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標題: | 探討小檗鹼誘導之微小 RNA 於肝細胞之作用機制 The functional mechanism of berberine-induced microRNA in hepatocytes |
作者: | Ting-Fang Lo 羅庭芳 |
指導教授: | 陳水田 |
關鍵字: | 小檗鹼,微小 RNA,肝癌,甲硫胺酸腺基轉移酵素,脂肪肝,乙醯輔? A 羧化酵素,二脂?甘油?基轉移?, berberine,microRNA,hepatocellular carcinoma,methionine adenosyltransferase,fatty liver disease,acetyl-CoA carboxylase,diglyceride acyltransferase, |
出版年 : | 2014 |
學位: | 博士 |
摘要: | 小檗鹼 (berberine) 是一種異喹啉類生物鹼,具有廣泛的藥用效果,包含了抗癌和降低脂肪的效果。我們利用微小 RNA 的微陣列晶片發現, 人類肝癌細胞株 (HepG2) 以及人類初代肝細胞 (primary human hepatocytes) 在經小檗鹼處理後,miR-21-3p (從前命名為 miR-21*) 之表現量上升。在論文的第一個部分,我們證明 miR-21-3p 會調控甲硫胺酸腺基轉移酵素 (methionine adenosyltransferase, 簡稱 MAT) 之表現並抑制肝癌細胞生長。 MAT 是細胞內的酵素,負責合成 S-腺甘甲硫酸胺 (S-adenosylmethionine, 簡稱 SAM),SAM 是生物上主要的甲基供應來源,可調控肝細胞的增生、死亡及分化。MAT1A 和 MAT2A 兩個基因編碼出兩個不同的 MAT 催化 isoform。第三個基因,MAT2B 則是編碼出 MAT2A 的調控單元。在肝癌中, MAT1A 負調控且 MAT2A 正調控的現象會發生,這個現象被稱為 MAT1A:MAT2A 轉換。這個轉換的過程伴隨著 MAT2B 表現量上升,最後導致 SAM 濃度下降並加速癌症細胞的生長。我們發現 miR-21-3p 可藉由其種子序列直接和 MAT2A 及 MAT2B 之3’非編碼區(3’UTR) 配對,抑制MAT2A 及 MAT2B 之表現。此外,過量表現 miR-21-3p 後,肝癌細胞內之 SAM 濃度會上升,而 SAM 濃度上升已經被證實是不利於肝癌細胞生長的。我們亦證實 miR-21-3p 過量表現,會抑制肝癌細胞生長且誘導細胞凋亡。以上結果指出, miR-21-3p 在肝癌細胞中有腫瘤抑制的功能並具有治療的潛力。 在論文的第二個部分,我們證明 miR-21-3p 會調控脂肪代謝相關基因進而降低肝細胞內脂肪的含量。非酒精性脂肪肝是病理性脂肪油滴 (主成分為三酸甘油脂) 累積在肝細胞中的疾病,在人類日漸過重以及肥胖的趨勢之下,非酒精性脂肪肝已經成為了普遍的肝臟疾病之一。我們發現 miR-21-3p 可使人類初代肝細胞內的脂肪油滴含量下降至控制組的 40% ~44%;且可使 in vitro 油酸誘導之脂肪肝疾病模式細胞內的脂肪油滴含量下降至控制組的 50% ~58%。我們驗證了 miR-21-3p 可抑制乙醯輔酶 A 羧化酵素 1 (acetyl-CoA carboxylase alpha, ACACA; 是脂肪合成的第一個速率決定步驟) 及乙醯輔酶 A 羧化酵素2 (acetyl-CoA carboxylase beta, ACACB; 是脂肪氧化的關鍵負調控酵素) 之表現量,而造成表現下降的機制為造成ACACA mRNA的不穩定與藉由種子序列針對 ACACB 的外顯子 23 配對。此外,miR-21-3p 藉由其種子序列直接和 DGAT2 之3’ UTR 配對,抑制二脂酰甘油酰基轉移酶 2 (diglyceride acyltransferase 2, DGAT2; 是三酸甘油酯生合成路徑的最後一個且是唯一的速率決定步驟) 之表現。我們亦發現 miR-21-3p會降低人類初代肝細胞內脂肪合成相關基因的表現,如HLCS、MTOR、RPTOR。綜合以上結果,我們證明了 miR-21-3p 可減少肝臟脂肪生合成、促進脂肪氧化,進而對減緩脂肪肝。 Berberine, an isoquinoline alkaloid, has a wide range of pharmacological effects including anti-cancer and lipid-lowering effects. We used microRNA microarrays to find that the expression level of miR-21-3p (previously named miR-21*) increased after berberine treatment in the HepG2 human hepatoma cell line and in primary human hepatocytes (PHHs). In the first part of the dissertation, we show that miR-21-3p regulated methionine adenosyltransferase (MAT) and inhibited hepatoma cell growth. MAT is the cellular enzyme that catalyzes the synthesis of S-adenosylmethionine (SAM), the principal biological methyl donor and a key regulator of hepatocyte proliferation, death and differentiation. Two genes, MAT1A and MAT2A, encode 2 distinct catalytic MAT isoforms. A third gene, MAT2B, encodes a MAT2A regulatory subunit. In hepatocellular carcinoma (HCC), MAT1A downregulation and MAT2A upregulation occur, known as the MAT1A:MAT2A switch. The switch is accompanied with an increasing expression of MAT2B, which results in decreased SAM levels and facilitates cancer cell growth. Our findings provide the first evidence that miR-21-3p directly reduces the expression of MAT2A and MAT2B by targeting their 3' UTRs. In addition, an overexpression of miR-21-3p increased intracellular SAM contents, which have been proven to be a growth disadvantage for hepatoma cells. The overexpression of miR-21-3p suppresses growth and induces apoptosis in HepG2 cells. Overall, our results demonstrate that miR-21-3p functions as a tumor suppressor by directly targeting both MAT2A and MAT2B, indicating its therapeutic potential in HCC. In the second part of the dissertation, we show that miRNA-21-3p regulated lipid metabolism genes to reduce fat content in hepatocytes. Nonalcoholic fatty liver disease, which results from the accumulation of pathological lipid droplets in hepatocytes (primarily in triacylglycerol) is rapidly becoming a common liver disease because of the growing prevalence of overweight and obesity. Here we found that miR-21-3p caused a decrease in lipid droplet content in PHHs amongst the control group (from 40% to 44%), and in an oleic acid-induced in vitro fatty liver disease model (from 50% to 58%). We verified that miR-21-3p reduced the expression of acetyl-CoA carboxylase alpha (ACACA; the first rate-limiting step of fatty acid synthesis) and acetyl-CoA carboxylase beta (ACACB; the key negative regulator of fatty acid oxidation) by destabilising ACACA mRNA and targeting the exon 23 of ACACB, respectively. In addition, miR-21-3p directly reduced the expression of diglyceride acyltransferase 2 (DGAT2; the terminal and only committed enzyme in the biosynthesis of triacylglycerol) by targeting its 3' UTRs. Furthermore, miR-21-3p decreased the expression of fatty acid biosynthesis-related genes such as HLCS, MTOR, and RPTOR in PHHs. These findings demonstrate that miR-21-3p can alleviate fatty liver disease by reducing hepatic lipogenesis and promoting lipid oxidation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16968 |
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