| 摘要: | 目前基因表現的研究多集中於細胞核內轉錄與訊息核醣核酸 (mRNA) 合成的調控，但越來越多的文獻顯示不同基因的訊息核醣核酸在細胞內的半衰期都不同，說明了細胞內有一個機制來調控訊息核醣核酸的半衰期。因此後轉錄調控機制被認為在細胞的生理反應上扮演了一個重要的角色。先前有研究指出，凝血酶調節素(Thrombomodulin, TM)有調節發炎反應的功能，在Interleukin-1B (IL-1B的刺激下，核醣核酸結合蛋白HuR會與凝血酶調節素的訊息核醣核酸3端非轉譯區結合，藉此提升凝血酶調節素的訊息核醣核酸穩定性。 NF-κB是複合蛋白，也是一個轉錄因子，近年來許多研究指出，慢性發炎疾病轉變為癌症之關鍵蛋白即為NF-κB。而也有文獻指出TPCA-1 (2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide)會抑制NF-κB的作用，進而造成核醣核酸的分解，並使得訊息核醣核酸的降解機制改變。因此本論文欲探討 HuR 在發炎狀態下，來穩定凝血酶調節素 3’UTR，是否也會受NF-B的機制影響。 從實驗中得知在 IL-1B的刺激下，凝血酶調節素的訊息核醣核酸穩定度會增加，且與HuR的結合也會增加。於是利用NF-kB的抑制劑TPCA-1來抑制 NF-kB 的訊息傳遞路徑，並探討它是否能抑制 HuR與凝血酶調節素的結合。在 TPCA-1的刺激下，凝血酶調節素的核糖核酸表現減少了，同時也看到HuR與凝血酶調節素的結合減少，而從降低IkB kinase beta(IKK-B的表現中發現，HuR與凝血酶調節素的結合也降低，此結果說明了 NF-kB 的訊息傳遞路徑會調控HuR與凝血酶調節素的結合。此外我們也想知道這樣的現象是否也出現在其他的發炎相關基因中，所以我們看了發炎相關基因COX-2、cPLA2α，發現都有相同的結果，說明NF-kB參與在發炎相關因子核醣核酸的表現調控。 所以在發炎狀況下，NF-kB 的訊息傳遞路徑的確在後轉錄調控中扮演了一個重要的角色，且調控HuR與下游基因的結合。
Regarding the regulation of gene expression, most studies are：focus on transcriptional regulation and mRNA synthesis in cells. However, more and more literatures reveal that mRNA half-life are different, and indicate that a mechanism is responsible for regulating RNA half-life inside the cell. Therefore, post-transcriptional regulation mechanism plays an important role in the physiological function of cells. Previous study showed that thrombomodulin modulatid the inflammation reaction. Under IL-1 freatment, HuR can bind to the thrombomodulin mRNA 3'UTR and stabilize its mRNA.NF-κB protein complex is a transcription factor. In recent years, many studies point out that NF-κB is a key mediator to induce the progression of chronic inflammatory disease into cancers. Furthermore, some studies also indicate that TPCA-1 can inhibit NF-κB function, and change the mechanisms of mRNA turnover. Hence, the aim of this study was to investigate whether thrombomodulin mRNA 3’UTR stabilized by HuR is regulated by NF-κB activation under inflammation conditions.From our results, the stability of thrombomodulin mRNA and its interaction with HuR were increased under cells stimulated with IL-1B. The TPCA-1, a NF-κB inhibitor, was used to study whether the NF-κB signal pathway regulates the interaction between HuR and thrombomodulin mRNA. Under the treatment of TPCA-1, the thrombomodulin mRNA expression was decreased and the interaction between HuR and thrombomodulin mRNA was disrupted.In addition, the decrease of HuR-thrombomodulin mRNA interaction was also found in IKK- knockdown cells. It revealed that NF-κB signal pathway regulated the interaction between HuR and thrombomodulin mRNA. This phenomonem was also found in other inflammatory related genes , such as cox-2 and cPLA2a In conclusion, NF-κB signal pathway plays an important role in mRNA stability regulation, and regulates the interaction of HuR with its downstream genes under inflammation conditions. |
