臺灣博碩士論文加值系統

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.

中文摘要 I
Abstract III
誌謝 V
中英文縮寫對照表 (Abbreviations) VII
目錄 IX
表目錄 XIII
圖目錄 XIV
附錄 XV
壹、 緒論： 1
1.1 腎臟 (Kidney) 1
1.2 糖尿病 (Diabetic mellitus) 2
1.3 糖尿病之流行病學 3
1.4 糖尿病引發腎臟病變 4
1.5 糖尿病腎病變的檢測與分期 6
1.6 腎臟功能檢測方式 7
1.7 小分子核糖核酸　(MicroRNA, miRNA) 7
1.8 miR-30c 與腎臟疾病的關係 9
1.9 研究動機與目的 10
貳、 材料與方法： 12
2.1 檢體收集與處理 12
2.2 檢體分析 12
2.3 酵素免疫分析Enzyme-Linked ImmunoSorbent Assay (ELISA) 13
2.4 細胞培養 13
2.5 細菌培養 14
2.6 質體DNA萃取 14
2.7 染色體DNA萃取 15
2.8 聚合酶連鎖反應 (Polymerase chain reaction，PCR) 16
2.9 限制酵素處理 (digestion) 與接合作用 (ligation) 17
2.10 熱休克轉型法 (heat shock transformation) 17
2.11 純化DNA 18
2.12 轉染作用 (Transfection) 18
2.13 轉染之G-418抗生素濃度配置與篩選 19
2.14 RNA萃取 19
2.15 反轉錄聚合酶連鎖反應 (Reverse Transcription Polymerase Chain Reaction，RT-PCR) 20
2.16 即時定量聚合酶連鎖反應 (real-time quantitative PCR) 21
參、 結果： 22
3.1 臨床研究特點分析 22
3.2 ACR 值無法明確判定初期糖腎病檢測 22
3.3 糖腎病患血清中的β-trophin 含量較高 23
3.4 糖腎病患尿液中NO2-含量檢測 23
3.5 microRNA 表現量差異比較 23
3.6 構築pEGFP-N1-mir-30c 質體 24
3.7 以高濃度葡萄糖的培養基培養細胞後偵測mir-30c在細胞中表現量的差異 24
3.8 將帶有miR-30c之質體轉染至E11 細胞株 24
3.9 選取 miR-30c 相關的目標基因 25
3.10 大量表現miR-30c的細胞株在高濃度葡萄糖環境中DDAH-1 的表現量會下降 25
3.11 大量表現miR-30c的細胞株在高濃度葡萄糖環境中IRS-1 的表現量會下降 26
肆、 討論： 27
4.1 ACR值無法準確檢測出初期腎臟病變 27
4.2 葡萄糖濃度會影響足細胞中miR-30c 的表現量 27
4.3 β-trophin 會影響脂質的代謝並可用來檢測糖腎病 28
4.4 在糖腎病患中 NO 的減少會造成高血壓 28
4.5 miR-30c 會促進足細胞纖維化 29
伍、 結論： 31
陸、 參考文獻 33

1. Abdel-Rahman, E.M., Saadulla, L., Reeves, W.B., Awad, A.S., 2012. Therapeutic modalities in diabetic nephropathy: standard and emerging approaches. J Gen Intern Med 27, 458-468.

2. Abu-Farha, M., Al-Khairi, I., Cherian, P., Chandy, B., Sriraman, D., Alhubail, A., Al-Refaei, F., AlTerki, A., Abubaker, J., 2016. Increased ANGPTL3, 4 and ANGPTL8/betatrophin expression levels in obesity and T2D. Lipids Health Dis 15, 181.

3. Artunc, F., Schleicher, E., Weigert, C., Fritsche, A., Stefan, N., Haring, H.U., 2016. The impact of insulin resistance on the kidney and vasculature. Nat Rev Nephrol 12, 721-737.

4. Bhatwadekar, A.D., Yan, Y., Stepps, V., Hazra, S., Korah, M., Bartelmez, S., Chaqour, B., Grant, M.B., 2015. miR-92a Corrects CD34+ Cell Dysfunction in Diabetes by Modulating Core Circadian Genes Involved in Progenitor Differentiation. Diabetes 64, 4226-4237.

5. Bhensdadia, N.M., Hunt, K.J., Lopes-Virella, M.F., Michael Tucker, J., Mataria, M.R., Alge, J.L., Neely, B.A., Janech, M.G., Arthur, J.M., Veterans Affairs Diabetes Trial study, g., 2013. Urine haptoglobin levels predict early renal functional decline in patients with type 2 diabetes. Kidney Int 83, 1136-1143.

6. Boffa, J.J., Lu, Y., Placier, S., Stefanski, A., Dussaule, J.C., Chatziantoniou, C., 2003. Regression of renal vascular and glomerular fibrosis: role of angiotensin II receptor antagonism and matrix metalloproteinases. J Am Soc Nephrol 14, 1132-1144.

