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

描述（由申请人提供）：髓鞘形成的过程需要精确和及时表达几种蛋白质，这些蛋白质是轴突正确散热所必需的。中枢神经系统和周围神经系统的髓鞘形成细胞协调这一复杂过程的机制仍然是未知的。外周髓磷脂蛋白22（PMP 22）是主要在外周神经系统的髓鞘形成的雪旺细胞中表达的整合膜蛋白。PMP 22的错误表达与许多遗传性脱髓鞘性周围神经病有关，包括Charcot-Marie-Tooth病1A型，其估计患病率为每2500例活产中约1例。重复和基因缺失导致神经病表型，表明精确控制蛋白质表达的必要性。PMP 22似乎也在细胞周期调节中起作用，其中它首次被发现为在生长停滞期间上调的基因。PMP 22的异常表达也在几种癌症中被描述，包括骨肉瘤、乳腺癌和一些神经胶质瘤。有趣的是，PMP 22 mRNA在体内被广泛检测到，但蛋白质表达高度限于髓鞘形成的雪旺细胞、上皮细胞和选择的运动神经元，这表明存在转录后调节。已显示PMP 22基因的5'和3'-UTR影响mRNA的表达。5 '-UTR包含三个已知的启动子区域，导致三种不同的RNA转录物，但蛋白质相同。PMP 22的3 '-UTR似乎减少蛋白质翻译的机制仍然未知。microRNA（miRNAs）是小的内源性调节RNA分子，其通过结合RNA的3 '-UTR并通过几种可能的机制阻止翻译而在转录后发挥其作用。这是我的总体假设，PMP 22的表达是由特定的miRNAs在雪旺细胞调节。为了验证这一假设，我将证明稳定状态的PMP 22 RNA和蛋白质水平可以通过抑制miRNA生物合成途径来影响（目的1）。我还将使用PMP 22 3 '-UTR-荧光素酶报告基因构建体绘制出PMP 22 3'-UTR内的特异性功能性miRNA结合位点，并证明这些特异性PMP 22靶向miRNA的结合调节其表达（目的2）。最后，将证明特异性PMP 22靶向miRNA的不足和过表达将改变培养的雪旺细胞中PMP 22 mRNA和蛋白的稳态水平（目的3）。在这些实验的结论，我希望揭示新的机制，管理的PMP 22的调节，可能提供新的治疗目标，为相关的疾病状态。

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.