2015-01-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/654656摘要：膀胱癌是國人相當常見之腫瘤，鈣網酪蛋白calreticulin 被發現與膀胱癌之生成極具相關性。本實驗室已成功構築表達低量鈣網酪蛋白之穩定膀胱癌細胞株-J82-CalRNAi，顯示降低鈣網酪蛋白之表達會抑制膀胱癌細胞株之生長以及移行，並對細胞附著於基質之能力有重要之影響。此外，降低鈣網酪蛋白之表達亦會抑制膀胱癌細胞株於裸鼠中形成腫瘤，並明顯降低肺臟及肝臟之轉移，這些結果證明了鈣網酪蛋白可能是影響膀胱癌細胞癌化之重要調控因子。藉由核酸晶片比較J82-CalRNAi 細胞株與未轉染之細胞間基因表達之差異後，我們發現FUT-1 為其中表達差異性極大的一種蛋白質。FUT-1 為細胞內負責蛋白質醣基修飾的重要酵素，且J82-CalRNAi 細胞株之integrin beta1 醣基修飾亦出現缺失。此外，J82-CalRNAi 細胞株之FUT-1 表達降低之原因，可能是由於FUT-1RNA 穩定性下降之結果。本計畫之實驗目的，即為驗證FUT-1 為鈣網酪蛋白影響膀胱癌細胞轉移之重要因子之假設。我們計劃以操控FUT-1 基因表達之手段， 釐清其為鈣網酪蛋白調控下游影響細胞附著以及移行之操控基因；此外，我們亦希望釐清鈣網酪蛋白調控FUT-1RNA 穩定性之機制；我們將釐清FUT-1 於膀胱癌細胞附著以及轉移之機制，並測試以FUT-1 作為治療膀胱癌標的之可行性。本計劃之研究，應可對膀胱癌病人之治療方式提供線索。<br> Abstract: Bladder cancer is one of the top leading causes of cancer related death world wide and in Taiwan. Calreticulin is a multifunctional calcium binding protein. Increase calreticulin expression in various cancers has recently been reported. In addition, urinary calreticulin has been suggested to be a potential marker for bladder cancer. However, the roles of calreticulin in tumor development are not clear. In order to clarify the roles of calreticulin in bladder cancer, we have successfully generated a bladder cancer cell line stably transfected with shRNA of calreticulin (J82-CalRNAi). Compared to control vector transfected cell, J82-CalRNAi has a lower proliferation rate, migrate slower and also adhere poorly to type-I collagen. These results suggested that alteration of the levels of calreticulin expression in cancer cells might affect bladder tumor progression. The expression levels of VEGF-A, an important regulator for angiogenesis, were also inhibited in the cell lines, suggested that the angiogenic behavior of the cancer cells might also regulated by this protein. In addition, J82-CalRNAi cell generated a smaller tumor in nude mice when compare to its vector control counter part. Most importantly, the J82-CalRNAi derived tumor in nude mice has much less sites of metastasis in lung and liver. These results strongly suggested that the expression patterns of calreticulin may be a marker for bladder cancer and our model also provide an excellent tool to further investigate the roles of calreticulin in bladder cancer development. By using DNA arrays comparing the genes expression profiles between J82-CalRNAi and control cells, FUT-1 was identified as the gene affected by down regulating of CRT expression. We found that down regulation of FUT-1 is likely the cause of decreasing cell adhesion in J82-CalRNAi cell. In addition, we also demonstrated that CRT affect the FUT-1 expression is mediated through affecting its mRNA stability. In this proposal, we will first attempt to utilize our developed system to clarify the relationship between calreticulin and FUT-1 on bladder cancer cell adhesion. In addition, we also attempt to investigate the molecular mechanism of CRT in regulating the mRNA stability of FUT-1. Finally, we will test the possibility of targeting FUT-1 for future bladder cancer treatment.