| Abstract: | 第一型肌強直肌肉萎縮症 (Myotonic dystrophy type I )，簡稱 DM1，是屬於多系統性神經肌肉退化性疾病的一種單基因顯性的遺傳疾病，DM1 病人的致病機制是在DMPK 基因的3’-端非編碼區上的 CUG 重複序列擴增，而MBNL1 會被吸引結合到CUG擴增，形成 foci，造成下游的基因失調。先前我們發現在肌纖維母細胞 (C2C12) 表現 CUG 重複序列擴增，在分化後的myoD mRNA與控制組相比無明顯差異，但在 myoD 蛋白質表現量是下降的，推測可能為轉錄後修飾的調控。近年在許多篇文獻有提到在 DM1 疾病當中 ，miRNA 會參與其調控致病機制。我們利用Next Generation Sequencing (NGS) 來偵測C2C12-(CUG)200 重複序列細胞中的 miRNA 表現量，分化前總共偵測1061個 miRNA ，分化後偵測到975個，經過數據的分類、生物資訊的分析 (mirTarBase) 尋找下游基因與 qRT-PCR 驗證後最後我們找出幾個在 C2C12-(CUG)200 分化後表現量上升的 miRNA，有 miR-22-5p, miR-26a-1-3p, miR-29c-5p, miR-100-5p, miR-133b-5p, miR-132-5p, miR-132-3p, miR-218-5p, miR-218-1-3p, miR-335-5p, miR-335-3p ，在C2C12 細胞中過度表現 miRNA，發現在分化後第三天 miR-22, miR-29C, miR-100 會抑制myotube 的形成，此結果顯示miR-22, miR-29C, miR-100 可能會調控肌肉細胞的分化。並且將這些 miRNA 已知的下游基因，透過生資軟體 (DAVID)，預測到許多的 GO pathway 與 skeletal system development, regulation of cell proliferation, regulation of cell differentiation, cell cycle arrest 相關。可能受到 miRNA 調控的下游基因，包括MYC, SMAD3, MP2, RUNX2, TGFB2, SHH, ZEB1, KLF4, LIF, KIT。經由西方點墨法驗證miR-29c 可能會調控 MYC, SMAD3，miR-22 可能會調控 MYC。另外，我們也利用 microRNA.org, Microcosm Targets, TargetScan Human 三種生物資訊預測網站預測可以結合在 myoD 3’UTR 的 miRNA，包括 mmu-miR-759, mmu-miR-762, mmu-miR-339-3P, mmu-miR-682，以及miR-337-5P。我們利用 Luciferase reporter 和qRT-PCR分析，過度表現miRNA 或在MBNL1 knockdown 細胞及在C2C12-(CUG)200細胞抑制 miRNA， 觀察細胞分化形態與 myoD、 MHC 蛋白質表現量，結果顯示 miR-682, miR-337-5P 可參與調控 myoD 3’UTR，並且可能還有其他的 miRNA 也參與調控。
Myotonic dystrophy type 1 (DM1) is a multisystemic dominant disease caused by expansion of CUG repeats in the 3’-UTR of the myotonic dystrophy protein kinase (DMPK) gene. In DM cells, the muscleblind-like (MBNL) proteins are sequestered by the double- stranded CUG repeats to form RNA foci, leading to defects in alternative RNA splicing that are linked to the pathologic features. Previously, we found myoD protein levels, but not RNA, were decreased in C2C12 myoblasts expressing CUG expansion or in myoblasts with mbnl1 knockdown. Because miRNAs are involved in the pathogenesis of DM1, we test the hypothesis that myoD can be regulated by miRNA. We have monitored miRNA expression in C2C12-(CUG)200 cells by Next Generation Sequencing (NGS). Before differentiation, 1061 miRNA were detected, whereas a total of 975 miRNA were detected after cell differentiation. By bioinformatics software (mirTarBase) analysis and qRT-PCR validation, we selected several miRNAs that are up-regulated during the differentiation course in C2C12-(CUG)200 cells. These include miR-22-5p, miR-26a-1-3p, miR-29c-5p, miR-100-5p, miR-133b-5p, miR-132-5p, miR-132-3p, miR-218-5p, miR-218-1-3p, miR-335-5p, miR-335-3p. Overexpression of miR-22, miR-29C, miR-100 in C2C12 cells can suppress myotube formation, indicating that these miRNAs might regulate the differentiation of myoblasts. GO pathways predicted by bioinformatics software (DAVID) reveal that the target genes are involved in skeletal system development, regulation of cell proliferation, regulation of cell differentiation, and cell cycle arrest. The downstream targets include MYC, SMAD3, MP2, RUNX2, TGFB2, SHH, ZEB1, KLF4, LIF, KIT. By Western blot validation, we found that miR-29c may regulate MYC and SMAD3, and miR-22 may regulate MYC. In addition, we used microRNA.org, Microcosm Targets, TargetScan bioinformatics softwares to predict miRNAs that may target myoD 3’UTR. Among them mmu-miR-759, mmu-miR-762, mmu-miR-339-3P, mmu-miR-682 and mmu-miR-337-5p were selected for further analysis. Using Luciferase reporter and qRT-PCR assays, overexpression of miRNA or repression of miRNA in MBNL1 knockdown cells and C2C12-(CUG)200 cells, we examined the myotube formation and expression of myoD and MHC protein levels. The results indicate that miR-682 and miR-337-5p can regulate myoD 3’UTR, together with other miRNAs. |
