MiR-216b Targeting SDCBP Regulates Pancreatic Cancer Proliferation,Migration And Epithelial-mesenchymal Transformation

Objective:Tumor invasion and metastasis is the greatest challenge impeding effective therapy of cancer and the leading cause of cancer-associated morbidity in pancreatic cacner(PC).Defining the genes and pathways that govern migation and metastasis can provide new targets for therapeutic intervention.In previous studies,we identified 161 differentially expressed proteins(DEPs)through secretory proteomics in two homologous PC cell lines with completely different in invasion and metastasis abilities(PC-1.0 vs PC-1).Further verification revealed SDCBP was significantly overexpressed in high metastasis PC cells(PC-1.0).SDCBP is an established Syndecan binding protein that functions as an adaptor protein.Expression of SDCBP is elevated in a wide-range of cancers and closely associated with tumor metastasis.But its role in PC remains unknown.Therefore,we aim to explore the roles of SDCBP in PC and its molecular regulatory mechanism,and to provide a new target for the treatment.Methods:1.Employing proteomic analysis,we identified 161 DEPs with a ≥1.5-fold difference in expression between PC-1.0 with high metastasis and PC-1 with less metastasis.To comprehensively clarify their roles in PC,we detected their expression and prognosis through bioinformatic analysis.The expression of DEPs was validated in multiple PC databases,and their prognosis was verified by survival analysis,univariate Cox analysis and multivariate Cox analysis,respectively.2.The expression of SDCBP in PC was verified by immunohistochemistry,Western blot and qPCR.The effect of SDCBP on prognosis was verified by survival analysis.Univariate Cox analysis and multivariate Cox analysis were used to explore the relationship between SDCBP and clinicopathological parameters.3.The lentivirus of SDCBP knockdown and overexpression plasmid were constructed.We detected cell changes in proliferation,clonal formation,migration and invasion through CCK8 assay,clone formation assay,Transwell assay and wound-healing assay after SDCBP knockdown or overexpression,respectively.Based on the functional prediction of GSEA analysis,we investigated the relationship between SDCBP and PI3K/AKT pathway and epithelial-mesenchymal transformation(EMT)by Western blot.4.The upstream micrornas(miRNAs)of SDCBP was predicted by TargetScan and miRNet database,and their expression was validated in TCGA database.Only miRNAs with low expression in PC were selected for further analysis.Then,we validated their correlation between miRNAs and SDCBP via co-expression analysis.Additionally,we verified miR-216b expression in PC tissues and evaluated its correlation with SDCBP expression.Next,we predicted the binding sites of miR-216b and SDCBP by TargetScan database,and constructed the SDCBP wild-type and mutant plasmids,and verified the direct binding by luciferase reporter assay.5.The mimics(agomiR-216b)and inhibitors(antagomiR-216b)of miR-216b were constructed.We detected cell changes in proliferation,clonal formation,migration and invasion using CCK8 assay,clone formation assay,Transwell assay and wound-healing assay after agomiR-216b and antagomiR-216b transfection,respectively.Furthermore,western blot was applied to detect the changes of PI3K/AKT pathway and EMT-related proteins.Moreover,antagomiR-216b was simultaneously co-transfected into SDCBP knockdown cells,and the changes of proliferation,invasion,migration,PI3K/AKT pathway and EMT were detected by the above experiments.6.Finally,we observed the effects of miR-216b and SDCBP on tumorigenesis in nude mice.The difference in tumor growth was observed in four groups including,NC,agomiR-216b,antagomiR-216b,antagomiR-216b+SDCBP knockdown through tumor xenografts experiment.Results:1.There were 21 highly expressed DEPs among TCGA_PAAD,GSE62165 and GSE15471 database.Univariate Cox analysis and multivariate Cox analysis showed that only 3 DEPs were associated with prognosis of pancreatic cancer.Patients with high expression of SDCBP had a poor prognosis.We defined SDCBP as an independent risk factor for PC prognosis with the hazard ratio of univariate Cox and multivariate Cox of 1.349(1.15-1.58)and 1.763(1.19-2.61)separately.GSEA functional enrichment analysis showed that SDCBP was related to cell adhesion and extracellular matrix and was more frequently enriched in Jak/STAT and PI3K/Akt pathways.2.The expression of SDCBP in 61 pairs of PC tissues was significantly higher than that in normal pancreatic tissues(67.2%vs 29.5%,P<0.05).High expression of SDCBP is associated with poor prognosis and advanced TNM staging and is a risk factor for PC prognosis.HR and 95%confidence interval(CI)of univariate