凝血酶調節素(Thrombomodulin)是第一型膜醣蛋白(glycoprotein)，在細胞細胞間的黏附以及癌症生成(tumorigenesis)上扮演重要角色。癌症生成是個極為複雜的過程，其中又包括血管新生(angiogenesis)和癌症轉移(metastasis)。細胞的非貼附性生長(anchorage-independent growth)被認為是細胞變形(transformation)的特徵，且能夠幫助癌症轉移。可溶性凝血酶調節素(soluble TM)已被發現在癌症病人的血漿中有上升的情形，但究竟可溶性凝血酶調節素的生理功能為何目前還不清楚。扁菱蛋白(Rhomboid)已被證實會辨識凝血酶調節素並且在其穿膜區做切割，進而產生可溶性的凝血酶調節素。在本篇研究當中，我們將探討可溶性凝血酶調節素是否能夠促進癌細胞的非貼附性生長。不同的子宮頸癌細胞株均可以在軟瓊脂培養基(soft agar assay)當中形成群落(colony)並且存活到第14天。利用慢病毒(lentivirus)感染細胞，送入凝血酶調節素的短髮夾核醣核酸(short hairpin RNA)來抑制HeLa229細胞中凝血酶調節素蛋白表現，並且使用西方墨點法確定凝血酶調節素的表現量。將HeLa229上的凝血酶調節素蛋白抑制後會降低細胞懸浮培養時的群落數目與群落大小。除此之外，和對照組細胞相比，降低凝血酶調節素的表現會降低ERK的磷酸化以及增加caspase 3活化。在對照組細胞當中，扁菱蛋白在懸浮性培養24和48小時後有大量的表現，然而在凝血酶調節素被抑制的細胞中則無此現象。可溶性凝血酶調節素的蛋白量在凝血酶調節素被抑制時也有明顯的下降。利用DCI(扁菱蛋白的抑制劑)處理細胞後，ERK的磷酸化以及可溶性凝血酶調節素的蛋白量都會減少。利用重組蛋白TMD123處理細胞後，細胞懸浮培養的存活率和ERK的磷酸化都有上升的現象；而caspase 3則有被抑制的情形。總結以上，凝血酶調節素在癌細胞懸浮時會被扁菱蛋白切割釋放可溶性凝血酶調節素，而可溶性凝血酶調節素可以促進癌細胞的非貼附性生長。

Thrombomodulin (TM), a type I transmembrane glycoprotein plays important roles in cell adhesion and tumorigenesis. Tumorigenesis is a complicated process including angiogenesis and metastasis. Anchorage-independent growth has been recognized as a hallmark of cell transformation which promotes tumor metastasis. Increase of soluble TM was found in plasma of cancer patients; however, the biological function of soluble TM remains unclear. Moreover, rhomboid, an intramembrane protease specifically cleaves TM at its transmembrane domain, and cause release of soluble TM from cell membrane. In this study, we investigated whether soluble TM promotes anchorage-independent growth in tumor cells. Human cervical cancer cell lines formed colonies in soft agar and survived until day 14th. Lentivirus transduction of short hairpin RNA was used to knockdown TM expression in HeLa229 cells (HeLa229-shTM), and the efficiency of TM knockdown was confirmed by Western blotting. HeLa229-shTM showed lower colony numbers and smaller size in suspension culture. Moreover, knockdown of TM in HeLa229 decreased extracellular signal regulated kinase phosphorylation and increased caspase 3 activation compared with vector control (HeLa229-shLuc). The level of rhomboid was upregulated in HeLa229-shLuc in suspension culture for 24 and 48 hour; however, TM-knockdown cells had no such effect. After treatment with dichloroisocoumarin, a rhomboid inhibitor, the levels of soluble TM and ERK phosphorylation were decreased. Recombinant TM domains 1, 2 and 3 increased cell viability, ERK phosphorylation and decreased caspase 3 activation in suspension. In summary, TM was shedded by rhomboid, and soluble TM promoted anchorage-independent growth of tumor cells.

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