microRNA对黄牛和水牛朊病毒基因转录后调控机制的研究

Bovine spongiform encephalopathy (BSE) is one of prion diseases caused by prion protein (PrP). PrP levels play a crucial role in susceptibility and development of clinical BSE. Since no case of BSE has ever been reported in buffalo, compared to more than 190,000 cattle infected by BSE, we hypothesized that differences in expression of PrP may relate to these species-specific susceptibility. Recently, our findings revealed that buffaloes have significantly lower relative expressions of PrP in several tissues belonging to the central nervous system and the lymphoreticular system. Although the results support our primary assumption, PrP expressions did not synchronize with the corresponding mRNA expression levels. It was suggested that the biological modulations of PrP are at a post-transcriptional level. The mechanism remains to be clarified. More and more studies report microRNAs (miRNAs) are kind of non-coding small RNAs that function as regulators of post-transcriptional gene expression. In this study, we will screen differentiated miRNA cascade relate to differentiated 3′UTR of PRNP between cattle and buffalo. Luciferase reporter assays will be performed to confirm the relationship between target and predicted miRNA.The different expressions of miRNAs in various tissues between cattle and buffaoes will be analyzed by real-time PCR. Moreover, the evolutionary analysis of PRNP and miRNA genes will be performed in Bovidae. Finally, the different mechanisms of post-transcriptional regulation of PRNP between cattle and buffalo by miRNAs will be revealed. These findings may prove insight into better understanding the mechanism of susceptibility to prion diseases, and prove drug discoveries/development.

疯牛病是一种朊病毒疾病，朊病毒蛋白表达量与疾病的易感性正相关。申请人的前期研究发现：在中枢神经系统和淋巴系统的几个组织中，黄牛朊蛋白表达量显著高于水牛，这个结果与"黄牛易感疯牛病而水牛不易感"的现象相吻合，但机制尚不清楚。近来我们发现朊蛋白表达的差异并不是由朊病毒基因（PRNP）转录水平的差异引起，而是由两个物种中该基因在转录后水平调控的差异引起。因此，本项目拟从对基因转录后调控起重要作用的miRNA入手，研究两个物种3′UTR序列的差异及其结合miRNA类群的异同，鉴定引起种间和组织间朊病毒表达差异的miRNA类群，通过双荧光素酶报告技术验证PRNP与miRNA的靶标关系，用定量RT-PCR技术确认miRNA的表达情况，并分析牛科物种PRNP和miRNA基因的进化特征，最终阐明黄牛和水牛PRNP基因转录后调控机制的差异。本研究结果将为朊病毒疾病易感性的研究和疾病的治疗提供新思路和新靶点。

疯牛病是一种传染性海绵状脑病。虽然在黄牛中发现了大量的疯牛病病例（> 190,000），但是在水牛中还未见报道。以往的研究揭示，朊病毒基因PRNP及其编码的朊病毒蛋白是该病发生和发展的基础。我们的前期研究表明，与水牛相比，黄牛几个重要组织（如脑闩）表达了更多的朊病毒蛋白，但是转录水平PRNP mRNA的表达趋势却是相反的。因此，本项目我们试图解析水牛和黄牛PRNP基因3’UTR的遗传差异，考察由miRNA转录后调控作用引起的朊病毒蛋白表达的种间差异。首先，通过3’RACE和测序技术，我们界定了水牛PRNP基因3’UTR及其序列组成。接下来，通过对13个黄牛和13个水牛样品的全长3’UTR（~3 kb）的测序，我们一共发现92个种间固定差异位点。为了确定几个重要位点的变异是否在种间固定下来，我们分别对147个黄牛样品的UTR-C区段（g.786-1436）和146个水牛样品的UTR-B区段（g.778-1456）进行重测序，结果显示除了g.1022T这个位点外，在UTR-C和UTR-B区段中所有的差异位点均在两个物种中固定下来。通过预测这些种间固定差异位点与miRNA结合的自由能（ΔG），我们发现几个值得关注的位点：两个水牛特异性的插入（位于g.970-997的28-bp片段插入和位于g.1088-1089的AG插入），以及两个种间突变位点（g.1007-1008 TG→CC），因为这些位点的变异给水牛3’UTR提供更多与miRNAs结合的机会。通过荧光双报告实验，我们证实miR-125b-5p, miR-132-3p, miR-145-5p, miR-331-3p和 miR-338-3p可特异性地结合水牛PRNP 3’UTR，并转录后调控水牛PRNP的表达。进一步的表达分析结果显示，在牛的脑闩组织中这五个miRNAs 与PRNP 共表达，而且PRNP mRNA的表达与PrP蛋白的表达呈相反趋势。该研究结果从miRNA调控入手解释黄牛和水牛PRNP 基因转录后调控机制的差异，为朊病毒疾病易感性的研究和疾病的治疗提供新思路和新靶点。发表SCI论文5篇。