- 學位論文
沒食子酸藉由增加miR-1247-3p調控目標基因抑制大腸直腸癌轉移之作用
Gallic acid inhibits colorectal cancer metastasis by enhancing miR-1247-3p targeting
摘要
根據WHO的統計結果顯示，大腸直腸癌在全球癌症造成的死亡率中排名第三，由於早期大腸直腸癌並無症狀，直到癌細胞惡化或轉移後才會出現臨床表徵。治療上，主要以手術切除並以藥物治療、放射線治療為輔助；然而傳統化療藥物對於許多大腸直腸癌患者產生嚴重副作用，甚至在切除病灶後有復發的可能，因此尋找有效的成份做為輔助藥物抑制或減緩癌症的發展，直至今日依然是被爭相研究的議題。 沒食子酸(Gallic acid, GA)是種廣泛存在自然界中的酚酸，在許多草本、木本植物及蕈類中都有GA存在。GA在醫學上應用廣泛，除了毒性低之外，更具有很好的抗氧化功能，且對於癌細胞也有較優越的選擇性，使GA在癌症上的應用有許多成效。在本實驗中使用人類大腸癌細胞株在經過GA處理後，經過MTT毒性測試選用不會對癌細胞產生毒性的劑量，在經細胞功能測試後發現GA具有抑制腸癌細胞侵犯、侵襲和黏附的能力。而研究分子機制調控中，GA會透過抑制integrin αv和β3蛋白表現，進而抑制細胞內FAK、Src、Paxillin以及PI3K/Akt磷酸化和Epithelial-mesenchymal Transition (EMT)相關轉錄因子表現，抑制癌細胞EMT的現象。為進一步探討GA如何調控癌細胞的EMT，我們將處理過GA的腸癌細胞進行microRNA晶片分析比對，其中在加入GA後改變量提升最多的是miR-1247-3p，而透過預測網站預測目標基因後發現miR-1247-3p會與integrin β3的3′ UTR結合，使integrin β3下游訊息傳遞受抑制而影響細胞EMT能力。在反證的實驗中使用miR-1247-3p抑制劑之後回復了integrin β3 和下游蛋白paxillin tyrosine118磷酸化蛋白表現量。透過以上實驗，沒食子酸具有促進miR-1247-3p表現並抑制integrin β3及其下游訊息傳遞達到抑制腸癌細胞EMT的功能。
並列摘要
Colorectal cancer has been the 3rd cancer-leading death worldwide, because at the early colorectal cancer didn’t show any symptoms till the cancer cell became malignant or been metastasized to secondary organ. The treatment of colorectal cancer majorly toward surgery also collocation with chemotherapy and radiation therapy, however the severe side effect on tradition drugs droved the development of new target drugs to deal with cancer metastasis. Gallic acid had been wildly used in medication for a long time, especially for its anti-oxidation activity and low cytotoxicity as well as the great cancer cell selectivity. In this study, gallic acid performed the effect on inhibits DLD-1 cell migration, invasion, adhesion and colony formation. The mechanism on how gallic acid work in cancer EMT was next tested by western blotting. The result showed that gallic acid downregulated the integrin αv and β3 protein expression and downstrem phosphorylation FAK, Src, Paxillin, PI3K/Akt signaling and also reduce EMT-related transcription factors, Snail and Twist. To further investigate how gallic acid regulate cell adhesion molecular integrin expression, the usage of microRNA chip demonstrated the difference more than 0.5 log2 fold change microRNA as significant. Among all microRNAs, miR-1247-3p showed the most increase one after gallic acid treated for 24 hours and even more importantly thing was it has a predictable integrin β3 binding site at 3′ UTR. In the usage of anti-miR-1247-3p inhibitor had restored the repression protein expression by GA. In summary, GA treated colorectal cancer cell -will induce miR-1247-3p and reduce integrin β3 as well as its downstream signaling
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