广西巴马小型猪T2DM相关miRNAs表达差异及调控机制初步研究

发布时间：2018-02-04 22:02

本文关键词： 广西巴马小型猪 Ⅱ型糖尿病 miR-103/107 miR-122Caveolin-1　出处：《广西大学》2015年硕士论文　论文类型：学位论文

【摘要】：糖尿病是人类三大慢性疾病之一,以Ⅱ型糖尿病居多。Ⅱ型糖尿病主要是由于机体的胰岛素抵抗作用进而导致全身能量代谢紊乱的疾病,患者在患病后期会出现糖尿病肾病、心脏病等并发症,严重威胁人类健康。近年来研究发现,小分子miRNAs能够参与Ⅱ型糖尿病的调控。本研究的目的是测定广西巴马小型猪miRNAs的组织表达谱;对广西巴马小型猪Ⅱ型糖尿病模型不同组织：niRNAs的表达差异进行分析；探讨miR-103/107在体外和体内对Ⅱ型糖尿病的调控机制,为人类糖尿病的研究、治疗提供新的靶点。参照miRBase公布的miR-103、107、122、143-5p基因序列,设计特异性的扩增引物,克隆广西巴马小型猪miR-103、107、122、143-5p基因的成熟序列。利用实时荧光定量PCR测定巴马小型猪miRNAs的组织表达谱。结果显示巴马小型猪miR-103、107、122、143-5p与miRBase公布的猪、人类、小鼠、绵羊、鸡等物种序列一致,未出现碱基突变；miR-122在肝脏组织中特异性的表达,且表达量显著高于其他miRNAs(P0.05);miR-103、107、143-5p在肝脏、骨骼肌、脂肪组织中均有表达,且miR-103在骨骼肌和脂肪组织中的表达量显著高于miR-143-5p和miR-122(P0.05)。通过实时荧光定量PCR的方法测定T2DM模型发病组、未发病以及对照组的肝脏、骨骼肌、脂肪组织中miR-103、107、122的表达水平,结果显示发病组肝脏组织中miR-122的表达量上调,miR-103/107在发病组三个组织中的表达量均上调。因此筛选miR-103/107进行后续研究。利用miRDanda、Targetscan网站以及文献报道预测miR-103/107能够与人和小鼠Caveolin-1基因的3'UTR区结合,存在三个结合位点,与NCBI公布的猪Caveolin-1基因序列进行比对,猪Caveolin-1基因3'UTR区存在一个miR-103/107结合位点。本试验克隆巴马小型猪Caveolin-1基因包含3'UTR区域的1885 bp基因序列,与GenBank中公布的猪Caveolin-1基因3'UTR序列比对,结果显示巴马小型猪Caveolin-1基因3'UTR存在8处碱基突变,1处碱基缺失,1处碱基插入,同源性为99.6%,巴马小型猪Caveolin-1基因3'UTR序列在1658-1715 bp存在一处miR-103/107的结合位点。本试验构建pEGFP-C1-pre-103、pcDNA3.1(+)-EGFP-pre-103、pEGFP-C1-pre-107、 pcDNA3.1 (+)-EGFP-pre-107真核表达载体,转染小鼠成肌细胞C2C12,实时荧光定量PCR检测miR-103/107、Caveolin-1基因在细胞内的表达情况。结果显示C2C12细胞内转染pEGFP-C1-pre-103、 pcDNA3.1(+)-EGFP-pre-103真核表达载体后miR-103的表达量上调,C2C12细胞内转染pEGFP-C 1-pre-107、pcDNA3.1(+)-EGFP-pre-107真核表达载体后miR-107的表达量上调；在C2C12细胞内过表达miR-103/107后,Caveolin-1基因的表达量均下调。结果表明,在体外过表达miR-103/107能够直接抑制靶基因Caveolin-1的表达水平。采用实时荧光定量PCR测定巴马小型猪T2DM模型肝脏、骨骼肌、脂肪组织中Caveolin-l的表达水平。结果表明,巴马小型猪发病组Caveolin-l在三个组织中的表达量均下调。对巴马小型猪T2DM模型中发病组和未发病组Caveolin-l基因与miR-103/107的结合区域进行PCR扩增并测序,结果显示在Caveolin-l基因与miR-103/107结合区域的1683 bp位点存在A/T突变,发病组有AA基因型猪3头、AT基因型猪3头,未发病组AT基因型猪1头、TT基因型猪2头。综上所述,巴马小型猪T2DM模型发病组肝脏组织miR-122表达量上调；miR-103/107在发病组肝脏、骨骼肌和脂肪组织中的表达量均上调。miR-103/107表达量的上调,可以直接作用于靶基因Caveolin-l基因的3'UTR区,抑制靶基因Caveolin-l基因mRNA水平的表达。Caveolin-l基因与miR-103/107结合区域内1683 bp位点的A/T突变可能与Ⅱ型糖尿病的易感性有关。
[Abstract]:Diabetes is one of the three major chronic diseases in type II diabetes. Type II diabetes is mainly due to systemic energy metabolism disorder effect of insulin resistance in the body, can occur in patients with diabetic nephropathy in later stage of disease, heart disease and other complications, a serious threat to human health. In recent years, the study found that the regulation of small molecule miRNAs to be able to participate in type II diabetes. The purpose of this study is to express spectrum determination of Guangxi Bama miniature pig miRNAs organization; the Guangxi Bama miniature pig model of type II diabetes in different tissues: analysis of differentially expressed niRNAs; discuss the regulation mechanism of miR-103/107 on type II diabetes in vitro and in vivo, for the study of human diabetes, provide the target a new treatment. According to miR-103107122143-5p gene sequence published by miRBase, the specific primers design, cloning of Guangxi Bama minipig mi The mature sequence of R-103107122143-5p gene. The expression spectrum determination of Bama minipig miRNAs using real-time fluorescence quantitative PCR. Results showed that miR-103107122143-5p and miRBase released a pig, Bama miniature pig human, mouse, sheep, chicken and other species sequence, no base mutation; the expression of miR-122 in liver tissue specificity, and the expression of significantly higher than the other miRNAs (P0.05); miR-103107143-5p in