7. Chen, C.C., Susanto, H., Chuang, W.H., Liu, T.Y., Wang, C.H., 2016. Higher serum betatrophin level in type 2 diabetes subjects is associated with urinary albumin excretion and renal function. Cardiovasc Diabetol 15, 3.

8. Chien, H.Y., Chen, C.Y., Chiu, Y.H., Lin, Y.C., Li, W.C., 2016. Differential microRNA Profiles Predict Diabetic Nephropathy Progression in Taiwan. Int J Med Sci 13, 457-465.

9. Chou, P.-S., Tung, Tao-Hsin,Li, Chia-Lin,Chuang, Shao-Yuan,Lin, Cheng-Heng, Yang, Nan-Ping, 2002. Epidemiology of Diabetes Mellitus in Taiwan. Taiwan Journal of Public Health.

10. De Muro, P., Lepedda, A.J., Nieddu, G., Idini, M., Tram Nguyen, H.Q., Lobina, O., Fresu, P., Formato, M., 2016. Evaluation of Early Markers of Nephropathy in Patients with Type 2 Diabetes Mellitus. Biochem Res Int 2016, 7497614.

11. Du, T., Zamore, P.D., 2005. microPrimer: the biogenesis and function of microRNA. Development 132, 4645-4652.

12. Duisters, R.F., Tijsen, A.J., Schroen, B., Leenders, J.J., Lentink, V., van der Made, I., Herias, V., van Leeuwen, R.E., Schellings, M.W., Barenbrug, P., Maessen, J.G., Heymans, S., Pinto, Y.M., Creemers, E.E., 2009. miR-133 and miR-30 regulate connective tissue growth factor: implications for a role of microRNAs in myocardial matrix remodeling. Circ Res 104, 170-178, 176p following 178.

13. Fiseha, T., Tamir, Z., 2016. Urinary Markers of Tubular Injury in Early Diabetic Nephropathy. Int J Nephrol 2016, 4647685.

14. Garber, A., 1998. Diabetes mellitus, in: Stein, J. (Ed.), Internal Medicine. Mosby, St Louis, pp. 1850–1854.

15. Gusarova, V., Alexa, C.A., Na, E., Stevis, P.E., Xin, Y., Bonner-Weir, S., Cohen, J.C., Hobbs, H.H., Murphy, A.J., Yancopoulos, G.D., Gromada, J., 2014. ANGPTL8/betatrophin does not control pancreatic beta cell expansion. Cell 159, 691-696.

16. Gutierrez-Escolano, A., Santacruz-Vazquez, E., Gomez-Perez, F., 2015. Dysregulated microRNAs involved in contrast-induced acute kidney injury in rat and human. Ren Fail 37, 1498-1506.

17. Hagiwara, S., McClelland, A., Kantharidis, P., 2013. MicroRNA in diabetic nephropathy: renin angiotensin, aGE/RAGE, and oxidative stress pathway. J Diabetes Res 2013, 173783.

18. Hanson, R.L., Leti, F., Tsinajinnie, D., Kobes, S., Puppala, S., Curran, J.E., Almasy, L., Lehman, D.M., Blangero, J., Duggirala, R., DiStefano, J.K., 2016. The Arg59Trp variant in ANGPTL8 (betatrophin) is associated with total and HDL-cholesterol in American Indians and Mexican Americans and differentially affects cleavage of ANGPTL3. Mol Genet Metab 118, 128-137.

19. Hosoya, K., Minakuchi, H., Wakino, S., Fujimura, K., Hasegawa, K., Komatsu, M., Yoshifuji, A., Futatsugi, K., Shinozuka, K., Washida, N., Kanda, T., Tokuyama, H., Hayashi, K., Itoh, H., 2015. Insulin resistance in chronic kidney disease is ameliorated by spironolactone in rats and humans. Kidney Int 87, 749-760.

20. Huang, H.C., Preisig, P.A., 2000. G1 kinases and transforming growth factor-beta signaling are associated with a growth pattern switch in diabetes-induced renal growth. Kidney Int 58, 162-172.

21. Huang, J., Yao, X., Zhang, J., Dong, B., Chen, Q., Xue, W., Liu, D., Huang, Y., 2013. Hypoxia-induced downregulation of miR-30c promotes epithelial-mesenchymal transition in human renal cell carcinoma. Cancer Sci 104, 1609-1617.

