| Objective:Pancreatic adenocarcinoma(PAAD)is one of the most common gastrointestinal malignancy and has an extremely poor prognosis,with overall 5?year survival rate of<5%.Human far upstream element ? binding protein 1(FUBP1)is involved in regulating the transcription,m RNA splicing and translation of target genes by binding to the far upstream element(FUSE).Many studies have reported that FUBP1 is involved in the occurrence,development and prognosis of tumors,but its study in pancreatic cancer has not been reported.Therefore,the biological functions and molecular mechanisms of FUBP1 in PAAD remain unknown and require further investigations.Methods:1、We analyzed the m RNA levels of FUBP1 in pancreatic adenocarcinoma and the correlation between gene expression and overall survival via TCGA database and star Base database.2、RT?q PCR was used to detect the relative m RNA expression levels in PAAD cell lines.3 、 Overexpression plasmid and si RNA were designed and transfected into pancreatic cancer cell lines,respectively.The up-regulation and down-regulation of FUBP1 expression were conducted respectively,and untreated cells were served as negative control.4、To investigate the biological functions of FUBP1 in PAAD,the expression of FUBP1 was up-regulated or down-regulated.The CCK-8 assay and the Transwell assay were used to detect cell proliferation,migration and invasion.5、To investigate the molecular mechanisms of FUBP1 in PAAD,we used western blot and immunofluorescence assays to analyze the effect of FUBP1 on EMT related proteins.And the expression levels of p-Smad2/3 and TGFβ1,which are pivotal signaling molecules in the TGFβ/Smad signaling pathway were analyzed using western blot.Results:1、It was found that the expression levels of FUBP1 were upregulated in human PAAD tissues compared with adjacent normal tissues from TCGA database,and the upregulated expression was significantly associated with poor survival.Similarly,RT?q PCR analysis showed that the expression of FUBP1 in 7 pairs of pancreatic cancer tissues was higher than that in adjacent normal tissues.2、Among the four human PAAD cell lines,the expression level of FUBP1 was the highest in Pa Tu8988 cells and the lowest in SW1990 cells.Thus,we selected Pa Tu8988 cells to investigate the effects of the knockdown of FUBP1 in PAAD,while SW1990 cells were selected to determine the effects of the overexpression of FUBP1.3、Knockdown of FUBP1 represses proliferation,migration and invasion and metastasis of PAAD cells,whereas the overexpression of FUBP1 demonstrated the opposite trend.4、Following knockdown of FUBP1,western blot showed that FUBP1 increased E-cadherin protein levels,and reduced N-cadherin,vimentin and β-catenin protein levels.In contrast,the overexpression of FUBP1 demonstrated the opposite trend.Similarly,immunofluorescence analysis showed consistent results with western blot.5、After knockdown of FUBP1,western blot showed that FUBP1 reduced p?Smad2/3 and TGFβ1 protein levels,while the overexpression of FUBP1 significantly increased p?Smad2/3 and TGFβ1 protein levels.Conclusion:In conclusion,our study revealed that FUBP1 expression levels were upregulated in patients with PAAD and related to the poor prognosis of PAAD patients through bioinformatics analysis.Moreover,we demonstrated that FUBP1 promoted PAAD cells growth and metastasis in vitro.And FUBP1 could enhance Epithelial-Mesenchymal Transition(EMT)by Transforming Growth Factor-β(TGF-β)/Smad pathway.Collectively,FUBP1 may serve as a clinically relevant prognostic biomarker or represent a novel therapeutic target for PAAD.Figure [12] table [23] reference [61]... |
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