||To study the regulation mechanism of thrombomodulin protein synthesis under inflammation
||Department of Pharmacology
凝血脢調節素（TM）為重要的抗凝血因子，在心血管疾病（例如：血栓、動脈粥狀硬化和心肌梗塞等…）的病程中，TM也扮演一個重要的角色。過去文獻對於TM 的mRNA轉譯成蛋白質過程的調控是未知的，本實驗發現TM mRNA的五端未轉譯區（5’UTR）其第36到160核甘酸的位置具有internal ribosome entry site ( IRES）的結構與活性。此外我們也發現RNA結合蛋白HuR會與TM mRNA 5’UTR結合進而影響TM IRES的活性，利用reporter assay的實驗方式分析，我們發現持續大量表現HuR蛋白質會抑制TM IRES的活性，相反地利用siRNA專一性抑制HuR蛋白質的表現則增加IRES的活性。同時我們也發現到HuR蛋白質表現增加會抑制內生性TM蛋白質的表現、反之亦然。過去文獻也報導在發炎反應下TM的蛋白質表現量會下降，我們發現在細胞處理促發炎激素（proinflammatory cytokine） TNFα或IL-1β皆會抑制TM IRES的活性，在RNA-IP（immunoprecipitation）的實驗結果中，我們證實HuR與內生性TM mRNA的作用增加而降低TM IRES的活性。綜合以上的結果我們發現HuR對於TM蛋白質表現扮演負向調控的角色，其中HuR參與TM IRES的活性調控上具有重要性的影響。
Thrombomodulin (TM), an important anticoagulant factor, is also involved in the development of cardiovascular diseases, such as thrombosis, arteriosclerosis, and myocardial infarction. Translational regulation of TM protein expression was unknown. In this report, we demonstrated that the 5’UTR of TM mRNA contains an internal ribosome entry site (IRES) spanned at nt. 36 to nt. 160. Searching for possible regulators of IRES activity, we have identified the RNA binding protein HuR interacted with the 5’UTR of TM. Using reporter assay analysis, we found overexpression of HuR protein inhibited TM IRES activity, whereas knockdown the HuR protein expression by SiRNA reversed this effect. Although this phenomenon was found that the expression level of HuR regulated the TM protein expression in vivo. It has been reported that TM protein expression was decreased under inflammatory condition. We observed TM IRES activity was decreased under IL-1 treatment. And the association of HuR with TM IRES or endogenous TM mRNA was elevated by RNA-IP assay. All together, our data suggest that HuR was negative regulator in TM protein expression, and IRES play a crucial role in HuR mediated down-regulation TM protein expression.
第一章 緒論 1
第一節 mRNA的轉譯機制 2
第二節 AREs在發炎反應下對轉譯活性的調控 4
第三節 Thrombomodulin（TM）介紹 5
第四節 HuR蛋白質的介紹 9
第五節 研究動機 10
第二章 實驗材料與方法 12
第三章 實驗結果 39
第一節 Thrombomodulin mRNA 5’UTR在TNF-α抑制TM蛋白質表現中扮演重要的角色 39
第二節 TM 5’UTR具有internal ribosome entry site（IRES） 40
第三節 參與在TM IRES活性的ITAFs（IRES trans-activate factors） 42
第四節 HuR、PCBP1與PTB在TM 5’UTR轉譯活性中的角色 43
第五節 HuR抑制TM IRES的活性需要RRM1、RRM2與RRM3 44
第六節 HuR蛋白質會影響TM蛋白質的表現 44
第七節 在發炎反應下會促進HuR與TM 5’UTR作用而抑制TM IRES的活性 45
第八節 在大鼠敗血症的實驗下HuR與TM mRNA之關係 46
第四章 討論 47
第五章 參考文獻 51
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