綠茶表沒食子兒茶素沒食子酸酯經由MicroRNA-let-7a/HMGA2訊息路徑抑制米色前脂肪細胞的生長

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陳雯婷(Wen-Ting Chen) 查詢紙本館藏

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生命科學系

論文名稱

綠茶表沒食子兒茶素沒食子酸酯經由MicroRNA-let-7a/HMGA2訊息路徑抑制米色前脂肪細胞的生長
(Green tea epigallocatechin gallate inhibits beige preadipocyte growth via the microRNA-let-7a/HMGA2 signaling pathways)

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至系統瀏覽論文 (2026-8-1以後開放)

摘要(中)

根據先前研究指出，綠茶表沒食子兒茶素沒食子酸酯 (Epigallocatechin gallate, EGCG) 和 microRNA (miR) 藉由抑制脂肪細胞分裂及脂肪分化過程，進而抑制脂肪細胞生長及脂肪生成，但鮮少研究探討EGCG 藉由調控miR影響脂肪細胞生長。雖然先前實驗結果發現EGCG可能藉由調控miR-let-7a及HMGA2表現影響3T3-L1白色前脂肪細胞生長，但需更進一步實驗驗證，而且目前也無研究指出EGCG是否藉由調控miR路徑影響米色前脂肪細胞生長，故本論文探討EGCG是否影響D12及X9米色前脂肪細胞的生長，以及證實EGCG是否藉由調控miR-let-7a/HMGA2路徑調控3T3-L1白色及D12及X9米色前脂肪細胞的生長。結果發現EGCG會抑制D12及X9米脂細胞的生長，且也會時間性及劑量依賴性地上調miR-let-7a及減少HMGA2基因表現。此外，大量表達miR-let-7a後，3T3-L1、D12及X9細胞生長明顯受到抑制，且HMGA2基因表現顯著性下降。而抑制miR-let-7a表現後，阻擋了EGCG對3T3-L1、D12及X9細胞生長之抑制作用，以及上調miR-let-7a及減少HMGA2基因表現之作用。接著根據雙冷光酵素報導基因檢測實驗結果得知，miR-let-7a可直接作用於HMGA2 3’UTR，但無法作用於mutant HMGA2 3’UTR site，由此可知HMGA2為miR-let-7a之標靶基因。進一步的發現，大量表達HMGA2後，D12及3T3-L1細胞數目明顯增加，且阻擋EGCG對D12及3T3-L1細胞生長及HMGA2基因表現之抑制作用。總之，EGCG 藉由調控miR-let-7a/HMGA2的路徑，進而影響D12及X9米色以及3T3-L1白色前脂肪細胞的生長。本篇研究成果可能有助於解釋 EGCG 介導脂肪細胞活性和脂肪細胞相關疾病的miR 信號傳導機制。

摘要(英)

Green tea epigallocatechin gallate (EGCG) and microRNA (miR) have been reported to inhibit the growth and fat synthesis of fat cell by suppressing mitogenic and adipogenic processes, respectively. Few studies have reported how EGCG affects fat cell growth by regulating miR molecules. Although previous report had shown that EGCG might affect 3T3-L1 white preadipocyte growth by regulating miR-let-7a and HMGA2 expressions, the contention remains to be demonstrated in further studies. No reports were found whether EGCG affects beige preadipocyte growth through the miR pathway. Thus, this study investigated whether EGCG affects beige preadipocyte growth, and demonstrated whether the miR-let-7a/HMGA2 pathways transduce EGCG signals to regulate growths of white and beige preadipocytes. Using D12 and X9 cells, we found that EGCG inhibited beige preadipocyte growth and that EGCG time- and dose-dependently upregulated miR-let-7a mRNA levels and downregulated HMGA2 gene expression. In addition, overexpression of miR-let-7a significantly inhibited growths of D12, X9, and 3T3-L1 cells, and decreased HMGA2 gene expression. Whereas, the miR-let-7a inhibitor antagonized the inhibitory effects of EGCG on cell number and cell viability of all three cell lines. Moreover, miR-let-7a inhibitor reversed the EGCG-increased level of miR-let-7a mRNA and the EGCG-decreased level of HMGA2 gene expression. Further dual-luciferase report assay indicated that miR-let-7a could directly bind to the HMGA2 3′-UTR site but not mutant HMGA2 3′-UTR site, suggesting that HMGA2 is the miR-let-7a target. Also, overexpression of HMGA2 induced increases in both cell number and cell viability and antagonized EGCG-suppressed growth and HMGA2 expression in D12 beige and 3T3-L1 white preadipocytes. We concluded that EGCG inhibits growths of D12 and X9 beige and 3T3-L1 white preadipocytes through modulations of the miR-let-7a and HMGA2 pathways. Results of this study may help explain the miR signaling mechanism through which EGCG mediates fat cell activity and fat cell-associated disease.

