先前研究發現IRSp53 (Insulin Receptor tyrosine kinase Substrate Protein of 53 kDa)為Eps8 (EGF receptor pathway substrate NO.8)的結合蛋白質，並且Eps8- IRSp53的結合會參與在v-Src細胞的轉型當中於是我們想進一步探討IRSp53是否會參與在大腸癌細胞的增生和運動過程中。目前已知IRSp53具有四種人類異構物 (IRSp53S, IRSp53T, IRSp58M, and IRSp53L)，然而對於這些異構物的功能尚未清楚，因此也想探討不同異構物在其中所扮演的角色。我們首先利用shRNA去抑制大腸癌細胞SW620中IRSp53的表現量，由實驗結果發現當降低IRSp53的表現量後，細胞生長情形有減緩的現象。此外，在SW620細胞中當降低IRSp53的表現量後，Pi-Y416 Src的表現量也跟著受到抑制。在WST-1 cell viability assay中，降低SW620細胞中IRSp53的表現量，可能增進其對化療藥物的化學敏感度，然而在wound healing assay結果發現，降低IRSp53蛋白表現量的SW620細胞株中其wound closure百分比相較於Ctrl細胞株並沒有顯著的差異。另一方面，我們也透過外送不同人類 IRSp53 異構物的質體進入IRSp53表達量少的SW480細胞中去觀察其對於細胞功能上的影響，由實驗結果發現到當外送Myc-tagged human IRSp53S和IRSp53S-YA mutant (缺乏C端與Eps8結合區域之序列)進入SW480細胞，Src，Pi-Y416 Src 和 Pi-Y861 FAK的表現量都有增加的現象，然而外送Myc-tagged human IRSp58M入SW480細胞，則沒有明顯變化。並且在外送IRSp53S入SW480細胞後，細胞生長速率有增加的趨勢，然而外送IRSp53S-YA mutant入SW480細胞，則沒有此現象。在wound healing assay結果發現，無論外送IRSp53S，IRSp53S-YA mutant以及IRSp58M皆使細胞移動爬行變慢，其中又以IRSp53S-YA mutant細胞株爬行速率最慢。因此我們認為IRSp53S可能參與在調控Src的活性以及影響相關細胞生長路徑，但是對於細胞的移動可能不是促進的角色。

Previous studies showed that IRSp53 (Insulin Receptor tyrosine kinase Substrate Protein of 53 kDa) was identified as one of the Eps8-binding partners. The interaction between Eps8 and IRSp53 contributed to Src-mediated transformation. There are at least four IRSp53 isoforms (IRSp53S, IRSp53T, IRSp58M, and IRSp53L) identified in human cells. However, the detailed functions of these isoforms remain largely unknown. Therefore, we wanted to address the importance of IRSp53 and these isoforms in cell proliferation and motility in colorectal cancer cells. First, we generated IRSp53 knockdown cells from SW620 colon cancer cells and observed both anchorage-dependent and -independent growth were reduced. In addition, IRSp53 attenuation resulted in reduced Src activity. In WST-1 cell viability assay, IRSp53 depletion might increase chemosensitivity in colon cancer cells under the treatment of anti-cancer drug oxaliplatin. In wound healing assay, there were no significant differences between IRSp53 knockdown cells and Ctrl cells. On the other hand, overexpression of myc-tagged human IRSp53S and IRSp53S-YA mutant (Eps8-binding detective mutant) but not IRSp58M in SW480 colon cancer cells resulted in elevated expression of Src，Pi-Y416 Src and Pi-Y861 FAK. However, IRSp53S, but not its YA mutant increased cell growth in SW480 cells. We also found decreased cell motility in both IRSp53S- and IRSp58M-overpressing cells. Overexpression of IRSp53S-YA mutant showed the slowest cell motility. In conclusion, IRSp53S may play a positive role in cell proliferation but not motility in colon cancer cells.

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