Dysbindin promotes PDAC metastasis and invasion in vitro and in vivo

 D. Zhu, et al. Cancer Letters 477 (2020) 107–121 Fig. 1. Dysbindin promotes PDAC metastasis and invasion in vitro and in vivo. (A) Western blot analysis showing dysbindin expression in PDAC and human pancreatic ductal epithelial cells. The data are presented as the mean ± SEM. (B) Western blotting and qRT-PCR analysis showing dysbindin expression in dysbindin-overexpressingcells(Aspc-1-LV-dysbindinandBxpc-3-LV-dysbindin)andcontrolcells(Aspc-1-LV-vectorandBxpc-3-LV-vector).Thedataarepresentedas themean ± SEM.(C)AfterPanc-1andCapan-2cellsweretransfectedwithLV-shDysbindin(sh1andsh2)orLV-shVector cck8 price,WesternblottingandqRT-PCRanalysis wereperformed tomeasuredysbindinexpression.Thedataarepresentedasthe mean ± SEM.(D)OverexpressionofdysbindinenhancedAspc-1andBxpc-3cell migrationandinvasion.Thedataarepresentedasthemean ± SEM.(E)DysbindinknockdowndecreasedPanc-1andCapan-2cellmigrationandinvasion.Thedata are presented as the mean ± SEM. (F) In vivo metastasis assays. 

Four stable cell lines (Aspc-1-LV-vector, Aspc-1-LV-dysbindin, Panc-1-LV-shvector and Panc-1- sh2dysbindin)wereinjectedintothetailveinofnudemice.RepresentativeH&Estainingofthelungsfromtheabovefourgroupsisshown.Scalebars:top,200μm; bottom,50μm.(G,H)Theincidenceoflungmetastasisandthenumberofmetastaticlungnodulesinnudemicefromtheabovefourgroupsafter8weeksareshown. *P < 0.05, **P < 0.01, ***P < 0.001. sequence-specifictranscriptionfactorsisinvolvedinregulatingcellular expression. growth, controlling apoptosis, and stimulating invasion/metastasis IHC staining was performed on formalin-fixed, paraffin-embedded [12,13]. The NF-κB signalling pathway is regulated by IκB and its ki- tissues. The tissue sections were heated for 1 h at 60 °C, dewaxed and nase, IκB kinase (IKK) [14–16]. rehydrated. Then, antigen retrieval was performed, and the sections MicroRNAs(miRNAs)aresmallnoncodingRNAsthatregulategene were blocked with 3% H O AWD 131-138. Subsequently, 10% goat serum was used 2 2 expression at the posttranscriptional level. MiRNAs play a crucial role to block nonspecific staining. Appropriate primary and secondary an- in diverse biological processes, such as cell growth, proliferation, de- tibodies were used, and DAB substrate was used for the chromogenic velopment and apoptosis [17,18]. MiR-342–3p is downregulated in reaction. humancancers,suchashepatocellularcarcinoma(HCC)[19]andcolon cancer [20] E-64-c. However, the relationship between dysbindin and miR- 2.3. Cell transfection 342–3p in PDAC has not been reported. OurpreviousstudyfoundthatdysbindinlevelswerehigherinPDAC AmiR-342–3pmimicandanegativecontrolmimicwerepurchased patientsthanincontrolindividualsandthatthisproteinregulatedcell from RiboBio (Guangzhou, China). siRNAs targeting dysbindin and growth via the PI3K pathway [21,22]. However, we recently de- MDM2 were synthesized by GenePharma (Shanghai, China). And the termined that dysbindin may be closely involved in PD###http://www.glpbio.com/simage/GA11233-L-NAME-hydrochloride-2.png####AC metastasis interference sequences are provided in the supplementary files. and invasion. However, the mechanisms by which dysbindin upregu- Transfections were performed with Lipofectamine 2000 according to lation and activation are involved in PDAC have not been elucidated. the manufacturer\'s instructions (Invitrogen). Thus, in this study, we investigated the signalling process leading to dysbindin upregulation and elucidated the underlying mechanism 2.4. Lentivirus through which dysbindin promotes PDAC metastasis and invasion. HEK293T were used for virus packaging with a mixture of 

2. Methods and materials pHelper 1.0 vector (packaging plasmid) and pHelper 2.0 vector (en- velopeplasmid)(GeneChem,Shanghai,China)andLipofectamine2000 2.1. Cell culture and tissue samples (Invitrogen) according to the manufacturer\'s instructions. After 48 h, thevirus-containingsupernatantswerecollected.TheshRNAsequences ThehumanPDACcelllinesAspc-1andBxpc-3werepurchasedfrom areprovidedin thesupplementaryfiles.TheGV493vector(hU6-MCS- iCell Bioscience Inc. (Shanghai). Human pancreatic ductal epithelial CBh-gcGFP-IRES-puromycin,GeneChem,Shanghai,China)wasusedto cells(HPDE6c-7)andotherPDACcelllineswereagiftfromDrQu.The generate dysbindin-shRNA. The GV341 vector (Ubi-MCS-3FLAG-SV40- cells were cultured in Dulbecco\'s modified Eagle\'s medium (DMEM, puromycin, GeneChem, Shanghai, China) was used to generate stably HyClone)supplementedwith10%foetalbovineserum(FBS,Biological transfected cells overexpressing dysbindin. An empty vector was used Industries) at 37 °C in 5% CO . asanegativecontrol.Stablecelllinesweregeneratedbyselectionwith 2 PDAC and paired adjacent normal tissues were collected from 20 2 μg/ml puromycin for 15 days. patients(XiJingHospital,Xi\'an,China)fromFebruary2018toJanuary 2019. The Ethics Committee of Xi Jing Hospital approved this study. 2.5. RNA extraction and qRT-PCR analysis 

2.2. Tissue microarray construction and immunohistochemistry TotalRNAwasextractedfromcellsandtissuesusingTRIzolReagent (Invitrogen),and1μgofRNAwasreversetranscribedtogeneratecDNA Atissuemicroarraycontaining63PDACspecimensand57adjacent using a PrimeScript™ RT Master Mix kit (Takara, Japan). mRNA ex- noncanceroustissueswasusedinthisstudy.Thepercentageofpositive pression levels were detected by qPCR using a SYBR Premix Ex Taq cellswasscoredas0(<10%),1(10–40%),2(40–60%)or3(>60%), reagent kit (Takara, Japan) following the manufacturer\'s instructions. andtheintensityoftheimmunostainingwasscoredas0(nostaining),1 Theprimersequencesareprovidedinthesupplementaryfiles.ThePCR (weak),2(moderate)or3(strong).Theproductoftheabovetwoscores conditionswereasfollows:95°Cfor30s,followedby40cyclesof95°C was used as the final immunohistochemistry (IHC) staining score. A for5sand60°Cfor30s.Bio-RadCFX96softwarewasusedtoanalyse score of 1–6represented low expression, and 7–9 wasconsidered high the PCR results.