【摘要】：奶山羊的乳脂代謝是通過(guò)由脂質(zhì)代謝相關(guān)基因組成的多條信號(hào)通路形成的復(fù)雜調(diào)控網(wǎng)絡(luò)進(jìn)行調(diào)節(jié)的。MicroRNAs(miRNAs)作為一種轉(zhuǎn)錄后調(diào)控因子,參與脂質(zhì)合成、分解過(guò)程中相關(guān)基因的表達(dá)調(diào)控,在維持脂質(zhì)代謝穩(wěn)態(tài)方面發(fā)揮著重要作用。繪制miRNAs在不同泌乳階段的動(dòng)態(tài)表達(dá)圖譜有助于研究脂質(zhì)代謝相關(guān)基因的調(diào)控。本文應(yīng)用靈敏特異的熒光定量PCR S-poly(T)plus檢測(cè)技術(shù),系統(tǒng)地繪制了泌乳空期、泌乳前期、泌乳盛期和泌乳后期,4個(gè)不同泌乳時(shí)期的奶山羊乳腺組織中miRNAs表達(dá)圖譜。在有效檢測(cè)的715個(gè)miRNAs中,共篩選出112個(gè)表達(dá)量上調(diào)或下調(diào)2倍以上(fold change≥2)的miRNAs。經(jīng)初步篩選,共發(fā)現(xiàn)20個(gè)差異表達(dá)的miRNAs可能參與調(diào)控脂質(zhì)代謝,其中9個(gè)未見報(bào)道。結(jié)合表達(dá)量及擴(kuò)增條帶的特異性,從這20個(gè)miRNAs中挑選出6個(gè)miRNAs(即let-7c、miR-19b、miR-497、miR-484、miR-25、mi R-17-5p)進(jìn)行后續(xù)研究。乳腺上皮細(xì)胞(mammary epithelial cells)是體外研究乳脂代謝的常用模型。通過(guò)在奶山羊乳腺上皮細(xì)胞上過(guò)表達(dá)上述6個(gè)miRNAs進(jìn)行功能分析,發(fā)現(xiàn)miR-25能夠顯著降低中細(xì)胞內(nèi)甘油三酯的水平及脂滴含量。這與miR-25在組織中的表達(dá)趨勢(shì)是相一致的:隨著泌乳期的增長(zhǎng),miR-25的表達(dá)水平逐漸下調(diào)并在泌乳盛期達(dá)到最低,而到泌乳后期則有所恢復(fù)。通過(guò)軟件預(yù)測(cè)、靶基因3’-UTR熒光素酶報(bào)告分析及Westernblot驗(yàn)證,發(fā)現(xiàn)在乳腺上皮細(xì)胞中,miR-25直接靶向過(guò)氧化物酶體增殖物激活受體γ共激活因子1β(peroxisome proliferator-activated receptor gamma,coactivator 1 beta,PPARGC1B又稱PGC-1β)。而PGC-1β是固醇調(diào)節(jié)元件結(jié)合蛋白(Sterol Regulatory Element-binding Protein 1c,SREBP-1c)發(fā)揮其轉(zhuǎn)錄活性所必需的,實(shí)驗(yàn)發(fā)現(xiàn),在mRNA水平上,mi R-25抑制PGC-1β的同時(shí)也能抑制SREBP-1c的表達(dá)。因此,推測(cè)miR-25在可通過(guò)靶向PGC-1β間接調(diào)控SREBP-1c從而調(diào)控SREBP-1c下游靶基因的表達(dá),抑制脂質(zhì)生成。綜上所述,本研究分析了不同泌乳時(shí)期的奶山羊乳腺組織中miRNAs的表達(dá)特征,并揭示miR-25-PGC-1β介導(dǎo)的脂質(zhì)代謝調(diào)控通路,為闡明miRNAs在乳脂代謝中的調(diào)控功能提供了實(shí)驗(yàn)依據(jù)。
[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，

本文編號(hào)：2201831