| MicroRNAs are evolutionarily conserved small non-coding RNAs that post-transcriptionally regulate gene expression and emerged as crucial regulators of skeletal muscle development. Preliminary results in our lab, using a Solexa deep sequencing approach, have shown that miR-543was differently expressed during fetal ongissimus dorsi muscle development in Chinese Qinchuan cattle(Sun et al.,2013). But relative functional studies about regulation of miR-543in myogenesis have been not reported in literatures. Hence, the miR-543expression profiling was analyzed in different developmental stages of different tissues in Chinese Qinchuan cattle; constructed the highly efficient overexpression vector of miR-543; studied the impacts of miR-543overexpression on proliferation and differentiation of C2C12cells; analyzed genetic variations of Wnt7a gene which would be the potential target gene of miR-543. The results were as follows:1. After verified the expression of miR-543in different periods of different tissues of Qinchuan cattle, we found that miR-543had the highest expression level in fetal cattle muscle and the expression was extremely low in muscle of neonatal cattle and adult cattle. In the part of tissue specificity, beside higher expression in liver and kidney which are the two main metabolic organs, miR-543expressed very low in other tissues. Hence, miR-543was a tissue specific expression miRNA.2. In this study, five different lengths of pri-miRNA-543(707bp,509bp,378bp,200bp and120bp) eukaryotic expression vectors (pAdTrack/CMV) were constructed. The expression of pri-miRNA-543(200bp) was the highest after transfected293T cells. However, compared with the707bp and378bp, there was no significant difference (P>0.05).Strangely, pri-miR-543of509bp expressed extremely low.3. C2C12cells were cultured in growth medium (GM) or differential medium (DM) after transfected with pAdTrack/CMV-pri-miR-543(200bp). Then expression of marker genes of cell proliferation (Ki67) and muscle differentiation (MHC, MyoD and Myf5) was analyzed by qRCR. The results showed that Ki67, MHC, MyoD and Myf5were up-regulated and the difference was significant or extremely significant (P<0.05or P<0.01). That means miR-543plays a positive role in skeletal muscle proliferation and development. 4. The3’UTR region of wnt7a gene was analyzed and there was a binding domain between3’UTR and miR-543. Therefore, Wnt7a gene may be the potential target gene of miR-543. Wnt7a gene regulates the regenerative potential of the muscle. However, similar researches about the bovine Wnt7a gene and its genetic variation are lacking. Therefore, in this study, we studied individuals of Chinese Qinchuan cattle to identify the effects of polymorphisms on Wnt7a by using DNA pooling, PCR-RFLP, and DNA sequencing analyses and then to establish an association between mutations of the bovine Wnt7a gene and growth traits.3novel SNPs were identified, two SNPs (g.T4926C and g.A21943G) were in the intron and the last one (g.C63777T) which was a missense mutation (Arg>Cys) was in the fourth exon. These three SNPs had moderate genetic diversity and the genotypic frequencies of all three loci were in Hardy-Weinberg disequilibrium. g.T4926C and g.A21943G did not show any significant correlation with growth traits. g.C63777T significantly or extremely significantly effected body height, body weight, chest width and height at hip cross (P<0.05or P<0.01). Five haplotypes involved in these three variant sites in the Wnt7a gene were identified and their effects on growth traits were analyzed. The results revealed that haplotype1had the highest haplotype frequencies and was highly significantly associated with body height, body weight, chest width and height at hip cross (P<0.01or P<0.05). |
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