The Role Of Cyclooxygenase-2 In Gastic Cancer Vasculogenic Mimicry And Its Molecular Machanism

Background and Objective: Gastric cancer(GC) is one of the most common malignancy cancer worldwide.Although its incidence is declined recent years,but the death rate of GC patients remains high.Vasculogenic mimicry(VM) is a new kind of tumor microcirculation pattern, is closely related to tumor invasion, metastasis and tumor-related death. Many studies have reported that a variety of malignant tumors exist VM, such as liver cancer, ovarian cancer, ect. This study aimed at confirming whether there is VM in gastric cancer, and clarifying the relation between VM and clinical pathological parameters,and further exploring the possible mechanism of Cyclooxygenase-2 in gastric cancer VM.Methods: 1) PAS/CD31 dual staning were used to detect VM, and analyzes the relationship between VM and clinical pathological date.2) IHC staning was used to detect the expression of cox-2, PGE2, Snail, analyzes the relationship between COX-2 and clinical pathological date and discuse the relationship between VM and cox-2,PGE2,Snail.3) COX-2 was upregulated in MKN74 cells and COX-2 was downregulated in MKN45 cells. Western blotting and RT-PCR were used to examine the transfection effect.4) Three-dimension culture(3D) was used to investigate the VM formation of MKN74 and MKN45 after COX-2 upregulated or downregulated. Western blotting was used to detect the expression of VE-cadherin.5) Wound healing and invasion assays were used to detect the change of cell movement and invasion after COX-2 upregulated or downregulated in MKN74 or MKN45 gastric cancer cells.6) Western blotting was used to detect the expression of PGE2 and EMT-related proteins.7) Snail was upregulated in MKN74 cells and Snail was downregulated in MKN45 cells. Western blotting were used to examine the transfection effect and detect the expression of EMT-related proteins.8) Three-dimension culture(3D) was used to investigate the VM formation of MKN74 and MKN45 cells after Snail upregulated or downregulated.9) Wound healing and invasion assays were used to detect the cell movement and invasion ability in MKN74 and MKN45 cells after Snail upregulated or downregulated.Results : 1) CD31/PAS dual staning were used to detect VM, VM was observed in gastric cancer. VM was identified in 35(35%) of the 100 gastric cancer specimens.The frequency of VM was found to be closely associated with Lauren, s classification, lymph node metastasis and distant metastasis,(p = 0.029; p = 0.020; p = 0.018). No association was found between the frequency of VM and age, sex, tumor size, TNM stage(p = 0.284; p = 0.946; p = 0.850; p = 0.232). The VM group average survival time was 54.9 months, no VM group average survival time was 25.7 months. Kaplan-Meier survival analysis showed that the total survival time for patients in the VM-present group was significantly shorter than the VM-absent group(p = 0.001).2) The expression of cox-2 in gastric cancer was found to be closely related to lymph node metastasis and distant metastasis(p = 0.025,p = 0.022). The staining for COX-2 had no significant association with gender, age, diameter of tumors, Lauren, s type and TNM stages(p = 0.191; p = 0.480; p = 0.368; p = 0.462; p = 0.514). The average survival time of patients with COX-2 expression was 61.9 months, whereas that for COX-2-negative patients was 31.9 months. Kaplan-Meier survival analysis showed that the total survival time for patients with COX-2 expression was found to be significantly shorter than that of patients without COX-2 expression.3) Immunohistochemical results showed that in gastric cancer the positive rate of COX-2?PGE2?Snail in VM-present group was siginificantly higher than that in VM-absent group(p = 0.032; p = 0.013; p = 0.012)4) The expression of COX-2 in MKN74 cells was low; The expression of COX-2 in MKN45 cells was high.5) The expression of COX-2 was upregulated in MKN74 and was downregulated in MKN45 obviously.6) The COX-2-upregulated MKN74 cells could form vessel-like structures in the 3D culture and expressed lower expression of VE-Cadherin. Knockdown of COX-2 induced higher expression of VE-Cadherin and destroy the property of vascular-like formation in MKN45 cells.7) COX-2 upregulated in MKN74 cells enhanced the ability to migrate and invade. COX-2 downregulated in MKN45 cells significantly inhibited the ability of migration and invasion.8) The COX-2-upexpressed MKN74 cells showed lower expression of E-cadherin and higher expression of PGE2, Vimentin, snail; The COX-2-downexpressed MKN45 cells showed higher expression of E-cadherin and lower expression of PGE2, Vimentin, snail.9) The expression of Snail was upregulated in MKN74 and was downregulated in MKN45 obviously by Lenti Virus technology.and significantly inhibited the ability of migration and invasion.10) The Snail-upexpressed MKN74 cells showed lower expression of E-cadherin and higher expression of Vimentin; The Snail-downexpressed MKN45 cells showed higher expression of E-cadherin and lower expression of Vimentin.11) The Snail-upregulated MKN74 cells could form vessel-like structures in the 3D culture and enhanced the ability to migrate and invade.; Knockdown of Snail destroy the property of vascular-like formation in MKN45 cells and decreased the ability of migration and invasion.Conclusions: 1) VM exist in gastric cancer. VM was related with gastric cancer tissue differentiation, lymph node metastasis and distant metastasis, and was an unfavorable prognostic indicator.2) The positive rates of COX-2 were 60%(60/100)in gastric cancer and related with lymph node metastasis and distant metastasis, and predicted a poor prognosis.3) Snail could induced the EMT, and promote the migration, invasion and VM formation ability in gastric cancer cells.4) COX-2 may through inducing the EMT and upregulated the expression of VE-Cadherin to promote VM formation and enhance the ability of migration and invasion in gastric cancer cells.