早期糖腎病患的生物標誌暨微小核糖核酸 miR-30c 透過調控DDAH1及IRS1 之表現量而造成腎臟細胞纖維化之影響

The biomarker for the early stage of diabetic nephropathy and microRNA miR-30c affects kidney fibrosis through regulating expression of DDAH1 and IRS1 proteins

陳書樊

糖腎病 ； 微小核醣核酸 ； 腎臟細胞纖維化 ； 生物標誌 ； diabetic nephropathy ； microRNA ； miR-30c ； DDAH1 ； IRS1 ； kidney fibrosis

交通大學生物科技學系學位論文

2017年

碩士

王志宏

繁體中文

糖尿病為一種慢性疾病，它會造成人體許多併發症，其中糖尿病患者會引發腎臟疾病造成腎臟功能損壞導致末期腎臟病，為了能有效控制糖腎病病患減緩腎臟功能喪失導致末期腎臟病，我們將進行糖腎病的研究與實驗。miRNA 為一小段非編碼的基因序列，在細胞的生長、增生、分化與免疫反應中扮演著重要的角色，也具有調節生理及病理的潛力，我們發現在腎臟中有許多特定的miRNA 能調控TFG-β的表現量，以及改善糖腎病患的高血糖及血脂，並且調控細胞外基質的不正常增生與增厚，減緩腎臟疾病引發末期腎臟病。首先我們將糖腎病病患檢體血清依照ACR 值進行分類，檢測血清中β-trophin 及尿液中NO 的含量，發現β-trophin 及 NO 在ACR 值較高的患者中此兩種特定代謝產物濃度較高，為了探討糖腎病患者中此兩種特定代謝物調控基因，我們將分組後的血清進行micrro arry 分析，找到 miR-30c 為上調控因子，根據先前研究發現 miR-30c 能減少脂質的合成與脂蛋白的分泌，一氧化氮 (NO) 與發炎反應及細胞凋亡有關，我們將進一步探討miR-30c 是否能減緩白蛋白尿及NO 的產生造成腎臟細胞基底膜不正常增厚而壞死，我們將 miR-30c 送入足細胞 (E11) 中，發現細胞中帶有 miR-30c 基因大量表現下其DDAH1 和 IRS1 的蛋白表現量下降，顯示出 miR-30c 能有效調控此兩種蛋白的形成，促進糖腎病造成腎臟功能損壞的可能。

Diabetes mellitus (DM) is a chronic disease that can result in many complications in people. One majority of them is kidney disease, which may finally lead to the end-stage renal disease (ESRD). However, the precise mechanism is still unclear. Hence, the better understanding of the disease may provide us novel therapeutic targets. MicroRNA is a short non-coding RNA that plays an important role in cell growth, proliferation, differentiation and immune response. Furthermore, it also has the potential to regulate several physiological and pathological processes. We found that many specific miRNA can regulate the expression of TGF-β in the kidney. Recent studies have highlighted the importance of miRNA in the regulation of glycaemia and the reduction of hyperlipidemia in DN patients. It can also modulate the abnormal extracellular matrix thickening, and prevent the chronic kidney disease from ESRD. First, according to the values of ACR, we separated the participants into four groups, H, DN1, DN2, and DN3. We detected the serum levels of β-trophin and urine levels of NO and found that β-trophin and NO were higher in DN3 and DN4 groups than in Healthy group. In order to explore the regulation of specific genes in diabetic nephropathy patients, we used micrroarray analysis to find the miRNA, which can regulate the β-trophin and NO production. In recent study, mir-30c has been found that can downregulate the lipid synthesis and the secretion of lipoprotein. It is also associated with the NO production, inflammatory response, and apoptosis. Most importantly, we found that miR-30c strongly upregulated in the development of DN. To further investigate the role of mir-30c in renal dysfunction, we overexpressed miR-30c in the podocyte cell line (E11). Upregulation of miR-30c can inhibit the DDAH1 and IRS1 expression in transcriptional levels. Our results suggest that miR-30c may play a critical role in the DN development and blockade of miR-30c may be a potential therapeutic option in DN.

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