The Mechanism Study Of PTBP3 Mediated TGF-β-induced Epithelial Mesenchymal Transition In Lung Adenocarcinoma

ObjectiveLung adenocarcinoma(LUAD)is the major pathological subtype of non-small cell lung cancer(NSCLC).Most patients have advanced to the middle and late stage of the disease by the time of clinical diagnosis due to early metastasis to distant organs.With the improvement of surgical techniques,combined application of chemoradiotherapy and immunotherapy,the survival of patients with LUAD has improved.However,tumor recurrence and distant metastasis are major causes of affecting prognosis.Epithelial mesenchymal transition(EMT)is a molecular remodeling and phenotypic transition from epithelial cells to mesenchymal cells,which can lead to increased cell mobility and is a major cause of tumor metastasis and drug resistance.Transforming growth factor-β(TGF-β)has been demonstrated to be a potent promoter of EMT and tumor metastasis through activation of the canonical Smad signaling pathway.However,the mechanisms underlying this alteration are largely unknown.Polypyrimidine tract-binding protein 3(PTBP3)is a RNA binding protein that plays a critical role in RNA splicing and transcriptional regulation.More and more studies show that dysregulation of PTBP3 closely correlates with malignant growth and metastasis in many types of cancers.Whether PTBP3 mediates TGF-β-induced EMT and metastasis in LUAD remains unknown.Therefore,we investigate the function and molecular mechanism that PTBP3 mediates TGF-β-induced EMT and metastasis of LUAD.MethodsThe expression levels and prognostic value of PTBP3 were analyzed by searching public LUAD databases,including GEO and TCGA,and performing IHC analysis in LUAD tissue microarray.To construct cell lines with transient knockdown or stable overexpression of PTBP3,si RNA and lentivirus-mediated vectors were used to transfect LUAD cell lines.We analyzed the invasion and migration of these cells in vitro by performing Transwell assays.Western-blot,q RT-PCR,a luciferase reporter assay and chromatin immunoprecipitation(Ch IP)assay were used to determine the molecular mechanism which PTBP3 mediates TGF-β-induced EMT.Meanwhile,in vivo metastasis model was established by injecting A549 cells into the tail vein of nude mice to analyze the effect of PTBP3 on LUAD cell metastasis in vivo.ResultsPTBP3 was markedly upregulated in LUAD tissues compared with para-carcinoma tissues and patients with high expression of PTBP3 had shorter overall survival and recurrence-free survival.Univariate and multivariate Cox analysis showed that PTBP3 was an independent risk factor for patient survival.TGF-β-induced p-Smad2/3 complex activated PTBP3 gene transcription and promoted PTBP3 expression.In turn,PTBP3 knockdown and overexpression significantly suppressed and increased Smad2/3 expression,and then regulated TGF-β-induced migration,invasion and EMT.In addition,the in vivo metastasis assays show that PTBP3 overexpression strikingly increased the number of lung and liver metastatic nodules in nude mice.ConclusionIn this study,we determined that PTBP3 is indispensable to TGF-β-induced EMT and metastasis of LUAD cells and identified a critical positive feedback loop between PTBP3 and Smad2/3 driving TGF-β-induced EMT.Thus,we determined that PTBP3 is a novel potential therapeutic target for the treatment of LUAD.