The Mechanism Of HIF-2? In Regulating Vasculogenic Mimicry In Pancreatic Cancer

Part? Expression and clinical significance of HIF-2? and VM in human pancreatic carcinomaObjective To study the expression of HIF-2? and VM in pancreatic cancer tissues,and further to observe the significance of HIF-2? and VM in human pancreatic cancer.Methods(1)Immunohistochemistry(IHC)was performed to detect the expression of HIF-2? in 70 pairs of pancreatic tumor tissues and adjacent non-tumor pancreatic tissues.(2)Then the relationship between HIF-2? expression and clinical pathological characteristics was analyzed.(3)PAS/CD34 double staining was performed to detect the expression of VM,and further to detect the relationship between VM expression and clinical pathological characteristics.(4)The relationship between the expression of HIF-2? and VM in 70 pancreatic cancer tissues was analyzed.(5)To assess the prognostic significance of HIF-2? and VM expression on survival of pancreatic cancer patients further,a Kaplan-Meier analysis was conducted.Then,the univariate analysis and multivariate analysis were performed to detect possible prognostic factors of pancreatic cancer patients.Results(1)In our immunostaining results,the location of HIF-2?protein was observed mainly in the cytoplasm and nucleus.HIF-2?protein was expressed positively in 67.1%(47/70)of pancreatic cancer tissues and 11.4%(8/70)of adjacent non-tumor pancreatic tissues.There was a significant difference in the positive rate of HIF-2? protein between the pancreatic cancer tissues group and non-tumor tissues group(?2=45.549,P<0.05).In addition,HIF-2?was correlated with tumor differentiation,clinical stage and lymph node metastasis(P<0.05,respectively).(2)Using PAS/CD34 double staining,we found that 51.4%(36/70)of the tissues contained VM channels within the pancreatic cancer tissues;however,none of the tissues in the non-tumor tissue group exhibited a VM pattern.Further,we found that the expression of VM channels was correlated with tumor differentiation,clinical stage,and lymph node metastasis(P<0.05,respectively).(3)There was a significant positive correlation between the expression of HIF-2? and VM(Spearman,r=0.294,P<0.05).(4)Using Kaplan-Meier analysis,we found that patients with HIF-2?-negative expression had a significantly longer survival time than those with HIF-2?-positive expression(log-rank test,P<0.05),similarly,the patients in the VM-positive group had a poorer prognosis than those in the VM-negative group(log-rank test,P<0.05).(5)The results of univariate analysis revealed that clinical stage(P=0.001),lymph node metastasis(P=0.001),HIF-2? expression(P=0.001)and VM expression(P=0.001)were significantly associated with patient survival.In addition,the results of the multivariate analysis using Cox's proportional hazards model revealed that HIF-2? expression(HR=4.694,P=0.001)and VM expression(HR=5.857,P=0.012)were independent and important prognostic factors in all patients.Conclusions HIF-2? protein and VM structures were expressed a high level in pancreatic cancer tissues,and high expression of HIF-2? and VM were correlated with tumor differentiation,clinical stage and lymph node metastasis.There was a significant positive correlation between the expression of HIF-2? and VM,and overexpression of HIF-2? or VM was correlated with poor prognosis.The results of the multivariate analysis using Cox's proportional hazards model revealed that HIF-2? expression and VM expression were independent and important prognostic factors in pancreatic cancer.These results indicated that HIF-2? and VM are closely associated with the prognosis of pancreatic cancer patients.Part? The effect and mechanism of HIF-2? in regulating Vasculogenic Mimicry in pancreatic cancer cellsObjective To investigate the effect of HIF-2? on VM formation,and further to study the possible mechanism of HIF-2? on the occurrence of VM in pancreatic cancer cells.Methods(1)Small interfering RNA(si RNA)expression vector and overexpressed fragment targeting HIF-2? gene was separately infected into pancreatic cancer cells,then Polymerase chain reaction(PCR)and western blot(WB)were conducted to examine the transfection efficiency of HIF-2?.(2)The ability of invasion and migration of pancreatic cancer cells via cell invasion assay and wound healing assay when regulating the level of HIF-2? in vitro.The expression of VM associated proteins were detected by western blot after regulating the expression of HIF-2?.(3)To confirm the role of HIF-2? in VM formation,3D tumor cell culture was used to observe VM formation after regulating the level of HIF-2? in vitro.