22. Huang, Y.Y., Lin, K.D., Jiang, Y.D., Chang, C.H., Chung, C.H., Chuang, L.M., Tai, T.Y., Ho, L.T., Shin, S.J., 2012. Diabetes-related kidney, eye, and foot disease in Taiwan: an analysis of the nationwide data for 2000-2009. J Formos Med Assoc 111, 637-644.
23. Inaguma, D., Imai, E., Takeuchi, A., Ohashi, Y., Watanabe, T., Nitta, K., Akizawa, T., Matsuo, S., Makino, H., Hishida, A., Chronic Kidney Disease Japan Cohort Study, G., 2016. Risk factors for CKD progression in Japanese patients: findings from the Chronic Kidney Disease Japan Cohort (CKD-JAC) study. Clin Exp Nephrol.

24. Ji, E., Kim, Y.S., 2016. Prevalence of chronic kidney disease defined by using CKD-EPI equation and albumin-to-creatinine ratio in the Korean adult population. Korean J Intern Med 31, 1120-1130.
25. Jiao, Y., Le Lay, J., Yu, M., Naji, A., Kaestner, K.H., 2014. Elevated mouse hepatic betatrophin expression does not increase human beta-cell replication in the transplant setting. Diabetes 63, 1283-1288.

26. Kato, M., Arce, L., Natarajan, R., 2009. MicroRNAs and their role in progressive kidney diseases. Clin J Am Soc Nephrol 4, 1255-1266.
27. Kitada, M., Koya, D., 2015. [Diagnosis and management of diabetic nephropathy]. Nihon Rinsho 73, 2037-2043.

28. Lai, L., Ghebremariam, Y.T., 2016. Modulating DDAH/NOS Pathway to Discover Vasoprotective Insulin Sensitizers. J Diabetes Res 2016, 1982096.

29. Lavin, D.P., White, M.F., Brazil, D.P., 2016. IRS proteins and diabetic complications. Diabetologia 59, 2280-2291.

30. Li, F., Li, L., Cheng, M., Wang, X., Hao, J., Liu, S., Duan, H., 2017. SHIP, a novel factor to ameliorate extracellular matrix accumulation via suppressing PI3K/Akt/CTGF signaling in diabetic kidney disease. Biochem Biophys Res Commun 482, 1477-1483.

31. Liu, L., Lin, W., Zhang, Q., Cao, W., Liu, Z., 2016. TGF-beta induces miR-30d down-regulation and podocyte injury through Smad2/3 and HDAC3-associated transcriptional repression. J Mol Med (Berl) 94, 291-300.

32. Liu, Y.F., Ding, M., Liu, D.W., Liu, Y., Mao, Y.G., Peng, Y., 2015. MicroRNA profiling in cutaneous wounds of diabetic rats. Genet Mol Res 14, 9614-9625.

33. Matsumoto, Y., Ueda, S., Yamagishi, S., Matsuguma, K., Shibata, R., Fukami, K., Matsuoka, H., Imaizumi, T., Okuda, S., 2007. Dimethylarginine dimethylaminohydrolase prevents progression of renal dysfunction by inhibiting loss of peritubular capillaries and tubulointerstitial fibrosis in a rat model of chronic kidney disease. J Am Soc Nephrol 18, 1525-1533.

34. Mauer, S.M., Barbosa, J., Vernier, R.L., Kjellstrand, C.M., Buselmeier, T.J., Simmons, R.L., Najarian, J.S., Goetz, F.C., 1976. Development of diabetic vascular lesions in normal kidneys transplanted into patients with diabetes mellitus. N Engl J Med 295, 916-920.

35. Maurer, L., Brachs, S., Decker, A.M., Brachs, M., Leupelt, V., Jumpertz von Schwartzenberg, R., Ernert, A., Bobbert, T., Krude, H., Spranger, J., Mai, K., 2016. Weight Loss Partially Restores Glucose-Driven Betatrophin Response in Humans. J Clin Endocrinol Metab 101, 4014-4020.

36. Max Ellenberg, H.R.a.D.P., 1983. Diabetes Mellitus: Theory and Practice. Elsevier Science Ltd; 4th edition

37. Navarro-Gonzalez, J.F., Mora-Fernandez, C., 2008. The role of inflammatory cytokines in diabetic nephropathy. J Am Soc Nephrol 19, 433-442.

38. Nolan, C.J., Damm, P., Prentki, M., 2011. Type 2 diabetes across generations: from pathophysiology to prevention and management. Lancet 378, 169-181.

39. Ozcan, S., 2009. MiR-30 family and EMT in human fetal pancreatic islets. Islets 1, 283-285.

40. Palm, F., Onozato, M.L., Luo, Z., Wilcox, C.S., 2007. Dimethylarginine dimethylaminohydrolase (DDAH): expression, regulation, and function in the cardiovascular and renal systems. Am J Physiol Heart Circ Physiol 293, H3227-3245.

41. Patrick L. Gentry., S.A.K., 2006. Mechanisms of Endothelial Dysfunction: Clues from Cyclosporine. Journal of Pharmacology and Toxicology.

