The Post-Transcriptional Regulation of Peripheral Myelin Protein 22 by MicroRNAs

描述(申请人提供)：髓鞘形成的过程需要准确和及时地表达几种蛋白质，这些蛋白质是适当加热轴突所必需的。中枢和外周神经系统的髓鞘细胞协调这一复杂过程的机制在很大程度上仍不清楚。外周髓鞘蛋白22(PMP22)是一种完整的膜蛋白，主要表达于周围神经系统的髓鞘形成施万细胞。PMP22的错误表达与许多遗传性脱髓鞘周围神经病有关，包括Charcot-Marie-Tooth病1A型，据估计，每2500名活产儿中约有1名患病率。复制和基因缺失都会导致神经病变表型，这表明有必要对蛋白质的表达进行精确控制。PMP22似乎还在细胞周期调节中发挥作用，它最初是作为一种在生长停滞期间上调的基因被发现的。PMP22的异常表达在几种癌症中也有报道，包括骨肉瘤、乳腺癌和一些胶质瘤。有趣的是，PMP22在体内被广泛检测到，但蛋白的表达高度局限于髓鞘雪旺细胞、上皮细胞和选定的运动神经元，提示转录后调控。研究表明，PMP22基因的5‘和3’-UTRs会影响该基因的表达。5‘-UTR包含三个已知的启动子区域，导致三个不同的RNA转录本，但蛋白质相同。PMP22的3‘-非编码区似乎减少蛋白质翻译的机制尚不清楚。MicroRNAs(MiRNAs)是一种小的内源性调节性RNA分子，通过与RNA的3‘-UTR结合，通过几种可能的机制阻止翻译，从而在转录后发挥作用。我的总体假设是，PMP22的表达受雪旺细胞中特定的miRNAs调控。为了验证这一假设，我将证明稳定状态的PMP22 RNA和蛋白质水平可以受到抑制miRNA生物发生途径的影响(目标1)。我还将使用PMP22 3‘-UTR-荧光素酶报告构建物在PMP22的3’-UTR内绘制特定的功能性miRNA结合位点，并证明这些针对miRNAs的特定PMP22的结合调节其表达(目标2)。最后，将证明针对miRNAs的特定PMP22的过低和过表达将改变培养的雪旺细胞中PMP22的mRNA和蛋白的稳态水平(目标3)。在这些实验的结论中，我希望揭示调控PMP22的新机制，可能为相关疾病状态提供新的治疗靶点。

DESCRIPTION (provided by applicant): The process of myelination requires the precise and timely expression of several proteins which are necessary for the proper esheatment of axons. The mechanisms by which the myelinating cells of the central and peripheral nervous systems coordinate this elaborate process still remain largely unknown. Peripheral myelin protein 22 (PMP22) is an integral membrane protein that is primarily expressed in myelinforming Schwann cells of the peripheral nervous system. The misexpression of PMP22 is implicated in a host of hereditary demyelinating peripheral neuropathies including Charcot-Marie-Tooth disease type 1A which has an estimated prevalence of approximately 1 in every 2500 live births. Both duplication and gene deletion result in neuropathic phenotypes indicating the necessity for precise control over protein expression. PMP22 also appears to serve a function in cell cycle regulation, where it was first discovered as a gene upregulated during growth arrest. Aberrant expression of PMP22 is also described in several cancers, including osteosarcoma, breast cancer, and some gliomas. Interestingly, PMP22 mRNA is widely detected in the body, but protein expression is highly restricted to the myelinating Schwann cells, epithelial cells and select motor neurons, suggesting post-transcriptional regulation. The 5' and 3'-UTRs of the PMP22 gene have been shown to influence the expression of the mRNA. The 5'-UTR contains three known promoter regions resulting in three distinct RNA transcripts, but same protein. The mechanism by which the 3'-UTR of PMP22 appears to reduce protein translation remains unknown. MicroRNAs (miRNAs) are small, endogenous regulatory RNA molecules that exert their action post-transcriptionally by binding to the 3'-UTR of RNA and preventing translation through several possible mechanisms. It is my overall hypothesis that PMP22 expression is regulated by specific miRNAs in Schwann cells. To test this hypothesis, I will demonstrate that steady-state PMP22 RNA and protein levels can be influenced by inhibition of the miRNA biogenesis pathway (Aim 1). I will also map out specific functional miRNA binging sites within the 3'-UTR of PMP22 using PMP22 3'-UTR-luciferase reporter constructs and demonstrate that the binding of these specific PMP22 targeting miRNAs regulate its expression (Aim 2). Finally, it will be demonstrated that under- and overexpression of specific PMP22 targeting miRNAs will modify the steady-state levels of PMP22 mRNA and protein in cultured Schwann cells (Aim 3). At the conclusion of these experiments, I hope to reveal novel mechanisms governing the regulation of PMP22 that may provide new therapeutic targets for associated disease states.