Cox analysis were 2.379(1.486-4.348),P<0.001;the HR and 95%CI of multivariate Cox analysis were 2.177(1.294-3.475),p<0.001,indicating that SDCBP was an independent risk predictor of PC prognosis.3.The expression of SDCBP in PC cells was significantly higher than that in normal pancreatic cells.Knockdown of SDCBP in high expression PC cells could significantly inhibit PC proliferation,clonal formation,invasion and migration.Overexpression of SDCBP significantly promoted the proliferation,invasion and migration of cancer cells.After SDCBP knockdown,the expression of phosphorylated PI3K,phosphorylated AKT and N-cadherin was significantly decreased,while the expression of e-cadherin was significantly increased,while the expression of PI3K and AKT was not changed.These results suggest that SDCBP induces PC epithelial-mesenchymal transformation through the PI3K/AKT pathway,thereby regulating cancer proliferation,migration and invasion.4.We validated 3 miRNAs interacting with SDCBP through TargetScan,miRNet and TCGA PAAD database.Only miR-216b was identified as significantly negative correlation with SDCBP expression(R=-0.34,P<0.001)through co-expression analysis.The low expression of miR-216b was associated with a dismal prognosis in PC(P=0.016).Additionally,miR-216b expression was verified in PC tissues.The expression of miR-216b was relatively low both in PC tissues and cells.There was a negative correlation between miR-216b and SDCBP expression,and the relation index was-0.50,P=0.009.We predicted the binding site of miR-216b to SDCBP.Luciferase reporter assay indicated that miR-216b reduced luciferase activity of wild-type SDCBP,while had no change in SDCBP mutation.And WB results suggested that miR-216b can significantly inhibit the expression of SDCBP protein.5.We transfected PC cells with miR-216b mimics(agomiR-216b)and inhibitors(antagomiR-216b).The results indicated that agomiR-216b transfection significantly inhibited PC proliferation,clonal formation,migration and invasion.While antagomiR-216b transfection promoted the proliferation,invasion and migration of cancer cells.Co-transfection of antagomiR-216b and SDCBP knockdown could counteract the promoting effect of miR-216b inhibitor on the proliferation,migration and invasion.WB results showed that agomiR-216b transfection could decrease the expression of phosphorylated PI3K,phosphorylated AKT and N-cadherin,and increase the expression of e-cadherin,without changing the expression of PI3K and AKT.While the effect of antagomiR-216b transfection was opposite.The co-transfection of antagomiR-216b and SDCBP knockdown restored the effect of miR-216b inhibitor on PI3K/AKT and EMT proteins.These results indicate that miR-216b targeting with SDCBP induce EMT through PI3K/AKT pathway,thus regulating PC proliferation,migration and invasion.6.Tumor volume and weight were dramatically increased and decreased after antagomiR-216b and agomiR-216b transfection,respectively.And tumor volume and weight with antagomiR-216b promotion were significantly reduced after SDCBP knockdown.Those findings revealed that miR-216b directly targeted with SDCBP to regulate tumorigenesis.Conclusion:1.SDCBP is widely over expression in PC.High expression of SDCBP is significantly associated with poor prognosis and advanced TNM stage,which is an independent risk factor for PC prognosis.Enrichment analysis suggested that SDCBP might involve in EMT process and PI3K/Akt pathways.2.SDCBP konckdown can inhibit PC proliferation,migration and invasion,while SDCBP overexpression promote the proliferation,migration and invasion of cancer cells.SDCBP can activate the phosphorylation of PI3K and AKT,affecting the expression of N cadherin and E cadherin,and inducing the occurrence of EMT in PC.These results indicate that SDCBP induces EMT through PI3K/AKT pathway,and thus promotes PC proliferation,invasion and migration.3.The miR-216b is negatively correlated with SDCBP expression and can bind to SDCBP 3’-UTR sequence to regulate its expression.As a tumor suppressor miRNA,miR-216b overexpression can inhibit PC proliferation,migration,invasion,and impede EMT process through inhibiting the PI3K/AKT pathway.While miR-216b interference promotes PC progression and induces EMT through the PI3K/AKT pathway.And the promotion effect of antagomir-216b on PC could be offset by SDCBP knockdown.4.In vivo experiments,tumor growth in nude mice was also increased or decreased with antagomiR-216b and agomiR-216b transfection,and SDCBP knockdown counteracted the promoting effect of antagomiR-216b on tumor weight and volume.These results indicate that miR-216b can directly interact on SDCBP and induce PC EMT via PI3K/AKT pathway,promoting the proliferation,invasion and migration of cancer cells.