肉牛运输应激相关microRNA的筛选及其功能验证

The transport stimulation is a common stressor in livestock production, and it prevent the rapid development of beef breeding industry which is due to the transport stimulation could reduce the quality of animal products and increase animal mortality. Scientist pay attention to the mechanism research on transport stress. Among human, mouse and other model organisms, miRNA has been regarded as an valid target tag on the early diagnosis and prognosis in the related diseases, while there still have no such study around the early diagnosis and mechanism on beef transport stress. In this study, beeves are as the objects, and to construct a differential expression profiling of serum miRNAs on transport stressed beeves compared with normal beeves by high-throughput microarray, qRT-PCR and Northern blot technology. Then to analyse differentially expressed miRNAs and its target genes which both are related to transport stress. Silencing and over-expression methods will be using to verify the functional and regulational mechanism of the target differentially expressed miRNAs and its target genes. By analyzing the above experimental results could obtain the miRNA diagnosis maker on transport stress and reveal the roles of target miRNAs when the beeves are on the state of transport stress. The study results could play a key role on clarifying the molecular mechanisms on transport stress.

运输刺激作为畜牧生产中常见的应激原，可导致畜产品质量降低，动物死亡率增加，已成为制约我国肉牛养殖业快速发展的关键因素之一，其发生机制的研究受到国内外科研工作者的广泛关注。目前，在人和小鼠等模式生物中，miRNA已经作为一种有效标靶，用于相关疾病的早期诊断和预后研究，但在家畜尤其是涉及肉牛运输应激诊断及发生机制的研究国内未见报道。因此，本研究以肉牛为研究对象，采用高通量的全基因组芯片、qRT-PCR、Northern blot等现代分子生物学技术，拟构建运输应激前后肉牛血清miRNA差异表达谱，结合生物信息学技术，分析运输应激相关的差异表达miRNAs及其靶基因，应用基因沉默和过表达等技术，对目标miRNA和靶基因进行鉴定和功能验证，以期获得运输应激miRNA诊断标志物，并揭示目标miRNA在调控肉牛运输应激反应的作用，为明晰运输应激发生的分子机制奠定理论基础。

运输刺激作为畜牧生产中常见的应激原，可导致畜产品质量降低，动物死亡率增加，已成为制约我国肉牛养殖业快速发展的关键因素之一，其发生机制的研究受到国内外科研工作者的广泛关注。为探究运输应激对肉牛肝脏miRNA的影响，本研究对肉牛进行运输刺激，构建肉牛运输应激模型。使用ELISA方法检测血清中IL-1、IL-6、IL-8、IL-10、TNF-α、CORT、SAA、HP、CRP、HSP70的含量，同时检测肝脏和脾脏中SAA、HP、CRP、HSP70的含量。使用qRT-PCR检测肝脏和脾脏中SAA、HP、CRP、HSP70的表达量，评估肉牛运输应激状态。应用miRNA芯片技术构建运输应激肉牛肝脏miRNA差异表达谱。采用生物信息学技术进行差异表达miRNAs的靶点预测以及miRNAs靶基因所涉及的应激相关信号通路的分析，通过qRT-PCR方法对芯片结果的可靠性予以验证，并筛选运输应激肉牛肝脏miRNA候选诊断标志物。结合生物信息学分析结果，针对目标miRNA在细胞水平进行功能研究。结果表明，本项目成功构建运输应激肉牛动物模型，并筛选出3619个差异表达的miRNA，其中2029个上调，1590个下调。RNAhybrid和miRanda软件预测差异表达miRNA的靶基因，分别为22914，22896个，过滤后得到21946个。GO富集分析分类注释显示，56个有效的GO term分别富集于生物学进程、分子功能和细胞组分三大部分。以KEGG公共数据库对预测靶基因作Pathway富集分析，富集在癌症，MAPK和斑点通路较多，其结果表明，miRNA靶基因参与的信号转导通路，代谢信号通路、免疫信号通路等多个通路，都可能与运输应激相关。在差异表达的miRNA中，选取12个目标miRNA（miR-31, miR-122, miR-30a, miR-125b, miR-152, miR-186, miR-199a, miR-455, novel-1624, novel-2108）进行qRT-PCR验证，得到结果与高通量测序结果相一致，且确定它们为运输应激肉牛肝脏miRNA候选诊断标志物。通过目标miRNA的功能验证，推测miR-31在肉牛肝脏细胞氧化应激发生过程中发挥重要功能。本项目公开发表科研论文9篇，其中SCI论文1篇，国家级核心期刊论文7篇，省级期刊论文1篇；培养硕士研究生1名。