moderately expressed in liver, the large intestine, spleen and weakly expressed in the Longissimus muscles and leg muscles. And the SFRP2gene were abundantly expressed in the stomach, lung, colon and small intestine, moderately expressed in the Longissimus muscles and leg muscles and weakly expressed in the heart and spleen. The expression levels of SFRP1and SFRP2genes in the embryonic skeletal muscle development stages were significantly higher than that in the postnatal stages birth in both Tongcheng pigs and Landrace pigs. These results indicated that SFRP1and SFRP2genes may involved in the formation of primary and secondary fibers of the skeletal muscle. Between Tongcheng and Landrace pigs, the expression pattern of SFRP1and SFRP2showed significantly differences. It suggested that SFRP1and SFRP2might be related to the muscle phenotypic differences between these two pig breeds.