MiR-342–3p is downregulated in PDAC tissues

 D. Zhu, et al. Cancer Letters 477 (2020) 107–121 Fig. 5. Dysbindin,a directtargetofmiR-342–3p, isnegatively regulatedbymiR-342–3pinPDAC.(A)Thetop5predictedcandidate miRNAs(miR-199a-5p,miR- 199b-5p,miR-182–5p,miR-377–3pandmiR-342–3p)targetingdysbindinaccordingtoTargetScansoftware.(B)Predictedpairingtothetargetregioninthe3′UTRof dysbindin(top)andthefivepredictedcandidatemiRNAs(bottom).(C)WesternblotanalysisshowingdysbindinproteinlevelsinAspc-1cellstransfectedwiththe fivecandidatemiRNAmimics(miR-199a-5p,miR-199b-5p,miR-182–5p,miR-377–3pandmiR-342–3p).Thedataarepresentedasthemean ± SEM.(D)AfterPDAC cells(Aspc-1,Capan-2andBxpc-3)weretransfectedwithmiR-342–3pmimicormimiccontrol,Westernblotanalysiswasperformedtodeterminedysbindinprotein levels.Thedataarepresentedasthemean ± SEM.(E)ThreepredictedbindingsitesofmiR-342–3pinthe3′UTRofdysbindinmRNA.MiR-342–3pdecreasedthe luciferaseactivityoftheWTdysbindinreporterbutnotthemutantreporter.Thedataarepresentedasthemean ± SEM.*P < 0.05,**P < 0.01,***P < 0.001. cells but decreased in dysbindin-silenced cells, while IKKβ, IκBα and protein levels were lower in PDAC cells transfected with miR-342–3p p65 expression did not change significantly (Fig. 4B–D). These results mimic than in control cells (Fig Rottlerin. 5D). Taken together, the data sup- indicate that dysbindin promotes activation of the NF-κB signalling portedtheselectionofmiR-342–3pforfurtherexperiments.Luciferase pathway in PDAC cells. assayswereperformedtovalidatethedirectbindingofmiR-342–3pto ToinvestigatewhethertheNF-κBsignallingpathwayisinvolvedin the 3′UTR of dysbindin. As shown in Fig. 5E, we identified three pu- dysbindin-induced MDM2 upregulation, we first examined the re- tative miR-342–3p in the 3′UTR of dysbindin mRNA. Lu- lationship between this pathway and MDM2. As shown in Fig. 4E, ciferase assays showed that miR-342–3p overexpression significantly MDM2proteinlevelsdecreasedinPanc-1andAspc-1cellscomparedto suppressed the WT dysbindin 3′UTR reporter construct but not the control cells after treatment with curcumin, an inhibitor of the NF-κB mutant construct. These data suggest that dysbindin is negatively signalling pathway. Conversely, MDM2 protein levels were higher in regulated by miR-342–3p in PDAC and a direct target of miR-342–3p. Panc-1 and Capan-2 cells than in control cells after treatment with in- creasing concentrations of TNF-α, a known activator of the NF-κB sig- 3.6. MiR-342–3p is downregulated in PDAC tissues, and dysbindin is nalling pathway (Fig. 4F). To determine whether NF-κB signalling significantly negatively correlated with miR-342–3p pathway activation is required for dysbindin-induced MDM2 over- expression,weblockedtheNF-κBsignallingpathwaywithcurcuminin TheCancerGenomeAtlas(TCGA)datashowedthatinPDAC,miR- dysbindin-overexpressing cells (Aspc-1-LV-dysbindin and Bxpc-3-LV- 342–3p is downregulated (Fig. 6A), and dysbindin is upregulated dysbindin cells) and control cells. We found that MDM2 protein ex- (Fig. 6B). To validate miR-342–3p and dysbindi###http://www.glpbio.com/simage/GA11366-H-D-Leu-Thr-Arg-pNA-acetate-salt-1.png####n expression in PDAC, pression levels did not change considerably in dysbindin-over- we measured miR-342–3p expression and dysbindin mRNA levels in 8 expressing compared with control cells (Fig. 4G).

 These findings pairedPDACandadjacentnoncanceroustissuesbyqRT-PCR.Asshown indicate that the NF-κB signalling pathway can regulate MDM2 ex- inFig.6C,miR-342–3pexpressionwashigherinadjacentnoncancerous pression and that dysbindin-induced MDM2 overexpression is depen- tissues than in PDAC tissues. Conversely, dysbindin expression was dent on the NF-κB signalling pathway. higher in PDAC tissues than in adjacent noncancerous tissues. Subse- quently, we measuredmiR-342–3p expressionin three PDAC cell lines 3.5. Dysbindin, a direct target of miR-342–3p, is negatively regulated by (Panc-1 cck-8 ic50, Bxpc-3 and Aspc-1) and a normal pancreatic ductal epithelial cell line (HPDE6c-7). As shown in Fig. 6D, miR-342–3p was down- miR-342–3p in PDAC regulated in PDAC cell lines and upregulated in HPDE6c-7 cells. Dys- Because dysbindin plays an important role in PDAC development, bindin expression was significantly negatively correlated with miR- wewantedtodeterminehowdysbindinisregulatedinPDAC.MiRNAs 342–3p(Fig.6E).Inaddition,TCGAdatashowedthatlowmiR-342–3p have been reported to be closely involved in PDAC, but there is no expression levels were correlated with poor overall survival among publishedresearchontherelationshipbetweenmiRNAsanddysbindin. PDAC patients (Fig. 6F). 

These data indicate that miR-342–3p is Therefore, we aimed to elucidate whether miRNAs are involved in the downregulated in PDAC and that dysbindin is significantly negatively regulation of dysbindin. We predicted candidate miRNAs targeting correlated with miR-342–3p. dysbindinusingTargetScansoftware.AsshowninFig.5A,wescreened the top 5 miRNAs that potentially regulate dysbindin according to the 3.7. The antitumour effect of miR-342–3p on PDAC cell lines is decreased context++ score generated by TargetScan software. Of these, two by dysbindin overexpression, and miR-342–3p decreases MDM2 expression miRNAshadconservedtargetsitesinthe3′UTRofdysbindin,andthree had poorly conserved target sites. The predicted putative miRNA To determine the effect of miR-342–3p on PDAC metastasis and bindingsitesinthe3′UTRofdysbindinareshowninFig Paclitaxel (Taxol).5B.Basedon invasion, transwell assays were conducted with Aspc-1, Capan-2 and these data, we transfected Aspc-1 cells with five candidate miRNA Panc-1 cells transfected with mimic control or miR-342–3p mimic. As mimics (miR-199a-5p, miR-199b-5p, miR-182–5p, miR-377–3p and shown in Fig. 7A, the miR-342-3p-transfected PDAC cells (Aspc-1, miR-342–3p).AsshowninFig.5C,WesternblottingshowedthatmiR- Capan-2andPanc-1)werelessmetastaticandinvasivethanthecontrol 342–3phadthemostsignificantinhibitoryeffectondysbindin.Weused cells. We confirmed that miR-342–3p decreased dysbindin expression, differentcells(Aspc-1, Capan-2andBxpc-3)tovalidatethatdysbindin and we next wondered whether dysbindin overexpression can reverse 117