MiRNA及PDK1/TACE在朊病毒病致病及诊断治疗中的潜在应用研究

Prion disease was caused by a pathological protein prion which was misfolded by a normal cellular isoform. Prion α-cleavages was consider as a protective factor in prion disease. TACE (tumor necrosis factor-α-converting enzyme) was one of the PrP α-secretases whose activity was regulated by PDK1 (3-phosphoinositide-dependent kinase-1). Survival time was increased when TACE activity was increased by PDK1 inhibitor in animal experiment. miRNAs are small non-coding RNA molecules, which function in transcriptional and post-transcriptional regulation of gene expression. MiR-375 was a regulator of PDK1. Therefore, we speculate that formation of PrPSc can be effected by miR-375 through PDK1/TACE way. In this project, cell lines and brain of prion-infected mouse will be used to detect the miR-375 and PDK1/TACE during disease progression.Their functions on the fomation of PrPSc were also evaluated. More miRNAs, which were changed in disease progression, involved in PrPC α-cleavages were also focused on. These research work will not only make us better understand the pathogenesis of prion disease, but also provide clues for diagnosis and treatment of this disease.

朊病毒病(Prion disease）的病因为体内正常的朊蛋白(PrPC)发生构象改变形成异常朊病毒(PrPSc)所致。朊蛋白的α切割被认为是在发病过程中对机体起着保护作用。TACE为α切割主要内切酶之一，PDK1是影响着TACE活性的重要因子。抑制PDK1活性能够提高TACE活性，从而延长实验动物生命。MiRNAs是一种体内调节蛋白表达的保守小分子。MiR-375能够调控PDK1的表达及活性。因此，我们推测MiR-375很可能通过调控TACE从而调控着PrPSc的形成。本研究拟以细胞/动物中的miR-375对PDK1/TACE活性和PrPSc形成的影响进行研究，同时以建立的小鼠感染脑组织中与PrPC α-内切酶相关的miRNA时间的变化谱，分析并寻找靶标蛋白/miRNA，研究其在朊病毒感染过程中的机制，并为寻找潜在的朊病毒病诊断及治疗靶标提供科学依据和线索。

本课题利用二代测序平台，对不同羊瘙痒因子毒株139A、ME7和S15感染小鼠脑组织中的miRNA表达谱进行了检测，结果显示三毒株具有类似的miRNA表达谱，在此基础上利用qRT-PCR方法对其进行了体外的检测，筛选出了共同变化的miRNA 12个；通过研究miR-375和PDK1的变化规律，并在体外构建报道质粒，证实了朊病毒病中PDK1会明显增加，且受到miR-375的调控；利用二代测序方法，对克雅氏病人和正常人血液中的循环miRNA进行了表达谱的检测，结合临床表现以及非克雅氏病神经系统疾病病人的miRNA表达谱进行分析并验证，初步结果表明hsa-miR-4508在sCJD人群中的表达明显上调，hsa-miR-4657表达下调；提取克雅氏病患者、神经系统疾病患者和正常献血员血液DNA，并进行SNP位点（rs57095329，rs2910164和rs295301）的序列测定，结果显示FFI与rs2910164相关。这些研究结果都为研究朊病毒致病机制提供了科学线索。