| BackgroundPancreatic cancer is well acknowledged as one of malignancies with extremely dismal prognosis,because the mortality is close to the incidence.The mechanisms of proliferation,invasion and metastasis of pancreatic cancer is currently unclear.Therefore,investigations concerning development and progression of pancreatic cancer are of great significance.STUB1(STIP1 homology and U-box containing protein 1)',promotes the ubiquitination and degradation of numerous crucial cancer-related proteins.Previously,we found that STUB1 suppressed pancreatic cancer cell proliferation,anchorage-independent growth,migration and invasion.However,the mechanisms of the regulation of STUB1 expression in human cells remain unknown.In the current study,we performed an screen of micro RNAs(miRNAs)to identify possible regulators of STUB1 expression and explored the function of the relative miRNAs.ObjectiveThe current study aimed to investigate the regulatory roles and mechanisms of miR-1178,which is a regulator of STUB 1.We also investigated the effects of miR-1178 on regulating proliferation,cell cycle,migration and invasion in pancreatic cancer cells.Then we studied the mechanisms of miR-1178 in regulating the progression of pancreatic cancer.In addition,we detected the expression levels of miR-1178 in pancreatic cancer tissues,and then assessed its prognostic values.MethodsLuciferase reporter assays were used to verify the candidate regulators of STUB1.For the in vitro experiments,proliferation was analyzed using a cell count kit(CCK-8).Cell-cycle analysis were performed by fluorescence-activated cell sorting(FACS)flow-cytometry.Real-time PCR was used to detect the expression levels of miRNA and mRNA.Protein levels were detected by western blot.For the in vivo experiment,BxPC-3 cells steadily expressing miR-1178 were established.Subcutaneous transplantation models of pancreatic cancer in nude mice were established to observe the tumor growth.The expression levels of miR-1178 in tissues was detected by in situ hybridization(ISH),respectively.The prognostic values of miR-1178 was assessed by univariate and multivariate survival analysis.Results1.The prediction and filtering of miRNAs in regulating STUB1 expressionWe first used 2 target prediction programs,miRBase and TargetScan,to screen for miRNAs that target STUB1.Our analysis predicted 4 potential STUB 1-targeting miRNAs,hsa-miR-4716-3p,hsa-miR-1178,hsa-miR-4690-3p and hsa-miR-556-3p.We next investigated the effects of mimics of the 4 putative miRNAs on STUB1 expression in PANC-1 cells.Mimics of hsa-miR-4716-3p,hsa-miR-1178,hsa-miR-4690-3p and hsa-miR-556-3p did not downregulate STUB1 mRNA expression.In addition,mimics of hsa-miR-4716-3p and hsa-miR-1178 downregulated STUB1 prote:in expression.We further explored whether STUB1 could be directly regulated by hsa-miR-4716-3p and hsa-miR-1178 using a luciferase assay.After co-transfection with hsa-miR-4716-3p/hsa-miR-1178 and STUB1-wild vectors,the luciferase activity was significantly decreased only in hsa-miR-1178 and STUB 1-wild vectors.This effect was completely abolished by substituting this vector with its mutant version.Taken together,these results suggest that hsa-miR-1178 inhibits STUB1 expression by targeting its 3'-UTR.2.The effects of miR-1178 on the malignant phenotype of pancreatic cancerAfter transfection with the miR-1178 mimics or the inhibitor,the expression of miR-1178 was significantly up-regulated or down-regulated,respectively.A CCK-8 assay showed that the proliferation rates of PANC-1 and BxPC-3 cells were increased after miR-1178 over-expression.In contrast,miR-1178 down-regulation inhibited the proliferation of pancreatic cancer cells.Furthermore,the up-regulation of miR-1178 promoted the G1/S checkpoint transition in both PANC-1(S phase 30.17±1.31%vs.39.31±1.51%,P<0.01)and BxPC-3(S phase 29.89 ±1.56%vs.42.57 ± 3.74%,P<0.05)cells,while the down-regulation of miR-1178 led to an elongation of the G1 