| Objective:Pancreatic cancer is a formidable disease that often presents with insidious symptoms,limited options for radical surgery,and dismal outcomes.However,recent advances in the understanding of the molecular mechanisms underlying pancreatic cancer and the development of comprehensive therapeutic approaches,such as targeted immunotherapy,have brought a glimmer of hope,with a 5-year survival rate of 12%.Consequently,it is particularly important to explore the relevant molecular mechanisms of pancreatic cancer occurrence and development,and to find early diagnostic markers and suitable therapeutic targets for pancreatic cancer.Glia Maturation Factor Beta(GMFB),a member of the Actin Depolymerizing Factor(ADF)family,has been found to play a critical role in the progression of a variety of tumors.In our group,human tissue arrays screening suggested that GMFB was up-regulated in pancreatic cancer,but its role in pancreatic cancer is still unclear.Therefore,our study aims to elucidate the relationship between expression GMFB and the prognosis of pancreatic cancer patients,and to explore its underlying mechanism in pancreatic cancer development,with the ultimate goal of identifying novel diagnostic and therapeutic strategies for this devastating disease.Methods:1.The expression of pancreatic cancer GMFB in TCGA database were analyzed by Gene Expression Profiling Interactive Analysis(GEPIA).Meanwhile,the expression of GMFB protein in pancreatic cancer tissues and adjacent tissues in human tissue arrays was detected by immunohistochemical(IHC)staining.The relationship between the expression level of GMFB and the clinicopathological characteristics and prognosis of pancreatic cancer patients was further analyzed.Additionally,the GMFB expression levels of 4 pairs of pancreatic cancer tissue/corresponding adjacent tissues and pancreatic normal ductal epithelium(HPDE6-C7)/5 pancreatic cancer cell lines were detected using q RT-PCR and Western Blot.The localization of GMFB in PANC-1 and As PC-1 cells was detected by immunofluorescence(IF).2.GMFB overexpressing lentivirus(LV-GMFB)and interfering lentivirus(sh-GMFB #1 and sh-GMFB #2)PANC-1 and As PC-1 cells were constructed.The effects of GMFB on the proliferation,invasion,and metastasis of pancreatic cancer were detected by CCK-8,Colony formation,Ed U,Wound healing,and Transwell assays in vitro.In vivo,the effect of GMFB on the proliferation and metastasis of pancreatic cancer cells was verified by subcutaneous tumorigenesis,caudal vein metastasis,and spleen metastasis model in nude mice.HE staining was further used to evaluate lung and liver metastases,and IHC staining was used to assess the expression of proliferation markers(Ki67 and PCNA)in subcutaneous xenografts tissue.Finally,it was further predicted using GEPIA verified using Western Blot.The relationship between GMFB expression and proliferation markers,Epithelial-mesenchymal transition(EMT)markers.3.GEPIA and Gene Set Enrichment Analysis(GSEA)were used to predict the downstream signaling pathways and target genes that GMFB might regulate,and then Western Blot was used to verify.Furthermore,co-immunoprecipitation and LC-MS/MS analysis were used to identify the bridging molecule CBX4 between GMFB and β-catenin,a key molecule in Wnt/β-catenin signaling pathway.The co-localization of GMFB and CBX4 was further determined by IF.Meanwhile,the expression of CBX4 in pancreatic cancer tissues and adjacent tissues was detected by IHC and Western Blot.After knockdown of GMFB and overexpression of CBX4,overexpression of GMFB and knockdown of CBX4,or addition of Wnt/β-catenin inhibitor(XAV939),the expression of downstream effectors of Wnt/β-catenin signaling pathway was detected by Western Blot.Finally,CCK-8,Colony formation,Ed U,Wound healing,and Transwell assays were used to verify the regulatory relationship among GMFB,CBX4,and β-catenin.Results:1.The relationship between the expression level of GMFB and the prognosis of patients with pancreatic cancer.According to the IHC staining of human tissue arrays and online analysis of GEPIA database,we found that the expression level of GMFB was significantly higher in pancreatic cancer tissues compared to their corresponding adjacent tissues(P<0.05).Additionally,q RT-PCR and Western blot analyses showed higher levels of GMFB expression in four fresh pancreatic cancer tissues than in the adjacent tissues.We also observed higher levels of GMFB expression in five pancreatic cancer cell lines compared to normal pancreatic ductal epithelium(HPDE6-C7).Immunofluorescence staining further confirmed that GMFB was predominantly localized in the cytoplasm of pancreatic cancer cells.Furthermore,we scored the results of human tissue arrays and conducted Spearman correlation analysis,Kaplan-Meier analysis,Cox univariate and multivariate analysis to explore the correlation between the score data and the clinicopathological characteristics of the patients.Our results showed a positive correlation between GMFB expression and tumor differentiation(P=0.013),lymph node stage(P=0.024)and