奶牛microRNA与转录因子调控网络解析及其在乳腺炎抗性中的作用

Mastitis is a complex mammary gland inflammation caused by pathogens,etc,and results in the greatest loss in profit for dairy producers. Mastitis susceptibility or resistance differs among cows due to environmental, physiological and genetic factors. Interestingly, different bacteria and even different strains of the same bacteria can cause diverse immune responses in the mammary gland and affact subsequent resistance. So far, molecular mechanisms of mastitis resistance or susceptibility have remained poorly understood..MicroRNAs (miRNAs) and transcription factors (TFs) are main regulators of gene expression, which share the common logic and act as key players in the immunity system. MiRNA and TF may reciprocally regulate one another. We hypothesized that miRNAs and TFs might play combinatory regulatory roles for genes linked to mastitis resistance. .Inflammation occurs when bacteria enter the mammary gland and interact with host cells resulting in release of inflammatory mediators which guide immune cells, e.g, neutrophil, towards the site of infection. Neutrophils are commonly called the "first responders" to the infection. The neutrophil migration into the mammary gland is essential for effective resolution of mastitis in dairy cattle. .Base on our previous findings and reported advances regarding the mastitis resistance, we hypothesized that different pathogens invade into the mammary gland and up-regulate miRNAs expression,then active many miRNA-TF regulatory networks and pathways, cause various migration patterns of neutrophils, subsequently increase the incidence of clinical and subclinical intramammary infections. To testify the hypothesis, several experiments were carried out as follows: (1) to identify the differential expressed pattern of miRNAs between the healthy and mastitis-infected mammary glands using the miRNAs chips and Solexa methods; (2) to investigate the differential expressed pattern of proteomes between the healthy and mastitis-infected mammary glands using iTRAQ and Western-bolt methods, etc; (3) to predict and validate the target genes of miRNAs and TFs; (4) to explore and construct miRNA-TF regulatory networks in the mastitis; (5) to analyze the relationships between bovine neutrophils migration pattern with different genotypes of key genes or TFs and mastitis resistance or susceptibility to the mastitis.. The present study will provide insights to the underlying molecular mechanism for the different pathogens in vivo challenge can cause differential expressed genes in mammary glands. Finding what makes certain cows more susceptible or resistant to mastitis could lead to new ideas and strategies aimed at molecular breeding for mastitis resistance and improved prevention and treatment of mastitis.

乳腺炎是病原体感染等引发的复杂炎症。抗性形成的机制尚不清楚。MicroRNAs(miRNAs)和转录因子(TFs)是重要的基因表达调控因子，二者有着共同的调控逻辑，在免疫系统中扮演关键角色。基于本课题组前期研究及已有的报道，我们提出"不同病原体侵入乳腺上调miRNAs表达，激活众多miRNA-TF调控通路，产生不同的嗜中性粒细胞迁移模式，促进奶牛乳腺炎发生"的假设，认为miRNAs和TFs可形成调控网络，在奶牛乳腺炎抗性/易感性中发挥重要作用。拟采用miRNA芯片、iTRAQ、基因沉默、流式细胞、免疫荧光等技术研究和验证奶牛乳腺miRNA表达模式，预测和验证靶基因与miRNA的转录因子，构建miRNA-TF调控网络，探讨关键miRNA在乳腺炎抗性中的作用，分析嗜中性粒细胞迁移模式。本课题将从新的视角解析奶牛乳腺差异表达基因存在病原体特异性的分子机制，为乳腺炎抗性分子育种和防治提供新的思路。

奶牛乳腺炎是由微生物侵袭、物理、化学刺激等引起的复杂的炎症反应，也是奶业中的常见病、多发病。乳腺炎的发生及其抗性（易感性）形成的分子机制是一个亟待解决的科学问题。随着对miRNAs功能研究的逐渐深入，发现转录因子与miRNA之间存在着直接或间接的相互调控作用，这为了解miRNA 参与的复杂基因调控网络提供了新内容，同时也为miRNA在相关疾病的治疗提供重要线索。本项目首先利用Solexa和miRNA微流体芯片技术研究了健康和乳腺炎奶牛的乳腺组织差异表达的miRNAs，结果共鉴定出85个2倍以上的差异表达的miRNAs，如证明miR-2898，miR-2373和bta-miR-223等miRNA可分别靶向A2M，CLU和HMGB1等基因3’UTR区，并发现它们结合区域内的SNPs可影响其结合能力；其次，采用同位素标记相对和绝对定量（iTRAQ）技术测定了3头健康奶牛乳腺组织（对照组）和3头金黄色葡萄球菌导致的乳腺炎奶牛乳腺组织蛋白表达谱。结果共鉴定出768种蛋白质，36种蛋白在患乳腺炎组织中比健康乳腺组织有高表达（大于1.5倍），19种蛋白出现了下调。其中上调表达的COL1A1和ITIH4蛋白已经通过Western blotting和免疫组化实验得到了进一步的实验验证。通过miRNAs和蛋白质组的整合分析，构建了与免疫、组织损伤与修复有关的miRNA-TF调控网络，验证了几个关键miRNAs和靶基因在乳腺组织和血液嗜中性粒细胞中的表达。同时，本研究还鉴定出影响成熟miRNA合成且与乳腺炎易感性有关的功能性分子标记。本项目通过以上的研究，证明了miRNAs在乳腺炎抗性中发挥作用的部分机制，丰富对奶牛乳腺炎抗性分子机制的认识，为乳腺炎的抗性分子育种和治疗提供新的科学依据。