| Diabetes mellitus is one of the three major chronic diseases in the world, and type 2 diabetes mellitus(T2DM) consists of the major proportion in diabetes disaeases. The T2DM seriously affecting human health mainly is due to the effects of insulin resistance of the organism leading to the disorder of energy metabolic of the whole body, and finally patients appear diabetic kidney disease, heart disease and other complications in the later stage of disease. In recent years, many studies showed that miRNA had a very important role in the development of T2DM. The objective of current study is to analyze expression levels of miR-103,107,122,143-5p in different tissues from Guangxi Bama mini-pig, and elucidate the regulatory mechanism of miR-103/107 in T2DM in vitro and vivo, and provide a new target for the research and treatment of T2DM.According to mature sequences of miR-103,107,122,143-5p in miRBase, we designed primers and cloned these miRNAs from Guangxi Bama mini-pig. Expression profiles of miRNAs in different tissues in Guangxi Bama mini-pig were analyzed by real time quantification PCR(QRT-PCR). The results indicated that there were none any changes of base pair in miR-103,107,122,143-5p in Bama mini-pig compared with sus scrofa, human, mouse, chicken, sheep. MiR-122 was expressed specifically higher in liver compared with other miRNAs(P<0.05). MiR-103,107, miR-143-5p expressed in liver, skeletal muscle, and fat tissues. Expression level of miR-103 was significantly higher in skeletal muscle and fat than that of miR-122 and 143-5p (P<0.05). Expression levels of miR-103,107 and 122 in T2DM group, non-T2DM group and negative control group were testeded by QRT-PCR in liver, skeletal muscle, and fat. Results showed that expression level of miR-122 in T2DM was up-regulated only in liver, but the expression level of miR-103/107 was up-regulated in liver, skeletal muscle, and fat. Therefore, we supposed that miR-103/107 play a more important role in T2DM compared with miR-122.We analyzed the binding site of miR-103/107 using online software of miRanda and Targetscan and found that there was a site where miR-103/107 can bind with 3’UTR of Caveolin-1 gene in human and mouse. Caveolin-1 has three binding sites of miR-103/107 in human and mouse, however there are only one binding site in the 3’UTR of Caveolin-1 in sus scrofa released by GenBank. We cloned and obtained the 3’UTR sequence of Caveolin-1 with 1885bp in Bama mini-pig. There were eight mutations, one deletion and one insertion compared with 3’UTR sequence of Caveolin-1 of sus scrofa released by NCBI. The homology was 99.6%. In this study, we constructed four eukaryotic expression vectors, pEGFP-Cl-pre-103, pcDNA3.1(+)-EGFP-pre-103, pEGFP-Cl-pre-107, pcDNA3.1(±)-EGFP-pre-107, respectively. The plasmids were extracted and transfected into C2C12 cell. Expressional levels of miR-103/107 and Caveolin-1 in C2C12 cell were analyzed by QRT-PCR. The results showed that expression levels of miR-103 were up-regulated in cells transfected with pEGFP-Cl-pre-103 or pcDNA3.1(+)-EGFP-pre-103. While expression levels of miR-107 also were up-regulated in cells transfected with pEGFP-Cl-pre-107 or pcDNA3.1(+)-EGFP-pre-107. When expression levels of miR-103/107 was up-regulated, expression levels of Caveolin-1 were decreased. These results suggested that the over expression of miR-103/107 could inhibit expression of target gene, Caveolin-1, in vivo.Expression profiles of Caveolin-1 in liver, skeletal muscle, and fat were analyzed with QRT-PCR in T2DM group, non-T2DM group and negative control group. The results showed that expression levels of Caveolin-1 were decreased in T2DM group compared with in non-T2DM group and negative control group in liver, skeletal, and fat. Binding region of Caveolin-1 with miR-103/107 in Guangxi Bama mini-pig was amplified and sequenced in different three groups. Results showed that there was an A/T mutation in 1683 bp of Caveolin-1 gene. Among testing pig, there were 3 AA genotypes,3 AT genotypes in T2DM group. While there were 1 AT genotype and 2 TT genotypes in non-T2DM group.In conclusion, expression level of miR-122 was up-regulated in liver in Guangxi Bama mini-pig with T2DM, while expression level of miR-103/107 were up-regulated in liver, skeletal muscle, and fat in Guangxi Bama mini-pig with T2DM. Over expression of miR-103/107 could inhibit the expression of Caveolin-1. A/T mutation at 1683 bp of Caveolin-1 might associate with the susceptibility of T2DM in Guangxi Bama mini-pig. |
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