| Pork is an important source of animal protein for our residents, with the continuous improvement of living standards, people’s requirements on pork quality and production are also increasing. Therefore, from the perspective through improving muscle development to improve the quality and yield of pork has become the focus of swine genetic improvement.At present, many reports have confirmed that imprinted genes has played an irreplaceable role in skeletal muscle during development, among them was the H19-IGF2imprinting gene cluster. Previous studies have indicated that H19, IGF2inversely expressed,and miR-675generated from H19could inhibit C2C12cell proliferation, siH19and overexpression let7could promote C2C12cell differentiation, but it’s still not clear that how the H19/IGF2imprinted gene cluster regulate the skeletal muscle growth, and H19/IGF2have not been reported in pigs up to now. This study systematically revealled the expression pattern of H19, IGF2, miR-675and let7family genes in Large White,Meishan pig skeletal muscle and in the process of C2C12myoblast differentiation, then studied the promoter of H19’s methylation levels and the transcriptional regulation mechanism of H19.The main results are as follows:(1) We confirmed that H19, IGF2shared the same expression pattern in the skeletal muscle development of Large White and Meishan pig. H19, IGF2expression levels in embryonic50d were slightly lower than in95d skeletal muscle, but the expression level in embryonic were significantly higher than in adulthood. Inter-species comparison, the expression level of H19, IGF2in embryonic50d and95d Meishan pig were significantly higher than the Large White pig;but the expression level of H19, IGF2in adulthood Large White pig were significantly higher than Meishan pigs.(2) In the development of Meishan pig skeletal muscle, let7-a showed a upregulated expression pattern;The expression level of let7-f and let7-g first declined the rised;In Large White pig,let7-a, let7-f, let7-g showed a gradual upregulated expression pattern,and H19decreased significantly from the embryonic stage to adulthood, while let7-a, let7-f, let7-g were significantly increased. Interspecies comparison,we found that the expression level of let7-a in three periods of Large White pig were significantly higher than Meishan pigs, and the expression level let7-f, let7-g in embryonic50d,95d of the Large White pigs were significantly lower than Meishan pigs, but the expression level in adulthood were significantly higher than Meishan pigs.(3) Let-7was associated with meat quality traits--pH. In Large White pigs with high pHu (pH measured at24h after post-mortem), the expression level of let7-a, let7-f, let7-g were higher than in low pHu pigs. And the expression level of H19was significantly lower than low pHu pigs. In Large White pigs with high pHl (pH measured at45min after post-mortem), the expression level let7-a, let7-f, let7-g were lower than in low pHu pigs.(4) The mouse tissue expression profile showed that H19and miR-675-3p were higher expressed in muscle. And in the related genes of H19/IGF2gene cluster, the expression level of H19, IGF2, INS2first raised then declined in the process of C2C12differentiation,NCTC1had been rising.(5)The result of dual luciferase reporter gene analysis showed that, the sequence in the upstream of H19from0-4K didn’t have transcriptional activity in C2C12cells.(6) We found34differentially methylated sites in the promoter region of H19by Comparing the degree of methylation in C2C12cells differentiated for Od,4d, and these sites were mainly near the fourth CTCF binding sites of H19.And the whole extent of methylation of the fourth CTCF binding sites was significantly higher than Od. The level of methylation in the fourth CTCF binding sites was gradually upregulated in0d,1d,4d,while2d was the lowest. And the level of methylation and the expression level of H19was inversed in1d,2d,4d in C2C12cells. |
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