血纖維蛋白溶酶原激活系統是細胞表面重要的絲氨酸蛋白酶系統，其透過分解膜蛋白的功能來進一步調控細胞的爬行和腫瘤內的血管新生。許多研究指出系統當中所參與的蛋白在腫瘤惡化的過程扮演重要的角色。凝血酶調節素是一個表現在血管內皮細胞上的抗凝血因子，除此之外在許多腫瘤細胞上也有被發現。本實驗室先前的研究已證實凝血酶調節素功能區域1具有調控細胞間黏附的功能，而凝血酶調節素功能區域2和3具有促進血管新生的能力。許多研究指出凝血酶調節素的表現與腫瘤細胞的惡化具有負相關性。然而，腫瘤細胞利用抑制凝血酶調節素來促進腫瘤惡化的詳細機制仍不清楚。因此，在本篇研究裡我們擬探討血纖維蛋白溶酶原激活系統是否會去修飾剪切凝血酶調節素，釋放出凝血酶調節素的片段來促進血管新生，進一步影響腫瘤的生長。我們發現凝血酶調節素在人類肺腺癌細胞上的表現量相對於皮膚角質細胞來的低。此外，我們也發現凝血酶調節素在人類肺腺癌細胞上的表現呈現斷片的型式且在細胞培養液裡也有偵測到凝血酶調節素的片段。若把培養過人類肺腺癌細胞的培養液拿去培養牛動脈內皮細胞，會發現全長凝血酶調節素的表現明顯的下降。同時，以蛋白酶抑制劑處理人類肺腺癌細胞，可以看到在人類肺腺癌細胞裡凝血酶調節素由原本斷片形式回復成完整長度。進一步也利用不同的基質金屬蛋白酶抑制劑排除基質金屬蛋白酶參與作用的可能性。我們也偵測到纖溶酶原激活因子存在於人類肺腺癌細胞的培養基當中。以血纖維蛋白溶酶抑制劑處理人類肺腺癌細胞，觀察到凝血酶調節素由原本斷片形式回復成完整長度。另外，我們利用純化出來的凝血酶調節素與人類血纖維蛋白溶酶作用，可以發現凝血酶調節素可被切在兩個位點上。進一步我們利用核醣核酸干擾技術來抑制血纖維蛋白溶酶原、尿激酶型纖溶酶原激活因子和尿激酶型纖溶酶原激活因子受體在人類肺腺癌細胞中的表現，這些細胞的外觀變得較規則且緊密聚集在一起、全長凝血酶調節素的表現量明顯回復且腫瘤的生長受到抑制。另外，同樣利用核醣核酸干擾技術抑制凝血酶調節素的表現後，腫瘤生長的情形也有顯著的趨緩，而細胞內血管新生的情況也有明顯的減少。這些實驗結果顯示，在肺癌細胞中凝血酶調節素會被血纖維蛋白溶酶激活系統修飾剪切，進而轉換成血管新生因子來調控血管的生成，進一步幫助腫瘤生長。

Plasminogen activator system (PAS) functions as a major serine protease system for membrane protein degradation, and participates in cell migration and tumor angiogenesis. The components of this system have been postulated to play a role in tumor progression. Thrombomodulin (TM), a well recognized anticoagulant factor in endothelium, is also expressed in a wide range of tumor cells. Previously, we have demonstrated that TM mediates cell to cell adhesion through lectin-like domain, and epidermal growth factor-like domains to serine/threonine rich domain of TM can function as an angiogenic factor. Several studies indicated that cell malignancy is negatively correlated with the expression level of TM. However, the molecular mechanism by which down-regulation of TM in cancer cells promotes tumor progression is unclear. Therefore, we hypothesized that the lower expression of TM in tumor cells could be due to protease cleavage by PAS, and the proteolytic TM fragments may promote tumor growth by enhancing tumor angiogenesis. According to the hypothesis, we generated stable knockdown of plasminogen, urokinase-type plasminogen activator (uPA), uPA receptor, and TM in A549 cells, a human lung adenocarcinoma cell line, respectively using RNA interference technology. The expression of full-length TM in A549 cells was lower in comparison with HaCaT cells. However, the cleaved form of TM was presented in A549 cells, and the TM fragments were detected in conditioned medium. When bovine aortic endothelial cells were incubated with the conditioned medium of A549 cells, the expression level of TM in bovine aortic endothelial cells decreased. After treatment with protease inhibitors cocktail, the expression of full-length TM in A549 cells was increased. The possibility of matrix metalloproteinase involved in the TM shedding was excluded after different matrix metalloproteinase inhibitors treatment. In plasminogen activator activity assay, we showed that uPA was present in A549 cells. Treatment with plasmin inhibitor resulted in an increase in full-length TM expression in A549 cells. In addition, recombinant TM could be cleaved by plasmin at two sites. The morphology of plasminogen, uPA, and uPA receptor-knockdown cells became epithelial phenotype, and strikingly, the level of full-length TM increased. In xenograft mice model, the tumor volume of these short hairpin RNA-transfected cells was significantly suppressed, and the tumor angiogenesis of sh-TM cells was significantly decreased. In conclusion, we proposed that TM is converted to an angiogenic factor after modification by PAS, and it further promotes tumor growth in lung cancer cells.

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