| 摘要: | 近來的研究顯示，由三聯核酸重複序列在非編碼區造成的顯性遺傳疾病如強直型肌肉萎縮症第一型(DM1)，RNA扮演重要的致病角色。擴增的CUG重複序列RNA形成穩定的髮夾型二級結構，吸引MBNL蛋白質結合並堆積在核內形成聚集。此MBNL隔離伴隨著CUGBP上調，使得其下游基因的差異性剪輯異常而造成DM1多系統的臨床症狀。除了此RNA致病機轉外，最近亦有研究指出miRNA可能會參與DM1的致病。miRNA是小的非編碼RNA，可以在轉錄後層次調控基因表達。它們被預測可調控三分之ㄧ的基因體，且可調控廣泛的生物程序包括細胞分化和疾病生成。然而，它們如何具體的與CUG重複擴增或MBNL蛋白作用目前仍大部分不清楚。之前為了研究重複序列擴增致病的機制，我們已建立了過量表現CUG重複序列(CUG200)以及MBNL1 knockdown (shMBNL1)的C2C12肌母細胞株。這些細胞表現出與DM1細胞類似的病理特徵，例如形成核內RNA聚集(CUG200細胞)、降低分化能力、下調myoD蛋白表現和其他肌肉分化因子、以及基因剪輯的異常。我們先前的研究顯示，myoD可能會藉由其3’非轉譯區的序列被轉錄後調控。在本計劃中，我們首先將探討myoD是否可被miRNA調控，藉由冷光報導分析、生物資訊預測、表達分析、以及gain-of-function 和loss-of-function分析合併進行。其次，我們將研究是否CUG擴增或MBNL1會影響myoD miRNA的調控，以及CUG擴增或MBNL1如何影響其調控，包括分析miRNA的表達、剪切、及轉錄調控等。另外，我們將找出在CUG200或shMBNl1細胞中異常表達的miRNA並研究其調控和功能。我們已經有一些候選miRNA在進行研究，因此我們將不會對所有的miRNA進行微陣列分析，而是針對特定的一些肌肉細胞表現量多且有明顯異常表達的miRNA進行分析。期望這些研究有助於了解miRNA如何參與在DM1疾病的肌肉病變過程，並能提供未來疾病治療一些新的洞見。
Recent studies have revealed a key role for RNA in the pathogenesis of the dominantly inherited non-coding triplet repeat disorders such as myotonic dystrophy type 1 (DM1). The transcripts of expanded CUG repeats form stable hairpin structures that recruit the muscleblind-like (MBNL) proteins binding and accumulated as nuclear foci. MBNL sequestration, accompanied with CUGBP up-regulation, results in splicing defects leading to multisystemic clinical features. In addition to this RNA-pathogenesis mechanism, recent lines of evidence suggested that miRNA may also participate in the pathogenesis of DM1. miRNAs are small non-coding RNAs that regulate gene expression post-transcriptionally. They are predicted to target one-third of the genome and have been shown to play a key role in regulating a wide range of biological processes including cell differentiation and disease pathogenesis. However, exactly how miRNA interplays with repeat expansion or MBNL proteins remains largely unknown. Previously, in order to unravel the pathogenic mechanisms underlying the repeat expansions, we generated C2C12 myoblast cell lines with overexpression of CUG repeats (CUG200) and with MBNL1 knockdown (shMBNL1). These cells displayed characteristic features of DM1 cells such as nuclear RNA foci formation (in CUG200 cells), reduced differentiation capacity with down- regulation of myoD protein and other myogenic factors, and defects in splicing. Our previous data suggest that myoD may be regulated post-transcriptionally by its 3’UTR sequence. In this study, we will first address whether myoD expression can be regulated by miRNA, by a combination of luciferase reporter assay, bioinformatic prediction, expression profiling, and gain-/loss-of-function analysis. Secondly, we will investigate whether and how CUG expansion or MBNL1 affects regulation of the putative myoD miRNAs, including analysis of expression, processing and transcriptional control of the miRNAs. Thirdly, we will identify the miRNAs that are misexpressed in CUG200 or shMBNL1 cells and study their regulation and function. We already have some candidate miRNAs under analysis. Instead of profiling the expression of total miRNAs by microarray, we will focus on specific muscle-enriched miRNAs that show a roust misexpression pattern. We hope that the study will contribute to the understanding of potential miRNA involvement in muscle pathogenesis of DM1, and will provide new insights into future therapeutics. |
