CAV2 Promotes Proliferation,Invasion,and Metastasis Of Pancreatic Cancer Cells By Up-regulation MiR-4723/Wnt7A Signalling Axis

Objective:To investigate the extent of CAV2 gene expression in pancreatic cancer cells and its effect on proliferation,invasion and metastasis.To uncover the signalling axis associated with the CAV2 gene in this effect.And further explore its relationship with endocytosis and epithelial-mesenchymal transition to uncover the deeper mechanisms of action of CAV2 gene affecting pancreatic cancer cell progression.This will provide a theoretical basis for the research and treatment of pancreatic cancer in basic medicine and clinical medicine to find effective targets and relevant drugs.Methods:Bioinformatics analysis was performed through THE CANCER GENOME ATLAS(TCGA)and GENE EXPRESSION OMNIBUS(CEO)databases to preliminarily identify the pro-carcinogenic role of CAV2 in pancreatic cancer and its potential target Wnt7 A.q RT-PCR studies were performed on the expression of CAV2 in multiple pancreatic cancer cell lines,and the two cell lines with the highest expression were selected for subsequent experiments,namely: PAC-1 and BXPC-3.The two cell lines with the highest expression levels were selected for subsequent experiments,namely PAC-1 and BXPC-3,and Western Blot was used to verify the difference in protein expression between HPC-Y5 and the two pancreatic cancer cell lines.The pancreatic cancer cell lines were stably transfected by lentiviral vector to overexpress the CAV2 gene.After successful transfection,the experimental groups were divided into three groups: the original pancreatic cancer cells without any treatment,the pancreatic cancer cells overexpressing the CAV2 gene,and the control group of pancreatic cancer cells transfected with the empty vector lentivirus.The effect of CAV2 on the proliferation,invasion and metastasis of pancreatic cancer cells was verified by scratch healing assay,CCK-8 assay,Trans-Well assay and plate cloning assay.The expression of Wnt7 A and β-Catenin in the target cell lines was verified by applying q RT-PCR,Western Blot and immunofluorescence techniques,and CAV2 was verified as a positive regulator of Wnt7A/β-Catenin.Bioinformatics analysis,CCK-8 assay,q RT-PCR,and immunoluciferase assay showed that MiR-4723 was a negative regulator of Wnt7A/β-Catenin.To further investigate the mechanism of action of CAV2 on the effect of MiR-4723/Wnt7 A,the gene expression of CAV2 was detected by q RT-PCR in pancreatic cancer cells after overexpression of MiR-4723 by lentivirus.The expression of a series of several genes related to endocytosis and epithelial-mesenchymal transition(EMT)was subsequently analysed by immunofluorescence techniques,Western Blot and q RT-PCR.Results:1.Analysis of the TCGA and GEO databases revealed that CAV2 gene expression was low in normal pancreatic tissues and high in pancreatic cancer tissues.Moreover,in pancreatic cancer patients,the expression of CAV2 increased with age and its prognosis decreased with its increasing expression.In the TNM stage,it was also shown that CAV2 expression was higher in T3 and T4 stages than in T1 and 2stages.The expression of CAV2 was known to be highest in PAC-1 and BXPC-3 in pancreatic cancer multistage cells by q RT-PCR assay and in Western Blot assay showed that the expression of CAV2 was significantly higher in pancreatic cancer cell lines than in normal pancreatic cells HPC-Y5.2.In wound healing assay,CCK-8assay,Trans-Well assay and colony-forming assay,the ability of pancreatic cancer cells overexpressing CAV2 to proliferate,invade and metastasize was significantly increased compared to controls.3.The TCGA database was screened for expression trends of 182 pancreatic cancers and several related genes.The results from these analyses showed a significantly higher correlation trend for the expression of CAV2 and Wnt7A in pancreatic cancer tissues and their paracancerous tissues compared to other gene combinations.To verify the correlation,results were again obtained by q RT-PCR,and gene expression of Wnt7 A and β-catenin was significantly increased in pancreatic cancer cells overexpressing CAV2.Western Blot experiments showed that protein expression of Wnt7 A and β-catenin was significantly increased in pancreatic cancer cells