蔡菁華林毓瑩Yu-Ying醫學研究所2026-10-289999-12-312011https://handle.ncl.edu.tw/11296/j5w4jqhttps://scholars.csmu.edu.tw/handle/123456789/35435Slit2是ㄧ個分泌型蛋白，除了引導神經軸突的遷徙外，Slit2在細胞的移動、腫瘤形成的過程中扮演著重要的角色。然而，Slit2對細胞的生長、侵犯能力、及血管新生的影響仍意見分歧。我們發現Slit2在肺癌的表現被高度的抑制，有趣的是Slit2的表現與CL1系列的肺癌細胞之侵犯能力成反比。在建構Slit2全長cDNA時發現了exon 15的剪接變異型cDNAs。當CL1-5細胞過度表現缺乏exon15的Slit2-E15時，細胞的生長及侵犯能力明顯的被抑制。但是，過度表現具有exon 15的Slit2-WT時，只有細胞的侵犯能力被抑制。這結果顯示exon 15的存在與否會調控Slit2蛋白抑制細胞生長的能力，但對細胞的侵犯能力沒有影響。過去的研究指出，Robo蛋白是Slit2的受體。Slit2以Leucine Rich Repeat 2 (LRR2)的區域與Robo1的Immunoglobulin 1 (Ig1)區域直接結合，而exon 15正緊接於LLR2之後。因為CL1-5細胞中表現Robo1及Robo4，因此我們以siRNA抑制細胞中Robo1或Robo4的表現，然後偵測Slit2抑制細胞的生長或侵犯能力是否有紓解的現象。結果發現，當CL1-5細胞的Robo1或Robo4表現被抑制時，Slit2-E15失去抑制細胞生長的能力，暗示著Robo1和Robo4可以傳遞Slit2-E15抑制細胞生長的訊息。但是，抑制Robo1的表現不影響Slit2-WT或Slit2-E15抑制細胞侵犯的能力，表示Robo1在Slit2抑制侵犯的能力上不扮演任何角色。有趣的是，抑制了Robo4的表現明顯的消除了Slit2-WT抑制細胞侵犯的能力，但對於Slit2-ΔE15抑制侵犯能力沒有影響，這結果顯示，除了Robo1及Robo4，尚有其他的受體可以傳遞Slit2-E15的訊息。因此，我們的結果顯示exon 15的存在與否可能會決定Slit2與不同受體的親和力，進而影響Slit2的生物功能。Slit2 is a secreted protein. It plays important roles in guidance of commissural axon, cell migration and tumorigenesis. However, the effects of Slit2 on cell growth, cell invasion, and angiogenesis were not consistent among different systems. We found that the expression of Slit2 was highly repressed in lung cancer, and the expression of Slit2 is negatively correlated with the invasiveness of CL1 series lung cancer cell lines. In the process of cloning the full-length slit2 cDNA, we identified exon 15 splicing variants. In the absence of exon 15, Slit2-ΔE15 exhibited growth and invasion inhibition in CL1-5 cells. However, in the presence of exon 15, Slit2-WT only possessed invasion inhibition but not growth inhibition activity. These results suggested that exon 15 modulates the ability of Slit2 to inhibit cell growth but not cell invasion. Previous studies have shown that Robo receptors transduce Slit2 signals in various systems. It has been demonstrated that the Leucine Rich Repeat 2 (LRR2) domain of Slit2 directly binds to the immunoglobulin 1 (Ig1) domain of Robo1. Interestingly, exon 15 is located at the end of the LLR2 domain. Therefore, we use siRNA to interfere with the expression of Robo1 or Robo4, and determine the ability of Slit2 in growth and invasion inhibition. When the expression of Robo1 or Robo4 was interfered, the growth inhibition activity of Slit2-ΔE15 was reduced, suggesting that Robo1 and Robo4 mediates growth inhibition signal of Slit2-ΔE15. Our result suggested that Robo1 is not involved in Slit2-mediated invasion inhibition activity, since neither the invasive inhibition activity of Slit2-WT nor that of Slit2-ΔE15 was abolished by knockdown the expression level of Robo1. Surprisingly, RNA interference and antibody neutralization results demonstrated that Robo4 is involved in Slit2-WT but not Slit2-ΔE15 mediated invasion inhibition in CL1-5 lung cancer cells. These results revealed that other receptor is involved in Slit2-ΔE15-mediated invasion inhibition in CL1-5 lung cancer cells. Based on our results, we hypothesized that exon 15 may modulate the affinity of Slit2 to its various receptors thus altering its biological functions.thesis