The Mechanism Of HMGA2 On The Regulation Of Gastric Cancer Vasculogenic Mimicry

Objective: Gastric cancer(GC)is one of the most common malignancies in the world and its lethality rate is the second in the world,which is related to the metastasis and the poor prognosis of anti-vascular therapy.The term vasculogenic mimicry(VM)refers to the unique ability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks.VM has been widely associated with tumor progression and poor prognosis,and recent studies have shown that cancer stem cells(CSCs)and epithelial-to-mesenchymal transition(EMT)accelerate VM formation by stimulating tumor cell plasticity.HMGA2 has emerged as a tumor biomarker due to its tumor-specific overexpression in many human cancers,which mediates EMT and self-renewal in cancer stem cells.However,the relationship between HMGA2 and VM formation in GC remains unclear.This study was to explore the role of HMGA2 in the VM formation of gastric cancer and its molecular mechanism.Immunohistochemical technique was used to analyze the relationship between HMGA2 and VM in 228 cases of GC.In vitro and in vivo,the results showed that HMGA2 could promote the formation of VM and EMT,and enhance the growth of cancer stem cells.By using Ch IP and Co-IP to elucidate the mechanism of HMGA2 on VM formation,and which provide a new idea for the treatment of anti-angiogenesis of tumor.Methods: 1.Resected specimens of 228 patients identified as gastric cancer with complete follow-up data were obtained from General Hospital and Tumor Tissue Bank of Tianjin Cancer Hospital.The expression of HMGA2 and VM in gastric cancer were detected by immunohistochemistry and CD34 / PAS double staining.Meanwhile,we analyzed the relationship of HMGA2 and the clinicopathological parameters,VM.Kaplan-Meier survival analysis was performed to analyze their relationship with the prognosis of patients.2.To further analysis the relationship of HMGA2 and EMT?cancer stem cells,we detected the expression of EMT related markers(E-cadherin?Vimentin?Twist1??-catenin)?VM related markers(VE-cadherin?VEGF? MMP2)and cancer stem cell markers(CD44)by immunohistochemistry in human gastric cancer.3.The expression of HMGA2 in a series of gastric cancer cell lines was analyzed by Western Blot.The selected cell lines with low or high expression of HMGA2 were stably transfected with HMGA2 and sh-HMGA2 plasmids,respectively.The expression of HMGA2 in these cells were confirmed by Western Blot.Then,the expression of EMT-related markers(E-cadherin?N-cadherin?Vimentin??-catenin?Twist1?Snail),VM-related markers(VE-cadherin?VEGF?MMP2?MMP9)and cancer stem cell markers(CD44?ALDH1?Sox2?OCT4?c-Myc)were detected in the selected GC cell lines by RT-PCR,Western Blot and immunofluorescence assay.4.Scrape assays,invasion and migration assay,3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT),Cell maintenance of tumor spheres,Colony formation and Three-dimensional(3D)cultures were performed to analysis the role of the HMGA2 in GC cells.5.The relationship between HMGA2 and Twist1 was tested by a Dual Luciferase Reporter Assay,and the interaction between HMGA2 and Twist1 was verified by Co-IP and Ch-IP.In MKN74 cell line,we also transfected the HMGA2 overexpression plasmid and the sh-Twist1 expression plasmid.The interaction between HMGA2 and Twist1 was confirmed by western blot and functional assay(migration assay and three-dimensional culture)in GC cell line with stable transfected HMGA2-expression plasmids and sh-Twist1 plasmid.6.The effect of HMGA2 on gastric cancer was confirmed by inoculating gastric cancer cell lines with stably overexpressing and down-expressing HMGA2 in nude mice to construct gastric cancer transplanted tumor model.The effect of HMGA2 on tumor volume and weight was measured by measuring and plotting the tumor growth curve.The expressions of EMT and VM-related proteins and the formation of VM in the tumor tissues was detected by Immunohistochemistry and Endomucin / PAS double staining to analysis the influence of HMGA2 on EMT/VM in GC.Results:1.Among the 228 cases of gastric cancer,65 cases with typical VM structure,88 cases(38.6%)with HMGA2 positive expression.There were 42 cases of VM-positive patients with HMGA2 positive(64.6%),and HMGA2 had positive correlation with VM-PAS(r = 0.4194,P <0.001);The expression level of HMGA2 and VM were correlated with histological type,clinical stage,distant metastasis and recurrence of tumor.Kaplan-Meier Survival analysis showed that the expression of HMGA2 and VM were correlated with the prognosis of GC.2.In GC tissues,the results of Pearson test showed that there was correlation between HMGA2 and Twist1,VE-cadherin,VEGF,E-cadherin,Vimentin,?