| Summary: | 碩士 === 中山醫學大學 === 醫學研究所 === 103 === Slit2 is a secreted glycoprotein, which inhibits tumor metastasis, neuronal and endothelial cell migration, as well as affects inflammation and the development of kidneys and lungs. We identified two splicing forms of Slit2 at exon 15, exon 15-containing Slit2-WT and exon 15-deleted Slit2-ΔE15, respectively. Slit2-ΔE15 inhibits both growth and invasion of lung cancer cells, but Slit2-WT inhibits lung cancer cell invasion only. Previous studies have shown that Slit2 plays a role in angiogenesis, so we used human umbilical vein endothelial cells (HUVECs) to study the effect of Slit2 splicing variants on angiogenesis. We found that the conditioned medium (CM) of lung cancer cells increased permeability and formed poor tubes in HUVECs. The CM containing Slit2-ΔE15 inhibited CM-induced permeability of HUVECs and restored the quality of tubes, but Slit2-WT CM only inhibited CM-induced permeability of HUVECs. In chorioallantoic membrane (CAM) assay, the CM collected from lung cancer cells blocked angiogenesis on CAM, Slit2-ΔE15 CM restored the size of vessels, but Slit2-WT did not have this ability. Thus, our results indicated that Slit2-ΔE15 not only reduces cancer cell-induced permeability but also enhances the quality of vessels, while Slit2-WT inhibits cancer cell-induced permeability only. These results futher suggested that decreasing in permeability and enhancing vessel quality may be mediated by different pathways. When the expression level or the function of Robo4 was interfered by RNAi or anti-Robo4, respectively, Slit2-WT- or Slit2-ΔE15-mediated inhibition of CM-induced permeability was disappeared. But, knockdown Robo1 expression or function did not relieve Slit2-WT- or Slit2-ΔE15-mediated inhibition of permeability, indicating that Robo4 but not Robo1 is involved in Slit2-mediated inhibition of CM-induced permeability. Unexpectedly, knockdown the expression or function of either Robo1 or Robo4 did not block Slit2-ΔE15-enhanced tube quality. This result revealed the presence of unknown receptor(s) of Slit2-ΔE15 that transduce(s) the signal in the regulation of angiogenesis. Normalization of vessels will increase oxygen content, decrease acidity and reduce ROS in tumor microenvironment, which not only reduces malignancy of tumor but also enhances efficacy of chemotherapy or radiotherapy. Thus, identification of receptors involved in Slit2-ΔE15-mediated angiogenesis would provide the opportunity to develop strategy in normalizing blood vessel around tumor microenvironment.
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