The Function And Molecular Mechanisms Of Asporin On Metastasis Of Colorectal Cancer

Objection:We detected the m RNA and protein expression of Asporin in colorectal cancer(CRC)tissues and adjacent non tumor tissues.The relationship between Asporin expression and the clinicopathological features was analyzed.Further we constructed Asporin overexpression and interference cell lines in colorectal cancer,and performed a series of biological function experiments and animal experiments.Finally,we investigated the role of Asporin in metastasis of colorectal cancer by Western-blotting.Methods: We detect the Asporin expression of 200 colorectal cancer tissues at the m RNA and protein level and analyzed the relationship between the Asporin expression and the clinicopathological features.Western-blotting technology was used to explored the Asporin expression in a series of C RC cell lines,including HT-29,Lo Vo,Caco2,HC T116,SW1116,SW480,RKO and SW620.Immunohistochemistry staining of CRC cells verified the results of Western-blotting.We successfully upregulated Asporin expression in HT-29 and Lo Vo cells and obtained stable HT-29/Asporin and Lo Vo /Asporin by puromycin screening.Wound healing assays,Transwell assays and tube-formation assays were used to detect the healing,invasion,migration and tubule formation ability of HT-29/Asporin and Lo Vo /Asporin cells.Western-blotting was used to detect the expression of Src signaling protein and VEGF protein in HT-29/Asporin and Lo Vo /Asporin cells,and the co-expression of Asporin and P-cortactin(Tyr421)was detected in colorectal cancer tissues.HT-29/Asporin cells and Vector control cells were injected subcutaneously into nude mice,and the expression of VEGF was detected by immunohistochemistry and Western-blotting.We applied the portal vein injection model to determine the functions of Asporin in the liver metastasis of the CRC cells in vivo.We successfully constructed Asporin sh RNA interference sequence and obtained stable RKO/sh RNA and SW620/sh RN A by G418 screening and cloning,the biological function and molecular mechanism of Asporin were studied.Results: The expression of Asporin in CRC tissues was significantly higher compared to the adjacent non-tumor tissues(P<0.0001);in the analysis of clinical and pathological data of the 200 cases of patients with colorectal cancer,we found that overexpression of Asporin were significantly associated with TNM stage and lymph node metastasis of the CRC patients.Wound healing assays,Transwell assays and tube-formation assays showed that Asporin could enhance the healing,invasion,migration and tubule formation ability of colorectal cancer cells.Further more,with the xenograft model,we found that Asporin could promote the tumor liver metastasis and stimulate the VEGF signaling pathway.In addition,Asporin was found to augment the phosphorylation of EGFR/Src/cortactin signaling pathway.Conclusion: Asporin was high expressed in colorectal cancer tissues,and its expression level was related with TNM staging and lymph node metastasis;Asporin can promote the metastasis of colorectal cancer through the EGFR/Src/Cortactin signaling pathway.