| ObjectivePancreatic ductal adenocarcinoma(PDAC)is one of the most lethal malignant tumors,known as the king of cancer,with a very low 5-year survival rate.Therefore,it is of great significance to explore the causes of rapid proliferation and high invasion and metastasis of PDAC and find its important pathogenic factors for early stage of PDAC,which will contribute to the research and development of effective therapeutic drugs,and improving the prognosis of patients.IQGAP1(IQ-motif-containing GTPase-activating protein 1)is a kind of scaffold protein,which has been found to be highly expressed in various solid tumors,such as thyroid,colon and lung cancer,to promote the progress of tumors.However,little has been kown about IQGAP1 in PDAC,and its mechanism is still unclear.This study elaborated the mechanism of PDAC progression from clinical specimens,in vitro cell experiments and animal models,and provided a new target for the treatment of PDACMethods1.To investigate the effects of IQGAP1 on the proliferation of PDAC in vitro and in vivo:Firstly,the different expression of IQGAP1 in cancerous and adjacent tissues was compared by using clinical specimens of PDAC.The relationship between IQGAP1 expression and clinicopathological characteristics was evaluated,and the prognosis of PDAC patients was also analyzed.Then,two interfering RNAs(siRNAs)and overexpression plasmids were designed for IQGAP1 gene.siRNA with the best interference efficiency was used to transfect cells with high expression of IQGAP1,and cells with low expression of IQGAP1 were transfected by over-expression plasmid.Secondly,the effects of IQGAP1 on the growth of PD AC cells in vitro were demonstrated by cell proliferation and plate cloning experiments respectively.Finally,nude mice models of transplantation of PD AC were established by stable expression of shIQGAP1,and the effects of IQGAP1 on the growth of PD AC were analyzed2.To investigate the effects of IQGAP1 on invasion and metastasis of PD AC in vitro and in vivo:Firstly,the siIQGAPl was transfected into high-IQGAP1 PDAC cell lines,and the over-expression plasmid was transfected into low-IQGAP1 cell lines Wound healing assay and Transwell migration assay were used to detect the changes of migration and invasion ability of PDAC cells.Then,EMT-related proteins and transcription factors were detected by Western-blotting and PCR,respectively Secondly,the relationship between IQGAP1 and the key signaling pathways regulating the progression of PDAC was determined.Finally,liver metastasis models constructed by orthotopically injecting shIQGAPl and shNC cells into the pancreatic tails of nude mice to analyze the effect of IQGAP1 on the metastasis of PDAC cell lines.Results1.Immunohistochemical staining,Western-blotting and PCR methods showed that the positive expression of IQGAP1 in PDAC tissues was significantly higher than that in adjacent tissues,which was related to the degree of differentiation,lymph node metastasis.The high expression of IQGAP1 suggested poor prognosis.Multivariate analysis suggested that IQGAP1 was an independent prognostic predictor for PDAC patients.At the same time,the expression of IQGAP1 mRNA and protein in human PDAC cell lines were significantly higher than that in normal pancreatic ductal epithelial cell.Knockdown of IQGAP1 could inhibit the proliferation of SW1990 and CFPAC-1 cells,while over-expression of IQGAP1 in PANC-1 cells had the opposite effect.The transplantation model of PDAC in nude mice was successfully established The volume and weight of tumors in shIQGAP1 group were lower than those in the control group.2.Knockdown of IQGAP1 inhibited the migration and invasion of PDAC cells.IQGAP1 knockdown by siRNA suppressed the expression of DVL2,beta-catenin and downstream target genes,and also inhibited the expression of EMT-related proteins and transcription factors.Conversely,overexpression of IQGAP1 led to the opposite effects.Co-immunoprecipitation studies demonstrated that IQGAP1 interacted with both DVL2 and beta-catenin.Knockdown of IQGAP1 also reduced the interaction between DVL2 and beta-catenin by Co-IP assay.Knockdown of DVL2 reversed the EMT process induced by overexpression of IQGAP1.The inhibition of IQGAP1 expression suppressed the hepatic metastasis of PD AC cells in vivoConclusions1 IQGAP1 is highly expressed in PD AC tissues and cell lines.It is significantly correlated with pathological grade,lymph node metastasis and poor prognosis of PD AC patients.IQGAP1 expression is an independent prognostic factor for PD AC patients.2 IQGAP1 promotes EMT,proliferation and migration of PDAC,and hepatic metastasis of PD AC3 IQGAP1 forms complex with DVL2 and beta-catenin,which significantly reduces the binding of DVL2 to beta-catenin after interfering with IQGAP1.Inhibition of DVL2 reverses the EMT process induced by overexpression of IQGAP1. |
