探討miR-122a 和Betatrophin在代謝症候群與癌症發展初期是否可作為具潛力的生物指標

Abstract

代謝症候群是主要的世界健康議題之一，而肥胖和第二型糖尿病是兩個造成代謝症候群的潛在因子。長期的糖尿病或者低血糖耐受性所造成的肥胖被視為微血管併發症與癌症發展的一個重要過程。近期研究發現，betatrophin，一種由肝臟製造的激素，可以改善血糖耐受性與脂質代謝；miR-122a，也是一個脂質代謝的潛在調節者，在脂肪代謝失調中扮演了一個重要的角色。然而，miR-122a與betatrophin在代謝症候群與癌症發展中的潛在功能尚未被完全釐清。本論文的目的是要闡明betatrophin與miR-122a在此二病症中代謝失調部分的基礎角色。在此，我們將實驗分為兩方面，分別是人類的病例對照研究與使用miR-122a剔除小鼠所呈現的小鼠模型。 在臨床調查部分，32個健康人體樣本與109個第二型糖尿病患者樣本，可依照不同程度的白蛋白尿(albuminuria) 區分糖腎病的嚴重性。根據尿液中白蛋白與肌酸酐的比例(ACR)，樣本可分成正常白蛋白尿期(normoalbuminuria，ACR < 30 mg/g)、微白蛋白尿期(microalbuminuria，ACR 30~300 mg/g)、巨量白蛋白尿期(macroalbuminuria，ACR > 300 mg/g)等三種。另外，血清樣本亦從15位健康人體與105位胰腺導管腺癌患者中取得。胰腺導管腺癌的血清可依照血糖耐受性的影響程度區分成正常血糖耐受性(normal glucose tolerance，PDAC-NGT)、受損的血糖耐受性(impaired glucose tolerance，PDAC-IGT)、糖尿病(diabetes mellitus，PDAC-DM)等三種。在研究中，血清的betatrophin和carbohydrate antigen 19-9 (CA19-9)濃度皆以酵素連結免疫吸附分析法測定。在動物實驗中，miR-122a基因剔除小鼠被用來設計成肝臟纖維化與肝細胞癌的小鼠模型。小鼠被分為4組，每組8~10隻。在3個月的高脂肪飼料餵食後，部分組別進行6週的腹腔注射，施打carbon tetrachloride-human recombinant erythropoietin (CCl4+rhEpo)。 在我們的臨床研究中發現，相較於健康人體，血清中betatrophin的濃度在不同白蛋白尿程度的第二型糖尿病患者中皆有顯著性地上升(P < 0.001)，且與ACR呈正相關，然而卻與eGFR呈負相關(P < 0.05)。較高的betatrophin可能更容易導致患者得到糖腎病[odds ratio (OR) = 5.65, 95 % confidence interval (CI) 2.17-14.57, P < 0.001]。重要而有趣的是，血清betatrophin在有血糖耐受性問題的胰腺導管腺癌患者中顯著增加。在胰腺導管腺癌造成的糖尿病中，Betatrophin的勝算比[OR=3.39; 95% CI (1.20 - 9.57); P=0.021)]更勝於傳統的癌症指標CA 19-9。因此，我們建議，在糖腎病與胰腺導管腺癌造成的糖尿病中，betatrophin可能成為一個新的潛在生物指標。 在另一方面，動物實驗中，抑制miR-122a可能會加速代謝症候群的惡化。高脂飲食誘發肥胖，並使胰島素阻抗變嚴重、肝臟中脂肪酸的代謝降低、發炎反應增強、肝臟纖維化加劇，而造成肝臟腫瘤生成的加速。有趣的是，miR-122a的表現缺失與棕色脂肪組織的產熱比例增加和造血功能增加相關。所以，在肥胖造成的第二型糖尿病以及與肝細胞癌有關的代謝症候群中，miR-122a可能可以作為一個早期的預測指標。

Metabolic syndrome is the major health problem worldwide caused by two potential inducers, obesity and T2DM. Obesity linked long-standing diabetes, or glucose intolerance is consider as a crucial event in the process of microvascular complication and cancer development. Recently, betatrophin, a novel liver-derived hormone, improves glucose intolerance and lipid metabolism. Moreover, another potential regulator of lipid metabolism, miR-122a is playing a pivotal role in lipid metabolism disorder. However, the potential function of miR-122a and betatrophin in the pathogenesis of metabolic syndrome and cancer development is not entirely understood. The aims of this study were to elucidate the basic role of betatrophin and miR-122a in metabolic syndrome and cancer development related metabolic perturbation. Here, we have separated our study into human case-control study and animal model by using the miR-122a-/- mice. On clinical investigation, this first investigation was conducted in diabetic subjects with a kidney disorder. Human samples were obtained from 32 healthy subjects and 109 T2DM subjects onset diabetic nephropathy with different level of albuminuria. Based on albumin/creatinine ratio (ACR), the samples were divided into normoalbuminuria (ACR < 30 mg/g), microalbuminuria (ACR between 30 and 300 mg/g), and macroalbuminuria (ACR > 300 mg/g). Moreover, for the second clinical study, the serum samples were obtained from 15 healthy subjects and 105 pancreatic cancer patients with serial glucose intolerance. Pancreatic ductal adenocarcinoma serum classified into normal glucose tolerance (PDAC-NGT), impaired glucose tolerance (PDAC-IGT), and diabetes mellitus (PDAC-DM). Serum betatrophin and carbohydrate antigen 19-9 (CA19-9) levels were analyze using enzyme-linked immunosorbent assay (ELISA). In animal study, liver fibrosis and hepatocellular carcinoma mouse model designed by inducing miR-122a-/- mice by a high-fat diet for three months (n = 8-10/groups) and the combination of carbon tetrachloride-human recombinant erythropoietin intraperitoneal injection (CCl4+rhEpo) for six weeks (n = 8-10 mice/groups). In our clinical study, we found that serum level of betatrophin significantly increased in T2DM patients with several degrees of albuminuria compared to healthy control (P < 0.001). Serum betatrophin positively correlated to ACR, while inversely associated with eGFR (P < 0.05) in DN patients. The betatrophin had higher odds of having DN [odds ratio (OR) = 5.65, 95 % confidence interval (CI) 2.17-14.57, P < 0.001]. Importantly, an interesting additional finding of our study showed that the serum level of betatrophin significantly increased in PDAC subjects with glucose intolerance. The odds-ratio of betatrophin [OR=3.39; 95% CI (1.20 - 9.57); P=0.021)] was higher than classical cancer marker CA 19-9 in PDAC-associated diabetes. Our findings suggest that betatrophin may serve as the novel potential biomarker for DN and PDAC associated diabetes. In particular, for an animal study, the silencing of miR-122a was able to accelerate metabolic syndrome. Diet induce obesity to exacerbate insulin resistance, decreased liver fatty acid metabolism, enhanced inflammation, and severe liver fibrosis to accelerate liver tumorigenesis. Interestingly, the absence of miR-122a expression associated with a higher rate of thermogenesis in brown adipose tissue and erythropoiesis. Therefore, miR-122a might become an early predictor for obesity-linked T2DM and hepatocellular carcinoma related to the metabolic syndrome.