vascular invasion(P=0.037).Notably,pancreatic cancer patients with low GMFB expression had longer overall survival(OS)than those with high expression,and median survival time(low expression group: high expression group =19.4 months vs.16.3 months)(Log-rank P=0.045).In addition,univariate analysis revealed that female gender(P=0.006),TNM stage III/IV(P<0.001),T3/T4(P=0.001),N1/N2(P<0.001),M1(P<0.001),and high GMFB expression(P=0.046)were associated with poor prognosis of pancreatic cancer patients.Multivariate analysis further demonstrated that T3/T4(P=0.019),M1(P=0.001),and high GMFB expression(P=0.013)were independent prognostic factors of pancreatic cancer patients.2.GMFB promotes the proliferation,invasion,and metastasis of pancreatic cancer.To determine the role of GMFB in pancreatic cancer cell biology,we constructed stable overexpression cells(LV-NC and LV-GMFB)and knockdown cells(sh-NC,sh-GMFB#1,and sh-GMFB#2)in PANC-1 and As PC-1 cell lines.The expression of GMFB was validated by q RT-PCR and Western blot,and our results showed that LV-GMFB cells had significantly higher GMFB expression levels than LV-NC cells(P<0.01),whereas sh-GMFB#1 and sh-GMFB#2 cells had significantly lower GMFB expression levels than sh-NC cells(P<0.01).Functional assays,including CCK-8,Colony formation,Ed U,Transwell invasion and metastasis assay,and Wound healing assay,were performed to evaluate the impact of GMFB on PANC-1 and As PC-1 cell proliferation,invasion,and metastasis.Our data demonstrated that overexpression of GMFB promoted cell proliferation and invasion,while knockdown of GMFB had the opposite effects.In vivo experiments using nude mice models of subcutaneous tumorigenesis,caudal vein metastasis,and spleen metastasis further supported our findings,as knockdown of GMFB effectively inhibited the proliferation,invasion,and metastasis of pancreatic cancer cells.IHC staining revealed that the proliferation markers Ki67 and PCNA were more highly expressed in the NC groups than in the GMFB knockdown groups.We further predicted the relationship between GMFB expression and markers of proliferation and EMT using GEPIA and verified the relationship by Western Blot.Interestingly,the results revealed that up-regulated GMFB levels promoted N-Cadherin,Vimentin,Snail,Cyclin D1,CDK4,and CDK6 expression and silenced E-Cadherin expression in PANC-1 and As PC-1 cells.Conversely,GMFB knockdown inhibited the ability of PANC-1 and As PC-1 cells to acquire the EMT phenotype.3.GMFB activates CBX4/Wnt/β-catenin signaling pathway to promote the proliferation,invasion,and metastasis of pancreatic cancer.The Wnt/β-catenin signaling pathway was found to be significantly affected by the expression level of GMFB by GSEA prediction.Also GMFB was found to be positively correlated withβ-catenin,a key gene of Wnt/β-catenin signaling pathway,by GEPIA prediction(R=0.18,P=0.019).Validation by Western Blot revealed that the overexpression of GMFB promoted the expression of β-catenin in PANC-1 and As PC-1 cells,while GMFB knockdown inhibited the expression of β-catenin in PANC-1 and As PC-1 cells.A total of 72 differential proteins were further screened using immunoprecipitation techniques and LC-MS/MS analysis.The 72 target proteins were also searched and screened in the literature.Interestingly,CBX4 was found to promote β-catenin SUMOylation,inhibit its membrane localization and promote Wnt/β-catenin signaling.Therefore,we hypothesized that CBX4 may be a bridging gene between GMFB andβ-catenin.The interaction between GMFB and CBX4 was verified by co-immunoprecipitation.Further analysis using GEPIA revealed a positive correlation between GMFB and CBX4(R=0.18,P=0.015).The expression of CBX4 protein changed after overexpression or knockdown of GMFB.We also found that overexpression of CBX4 reversed the proliferation,invasion,and metastasis abilities of pancreatic cancer cells with knockdown of GMFB.Furthermore,we knocked down CBX4 or added a Wnt/β-catenin pathway inhibitor(XAV-939)based on the overexpression of GMFB As PC-1 stable cells.Through CCK-8,Colony formation,Ed U,Wound healing,and Transwell assays,it was found that knockdown of CBX4 or addition of XAV-939 inhibited the proliferation,invasion,and metastasis of pancreatic cancer As PC-1 cells.At the same time,it was found that after knockdown of CBX4 or addition of XAV-939,the expression of β-catenin protein and its target protein Cyclin D1,N-Cadherin,and Vimentin were also down-regulated.This is consistent with the results of the above functional experiments.Conclusions:1.GMFB is highly expressed in the pancreatic cancer tissues and human pancreatic cancer cell lines,and is mainly located in the cytoplasm,which is an independent predictor of the prognosis in pancreatic cancer patients.2.GMFB promotes cell proliferation,invasion and metastasis and EMT progression of PANC-1 and As PC-1 cells.3.GMFB promotes the proliferation,invasion,and metastasis of pancreatic cancer cells by activating the CBX4/Wnt/β-catenin signaling pathway. |
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