- 學位論文
Fibronectin經由AKT抑制miR-133a與miR-29a促進MES 13細胞的纖維化與發炎
Fibronectin induces fibrosis and inflammation and downregulation miR-133a and miR-29a via AKT in MES cells.
摘要
糖尿病腎病變(Diabetic nephropathy, DN)是糖尿病患者最常見的併發症，其主要的病理特徵包括：早期的腎臟細胞增生及後期的腎臟細胞肥大、細胞外間質(Extracellular matrix, ECM)堆積和上皮細胞轉變成間質細胞(Epithelial-to-mesenchymal transition, EMT)，最後導致腎臟纖維化，演變為末期腎病(End-stage renal disease, ESRD)。其中存在於ECM中的Fibronectin（FN）為醣蛋白質（Glycoprotein），可促進細胞增生、分化、修復受損細胞等刺激細胞的一種功能蛋白，已有許多研究顯示，Fibronectin會活化p-PI3K/AKT、p-ERK1/2 及p-STAT3導致EMT。另外，當microRNA失調時會導致癌症，心血管疾病，糖尿病和代謝性等疾病。因此本研究以Fibronectin刺激正常小鼠腎絲球間質細胞(MES 13)探討其效應及作用之分子機轉，並研究調控其效應之相關miRNA的表現與影響。 結果發現外加Fibronectin可抑制MES 13細胞生長且呈現劑量效應。當Fibronectin在15分鐘至4小時刺激下會促使ERK1/2、AKT、STAT3的磷酸化增加。而在24、48、72小時刺激下，纖維化相關分子collagen IV、TGF-ß1、CTGF、TGF-ß receptor II的表現都有明顯的增加，也會促進NF-κB次單元(p65) 、cox-2發炎因子表現。另外Fibronectin刺激下p27kipl、p21waf1/cip1 (A cyclin-dependent kinase inhibitor)蛋白表現增加，導致細胞肥大現象(Cell hypertrophy)。接著在Fibronectin刺激下分別加入STAT3、EKR1/2、AKT抑制劑，發現加入AKT抑制劑會逆轉相關纖維化分子以及發炎分子Cox-2與NF-κB次單元(p65) 的表現。而p27kipl、p21waf1/cip1蛋白則是加入STAT3、AKT抑制劑來逆轉Fibronectin所誘導的表現量。另外Fibronectin刺激下也會抑制調控相關纖維化分子之microRNA，例如：miR-133a (抑制CTGF和TGF-ß1)，miR-29a (抑制Collagen I、III 和IV)，而加入AKT抑制劑能逆轉其miRNA表現。 綜合以上結果，糖尿病腎病變導致ECM堆積時Fibronectin透過活化AKT訊息路徑抑制調控相關纖維化分子之microRNA表現而促進纖維化，另外Fibronectin也會透過活化AKT和STAT3訊息路徑促進發炎因子以及細胞週期抑制蛋白，進而影響正常腎臟細導致糖尿病腎病變。
並列摘要
Diabetic nephropathy (DN) is a common complication of diabetes mellitus. It is characterized by early renal cell proliferation, late renal cell hypertrophy, accumulation of extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT) to result in renal fibrosis and end-stage renal disease (ESRD). Fibronectin (FN) is involved in organizing ECM, mediating cell adhesion, proliferation, and differentiation. FN also promoted EMT by activating PI3K/AKT, ERK1/2, and STAT3. Dysregulation of microRNAs (miRNAs) is linked with cancer, cardiovascular disease, diabetes and other diseases. Thus, we studied the effects of fibronectin on Mes 13 cells in terms of miRNA and signal transduction pathways. We found that FN dose-dependently inhibited cell proliferation. FN also increased phosphorylation of ERK1/2, STAT3 and AKT during the period form 15min-4 hours, promoted fibrosis-related molecules, including collagen IV, TGF-ß, CTGF, TGF-ß receptor II at 24-72 hours. In addition, FN increased expression of NF-κB subunit (p65), Cox-2, p27cipl/kipl and p21waf1/cip1 (a cyclin-dependent kinase inhibitor), and hypertrophy in kidney cells. In FN stimulation, cells were treated with either STAT3, ERK1/2 or AKT inhibitors. An AKT inhibitor attenuated FN-induced expression of fibrosis-related molecules and inflammatory-related molecules (Cox-2, p65) while AKT and STAT3 inhibitors attenuatd FN-induced expression of p21 and p27. Furthermore, FN inhibits miR-133a (which inhibits CTGF and TGF-β1) and miR-29a (which inhibits collagen I, III and IV), and then AKT inhibitor reversed expression of the miRNA. All of these results demonstrated that FN increased pro-fibrotic molecules and inhibited miR-133a and miR-29a via the AKT pathway. In addition, FN also promoted inflammatory factors and cell cycle inhibitors via AKT and STAT3 pathway. These mechanisms may be involved in the pathogenesis of DN.