Influence Of The Expression Of L1CAM And ?5-integrin To The Secrete Of Pro-angiogenic Factors In Pancreatic Cancer Cells

Objective: Pancreatic cancer is one of the most common malignancies in the world.Due to the susceptibility of pancreatic cancer to the superior mesenteric artery and portal vein during the diagnosis,the surgical removal rate of patients is low,and the 5-year survival rate remains at about 8%.In the process of proliferation and vascular invasion of pancreatic cancer,the formation of abnormal microvessels is essential,moreover,the production of tumor microvessels depends on VEGF and other pro-angiogenic factors.Therefore,it is of great significant to further explore the mechanism of pancreatic tumor microvascular generation and its related molecular pathways in the inhibition of pancreatic cancer vascular invasion,and to improve the radical resection rate and cure rate of patients.This study aimed to use plasmid transfection to change the expression of L1 CAM and ?5-integrin in human pancreatic cancer cell lines Patu8988 and Capan-2,and detect the expression of pro-angiogenesis factorssuch as,STAT3,pSTAT3 and VEGF-A Ang-2,and further explore the influence of liquid supernatant to the tube formation of human umbilical vein endothelial cells.The mechanism of action of L1 CAM and?5-integrin in the generation of tumor microangiogenesis in pancreatic cancer was preliminary discussed.Methods: Patu8988 and Capan-2 pancreatic cancer cell lines were chosen as the subjects in vitro.The two types of pancreatic cancer cells were divided into three groups respectively: blank group,control group and experimental group.In the experiment of increasing L1 CAM expression,the blank group doesn't transfect the plasmid,the control group transient transfected with pcDNA3.1,the experimental group transient transfected with recombinant plasmid pcDNA3.1-L1CAM;In the experiment of inhibiting?5-integrin expression,the blank group doesn't transfect the plasmid,the control group transient transfected with blank siRNA,the experimental group transient transfected with?5-integrin siRNA.The expression of proteins and mRNA of L1 CAM and?5-integrin in the cell lines of Patu8988 and Capan-2 pancreatic cancer cells were detected by real-time quantitative PCR and western blot,and the expression of proteins andmRNA pro-angiogenic factors such as STAT3,pSTAT3,VEGF-A and Ang-2 were evaluated.Tube formation test was conducted to determine the effect of the supernatant on the blood vessels formation in the HUVEC cell lines in vitro of the pancreatic cancer cells of Patu8988 and capan-2 after the changes in the expression levels of L1 CAM and?5-integrin to simulate microvessels formation in vivo.Results: 1.The expression of L1 CAM in Patu8988 pancreatic cancer cell line was upgraded,and the results of qPCR were shown that L1 CAM,STAT3,VEGF-A,Ang-2mRNA relative expression levels were 1.7385±0.0763,1.4984±0.0404,3.8413±0.1345,1.2864±0.0282 in experimental group.Western test results were shown that L1 CAM,VEGF-A,Ang-2,pSTAT3,STAT3 protein relative expression levels were 2.7104±0.1734,2.1100±0.1099,2.4451±0.2384,4.2470±0.2895,1.0145±0.0168.The relative total length of intact tubules in human umbilical vein endothelial cells was 1.35±0.06 in experimental group.Besides,in Capan-2 pancreatic cancer cell line,L1 CAM,STAT3,VEGF-A,Ang-2mRNA relative expression levels were 1.6348±0.0965,1.2195±0.0256,3.9340±0.2409,2.2987±0.3505 in experimental group.L1 CAM,VEGF-A,Ang-2,pSTAT3,STAT3 protein relative expression levels were 2.4932±0.2985,2.1515±0.1222,3.2373±0.3323,2.0736±0.2755,1.0217±0.0874.The relative total length of intact tubules in human umbilical vein endothelial cells was 1.31±0.05 in experimental group.2.Inhibition of?5-integrin expression in Patu8988 pancreatic cancer cell line,and the results of qPCR were shown that ?5-integrin,STAT3,VEGF-A,Ang-2 m RNA relative expression levels were 0.4922±0.0754,0.7194±0.0710,0.3018±0.0776,0.4779±0.0748 in experimental group.Western test results were shown that ?5-integrin,VEGF-A,Ang-2,pSTAT3,STAT3 protein relative expression levels were 0.3478±0.0264,0.4237±0.0186,0.2740±0.0291,0.5699±0.0432,0.9637±0.0397.The relative total length of intact tubules in human umbilical vein endothelial cells was 0.79±0.07 in experimental group.Besides,in Capan-2 pancreatic cancer cell line,?5-integrin,STAT3,VEGF-A,Ang-2 mRNA relative expression levels were 0.5425±0.3061,05802±0.0033,0.3065±0.0459,0.5649±0.0555 in experimental group.?5-integrin,VEGF-A,Ang-2,pSTAT3,STAT3 protein relative expression levels were 0.4392±0.0286,0.6896±0.0312,0.4698±0.0365,0.3554±0.0304,1.0146±0.0276.The relative total length of intact tubules in human umbilical vein endothelial cells was 0.73±0.08 in experimental group.The results showed statistically significant difference between the experimental group and thecontrol group(P< 0.05)except for the relative expression level of STAT3 protein in western experiment.Conclusion: In pancreatic cancer cell lines in vitro,increased level of L1 CAM induced the secrete of the pro-angiogenic factors and enhanced tube formation,and silencing the expression of ?5-integrin reduced the secrete of the pro-angiogenic factors and weaken tube formation.