-catenin,MMP2 and CD44.Furthermore,the expression of HMGA2 was positively correlated with the expression of VE-cadherin and Twist1(r = 0.3254,P <0.001;r = 0.208,P = 0.016).3.In GC cell lines(MKN45,MGC803,MKN28,MKN74),the expression of HMGA2 was significantly downregulated in MKN74 and MKN28,whereas the expression of HMGA2 was significantly upregulated in MGC803 and MKN45.The gastric cancer cell lines MKN74 and MKN28 were transfected with HMGA2 expression plasmid and MGC803/MKN45 were transfected with sh-HMGA2 plasmid.Stable cell lines with over-expressing and down-regulating HMGA2 were established and were tentatively designated MKN74-HMGA2 or MKN28-HMGA2,MGC803-sh-HMGA2 or MKN45-sh-HMGA2.The expression of HMGA2 in these cells was confirmed by Western blot.MKN74-HMGA2 or MKN28-HMGA2 exhibited high expression of Twist1,VE-cadherin,N-cadherin,Vimentin,?-catenin,Snail,VEGF and cancer stem cell markers(CD44?ALDH1?Sox2?OCT4?c-Myc)and low expression of E-cadherin.While,in the MKN45-sh-HMGA2 and MGC803-sh-HMGA2 cell lines,the expression of E-cadherin is increased and the expression of Twist1,VE-cadherin,N-cadherin,Vimentin,?-catenin,Snail,VEGF and cancer stem cell markers(CD44?ALDH1?Sox2?OCT4?c-Myc)is decreased.4.MKN74 and MKN28 cells that were transfected with HMGA2 displayed a much higher ability to promote invasion,migration,proliferation and tube formation compared with those that were transfected with empty vector or with non-transfected cells(P<0.05).Compared with those of the controls,MGC803-sh-HMGA2 and MKN45-sh-HMGA2 showed a lower ability to invasion,migration,proliferation and tube formation(P <0.05).5.The results of Dual luciferase reporter assay showed that HMGA2 could increase the promoter activity of Twist1 and c-Myc.The results of Co-IP and Ch-IP further validated the interaction between HMGA2 and Twist1.In order to further prove that HMGA2 regulates the formation of VM by Twist1,we down-regulated Twist1 in HMGA2-transfected cell line.The results showed that the ability of migration and tube formation were significantly decreased,meanwhile the expression of N-cadherin,Vimentin and VE-cadherin was decreased,but the expression of E-cadherin was not changed.6.Compared with the control group,the tumor derived from MKN74-HMGA2 cells displayed higher lower levels of tumorigenicity,showed the expression of EMT and VM-related proteins(Vimentin,Twist1,VE-cadherin,VEGF,MMP2,CD44)were increased and E-cadherin expression was decreased in tumor tissue.However,the tumors derived from MGC803-sh-HMGA2 cells displayed lower levels of tumorigenicity,showed the expression of EMT and VM-related proteins(Vimentin,Twist1,VE-cadherin,VEGF,MMP2,CD44)were decreased and E-cadherin expression was increased in tumor tissue.Conclusions: 1.VM exists in GC,and is related to the malignant biological behavior.HMGA2 is related to VM,and also affect the survival of GC patients.HMGA2 was associated with the expression of VM-related markers,which suggesting that HMGA2 may promote the formation of VM,leading to poor prognosis of gastric cancer.2.The expression of HMGA2 promotes EMT and increases the characteristics of cancer stem cells.3.In vitro,HMGA2 promotes cell migration,invasion,proliferation,colony formation and tube formation;in vivo,HMGA2 promotes tumor growth and tumor VM formation.4.Twist1 is the target gene of HMGA2,and the down-regulation of Twist1 attenuates HMGA2-mediated promotion of VM and facilitation of metastasis.5.HMGA2 directly targets with Twist1 and promotes the expression of Twist1,VE-cadherin,resulting in the formation of VM and enhanced tumor invasion and metastasis.