| Objective:To explore the mechanism of the malignant biological behavior of pancreatic cancer,especially the role of WNT signaling pathway in pancreatic cancer,and observe the effects of WNT receptor Fzd7 on the stemness,drug resistance and EMT of pancreatic cancer cell lines,so as to explain the role of WNT/Fzd7 in pancreatic Ductal adenocarcinoma cancer,and provide a new direction for the treatment of pancreatic cancer.Methods:1.Expression of Fzd7 and clinical sample analysis.Oncomine database,R2 database and CCLE database were queried to analyze the expression level and survival prognosis of Fzd7 in pancreatic cancer.Using bioinformatics data as clues,the expression levels of Fzd7 and Wnt7b in pancreatic cancer cells were detected by cell assay,and the co-binding of Fzd7 and Wnt7b in living cells was detected by Co-Immunoprecipitation(Co-IP).Surgical samples of pancreatic ductal adenocarcinoma were selected for histochemical analysis to confirm that Fzd7 was highly expressed in pancreatic cancer and co-expressed with Wnt7b.According to the immunohistochemical expression level of Fzd7 in pancreatic cancer tissue samples,patients were divided into the high expression group and the low expression group.Combined with clinical follow-up data(including CT images),the correlation between the expression of Fzd7 and the occurrence time of postoperative liver metastasis and the final clinical survival time was analyzed.2.Evaluation of human pancreatic cancer cell stemness.In order to evaluate the effect of Fzd7 and Wnt7b expression levels on the stemness of pancreatic cancer cells,the sphere-forming ability of pancreatic cancer cells after si RNA targeting Fzd7 and Wnt7b intervention were observed by cell suspension culture.The proportion of CD24~+CD44~+cells representing CSC subgroup was detected by flow cytometry,and the expression level of CSC specific phenotype molecule ABCG2 was detected by Western blot.3.Evaluation of drug resistance in human pancreatic cancer cells.The expression of Fzd7 was up-regulated and down-regulated and then treated with anticancer drugs.Adopting MTT to measure IC of gem,adopting Flow cytometry and TUNEL to detect the changes in the proportion of apoptotic cells and necrotic cell subgroup.The expression of ABCG2,Fzd7,Wnt7b was detected by Western blot.The effect of CSC on the resistance of pancreatic cancer cells was analyzed.4.The analysis of the correlation between Fzd7 and EMT of PDAC cells.Using the Wound healing experiment and Transwell experiment,evaluate the changes of the ability of the cell migration and invasion of the PDAC cells after downregulation Fzd7,Western blot was used to detect the expressions of mesenchymal phenotype proteins.The ability of migration and invasion capacity and EMT relating phenotype of the sphere forming cells were also detected,so the influence of CSC on the EMT of pancreatic cancer cell migration was analyzed.Results:1.Expression of Fzd7 and clinical analysis of human pancreatic ductal adenocarcinomaOncomine database information showed that among 11 Frizzled Receptors,Fzd7 is most expressed in pancreatic cancer compared to adjacent normal pancreatic tissue.Analysis on CCLE data indicated that Fzd7 had a significant co-expression correlation with Wnt7b,one of the 19 WNT proteins,and R2 database information indicated that the correlation coefficient between the two was 0.548 in pancreatic malignancies.Fzd7and Mesenchymal phenotypic expression,TGF-β/SMAD3 signaling pathway molecular expression,had high correlation coefficients,which was statistically significant.It provides the big data background support for the follow-up research.Different levels of Fzd7 and Wnt7b expression were detected in all 4 pancreatic cancer cells,and co-IP assay confirmed the co-binding of Fzd7 and Wnt7b in living cells.In the surgically resected pancreatic ductal adenocarcinoma tissues,immunohistochemical staining confirmed that more than 70%of the pancreatic cancer tissues showed different levels of Fzd7 expression,which was significantly different from normal paracancer tissues.At the same time,Fzd7 and Wnt7b had obvious co-positive stain.2.Evaluation of human pancreatic cancer cell stemnessSuspension culture of pancreatic cancer cells in specific medium is a common method to evaluate the stemness of the cell population,which is that the stronger the pellet-forming ability of cells means the stronger the stemness.In our experiment,we used si RNA to overexpress or decrease the expression of Fzd7 and Wnt7b in pancreatic cancer cells at first.In the following pellet-forming culture,we found that the expression level of Fzd7 and Wnt7b was positively correlated with the pellet-forming ability of the cell population.We also used flow cytometry to detect the proportion of CD24~+CD44~+cell subgroup in the total cell population.Many cell surface molecules such as CD24 and CD44,including ABCG2,were considered stem cell-specific phenotype molecules.The results indicated that,following the down-regulation of Fzd7and Wnt7b expression,the proportion of CD24~+CD44~+cell subgroup in the whole cell population decreased,and the expression of ABCG2 as a stem cell phenotype in many studies also decreased after the down-regulation of Fzd7 and Wnt7b expression.However,the cell spheres-forming by suspension culture of cancer cells were blown away again for adherant culture.It was found that the proportion of CD24~+CD44~+cell subgroup and ABCG2 levels in such cells enriching with CSC of pancreatic cancer cells were significantly higher than those in normal cancer cells,and Fzd7 and Wnt7b was also increased simultaneously.It is reasonable to speculate that Fzd7/Wnt7b plays an important role on cell stemness.3.Evaluation of drug resistance of human pancreatic cancer cellsWe used gemcitabine,a first-line chemotherapy drug for pancreatic cancer,as the intervention factor,and used flow cytometry and TUNEL technology to observe the efficacy of the intervention.The results showed that the proportion of necrotic and apoptotic cells after gemcitabine treatment was significantly increased after Fzd7/Wnt7b expression down-regulated.The gemcitabine resistant cells of SW1990 were induced with gemcitabine,and Western blot showed that ABCG2 level was significantly higher than that of normal culturing cells,and the proportion of CD24~+CD44~+cell subgroup in gemcitabine resistant cells was also significantly higher than that of normal culturing cells.4.Fzd7 and EMT in human pancreatic cancer cellsIn Wound-healing assay and Transwell assay,which reflecting the migration and invasion ability of cancer cells,the results both support that the expression level of Fzd7 is positively correlated with the migration and invasion ability of pancreatic cancer cells,with statistical significance.However,we blew the cell spheres formed in the suspension culture again for Wound-healing assay and Transwell assay founding that such sphere-forming cells showed stronger migration and invasion ability than normal adherent culture cells.In combination with the statistically significant positive correlation between Fzd7 expression and cell population stemness in the second part of the previous experiment,we speculated that the improvement of EMT degree by Fzd7was realized through CSC proportion in total cell population.The analysis of the correlation between Fzd7 and EMT of PDAC cells.Using the Wound healing experiment and Transwell experiment,evaluate the changes of the ability of the cell migration of the PDAC cells after downregulation Fzd7,Western blot was used to detect the expression of mesenchymal phenotype.The migration invasion capacity and EMT phenotype of the sphere forming cells were also detected,so the influence of CSC on the EMT of pancreatic cancer cell migration was analyzed.Conclusions:Fzd7 is highly expressed in human PDAC.Fzd7 high expression can enhance the stemness,drug resistance and EMT degree of human PDAC cells.Fzd7 can activate the canonical WNT/β-catenin pathway to enlarge cancer stem cell subgroup to affect the drug resistance and EMT of pancreatic cancer cells... |
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