2014-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/644269摘要：胎盤滋養層細胞的分化與侵入子宮蛻膜和血管是人類懷孕與胎兒發育重要過程，腫瘤細胞侵入與滋養細胞侵入有著許多相似之處，包括上皮間質細胞轉化(EMT)和表觀遺傳調控，然而，不像癌細胞，其黏附、遷移及侵入受到轉化生長因子 β(TGFβ)訊號嚴謹的控制，微核醣核酸(miRNA)作為關鍵的基因中介者，藉由使它的標靶訊息核糖核酸降解或阻斷其轉譯，達到轉錄後調節功能性基因表現，鑑定微核醣核酸標靶以及它們的功能，旨在描繪出滋養層細胞侵入之微核醣核酸的機制。 此前，我們已經證明滋養層細胞侵入是由轉化生長因子受器 I(TGFBR1)的表現所調控，另外，我們已確認了在滋養層細胞株其轉化生長因子受器 I誘導微核醣核酸-7 的表達，於基質膠侵入性試驗中，微核醣核酸-7 的外加表達降低了細胞的侵襲；相反，我們發現微核醣核酸-7 海綿可以提高滋養層細胞侵入水平，說明抑制了微核醣核酸-7 導致促進細胞運動能力，雖然微核醣核酸-7 在滋養細胞裡被激活，但其目標的身分仍然是難以捉摸的，我們假設微核醣核酸-7 將靠轉化生長因子受器 I 訊號來誘導並且藉由調控EMT 相關基因來參與滋養層細胞的侵襲。 在這研究中，我們運用一個嶄新的蛋白質體學的策略：在細胞培養中由胺基酸進行穩定的同位素標記技術(SILAC)，針對穩定的微核醣核酸過量表現以量化數千種蛋白質的表現水平，隨後，我們察覺這些蛋白質的表現水平顯著地降低於微核醣核酸-7 過量表達細胞，我們採用四項微核醣核酸標的預測程式，於微核醣核酸-7 過量表達細胞檢查範圍內那些被下調兩倍以上假定的微核醣核酸-7 直接標的蛋白質，使用報導基因分析，我們建立了微核醣核酸-7 標靶基因的 3’非轉譯區，由於 EMT 直接與細胞侵襲有關，我們於是專注於 EMT 相關的基因可能為微核醣核酸-7 的標靶，這將決定微核醣核酸的微調與 EMT 過程調節了人類滋養層細胞侵襲，以及能讓人們了解參與滋養層細胞運動的新機制，這項研究結果可能應用於子癲前症探索早期偵測生物標誌和微核醣核酸標靶基因治療策略。<br> Abstract: Invasion of placental trophoblast cells into decidua and vessels is an essential progression for human pregnancy and fetal development. There are many similarities between cancer cell invasion and trophoblast invasion, including epithelial-mesenchymal transition (EMT) and epigenetic regulation. However, unlike cancer cells, their adhesion, migration, and invasion are strongly controlled by transforming growth factor beta (TGFβ) signaling. MicroRNAs (miRNA) are critical gene mediators that post-transcriptionally regulate expression of functional genes through degradation of their target mRNAs or blocking their translation. Identifying miRNA targets as well as their functions aims to draw the mechanism of miRNAs on trophoblast invasion. Previously, we have demonstrated that the trophoblast invasion is regulated by the transforming growth factor beta receptor I (TGFBR1) expression. Furthermore, we have identified that TGFBR1 induces miR-7 in trophoblast cell lines. In matrigel invasive assay, ectopic expression of miR-7 decreased cell invasion. By contrast, we found that the miR-7 sponges can increase invasion level of trophoblast, indicating inhibition of miR-7 result in promote cell motility ability. Although, miR-7 is activated in trophoblast cells, but the identity of its targets remains elusive. We hypothesized that miR-7 would be induced by TGFBR1 signaling and participate in trophoblast invasion though regulating EMT-relative genes. In this study, we used a new proteomic strategy, stable isotope labeling by amino acids in cell culture (SILAC) technology, to quantify the expression level of thousands proteins in response to stable miRNA overexpression. Subsequently, we noticed that expression levels of these proteins were significantly decreased in miR-7-overexpressing cells. We employed four microRNA target prediction programs, to examine the putative miR-7-directly targeted protein within those that were downregulated more than two fold in miR-7-overexpressing cells. Using reporter assays, we established that miR-7 targets the 3' Untranslated Regions (3'UTR) of target protein. Since the EMT directly associated with cell invasion. We then focused on EMT-relative genes that could be targeted by miR-7. This will conclude modulating of miRNA and EMT process regulating human trophoblast invasion, as well as give insights into novel mechanisms involved in the trophoblast motility. The results of this study may apply to explore biomarker early detection and miRNA targets treatment strategy for preeclampsia.