Regulation Of MiRNA-423-5p On Muscle Development By Targeting SRF Gene In Pig

MicroRNA(miRNA) is a class of 21 nucleotides small RNA molecules in length, which conserve in evolution. MiRNA participates in life process by posttranscriptional regulation. Muscle development is regulated by abundant of genes in pig. Recent study showed that miRNA has a very important role in skeletal muscle development. Current study showed that the expression of miRNA-423-5p in porcine skeletal muscle was significant difference in different development stages. We speculate that miR-423-5p(miRNA-423-5p) may regulate skeletal muscle development by targeting specific genes. There were no reports on regulation of miR-423-5p on porcine skeletal muscle development in previous studies. Therefore, the objective of current study is to scan the target genes of miR-423-5p and investigate target genes’ function in porcine skeletal muscle growth and development. The main results are following:1. SRF gene was predicted as a possible target gene of miR-423-5p using online biological software such as TargetScan et al.2. Employing real time quantification (qRT-PCR) technology tested the expression of miR-423-5p and SRF in longissimus muscles in five different periods in Guangxi Bama mini-pig and drew the expression profiling. The results indicated that the expression level of miR-423-5p mRNA was gradually increased with aging, but the mRNA expression of SRF was gradually decreased. The miR-423-5p was expressed in all tissues. The expression level of ssc-miR-423-5p was significantly highest in heart than in kidney, brain, longissimus dorsi, liver, and spleen. While the expression level of ssc-miR-423-5p was significantly higher in kidney, brain, and longissimus dorsi compared with in liver and spleen.3. The recombinant vectors, pLV-miR-423-5p, had been successfully constructed by connecting a miR-423-5p precursor sequences to pLV Lentiviral vectors. The results of transfection and qRT-PCR showed that the pLV-miR-423-5p was successfully expressed in C2C12 cell.4. SRF 3’UTR with binding site or with no binding site of miR-423-5p was amplified using a pair of specific primer and cloned into the psi-CHECKTM-2 vector to construct normal plasmid SRF-WT and mutant plasmid SRF-MUT. The recombinant plasmids were transfected into PK15 cell lines. The results showed that luciferase activity of WT group was significantly decreased compared with NC and MUT group (P<0.01). The luciferase activity of miWT group was significantly decreased compared with miNC, inWT and inMUT group (P<0.01). There were no significant difference between MUT group and NC group, and among miMUT, miNC, and inWT (P>0.05). The results indicated that miR-423-5p might interact with the 3’UTR binding sites of SRF, and could inhibit the expression of SRF. SRF was a target gene of miRNA-423-5p and the mutant sites were the target sites.5. C2C12 myoblast was induced to differentiate and collected for qRT-PCR testing. The result showed that the mRNA expression levels of miR-423-5p were significantly up-regulated at the first day to fourth day after inducing differentiation, to fifth day, was decreased. On the contrary, the expression level of SRF was significantly down-regulated at the first day to fourth day after inducing differentiation, to fifth day, was increased. During the differentiation process of C2C12, the expression of MyoG mRNA was significantly up-regulated and the expression of MyoD mRNA was highest at first day after inducing differentiation and before inducing differentiation than among any day after inducing.6. The miR-423-5p mimic or inhibitor was transfected into C2C12 cell for 48 hours and the cells were collected for qRT-PCR. The results of qRT-PCR showed that the miR-423-5p was successfully overexpressed in C2C12 cells transfected with mimic, while the expression of miR-423-5p was inhibited in C2C12 cells transfected with inhibitor. Compared with the NC group, the SRF mRNA expression levels had no significant difference (P>0.05), but MyoD and MyoG mRNA dramatically were decreased when the expression of miR-423-5p was overexpressed (P<0.05). On the contrary, when the expression of miR-423-5p was inhibited, the expression of MyoD and MyoG mRNA dramatically increased (P<0.05). The ELISA result indicated that the SRF protein levels was significant down-regulated when the expression of miR-423-5p overexpressed (P<0.05). But SRF protein levels dramatically were up-regulated when the expression of miR-423-5p was inhibited (P<0.05). The results further indicated that SRF is the target gene of miR-423-5p and the expression of SRF are mainly regulated by miR-423-5p at the translate level.To sum up, miR-423-5p negatively regulates the skeletal muscle development by inhibiting the expression of SRF gene in protein level.