Dysbindin over expression increases MDM2 expression in PDAC ,and MDM2 knock down significantly attenuates dysbindin

 D. Zhu, et al. Cancer Letters 477 (2020) 107–121 Fig.2. DysbindinoverexpressionincreasesMDM2expressioninPDAC,andMDM2knockdownsignificantlyattenuatesdysbindin-enhancedmetastasisandinvasion. (A)Aheatmapwasgeneratedfromthewhole-transcripthumangeneexpressionarrayanalysisoftotalRNAextractedfromthePanc-1-dysbindin-siRNAandcontrol groups.Redrepresentsrelativelylowerexpression,andgreenindicatesrelativelyhigherexpression.(B)WesternblotanalysisshowingMDM2anddysbindinprotein levels in dysbindin-overexpressing (Aspc-1-LV-dysbindin and Bxpc-3-LV-dysbindin) and control cells (Aspc-1-LV-vector and Bxpc-3-LV-vector). The data are presentedasthemean ± SEM.(C)WesternblotanalysisshowingMDM2anddysbindinproteinlevelsindysbindin-knockdowncells(Panc-1-dysbindin-siRNAand Capan-2-dysbindin-siRNA)andcontrolcells(Panc-1-control-siRNAandCapan-2-control-siRNA).Thedataarepresentedasthemean ± SEM.(D)AfterCapan-2and Bxpc-3cellsweretransfectedwithMDM2-siRNAorcontrol-siRNA cck8,WesternblottingandqRT-PCRanalysiswereperformedtodetermineMDM2expression.Thedata arepresentedasthemean ± SEM.(E)MDM2knockdowndecreasedCapan-2andBxpc-3cellmigrationandinvasion.Thedataarepresentedasthemean ± SEM. (F)Afterdysbindin-overexpressingcells(Aspc-1-LV-dysbindinandBxpc-3-LV-dysbindin)andcontrolcells(Aspc-1-LV-vectorandBxpc-3-LV-vector)weretransfected withMDM2-siRNAorcontrol-siRNA,WesternblotanalysiswasperformedtodetermineMDM2anddysbindinproteinlevels.(G,H)MDM2knockdownattenuated dysbindin-enhanced metastasis and invasion. The data are presented as the mean ± SEM. **P < 0.01, ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 2.6. Western blot analysis and regents medium in the upper chamber (Millipore POM 1, USA), and medium con- taining20%FBSwasplacedinthebottomchamber.After24–48h,the Total protein lysates were harvested from PDAC cells using RIPA cellsontheuppersideofthemembranewereremovedcarefullywitha buffersupplementedwithproteinaseandphosphataseinhibitors.Total cotton swab, and those in the lower side of the membrane were fixed protein was quantified using a Pierce BCA Protein Assay Kit (Thermo with paraformaldehyde and stained with a crystal violet solution. Scientific, USA). Total protein (30 μg) was separated by 10–12% SDS- To investigate cell invasion, Matrigel (Sigma)-coated transwell 4 PAGE and transferred to polyvinylidene fluoride (PVDF) membranes chambers(Millipore,USA)wereused,and5×10 cellswereseededin (Millipore, USA), which were blocked in 5% nonfat milk for 1–2 h at serum-freemediumintheupperchamber.

After48–72hofincubation, room temperature and then incubated overnight with the following the cells were treated as previously described. primaryantibodies: dysbindinantibody(Abcam,monoclonal, 1:1000), MDM2 antibody (CST, monoclonal, 1:1000), IKKβ antibody (CST, monoclonal, 1:1000), phospho-IKKβ antibody (CST, monoclonal, 2.9. Animal experiments 1:1000), IκBα antibody (CST, monoclonal, 1:###http://www.glpbio.com/simage/GA11233-L-NAME-hydrochloride-3.png####1000), phospho-IκBα an- tibody (CST, monoclonal, 1:1000), NF-κB p65 antibody (CST, mono- A tail vein injection assay was performed to evaluate the in vivo clonal, 1:1000), phospho–NF–κB p65 antibody (CST, monoclonal, metastatic ability of dysbindin. A total of thirty-two male nude mice 1:1000) cck8 storage,andGAPDHantibody(CST,monoclonal,1:2000).Onthenext weredividedintofourgroupsof8ratseach:theAspc-1-vector,Aspc-1- day,themembraneswereincubatedwithsecondaryantibodiesfor1h. dysbindin, Panc-1-vector and Panc-1-shDysbindin groups. All nude All experiments were conducted in triplicate. PDAC cells were treated mice were housed in a specific pathogen-free (SPF) laboratory. 

Eight with curcumin (20 μM; Selleck Chemicals, USA) and increasing con- weeksaftermodelcreation,thenudemicewerekilled.Themouselungs centrations of TNF-α (10 or 20 ng/ml; Sigma-Aldrich). wereharvested,fixedwithformalinandsubjectedtohaematoxylinand eosin (H&E) staining to assess the development of metastatic nodules. AllmiceweremaintainedunderSPFconditions.Thisstudywascarried 2.7. Dual luciferase reporter gene assay outinstrictaccordancewiththerecommendationsintheGuideforthe CareandUseofLaboratoryAnimalsoftheNationalInstitutesofHealth. The 3′UTR of the human dysbindin gene was amplified using PCR All procedures performed in studies involving animals were in ac- andclonedintothepGL3vectortogeneratepGL3-dysbindin-wildtype cordance with the ethical standards of The Fourth Military Medical (WT). Themutated (Mut)dysbindin3′UTR wasconstructedusing site- University. directed mutagenesis to generate pGL3-dysbindin-Mut. Because we identified three putative miR-342–3p binding sites in the 3′UTR of dysbindin mRNA, we mutated all three putative binding sites at the 2.10. Statistical analysis sametimeandconstructedtworeporterplasmids:pGL3-dysbindin-WT and pGL3-dysbindin-Mut. HEK293T cells were grown to 70–80% con- Statistical analysis was performed using SPSS 22.0. Student\'s t-test fluenceinasix-wellplateinDMEM(HyClone)supplementedwith10% was used to analyse differences between two groups. 

Pearson\'s corre- FBS (Biological Industries) at 37 °C in 5% CO . Then, the cells were 2 lation coefficient was used to analyse the correlations involving ex- cotransfected with either pGL3-dysbindin-WT or pGL3-dysbindin-Mut pression. The Kaplan-Meier method and log-rank test were used to and miR-342–3p mimic or negative control for 48 h. The Dual analyse the overall survival rate. Correlations between MDM2 expres- Luciferase Reporter Assay System (Promega USA) was used to assess sion and clinicopathological characteristics were evaluated using the luciferase activity after 48 h of transfection according to the manufac- chi-square test. The Cox proportional hazards model was used for turer\'s instructions. univariate and multivariate analyses. P < 0.05 was considered to in- dicate statistical significance. 2.8. Cell migration and invasion assays 4 For cell migration assays, 5 × 10 cells were seeded in serum-free 111