關鍵字(中)

★ 綠茶
★ 表沒食子兒茶素沒食子酸酯
★ 米色前脂肪細胞

關鍵字(英)

★ Green tea
★ epigallocatechin gallate
★ beige preadipocyte

論文目次

中文摘要 i
Abstract ii
致謝 iii
目錄 iv
表目錄 vii
圖目錄 viii
縮寫表與全名對照表 x
壹、前言 1
一、MicroRNA小分子核醣核酸 1
二、脂肪細胞 2
三、綠茶 3
四、研究動機及目的 5
貳、材料與方法 7
一、實驗材料 7
二、實驗方法 7
1. 細胞培養 7
2. EGCG配製 7
3. 細胞生長實驗 8
4. 大量表現miR-let-7a (miR-let-7a mimic) 與抑制miR-let-7a表現 (miR-let-7a inihibitor) 之試驗以及大量表現HMGA2之試驗 9
5. 基因分析 10
6. 蛋白質分析-西方墨點法 (Western blot) 12
7. 雙冷光酵素報導基因檢測方法 (Dual-Luciferase Reporter Assay) 14
8. 統計分析 14
參、實驗結果 15
一、 EGCG對D12及X9米色前脂肪細胞生長之影響 15
二、 EGCG對D12及X9米色前脂肪細胞內miR-let-7a及HMGA2表現之影響 16
三、大量表現miR-let-7a對D12及X9米色前脂肪細胞生長及細胞內HMGA2表現之影響 17
四、大量表現miR-let-7a對3T3-L1白色前脂肪細胞生長及細胞內HMGA2表現之影響 18
五、 miR-let-7a抑制劑對D12及X9米色前脂肪細胞生長及細胞內HMGA2表現之影響 19
六、 miR-let-7a抑制劑對3T3-L1白色前脂肪細胞生長及細胞內HMGA2表現之影響 21
七、 EGCG在D12米色前脂肪細胞中上調miR-let-7a作用於HMGA2 22
八、 EGCG在3T3-L1白色前脂肪細胞中上調miR-let-7a作用於HMGA2 23
九、大量表現HMGA2阻擋EGCG對D12米色前脂肪細胞生長及細胞內HMGA2表現之影響 23
十、大量表現HMGA2阻擋EGCG對3T3-L1白色前脂肪細胞生長及細胞內HMGA2表現之影響 24
肆、討論 26
伍、結論 30
陸、參考文獻 31
柒、附錄 65
附圖一、EGCG在不同時間點對 3T3 L1 前白色脂肪細胞內miR let 7a及其標靶基因HMGA2表現的影響 (陳嘉佩, 2020) 66
附圖二、不同時間的EGCG處理對D12及X9米色前脂肪細胞內miR-143基因表現之影響 67
附圖三、以Dual-luciferase Report assay證實HMGA2為miR-let-7a之標靶基因 68
附圖四、不同劑量的EGCG處理抑制D12米色前脂肪細胞內HMGA1基因表現 69
附錄五、使用試劑之配方 70
附錄六、MicroRNA mimic及inhibitor序列 73
附錄七、HMGA2及β-actin完整印跡圖 74
附錄八、Plasmid map 75

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指導教授

高永旭(Yung-Hsi Kao)

審核日期

2021-7-27

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