FBXO22 Promotes Pancreatic Cancer Progression By Targeting MCM5

Objective:To analyze and screen out FBXO family genes related to cell cycle regulation in pancreatic cancer.Methods:RNA sequencing results of pancreatic and pancreatic cancer tissues were obtained from TCGA and GTEx databases.The genes related to cell cycle were obtained from the MSigDB website.The prognostic risk models of pancreatic cancer were established by univariate Cox regression analysis.LASSO analysis and multivariate Cox regression analysis were used to screen cell cycle genes associated with pancreatic cancer prognosis and to construct risk score models.Pancreatic cancer patients were divided into high risk subgroup and low risk subgroup according to the median risk score.The intersubgroup differentially expressed genes were obtained by using R language limma and intersected with FBXO family,then FBXO22 and FBXO47 were obtained.The expression of FBXO22 and FBXO47 in pancreatic and paracancular tissues was analyzed by GEPIA website.Pancreatic cancer patients were divided into FBXO22 high expression subgroup and FBXO22 low expression subgroup according to the median expression of FBXO22 in the GSE57495 data set.Kaplan-Meier survival curve was used to analyze the predictive efficacy of the model.KEGG pathway enrichment analysis of FBXO22 low expression subgroup and FBXO22 high expression subgroup were analyzed by GSEA analysis.Moreover,the enrichment of FBXO22 high expression group genes in CELL_CYCLE pathway was analyzed.Results:Pancreatic cancer risk prediction model was constructed by cell cycle gene,and pancreatic cancer patients in the TCGA database were divided into high and low risk subgroups.The results of Kaplan-Meier survival analysis showed that the high-risk subgroup of pancreatic cancer patients had a lower five-year survival rate than the lowrisk subgroup.ROC curve evaluation results showed that the 1-year,3-year and 5-year predictive efficacy of this model in pancreatic cancer patients were 0.725,0.772 and 0.858,respectively.FBXO22 and FBXO47 were obtained by taking the intersection of differentially expressed genes between subgroups and FBXO family genes.The expression of FBXO47 was relatively low in pancreatic cancer and adjacent tissues,and the expression of FBXO22 in pancreatic cancer tissues was higher than that in adjacent tissues by GEPIA website.The results of Kaplan-Meier survival curve analysis showed that the overall survival rate of the high expression group of FBXO22 was lower than that of the low expression group of FBXO22.Enrichment analysis of KEGG pathway was performed for low expression group of FBXO22 and high expression group of FBXO22,and the results of KEGG pathways showed that amino acyl_trna_biosynthesis,BASE_EXCISION_REPAIR,CELL_CYCLE and other pathways were found.FBXO22 high expression group genes were obviously enriched in CELL_CYCLE by GSEA analysis.Conclusion:FBXO22 high expression group genes were enriched in CELL_CYCLE pathway.Objective:To explore the expression of FBXO22 in pancreatic cancer tissues and cells,and examine its biological effect on pancreatic cancer cells.Methods:The effects of FBXO22 on overall survival and disease-free survival of pancreatic cancer were analyzed by GEPIA website.The mRNA expression of FBXO22 in pancreatic duct epithelial cells hTERT-HPNE and pancreatic cancer cell lines AsPC-1,BxPc-3,Capan-2,CFPAC-1,PANC-1,MIA PaCa-2 were detected by qRT-PCR.Western blot assays were used to detect the protein expression of FBXO22 in pancreatic ductal epithelial cells hTERT-HPNE and pancreatic cancer cell lines.The expression of FBXO22 in pancreatic and paracancer tissues was determined by immunohistochemistry.The relationship between the expression of FBXO22 and clinicopathologic features of pancreatic cancer was analyzed.To further explore the effect of FBXO22 on pancreatic cancer cells,FBXO22 overexpression lentivirus and FBXO22 RNA interference lentivirus were constructed,and the lentivirus were transfected into pancreatic cancer cells PANC-1 and MIA PaCa-2.Pancreatic cancer cells were divided into five groups:Vector group,FBXO22 group(FBXO22 overexpression group),shControl group,shFBXO22#1 group and shFBXO22#2 group.The transfection efficiency of lentivirus was verified by qRT-PCR and western blot assays.The effects of FBXO22 on the proliferation of pancreatic cancer cells were verified by colony formation assays,CCK8 assays and EdU staining assays.The effects of FBXO22 on the migration and invasion ability of pancreatic cancer cells were verified by wound healing assays,transwell migration and invasion assays.The differentially expressed genes of MIA PaCa-2 cells in Vector group and FBXO22(overexpressed FBXO22)group were screened by transcriptome sequencing.GO enrichment analysis and KEGG enrichment analysis were performed to analyze differentially expressed genes.The results of enrichment analysis showed that FBXO22 was related to PI3K/Akt signaling pathway.Western blot assays were used to detect the expression of Akt and p-Akt in different groups.The effect of FBXO22 on pancreatic cancer cells in vivo was detected by subcutaneous tumor formation in nude mice.Results:The results above indicate that FBXO22 is associated