奶山羊乳腺发育过程中microRNA的表达谱分析及miR-25的功能研究
发布时间：2018-08-24 19:41
【摘要】：奶山羊的乳脂代谢是通过由脂质代谢相关基因组成的多条信号通路形成的复杂调控网络进行调节的。MicroRNAs(miRNAs)作为一种转录后调控因子,参与脂质合成、分解过程中相关基因的表达调控,在维持脂质代谢稳态方面发挥着重要作用。绘制miRNAs在不同泌乳阶段的动态表达图谱有助于研究脂质代谢相关基因的调控。本文应用灵敏特异的荧光定量PCR S-poly(T)plus检测技术,系统地绘制了泌乳空期、泌乳前期、泌乳盛期和泌乳后期,4个不同泌乳时期的奶山羊乳腺组织中miRNAs表达图谱。在有效检测的715个miRNAs中,共筛选出112个表达量上调或下调2倍以上(fold change≥2)的miRNAs。经初步筛选,共发现20个差异表达的miRNAs可能参与调控脂质代谢,其中9个未见报道。结合表达量及扩增条带的特异性,从这20个miRNAs中挑选出6个miRNAs(即let-7c、miR-19b、miR-497、miR-484、miR-25、mi R-17-5p)进行后续研究。乳腺上皮细胞(mammary epithelial cells)是体外研究乳脂代谢的常用模型。通过在奶山羊乳腺上皮细胞上过表达上述6个miRNAs进行功能分析,发现miR-25能够显著降低中细胞内甘油三酯的水平及脂滴含量。这与miR-25在组织中的表达趋势是相一致的:随着泌乳期的增长,miR-25的表达水平逐渐下调并在泌乳盛期达到最低,而到泌乳后期则有所恢复。通过软件预测、靶基因3’-UTR荧光素酶报告分析及Westernblot验证,发现在乳腺上皮细胞中,miR-25直接靶向过氧化物酶体增殖物激活受体γ共激活因子1β(peroxisome proliferator-activated receptor gamma,coactivator 1 beta,PPARGC1B又称PGC-1β)。而PGC-1β是固醇调节元件结合蛋白(Sterol Regulatory Element-binding Protein 1c,SREBP-1c)发挥其转录活性所必需的,实验发现,在mRNA水平上,mi R-25抑制PGC-1β的同时也能抑制SREBP-1c的表达。因此,推测miR-25在可通过靶向PGC-1β间接调控SREBP-1c从而调控SREBP-1c下游靶基因的表达,抑制脂质生成。综上所述,本研究分析了不同泌乳时期的奶山羊乳腺组织中miRNAs的表达特征,并揭示miR-25-PGC-1β介导的脂质代谢调控通路,为阐明miRNAs在乳脂代谢中的调控功能提供了实验依据。
[Abstract]:Milk fat metabolism in dairy goats is regulated by a complex regulatory network composed of multiple signaling pathways associated with lipid metabolism. As a posttranscriptional regulator, microRNAs (miRNAs) is involved in lipid synthesis. The regulation of gene expression in the process of catabolism plays an important role in maintaining the homeostasis of lipid metabolism. Mapping the dynamic expression of miRNAs in different lactation stages is helpful to study the regulation of genes related to lipid metabolism. A sensitive and specific fluorescence quantitative PCR S-poly (T) plus technique was used to systematically map the expression of miRNAs in the mammary tissues of four different lactation stages: empty lactation, early lactation, peak lactation and late lactation. Of the 715 miRNAs detected effectively, 112 miRNAs. were screened for up-regulation or down-regulation of (fold change 鈮，
本文编号：2201831
[Abstract]:Milk fat metabolism in dairy goats is regulated by a complex regulatory network composed of multiple signaling pathways associated with lipid metabolism. As a posttranscriptional regulator, microRNAs (miRNAs) is involved in lipid synthesis. The regulation of gene expression in the process of catabolism plays an important role in maintaining the homeostasis of lipid metabolism. Mapping the dynamic expression of miRNAs in different lactation stages is helpful to study the regulation of genes related to lipid metabolism. A sensitive and specific fluorescence quantitative PCR S-poly (T) plus technique was used to systematically map the expression of miRNAs in the mammary tissues of four different lactation stages: empty lactation, early lactation, peak lactation and late lactation. Of the 715 miRNAs detected effectively, 112 miRNAs. were screened for up-regulation or down-regulation of (fold change 鈮，
本文编号：2201831
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