liver, skeletal muscle, expressed in adipose tissue, and the expression of miR-103 in skeletal muscle and adipose tissues was significantly higher than that of miR-143-5p and miR-122 (P0.05). Determination of T2DM model onset group by real-time quantitative PCR, and the control group without the disease of liver, skeletal muscle, the expression level of miR-103107122 in adipose tissue, results show that the expression of miR-122 in liver disease group, miR-103/107 group of three groups in the incidence of The expression of the fabric were raised. Therefore, further research of screening miR-103/107. Use of miRDanda, Targetscan and miR-103/107 can predict the site reported in the literature and the human and mouse Caveolin-1 gene 3'UTR binding region, there are three binding sites, and porcine Caveolin-1 based NCBI released by sequence comparison, there is a region of Porcine Caveolin-1 gene 3'UTR miR-103/107 binding sites. This experiment cloned Bama minipig Caveolin-1 gene contains 1885 BP gene sequences of the 3'UTR region, and porcine Caveolin-1 gene sequences of 3'UTR published in GenBank, shows that Bama miniature pig Caveolin-1 gene 3'UTR had 8 nucleotide mutations, 1 deletion, 1 bases insertion, the homology of 99.6% binding sites in Bama the small pig Caveolin-1 gene sequence of 3'UTR miR-103/107 in the presence of a 1658-1715 BP. The test construct pEGFP-C1-pre-103 (+) -EGFP-pre, pcDNA3.1 -103, pEGFP-C1-pre-107, pcDNA3.1 (+) -EGFP-pre-107 eukaryotic expression vector was transfected into mouse myoblast C2C12, real-time fluorescence quantitative PCR to detect miR-103/107 expression of Caveolin-1 gene in the cells. The results showed that C2C12 cells transfected with pEGFP-C1-pre-103, pcDNA3.1 (+) -EGFP-pre-103 eukaryotic expression vector miR-103 was up-regulated after C2C12 cells. Transfection of pEGFP-C 1-pre-107, pcDNA3.1 (+) -EGFP-pre-107 eukaryotic expression up-regulated miR-107 vector; overexpression of miR-103/107 in C2C12 cells, the expression level of Caveolin-1 gene was down regulated in vitro. The results showed that overexpression of miR-103/107 can directly inhibit the expression level of target gene Caveolin-1. Determination of T2DM model of liver, Bama miniature pigs by real time fluorescence quantitative PCR in skeletal muscle, adipose tissue Caveolin-l expression. The results show that the small group Caveolin-l Bama pig disease The expression in the three tissues were cut. The combination of regional onset group Bama miniature pig T2DM model and incidence of group Caveolin-l and miR-103/107 gene were amplified by PCR and sequenced. The results showed that in the Caveolin-l gene and miR-103/107 combined with 1683 BP sites in the presence of A/T mutations, the incidence of group AA genotype of 3 pigs. AT genotypes of 3 pigs, incidence of AT genotype TT genotype of 1 pigs, 2 pigs. In summary, Bama miniature pig T2DM model onset group liver tissue miR-122 expression; miR-103/107 expression in the pathogenesis of liver, skeletal muscle and adipose tissue was significantly up-regulated the expression of.MiR-103/107 increases, can direct effect of 3'UTR on the target region of Caveolin-l gene,.Caveolin-l gene expression and inhibition of miR-103/107 target gene Caveolin-l gene mRNA level combined with area of 1683 BP loci of A/T mutation may be associated with type II diabetes The susceptibility of the disease is related.

【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1

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