(4)The effect of HIF-2? on VM related proteins after regulating the expression of HIF-? through western blot.(5)Patch software was used to identify potential transcription factor binding sites in the promoter region of the VE-cadherin gene,then Chromatin immunoprecipitation assay(Ch IP)in pancreatic cancer cells was conducted to detect the effect and sites of Twist1/2 on VE-cadherin,which was concerned as a specified VM protein.(6)Luciferase reporter gene assays was conducted to understand the effect of Twist on VE-cadherin transcription.Results(1)Small interfering RNA(si RNA)expression vector and overexpressed fragment targeting HIF-2? gene were generated and the virus were produced successfully.RT-PCR and Western blot analysis confirmed the transfection efficiency after upregulating or downregulating of HIF-2?(P<0.05;P<0.05).(2)HIF-2? promoted cell invasion and migration when HIF-2? up-regulated in pancreatic cancer cells,and the opposite result was observed in down-regulated group.(3)The number of complete tubular channels formed by As PC-1 cells was significantly decreased in the si-HIF-2? cells(8±2/HP)compared with si-Scramble cells(37±5/HP)in 3D cultures(P<0.05).In contrast,the overexpression of HIF-2? in SW1990 cells promoted the formation of more typical ECM-rich vessel-like networks by tumor cells in the OE-HIF-2? group(51±6/HP)compared with the vector group(14±3/HP)(P<0.05).(4)The expression of VE-cadherin,Twist1,Twist2,MMP2,and MMP9 were significantly decreased in si-HIF-2? As PC-1 cells compared with si-Scramble cells(P<0.05).Similarly,after upregulating HIF-2? by transfecting HIF-2? c DNA,the expression of VM-associated proteins including VE-cadherin,Twist1,Twist2,MMP2,and MMP9 were all increased in the OE-HIF-2? group compared with the negative vector group in the SW1990 cells(P<0.05).(5)Four potential Twist protein binding sites,separately designated P1(-421bp),P2(-714bp),P3(-2000bp),and P4(-2110bp)were identified in the Patch transcription factor binding site database.Ch IP assay using As PC-1 cells overexpressing Twist1 and Twist2 proved that the Twist1 antibody only specifically immunoprecipitated a Twist1-DNA complex in the P1 and P4 regions of the VE-cadherin promoter.(6)Luciferase reporter gene assays revealed the P1 and P4 Twist1 binding sequence regions are positive regulatory elements for VE-cadherin transcription.Conclusion HIF-2? and VM could participate the occurrence or progress of VM to promote the ability of invasion and metastasis of pancreatic cancer cells.As an important transcription factor,Twist1 could bind to the promoter of VE-cadherin.HIF-2? participated the occurrence of VM through regulating Twist1 binding to the promoter of VE-cadherin.Part? The effect of HIF-2? in regulating Vasculogenic Mimicry in pancreatic cancer cells in vivoObjective To further investigate the effect of HIF-2? on Vasculogenic Mimicry(VM)in pancreatic cancer in vivo,and then to confirm the possible mechanism of HIF-2? on the occurrence of VM.Methods Afterestablishing subcutaneous tumor model and orthotopic tumor model in nude mice,the mice were treated with si-Scramble,si-HIF-2?,Vector and OE-HIF-2? in each group.Efficiency of regulating HIF-2? was verified by IHC.In orthotopic tumor model,several aspects were recorded,including the growth of xenografts,ascites,other abdominal cavity metastases,tumor volume,and the expression of VM,Twist1,and VE-cadherin in the murine pancreatic tumor tissues.Results(1)In orthotopic tumor model,the tumors were significantly smaller in the si-HIF-2? group than in the si-Scramble group(P<0.05),whereas the tumors in the OE-HIF-2? group were larger compared with the vector group(P<0.05).(2)None of four groups were found pulmonary metastasis.One case was found mesenteric metastases in si-Scramble group.OE-HIF-2? group was found as follows: one case hepatic metastases,mesenteric metastases and skin metastases;one case hepatic metastasis;two cases mesenteric metastases;one case no other metastases.(3)CD34/PAS staining revealed that VM was obviously reduced in the si-HIF-2? xenografts compared with the si-Scramble group(P<0.05).In contrast,VM was markedly increased in the OE-HIF-2? xenografts compared with the vector group(P<0.05).(4)Twist1 and VE-cadherin were more highly expressed in the OE-HIF-2? xenografts compared with the vector group(P<0.05);however,the expression of both Twist1 and VE-cadherin was lower in the si-HIF-2? group compared with the matched group(P<0.05).Conclusion HIF-2? could contribute to VM formation in pancreatic cancer in vivo,and HIF-2? could regulate VM related proteins.We also found that HIF-2? can promote invasion and metastasis in pancreatic cancer in vivo.