奶山羊乳腺组织乳脂代谢关键miRNA的筛选及功能验证

发布时间：2018-03-21 13:08

本文选题：奶山羊　切入点：miRNA筛选　出处：《西北农林科技大学》2017年博士论文　论文类型：学位论文

【摘要】：羊奶中蛋白质、维生素、钙、矿物质、总脂肪含量高,羊奶所含的有益脂肪酸含量(如短、中链脂肪酸,不饱和脂肪酸和共轭酸等)均显著高于牛奶,羊奶中的乳脂代谢非常复杂,它受多种因素调控。Mi RNA(micro RNA)可通过调控其靶基因的表达影响脂肪酸代谢过程。本研究对奶山羊不同泌乳时期(泌乳前期、泌乳盛期、泌乳后期和干奶期)乳腺组织进行mi RNA筛选后,对催乳素处理的乳腺上皮细胞做进一步筛选,发现6个表达差异的mi RNA。在此基础上,对这些筛选出来的mi RNA在功能、调控关系以及分子机制方面进行深入研究。得到如下主要研究结果:1.奶山羊乳腺组织mi RNA表达谱分析及乳脂代谢关键mi RNA筛选研究以奶山羊泌乳前期、泌乳盛期、泌乳后期和干奶期的乳腺组织进行mi RNA表达量检测,基于mi RBase数据库提供的793条牛的mi RNA和267条羊的mi RNA进行第一轮的筛选,以p值小于0.05且差异在4倍以上为标准,筛选得到156个表达差异的mi RNA。随后使用与发动泌乳密切相关的因子——催乳素(2.5μg/ml)处理乳腺上皮细胞,进行第二轮筛选。发现mi R-30e-5p,mi R-15a,mi R-181b,mi R-148a,mi R-17-5p和mi R-135b这6个mi RNA在催乳素处理的乳腺上皮细胞中表达差异显著。2.Mi R-181b通过IRS2(Insulin receptor substrate 2)抑制TAG合成并调控Hippo信号通路基因在乳腺上皮细胞过表达mi R-181b可以抑制乳脂代谢,而抑制mi R-181b可以促进乳脂代谢。荧光素酶报告试验和Western Blot试验证明了mi R-181b的靶基因为IRS2。此外,在乳腺上皮细胞中,mi R-181b可以调控Hippo信号通路中的多个作用元件,如LATS1和YAP1。简而言之,研究发现mi R-181b通过调控Hippo通路上的多个元件及靶向IRS2抑制脂代谢。3.Mi R-30e-5p和mi R-15a通过靶向LRP6和YAP1基因协同调控乳脂代谢QRT-PCR和Western Blot试验证明了mi R-30e-5p和mi R-15a分别通过靶向Yes相关蛋白(YAP1)和LDL受体相关蛋白6(LRP6)来促进脂质分化。其中,mi R-30e-5p可以调控β-catenin基因,β-catenin进而介导YAP1的表达。在乳腺上皮细胞脂代谢中YAP1基因的m RNA和蛋白水平表达受mi R-30e-5p调控。此外,在乳腺上皮细胞中过表达mi R-30e-5p和mi R-15a可以促进脂代谢,与之相反抑制mi R-30e-5p和mi R-15a可以减少脂代谢。此外,mi R-30e-5p通过调控mi R-15a表达水平来协同调控YAP1在乳腺上皮细胞中的乳脂代谢功能。4.PRL通过甲基化修饰抑制mi R-135b功能QRT-PCR和Western Blot试验证明LATS2作为mi R-135b的靶基因来行使乳脂代谢功能。PRL处理乳腺上皮细胞时,对下调mi R-135b的表达有一定的时间和浓度梯度效果。此外,mi R-135b的5'端发现一个Cp G岛,通过调控Cp G岛的甲基化来抑制mi R-135b表达。进一步对PRL处理的乳腺上皮细胞中mi R-135b的功能和作用途径进行研究发现,DNMT I(DNA甲基化转移酶I)表达水平显著提高,而mi R-135b表达水平下调。此外,当mi R-135b的表达受到抑制时,LATS2表达水平上调。这就导致乳腺上皮细胞中乳脂代谢水平提高,从而行使维持泌乳等功能。5.Mi R-148a和mi R-17-5p通过PPARGC1A和PPARA基因协同调控乳腺上皮细胞乳脂代谢表达谱结果显示mi R-148a、mi R-17-5p、PPARGC1A和PPARA在泌乳盛期和干奶期中表达差异显著。荧光素酶和Western Blot试验发现,PPARA(脂肪酸调控重要因子)和PPARGC1A(调控脂代谢基因)分别是mi R-148a和mi R-17-5p的靶基因,而mi R-148a可以调控PPARA,mi R-17-5p可以调控PPARGC1A。过表达mi R-148a和mi R-17-5p促进甘油三酯(TAG,Triglyceride)的合成,而抑制mi R-148a和mi R-17-5p降低TAG的产生。重要的发现是mi R-148a和mi R-17-5p的协同作用促进了TAG的合成。即在乳腺上皮细胞中,mi R-148a与mi R-17-5p分别调控PPARGC1A和PPARA来协同完成TAG的合成,。本研究筛选的6个mi RNA及其靶基因对乳脂代谢的影响,为深入阐明奶山羊乳脂代谢调控机制提供了理论和试验依据,为解析羊奶脂肪酸成分形成机理奠定了基础。
[Abstract]:Goats'milk protein, vitamins, minerals, calcium, total fat content is high, beneficial fatty acids goats' milk contains (such as short chain fatty acid, unsaturated fatty acid and conjugated acid) were significantly higher than that of milk, milk fat metabolism in goats'milk is very complex, it is regulated by a variety of factors (.Mi RNA micro RNA) can regulate the expression of their target genes that affect the metabolism of fatty acids. The study of dairy goats in different lactation periods (early lactation, lactation period, late lactation and dry period) of MI RNA breast tissue after screening, further screening of prolactin treatment of mammary epithelial cells, found 6 differentially expressed mi RNA. on the basis of these, the selected mi RNA in function, regulation relationship and molecular mechanism were studied. The main results are as follows: the expression spectrum analysis and fat metabolism key mi RNA 1. mi RNA of dairy goat mammary tissue Study on screening of dairy goat in early lactation, lactation stage, MI RNA expression detection in late lactation and dry period of breast tissue, screened in the first round of the MI RNA 793 mi RNA mi RBase cattle and 267 sheep database based on the p value of less than 0.05 and the difference in more than 4 times as a standard, screened 156 differentially expressed mi RNA. and then use the launch of lactation related factors: prolactin (2.5 g/ml) treatment of mammary epithelial cells, the second round of screening. Mi R-30e-5p, MI R-15a, MI R-181b, MI R-148a, MI R-17-5p and MI R-135b expression of the 6 mi RNA in prolactin treatment of mammary epithelial cells significantly.2.Mi R-181b