InPDACcells,dysbindinactivatestheNF-κBsignallingpathway

 D. Zhu, et al. Cancer Letters 477 (2020) 107–121 Fig.4. InPDACcells,dysbindinactivatestheNF-κBsignallingpathway,whichregulatesMDM2.Dysbindin-inducedMDM2overexpressionisdependentontheNF- κBsignallingpathway.(A)Thetop20significantlyenrichedbiologicalprocessesidentifiedbyKEGGpathwayanalysis.TheNF-κBsignallingpathwaywasoneofthe mostenrichedpathways.(B–D)WesternblotanalysisshowingtheproteinlevelsofNF-κBsignallingpathway-relatedgenes(IKKβ,p-IKKβ,IκBα,p-IκBα,p65andp- p65)indysbindin-knockdowncells(Panc-1-dysbindin-siRNAandCapan-2-dysbindin-siRNA),dysbindin-overexpressingcells(Bxpc-3-LV-dysbindin)andcontrolcells. Thedataarepresentedasthemean ± SEM.(E)AfterPDACcells(Panc-1andAspc-1)weretreatedwiththeNF-κBinhibitorcurcumin(20μM)for24h,Westernblot analysis was performed to determine MDM2 and p-p65 protein levels in the curcumin-treated and DMSO (control)-treated groups. The data are presented as the mean ± SEM. (F) AfterPDAC (Panc-1 and Capan-2) were treated with control or increasing concentrations (10or 20 ng/ml) of TNF-α Puromycin, an NF-κB signalling pathwayactivator,WesternblotanalysiswasperformedtodetermineMDM2andp-p65proteinlevelsineachgroup.Thedataarepresentedasthemean ± SEM.(G) Westernblot analysisshowingMDM2andp-p65proteinlevelsindysbindin-overexpressingcells(Aspc-1-LV-dysbindinandBxpc-3-LV-dysbindin)andcontrolcells (Aspc-1-LV-vector and Bxpc-3-LV-vector) treated with curcumin (20 μM) for 24 h cck-8 storage. The data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. PDAC metastasis and invasion, a whole-transcript human gene expres- PDAC tissue microarrays were used to visualise MDM2 expression in sion array was used to comprehensively identify differentially ex- PDAC and paired adjacent noncancerous tissues; the results showed pressed mRNAs in Panc-1 cells with and without dysbindin dysregula- higher MDM2 expression in PDAC tissues than in paired adjacent tion, with a particular focus on genes involved in cancer. Dysbindin noncancerous tissues (Fig. 3C–E). Kaplan-Meier survival curves re- downregulation substantially reduced the expression of numerous vealed that MDM2 overexpression was associated with shorter overall genes, and we selected one of the most downregulated metastasis-re- survival in PDAC patients (P < 0.05) (Fig. 3F). As shown in Table 1, lated genes, MDM2, for further analysis (Fig. 2A). MDM2 is a primary MDM2 expression was significantly correlated with PDAC tumour size inhibitor of p53, promotes tumour metastasis and is associated with and histological grade (differentiation). Cox regression analyses in- invasive ductal breast carcinoma and ovarian cancer [23,24]. Con- dicated that MDM2 overexpression and perineural invasion were in- sidering the important role of MDM2 in tumour metastasis, we won- dependent and significant predictors of prognosis in PDAC patients dered whether MDM2 participates in dysbindin-mediated PDAC me- (Table 2). These results reveal correlations of MDM2 expression with tastasis. 

As shown in Fig. 2B, dysbindin-overexpressing cells (Aspc-1- PDAC tumour size and histologi###http://www.glpbio.com/simage/GA11233-L-NAME-hydrochloride-5.png####cal grade (differentiation) and the LV-dysbindin and Bxpc-3-LV-dysbindin) had higher MDM2 expression overall survival of PDAC patients. Next, we further ascertained the as- than control (Aspc-1-LV-vector and Bxpc-3-LV-vector). Moreover, sociation between dysbindin and MDM2 expression in human PDAC MDM2 expression was lower in dysbindin-knockdown cells (Panc-1- tissues.IHCresultsshowedthatdysbindinandMDM2expressionwere dysbindin-siRNA and Capan-2-dysbindin-siRNA) than in control cells both increased in PDAC tissues but decreased in paired adjacent non- (Panc-1-control-siRNA and Capan-2-control-siRNA) (Fig. 2C). We si- cancerous tissues (Fig. 3H). Pearson\'s correlation analysis revealed a lencedMDM2expressionusingsiRNA,andtheresultingMDM2protein positivecorrelationbetweendysbindinandMDM2expressioninhuman and mRNA expression levels are shown in Fig. 2D. MDM2 knockdown PDAC tissues (r = 0.639, p < 0.01) (Fig. 3G). significantly decreased the metastasis and invasion of Capan-2 and Bxpc-3 cells (Fig Echinomycin. 2E). Dysbindin-overexpressing cells (Aspc-1-LV-dys- 3.4. Dysbindin activates the NF-κB signalling pathway in PDAC cells, and bindin and Bxpc-3-LV-dysbindin) and control cells (Aspc-1-LV-vector the dysbindin-induced overexpression of MDM2 is dependent on the NF-κB and Bxpc-3-LV-vector) were further transfected with control-siRNA or signalling pathway MDM2-siRNA(Fig.2F).WefoundthatMDM2knockdownsignificantly attenuated dysbindin-enhanced metastasis and invasion (Fig. 2G and To further study how dysbindin increases MDM2 expression in H). These results suggest that dysbindin overexpression increases PDAC, we carried out Kyoto Encyclopedia of Genes and Genomes MDM2 expression and that MDM2 knockdown decreases PDAC me- (KEGG) pathway enrichment analysis to determine the potential func- tastasisandinvasionandattenuatesdysbindin-enhancedmetastasisand tions of dysbindin. As shown in Fig. 4A, KEGG pathway analysis iden- invasion. tified 20 pathways enriched in the mRNAs dysregulated by dysbindin. Of these, one of the most enriched pathways was the NF-κB signalling 

3.3. MDM2 expression is upregulated in PDAC tissues and correlates with pathway, which is known to be involved in regulating cancer metas- clinicopathological characteristics; dysbindin expression levels are positively tasis, sowe focused on this signalling pathway. To validate the results correlated with MDM2 expression in PDAC tissues of the KEGG pathway enrichment analyses, we examined the protein levels of NF-κB signalling pathway-related genes in dysbindin-over- TofurtherdeterminetheassociationbetweenMDM2expressionand expressing cells (Bxpc-3-LV-dysbindin cells), dysbindin-knockdown PDAC,wefirstdetectedMDM2expressioninPDACandpairedadjacent cells(Panc-1-dysbindin-siRNAandCapan-2-dysbindin-siRNAcells)and noncanceroustissuesandfoundthatitwashigherinPDACtissuesthan control cells. The results showed that p-IKKβ, p-IκBα and p-p65 ex- in paired adjacent noncancerous tissues (Fig. 3A and B). Moreover, pressionlevelsweresignificantlyincreasedindysbindin-overexpressing 115

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