phase.A transwell assay was performed to determine the role of miR-1178 in pancreatic cancer cell migration and invasion.The over-expression of miR-1178 increased the number of PANC-1 cells that penetrated the ECM-coated membrane.In contrast,the invasiveness of PANC-1 cells was significantly decreased when miR-1178 expression was suppressed.Similar results were found in BxPC-3 cells.In agreement with this finding,the migratory abilities of these two cell lines were also enhanced after the cells were treated with the miR-1178 mimics,while the opposite results were observed when the cells were treated with the miR-1178 inhibitor.For the in vivo experiment,overexpression of miR-1178 significantly promoted tumor growth,compared with control group.Opposite effects were observed after downregulation of miR-1178.(P<0.05).3.The mechanisms of miR-1178 in regulating the progression of pancreatic cancer.In our previous study,we demonstrated that STUB1 is a novel tumor suppressor in pancreatic cancer via the degradation of EGFR.Given our observation that miR-1178 down-regulated the expression of STUB1,we hypothesized that miR-1178 might also regulate the activity of EGFR and its downstream signaling cascade.We found that the over-expression of miR-1178 increased the EGFR protein level and activated the downstream AKT/p21 pathway and the Src/E-cadherin pathway.In contrast,the inhibition of miR-1178 had the opposite effects.Many studies have indicated that the EGFR/AKT/p21 and EGFR/SRC/E-cadherin pathways play key roles in the proliferation,cell cycle control,migration and invasion of pancreatic cancer cells.Thus,we speculated that the ectopic activation of the EGFR/AKT/p21 and EGFR/SRC/E-cadherin pathways might partially account for the effects of miR-1178 in pancreatic cancer cells.To further verify whether miR-178 promotes a malignant phenotype by repressing STUB1 in pancreatic cancer cells,we adopted a"rescue" strategy to examine the functional relevance of the miR-1178/STUB1 interaction.The pcDNA-STUB1 or the pcDNA empty vector was transfected into PANC-1 cells over-expressing miR-1178.As shown,the level of STUB1 was increased when pcDNA-STUB1 was transfected.Furthermore,the over-expression of STUB1 inhibited the miR-1178-induced expression of EGFR,p-AKT and p-SRC.In agreement with the inactivation of EGFR and its downstream pathways,cell invasion,migration,proliferation and Gl/S transition were all suppressed.These results indicate that miR-1178 promotes cell proliferation,G1/S transition,migration and invasion through the down-regulation of STUB 1.4.The expression levels and clinical values of miR-1178 in pancreatic cancer tissuesThe expression levels of miR-1178 were significantly increased in pancreatic cancer tissues,compared with that in tumor-adjacent tissues(P<0.05).There was a positive correlation between the expression levels of miR-1178 and lymphatic metastasis(P<0.05).There was no correlation between the expression levels of miR-1178 and other clinicopathological parameters,including sex,age,tumor locations,differential degree,TNM staging,and perineuronal invasion.Univariate analysis showed TNM staging,differential degree and miR-1178 levels were the potential prognostic factors of pancreatic cancer.Multivariate analysis indicated that TNM staging(II/III/IV)and miR-1178 expression(High)were the independent adverse prognostic factor(P=0.049,HR=1.866;P=0.025,HR=2.364,respectively).ConclusionIn conclusion,the current study demonstrated that miR-1178 promoted pancreatic cancer cell proliferation,G1/S transition,migration and invasion through the down-regulation of STUB 1.Our data suggest that miR-1178 is an endogenous attenuator of STUB1 that facilitates pancreatic tumorigenesis.The levels of miR-1178 in tissues displayed prognostic values.Our results suggest that miR-1178 might serve as a novel therapeutic target for miRNA-based therapy in pancreatic cancer. |
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