42. Santamaria, B., Marquez, E., Lay, A., Carew, R.M., Gonzalez-Rodriguez, A., Welsh, G.I., Ni, L., Hale, L.J., Ortiz, A., Saleem, M.A., Brazil, D.P., Coward, R.J., Valverde, A.M., 2015. IRS2 and PTEN are key molecules in controlling insulin sensitivity in podocytes. Biochim Biophys Acta 1853, 3224-3234.

43. Sequeira-Lopez, M.L., Weatherford, E.T., Borges, G.R., Monteagudo, M.C., Pentz, E.S., Harfe, B.D., Carretero, O., Sigmund, C.D., Gomez, R.A., 2010. The microRNA-processing enzyme dicer maintains juxtaglomerular cells. J Am Soc Nephrol 21, 460-467.

44. Shi, S., Yu, L., Chiu, C., Sun, Y., Chen, J., Khitrov, G., Merkenschlager, M., Holzman, L.B., Zhang, W., Mundel, P., Bottinger, E.P., 2008. Podocyte-selective deletion of dicer induces proteinuria and glomerulosclerosis. J Am Soc Nephrol 19, 2159-2169.

45. Tai, T.Y., Yang, C.L., Chang, C.J., Chang, S.M., Chen, Y.H., Lin, B.J., Ko, L.S., Chen, M.S., Chen, C.J., 1987. Epidemiology of diabetes mellitus among adults in Taiwan, R.O.C. J Med Assoc Thai 70 Suppl 2, 42-48.

46. Tamaki, K., Okuda, S., Ando, T., Iwamoto, T., Nakayama, M., Fujishima, M., 1994. TGF-beta 1 in glomerulosclerosis and interstitial fibrosis of adriamycin nephropathy. Kidney Int 45, 525-536.

47. Tomita, H., Egashira, K., Ohara, Y., Takemoto, M., Koyanagi, M., Katoh, M., Yamamoto, H., Tamaki, K., Shimokawa, H., Takeshita, A., 1998. Early induction of transforming growth factor-beta via angiotensin II type 1 receptors contributes to cardiac fibrosis induced by long-term blockade of nitric oxide synthesis in rats. Hypertension 32, 273-279.

48. Wahab, N.A., Weston, B.S., Mason, R.M., 2005. Modulation of the TGFbeta/Smad signaling pathway in mesangial cells by CTGF/CCN2. Exp Cell Res 307, 305-314.

49. Wang, J., Duan, L., Guo, T., Gao, Y., Tian, L., Liu, J., Wang, S., Yang, J., 2016. Downregulation of miR-30c promotes renal fibrosis by target CTGF in diabetic nephropathy. J Diabetes Complications 30, 406-414.

50. Wei, Q., Mi, Q.S., Dong, Z., 2013. The regulation and function of microRNAs in kidney diseases. IUBMB Life 65, 602-614.

51. Welsh, G.I., Hale, L.J., Eremina, V., Jeansson, M., Maezawa, Y., Lennon, R., Pons, D.A., Owen, R.J., Satchell, S.C., Miles, M.J., Caunt, C.J., McArdle, C.A., Pavenstadt, H., Tavare, J.M., Herzenberg, A.M., Kahn, C.R., Mathieson, P.W., Quaggin, S.E., Saleem, M.A., Coward, R.J., 2010. Insulin signaling to the glomerular podocyte is critical for normal kidney function. Cell Metab 12, 329-340.

52. Wotherspoon, F., Browne, D.L., Meeking, D.R., Allard, S.E., Munday, L.J., Shaw, K.M., Cummings, M.H., 2005. The contribution of nitric oxide and vasodilatory prostanoids to bradykinin-mediated vasodilation in Type 1 diabetes. Diabet Med 22, 697-702.

53. Wu, J., Zheng, C., Fan, Y., Zeng, C., Chen, Z., Qin, W., Zhang, C., Zhang, W., Wang, X., Zhu, X., Zhang, M., Zen, K., Liu, Z., 2014. Downregulation of microRNA-30 facilitates podocyte injury and is prevented by glucocorticoids. J Am Soc Nephrol 25, 92-104.

54. Xu, Y., Zhang, Q., Luo, D., Wang, J., Duan, D., 2016. Low molecular weight fucoidan modulates P-selectin and alleviates diabetic nephropathy. Int J Biol Macromol 91, 233-240.

55. Zhang, N., Lawrence, D.A., 2011. Tissue factor and obesity, a two-way street. Nat Med 17, 1343-1344.

56. Zhao, C., Li, T., Han, B., Yue, W., Shi, L., Wang, H., Guo, Y., Lu, Z., 2016. DDAH1 deficiency promotes intracellular oxidative stress and cell apoptosis via a miR-21-dependent pathway in mouse embryonic fibroblasts. Free Radic Biol Med 92, 50-60.