overexpressing CAV2,and their grey scale values were also statistically significant.The intranuclear expression level of β-catenin was also found to be significantly higher in CAV2 overexpressing pancreatic cancer cells compared to the two control groups by immunofluorescence technique.4.According to the analysis of the GEO database,MiR-4723 was significantly downregulated in pancreatic cancer tumour tissues and the survival rate of pancreatic cancer patients was higher in the MiR-4723 high expression group compared to the corresponding low expression group.The proliferative capacity of MiR-4723 was examined by lentiviral overexpression in pancreatic cancer cells using CCK-8 technology,and we found that the value-added capacity of MiR-4723 was reduced after its upregulation compared to the control group.To further verify the relationship between MiR-4723 and Wnt7A/β-catenin,q RT-PCR was performed and it was concluded that the expression of Wnt7 A was down-regulated in pancreatic cancer cells after MiR-4723up-regulation.And the immunoluciferase assay showed that the luciferase activity of wild-type Wnt7 A was significantly reduced after overexpression of MiR-4723 in both pancreatic cancer cell lines.5.q RT-PCR was applied to conclude that gene expression of CAV2 was significantly down-regulated in pancreatic cancer cell lines overexpressing MiR-4723 compared to control cells.6.To explore the effect of CAV2 on the MiR-4723/Wnt7 A pathway,bioinformatics analysis and immunofluorescence assay techniques were used to obtain results that the expression level of EGFR was significantly upregulated in the nuclei of pancreatic cancer cells overexpressing CAV2 compared to the control group and the correlation between the expression trends of CAV2 and EGFR was significantly higher in pancreatic cancer and its paraneoplastic tissues,and the expression relationship between the two showed a positive correlation.The results of q RT-PCR and Western Blot assays for a range of genes related to endocytosis and EMT showed that PDGFR and EGFR protein and gene expression were significantly reduced in pancreatic cancer cells overexpressing MiR-4723 and significantly increased in cells overexpressing CAV2 or Wnt7 A.Wnt7A protein and gene expression was reduced in cells overexpressing MiR-4723 and increased in cells overexpressing CAV2.Wnt7 A protein and gene expression were reduced in pancreatic cancer cells overexpressing MiR-4723 and increased in cells overexpressing CAV2.In addition,CAV2 protein expression was reduced in MiR-4723 overexpressing pancreatic cancer cells and increased in Wnt7 A overexpressing cells.Expression of PAR3 protein and gene was significantly increased in pancreatic cancer cells overexpressing MiR-4723,while PAR3 protein and gene expression was decreased in cells overexpressing CAV2 or Wnt7A;Claudin-6 protein and gene expression was increased in pancreatic cancer cells overexpressing MiR-4723,while CAV2 or Wnt7 A cells overexpressing E-cadherin protein and gene expression were significantly increased in pancreatic cancer cells overexpressing MiR-4723,but decreased in cells overexpressing CAV2 or Wnt7A;Vimentin and Snail protein and gene expression were decreased in pancreatic cancer cells overexpressing MiR-4723,but decreased in cells overexpressing CAV2 or Wnt7 A in pancreatic cancer cells.Conclusions:1.The CAV2 gene is highly expressed in pancreatic cancer cells and pancreatic cancer tissues,and is closely associated with the survival prognosis of patients and the TNM stage of cancers.2.Overexpression of CAV2 in vitro significantly promotes malignant biological behaviours such as proliferation,invasion and metastasis of pancreatic cancer cells.3.The potential target of CAV2 gene is Wnt7 A and can activate Wnt7A/β-catenin signaling pathway through upregulation of CAV2.4.MiR-4723 is an oncogene in pancreatic cancer cells,and MiR-4723 can negatively regulate the Wnt7A/β-catenin pathway by targeting Wnt7 A,thereby inhibiting the proliferation of pancreatic cancer cells.5.Upregulation of CAV2 can inhibit MiR-4723 expression.Meanwhile,CAV2 can activate MiR-4723/Wnt7 A signaling pathway through a combination of inhibition of cellular endocytosis as well as promotion of cellular EMT,which ultimately promotes the development of pancreatic cancer cells.