The Role Of Hepatocyte Growth Factor In Pancreatic Stellate Cell-induced Invasion And Metastasis Of Pancreatic Cancer Cell

Objective Pancreatic cancer,which is known as the“king of cancer”,is one of the most malignant diseases in the world due to its fast proliferation,early metastasis and insensitivity to chemotherapy drugs.The traditional model of pancreatic cancer research focused on the pancreatic cancer itself,while ignored the impact of tumor microenvironment on pancreatic cancer cells,which may be one of the reasons for poor treatment of pancreatic cancer.In this study,we measured the effect of pancreatic stellate cells on the invasion /migration of pancreatic cancer cells and the role of HGF/c-met in this progression,to reveal the role of tumor microenvironment in tumor malignant behaviors.Methods 1.The effect of PSCs on invasion and metastasis of pancreatic cancer cell line Panc-1 and SW1990.The supernatant of PSCs was collected as conditioned medium,and the effects of the supernatant on the invasion and metastasis of two pancreatic cancer cells were determined by cell scratch assay;Using Tanswell co-culture system,PSCs and pancreatic cancer cells were co-cultured to determine the effect of PSCs on the invasion and metastasis of pancreatic cancer cells;2.The effect of PSCs on epithelial mesenchymal transition in pancreatic cancer cells.PSCs and pancreatic cancer cells were co-cultured with 24 h,and the morphological changes were observed;The expression of E-cadherin and Vimentin in pancreatic cancer cells was detected by RT-PCR method and Western blot method,respectively;The expression of E-cadherin and Vimentin in pancreatic cancer cells was detected by immunofluorescence technique;The effect of HGF on EMT and invasion and metastasis of pancreatic cancer cells;ELISA assay was used to detect the content of HGF in the supernatant of pancreatic stellate cells and pancreatic cancer cells.The expression of HGF in pancreatic stellate cells and pancreatic cancer cells was detected by RT-PCR and Western Blot.si RNA was used to interfere with the expression of HGF in pancreatic stellate cells,and the supernatant was collected to observe the effect on the invasion and metastasis of pancreatic cancer cells;HGF-antibody was added into the supernatant of pancreatic stellate cells and then cultured with pancreatic cancer cells to observe the effects of HGF-antibody on invasion and metastasis of pancreatic cancer cells;The exogenous HGF was added into the pancreatic cancer cell culture medium to observe the change of the invasion and metastasis of pancreatic cancer cells;4.The effect of HGF on the expression of survivin pancreatic cancer cells.The expression of survivin gene in pancreatic cancer cells was detected by RT-PCR method and Western Blot after co cultured pancreatic cancer cells with PSCs.The expression of survivin gene in pancreatic cancer cell line was observed after adding HGF-antibody into pancreatic stellate cell conditioned medium;Si RNA technology was used to interfere with the expression of survivin gene in pancreatic cancer cells,Then Pancreatic cancer cells were co cultured with PSCs to observe the effect of PSCs on the invasion and migration of PC cells.5.The effect of HGF on the expression of its receptor c-Met.The expression of c-Met and p-Met gene in pancreatic cancer cells was detected by RT-PCR method and Western Blot after co-cultured pancreatic cancer cells with PSCs;HGF-antibody was added into the culture medium of pancreatic stellate cells and cultured in pancreatic cancer cells.The expression of c-Met and p-Met in the cancer cells was observed;SU11274(C-Met specific inhibitor)was added into the culture medium of pancreatic stellate cells,and then cultured in pancreatic cancer cells.The expression levels of c-Met,p-Met and Survivin were observed;6.The effect of P53/P21 on the HGF/c-met/survivin signaling pathway.RT-PCR method and Western Blot were used to detect the expression of P53 and P21 in pancreatic cancer cells.The expression of P21,c-Met,p-Met,survivin gene in cancer cells was detected after treated pancreatic cancer cells with pifithrin-?(the inhibitor of P53 gene.P53 overexpression plasmid was transfected into cancer cells to increase the expression of P53 gene,and the expression level of P21,c-Met,p-Met,and Survivin were detected;si RNA-P21 was used to interfere with the expression of P21 in cancer cells,and the expression level of c-Met was detected;P21 overexpression plasmid was transfected into cancer cells to increase the expression of P21 gene,and the expression level of c-Met was detected.Results Pancreatic stellate cells can promote the occurrence of EMT in pancreatic cancer cells,and promote the invasion and metastasis of pancreatic cancer cell line Panc-1,but it has no significant effect on the invasion and metastasis of SW1990;A large amount of HGF was detected in the supernatant of pancreatic stellate cells,but there was little in the supernatant of pancreatic cancer cells.Specific blockade of HGF could attenuate the effect of pancreatic stellate cells on the invasion and metastasis of Panc-1cells;Exogenous HGF could induce the invasion and metastasis of Panc-1 cells,but had no effect on SW1990 cells.After treated with HGF,the expression level of c-Met in cancer cells did not change significantly,while the level of phosphorylation(p-Met)was significantly increased,and the expression level of p-Met decreased after specific blockade of HGF.The expression level of p-Met was decreased as well as survivin after treated Panc-1 cells with SU11274;The effect of pancreatic stellate cells on the invasion and metastasis of pancreatic cancer cells was significantly lower than before after decreased survivin gene expression in cancer cells by si RNA.Both P53 and P21 were deletion in Panc-1 cells,while higher in SW1990 cells.After transfection of Panc-1 cells into P53 overexpressing plasmid,the expression of P21 was up-regulated while the expression level of c-Met was down regulated.The expression of P53 in SW1990 cells was inhibited by si RNA,and the expression of P21 was decreased while the expression of c-Met was up-regulated.After treated SW1990 cells with pifithrin-?,the ability of invasion and metastasis of pancreatic stellate cells was significantly enhanced when Incubated with PSC-CM.Conclusion Pancreatic stellate cells promote the invasion and metastasis of pancreatic cancer cells via HGF/c-Met/survivin signaling pathway,which is negatively regulated by P53/P21.