with the prognosis of pancreatic cancer.The results of GEPIA analysis showed that overall survival and disease-free survival were lower in the FBXO22 high expression group than in the FBXO22 low expression group.The results of qPCR and western blot assays showed that the mRNA and protein expression of FBXO22 in pancreatic cancer cell lines BxPc3,Capan-2,CFPAC-1,PANC-1,MIA PaCa-2 were higher than those in pancreatic ductal epithelial cells hTERT-HPNE.The results of immunohistochemical showed that the expression of FBXO22 in pancreatic cancer tissues was higher than that in paracancer tissues.The results of tissue microarray showed that the expression of FBXO22 in pancreatic cancer tissue was higher than that in paracancer tissue,and the expression of FBXO22 was correlated with clinicopathologic staging and M staging.To explore the effect of FBXO22 on pancreatic cancer cells,pancreatic cancer cells were divided into Vector group,FBXO22 group(FBXO22 overexpression group),shControl group,shFBXO22#1 group,and shFBXO22#2 group.The results of colony formation assays,CCK8 assays and EdU staining assays showed that the proliferation rate of pancreatic cancer cells in FBXO22 group was significantly higher than that in Vector group,and the proliferation rate of pancreatic cancer cells in shFBXO22#1 and shFBXO22#2 group was significantly lower than that in shControl group.The results of wound healing assays,transwell migration and invasion assays showed that the migration and invasion ability of FBXO22 group was significantly stronger than that of Vector group,and the migration and invasion ability of shFBXO22#1 and shFBXO22#2 group was significantly weaker than that of shControl group.The results of GO enrichment analysis showed that FBXO22 was involved in biological processes such as cell adhesion,cell components were related to calmodulin binding,and molecular function was related to collagen Ⅱ-containing extracellular matrix.The results of KEGG enrichment analysis showed that FBXO22 is involved in many pathways,such as Metabolic pathways and PI3K-Akt signaling pathways.The results of western blot assays showed that FBXO22 could activate PI3K-Akt signaling pathway.The results of subcutaneous tumor formation in nude mice showed that high expression of FBXO22 can promote the proliferation of PANC-1 cells in vivo.Conclusion:FBXO22 promotes pancreatic cancer progression by activating the PI3K-Akt signaling pathway.Objective:To further study the molecular mechanism of FBXO22 promoting malignant progression of pancreatic cancer,providing a new direction for biomarkers and therapeutic targets.Methods:Immunoprecipitation combined with mass spectrometry was used to screen and identify MCM5,the interaction target of FBXO22.Western blot assays were used to determine the expression of MCM5 in pancreatic cancer cells.Co-localization of FBXO22 and MCM5 in pancreatic cancer cells was determined by immunofluorescence assays.Pancreatic cancer cells were divided into three groups:Vector group,FBXO22 group(FBXO22 overexpressed group),and FBXO22+siMCM5 group(FBXO22 overexpressed and MCM5 knock down group).Colony formation assays,CCK8 assays and EdU staining assays were used to verify the effect of MCM5 knockdown on FBXO22 promoting the proliferation of pancreatic cancer cells.Wound healing assays,transwell migration and invasion assays were used to explore the effect of MCM5 knockdown on FBXO22’s ability to promote the migration and invasion of pancreatic cancer cells PANC-1 and MIA PaCa-2.The expression of Akt and p-Akt in different experimental groups was detected by western blot assays.Results:In order to further investigate the mechanism of role of FBXO22 in pancreatic cancer cells,the targets of FBXO22 were detected by mass spectrometry,and the results showed that MCM5 may be the target of FBXO22.The results of coimmunoprecipitation showed that FBXO22 could interact with MCM5 in MIA PaCa-2 cells.The results of western blot assays showed that MCM5 expression was increased when FBXO22 was higher expressed in pancreatic cancer cells.The results of immunofluorescence experiments showed that FBXO22 and MCM5 were mainly localized in the nucleus of pancreatic cancer cells.To further explore the mechanisms of FBXO22 in pancreatic cancer cells,pancreatic cancer cells were divided into Vector group,FBXO22 group(FBXO22 overexpression group),and FBXO22+si-MCM5 group(FBXO22 overexpressed and MCM5 knock down group).The results of colony formation assays,CCK8 assays and EdU staining assays showed that the decreased expression of MCM5 in pancreatic cancer cells inhibited the ability of FBXO22 to promote the proliferation of pancreatic cancer cells.The results of wound healing assays,transwell migration and invasion assays showed that decreased MCM5 expression in pancreatic cancer cells inhibited the ability of FBXO22 to promote the migration and invasion of pancreatic cancer cells.The results of western blot assays showed that the decreased expression of MCM5 in pancreatic cancer cells could inhibit the activation of Akt.Conclusion:FBXO22 activates the PI3K/Akt signaling pathway by promoting MCM5 expression,thereby promoting the proliferation,migration,and invasion of pancreatic cancer cells.