利用乳腺上皮细胞模型解析热应激条件下奶牛乳腺差异表达miRNAs及靶基因的调控网络

Environmental heat stress evokes a series of drastic changes in biological functions of cows including disturbance in metabolism of water, energy, protein, mineral, and hormonal secretions. These changes affect several cow production parameters, such as fertility, feed intake, milk production, growth, and animal health negatively.. Extensive dairy industry losses are attributed to heat stress in summer in our country. It is important to clarify the molecular mechanism of regulation of heat stress response. A number of studies are focus on heat stress response genes and their transcription factors, but how microRNAs (miRNAs) regulate heat stress response in dairy cattle remains unknown. . In the present study, we will identify novel and differentially expressed miRNAs in the mammary gland in dairy cattle under heat stress condition by Solexa sequencing, Northern blot technology and bioinformatics. Luciferase reporter system, qRT-PCR and Western blot will be used to validate the interaction between key miRNAs and target genes. The co-expression of miRNAs and target genes in the mammary gland will be analyzed. miRNA mimic/inhibitor will be used to investigate the effects of miRNAs on the expression of target genes. The MTT and Flow Cytometry (FCM）will be used to study the effects of miRNAs on proliferation and apoptosis of mammary epithelial cells in dairy cattle. . This study will be helpful to elucidate the post-transcriptional molecular mechanism of regulation of heat stress response and provide theoretical support for breeding program in dairy cattle.

夏季奶牛发生热应激反应给我国奶业造成巨大经济损失，发生热应激反应的分子机制受到研究者的高度重视，人们对参与热应激反应的编码基因及其转录因子的研究取得了较大进展，但microRNAs(miRNAs)在热应激奶牛乳腺中的转录后调控机制尚不清楚。本研究拟通过Solexa高通量测序、Northern blot技术结合生物信息学方法，分析奶牛乳腺组织热应激前后miRNA差异表达情况，预测重要miRNA的靶基因并构建miRNA调控网络。利用荧光素酶报告基因系统、miRNA mimic/inhibitor、qRT-PCR和Western blot鉴定重要miRNA和靶基因间的相互作用。采用MTT法和流式细胞术在细胞水平上研究热处理后关键miRNA对细胞增殖、凋亡的影响，探讨miRNA在奶牛热应激反应中的调节功能，为阐明奶牛热应激反应的转录后分子调控机制提供科学依据，并为将来应用于奶牛育种提供理论支撑。

夏季奶牛发生热应激反应给我国奶业造成了巨大的经济损失，发生热应激反应的分子机制受到研究者的高度重视，本项目按照预定计划开展了各项研究工作并完成了相关任务。目前，已发表SCI收录论文2篇，投稿英文文章1篇。申报国家技术发明专利1项。采集8头热应激期和非热应激期奶牛乳腺组织，提取RNA，构建奶牛乳腺组织小RNA文库，利用HiSeq测序技术，对热应激前后奶牛乳腺组织miRNA进行了系统性分析，发现已知miRNAs 483个，新miRNAs 139个。差异表达分析发现27个miRNAs在热应激期和非热应激期表达差异达到显著水平，其中已知miRNAs 24个，新miRNAs 3个；上调miRNAs 20个，下调miRNAs 7个，丰富了奶牛miRNA数据库，利用茎环PCR技术对7个差异表达miRNAs表达量进行实验验证，包括bta-miR-21-5p、bta-miR-99a-5p、bta-miR-146b、bta-miR-145、bta-miR-2285t、bta-miR-133a和bta-miR-29c，发现与RNA-Seq结果一致。.利用生物信息学方法预测差异表达miRNAs靶基因，共预测到59个Go分类中的65625个靶基因，靶基因位点数为293949个。应用DAVID对差异表达miRNA调控的靶基因进行GO分析和Pathway分析。通过对奶牛热应激基因的调控网络分析发现，Wnt、TGF-β、MAPK、Notch和JAK-STAT信号通路等广泛参与了奶牛热应激。其中MAPK信号通路中相关的预测的靶基因占0.23%，Wnt信号通路占1.65%，TGF-β信号通路占0.71%，Notch信号通路占0.58%，JAK-STAT信号通路占1.22%，初步阐明了miRNAs在热应激条件下的转录后调控机制。这些靶基因所对应的miRNA将是下一步研究miRNA在奶牛热应激反应中功能的重要候选miRNA，这些结果为奶牛抗热应激反应的分子调控途径提供了科学依据，为应用于奶牛育种提供了理论支撑。