泌乳期差异表达microRNA对奶牛乳腺κ-酪蛋白的调控作用研究

MicroRNA can regulate milk composition. κ-casein(CSN3) is a kind of important proteins in milk. But it is not clear that which microRNAs can regulate kappa casein expression. In bovine mammary tissue of different lactating stages, expression of κ-casein is markedly different. It was speculated that differentially expressed microRNAs in lactating period played a certain role in regulating κ-casein expression. The differentially expressed microRNAs at different stages of lactation were screened in this project. Selected microRNAs were over expressed in bovine mammary epithelial cells(BMEC), respectively. Then, the change of κ-casein protein was detected in BMEC. In order to make the analysis of dual fluorescence reporter gene, microRNAs that significantly affected κ-casein expression were transfected into tool cells. Through this step, microRNAs that directly combined with CSN3 3’-UTR were detected and others were indirect acting microRNAs. In order to detect the expression change of mRNA and protein of key genes in JAK-STAT signal pathway and mTOR signal pathway, indirect acting microRNA singly or in cluster was transfected into BMEC. To synthesize the above-mentioned results, microRNAs which direct and indirect regulated κ-casein expression and its mainly regulatory pathways were revealed.

microRNA可以调节乳成分，κ-酪蛋白是牛奶中的一种重要蛋白质，但对κ-酪蛋白起调控作用的microRNA尚不清楚。在奶牛不同泌乳阶段的乳腺组织中，κ-酪蛋白表达显著不同，推测泌乳期差异表达的microRNA对κ-酪蛋白的表达起一定的调控作用。本项目筛选不同泌乳阶段奶牛乳腺组织差异表达的microRNA，在牛乳腺上皮细胞中分别过表达筛选出的microRNA，检测κ-酪蛋白的蛋白表达变化。将引起κ-酪蛋白显著变化的microRNA进行双荧光报告基因分析，检测出与κ-酪蛋白3’-UTR直接结合的microRNA，其它均为间接起作用的microRNA。将间接起作用的单个或成簇microRNA导入牛乳腺上皮细胞，检测信号调节通路JAK-STAT和mTOR中关键基因的mRNA和蛋白表达变化。综合上述各方面结果，解析对κ-酪蛋白表达起直接和间接调控作用的microRNA，及其主要的调节路径。

乳蛋白是乳中的重要营养成份，人们采用多种手段对乳蛋白进行调控，以提高乳品质。从microRNA的角度研究其对乳成份的调控才刚刚开始，大量的microRNA靶标还未被发现，以往的研究主要集中于不同泌乳阶段奶牛乳腺microRNA表达谱的检测，但并不清楚特定microRNA的具体功能。鉴于κ-酪蛋白本身在乳成份中的重要性，极有必要开展microRNA对κ-酪蛋白及其重要信号通路的调控机理研究。本项目筛选奶牛乳腺不同泌乳期差异表达的microRNA，以奶牛乳腺上皮细胞为研究对象，采用离体细胞培养、microRNA过表达、生物信息学分析、双荧光报告基因分析、荧光定量RT-PCR和Western-Blot等方法，逐步筛选出对κ-酪蛋白有直接和间接调控作用的microRNA，并揭示这些microRNA主要通过哪条信号通路调控κ-酪蛋白表达。通过荧光定量检测和ELISA检测发现CSN3的表达量随着产奶量下降而下降。通过高通量测序的方法从产奶60天、120天和240天的奶牛乳腺组织中共检测出1949个miRNA，其中三个时间共同表达的有1115个miRNAs，390个差异表达的miRNAs。其中位于前10位的高表达miRNA主要富集于PI3K-Akt，MAPK和蛋白质在内质网的加工等路径。根据Target Scan的预测结果结合高通量分析结果，主要锁定bta-miR-33a、 bta-miR-2901-1、bta-miR-2887-1、 bta-miR-2284ab、 bta-miR-2284k、bta-miR-193a-3p、bta-miR-193b开展双荧光检测。结果发现bta-miR-193a-3p、bta-miR-193b对CSN3的3‘UTR有显著的下调作用。本研究不仅发现了调控CSN3的主要miRNAs，而且为以后研究调节产奶量的miRNAs指引了方向。