探討MBNL1蛋白質對vinculin基因轉錄調節之機制

Muscleblind-like 1 (MBNL1)屬於一個調節RNA剪接之蛋白質家族並參與擴增CUG序列RNA造成之強直型肌肉萎縮症(Dystrophia myotonia, DM)的致病機制。Z-band結構的異常是DM病人肌肉組織的典型特徵，然而，目前對於此一效應的分子機轉並不了解。先前我們實驗室利用線蟲作為模式動物發現擴增的CUG序列RNA會破壞肌肉組織Z-band的結構和降低vinculin (組成Z-band的結構蛋白質)的表現。在此研究我們利用lentiviral system製備能穩定表達shRNA以knockdown MBNL1的C2C12細胞株並調查其對vinculin基因表達的影響。我們發現相較shRFP對照組，shMBNL1細胞分化後的vinculin RNA表現量明顯降低。接著，我們建構以vinculin promoter驅動luciferase報導基因的表達質體並將其轉染至shRFP細胞，證實在分化後vinculin promoter的活性較分化前有明顯上升，但若轉染至shMBNL1細胞其活性則無法上升，顯示MBNL1對vinculin基因表達的影響發生在轉錄層次。在vinculin promoter上存在有MyoD辨識的序列(CAAGTG，稱為E-box)，將此序列突變會使vinculin promoter的活性在分化時無法上升。EMSA實驗證實MyoD會直接與vinculin promoter上的E-box序列結合，而且Western blot分析顯示shMBNL1細胞的MyoD蛋白質表現量顯著降低。若將MyoD cDNA送入shMBNL1細胞則可提高其vinculin promoter的活性及RNA表現量。我們的結果顯示在DM病理狀態下，MBNL1功能的喪失可能藉由影響MyoD的表現而降低vinculin基因的轉錄。

Muscleblind-like 1 (MBNL1) belongs to a protein family of splicing regulators and is implicated in the pathogenesis of myotonic dystrophy (DM). Z-band disruption is a typical feature of DM muscle. However, the underlying molecular mechanism of this defect remains unclear. Recently, our laboratory showed that expanded CUG RNA induce Z-band disruption and reduce the expression of vinculin gene, a basal component of Z-band. To investigate if MBNL1 participates in the regulation of vinculin expression, we have here produced C2C12 cells stably express shRNA targeted to MBNL1 (shMBNL1) and RFP gene (shRFP, served as scramble control) using lentiviral system. Compared to scramble control, MBNL1 knockdown significantly reduced vinculin expression. By expressing a heterogeneous promoter-reporter construct in C2C12 cells, we found that vinculin promoter activity increased significantly during differentiation and this increase was abolished in shMBNL1 cells. These observations suggest that the effect of MBNL1 on vinculin expression occurs at the transcriptional level. Moreover, we found a conserved E-box (CAAGTG) at -640~-635 region of vinculin promoter. Mutation of this E-box abolished the increase of vinculin promoter activity during differentiation. Further EMSA experiments confirmed that MyoD may bind to the E-box in vinculin promoter, implying that MyoD may be involved in vinculin expression regulation by MBNL1. Consistently, Western blot analysis revealed that MyoD protein expression was abolished in shMBNL1 cells. Overexpression of MyoD in shMBNL1 cells significantly increased the vinculin promoter activity and RNA expression. These findings suggest that MBNL1 loss-of-function in DM pathological state may down-regulate vinculin transcription through abolishment of MyoD expression.