by IRS2 (Insulin receptor substrate 2) inhibit TAG synthesis and regulation of Hippo signaling pathway genes in mammary epithelial cells overexpressing mi R-181b can inhibit the fat metabolism, and inhibition of MI R-181b In order to promote fat metabolism. The luciferase reporter assay and Western Blot tests showed that the target genes of MI R-181b because of IRS2. addition in mammary epithelial cells, MI R-181b can be a role of regulatory element in the Hippo signaling pathway, such as LATS1 and YAP1.. In short, studies have found that MI R-181b by a plurality of elements on the regulation of the Hippo pathway and target to IRS2.3.Mi and MI R-30e-5p inhibited the lipid metabolism of R-15a gene to LRP6 and YAP1 by QRT-PCR and fat metabolism target synergistic regulation of Western Blot MI and MI R-30e-5p test proved that R-15a were targeted by Yes associated protein (YAP1) and LDL receptor related protein 6 (LRP6) to promote differentiation. Among them, MI R-30e-5p can regulate beta -catenin gene, expression of beta -catenin and YAP1 mediated by Mi R-30e-5p. The regulation of the expression of YAP1 gene in mammary epithelial cells of lipid metabolism in M RNA and protein level. In addition, in breast epithelial cells. Overexpression of MI in R-30e-5p cell and MI R-15a can promote lipid metabolism, and inhibition of MI R-30e-5p and MI in R-15a can reduce lipid metabolism. In addition, the MI expression level of R-30e-5p by regulating mi R-15a collaborative fat metabolism function of.4.PRL regulation of YAP1 in mammary epithelial cells through inhibition of MI methylation modification of R-135b function of QRT-PCR and Western the experimental results show that the LATS2 Blot as the target gene of MI R-135b to exercise fat metabolism function of.PRL treatment of mammary epithelial cells, there is a certain amount of time and concentration gradient effect on the expression of MI R-135b mi R-135b 5'. In addition, the end found a Cp G Island, G island methylation through the regulation of Cp to inhibit the expression of R-135b Mi study found that the further function and effect on the treatment of PRL in mammary epithelial cells mi R-135b pathway, DNMT I (DNA methyltransferase I) expression level increased significantly, while the MI R-135b table of water Flat down. In addition, the expression of R-135b when MI is inhibited, LATS2 expression level increased. This leads to fat metabolism in mammary epithelial cells increased, thereby maintaining lactation function.5.Mi R-148a exercise and MI R-17-5p by PPARGC1A and PPARA gene co regulation of mammary epithelial cells in milk fat metabolism expression showed that MI R-148a, MI R-17-5p PPARGC1A, and PPARA at peak lactation and dry milk period significantly. The expression of luciferase and Western found Blot test, PPARA (an important factor regulating fatty acid (PPARGC1A) and the regulation of lipid metabolism genes) are target gene mi R-148a and MI R-17-5p, and MI R-148a PPARA mi R-17-5p can control, can control the PPARGC1A.. The expression of MI R-148a and MI R-17-5p (TAG, Triglyceride) promotes triglyceride synthesis and inhibition of MI R-148a and MI R-17-5p decreased TAG. The important finding is the MI R-148a and MI R-17- The synergistic effect of 5P and promote the synthesis of TAG. Namely in mammary epithelial cells, MI R-148a and MI R-17-5p and PPARA PPARGC1A respectively control to execute TAG synthesis. The research of 6 mi RNA and its target gene screening on fat metabolism, and provides a theoretical and experimental basis for the elucidation of dairy goat milk the metabolic regulation mechanism, laid the foundation for the mechanism of acid composition analysis goats'milk fat formation.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S823

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