The Mechanism Underlying TGFβ/SMAD Regulating P4HA2 Expression And The Functions Of P4HA2 On EMT And Metastasis In NSCLC Cells

Background and Objectives:Primary lung cancer is a malignant tumor with high incidence and high mortality,of which NSCLC accounts for about 85%,and the 5-year survival is still poor.According to statistical analysis,metastasis is the main cause of death in lung cancer patients.During the events of cancer metastasis,epithelial-mesenchymal transition(EMT)is the key process.It is supported that transforming growth factor-β(TGF-β)signaling pathway promotes the occurrence of EMT,and many genes are responsible for maintaining the phenotype induced by TGF-β.Protein proline hydroxylation modification is one of the commonly post-translational modifications.As a proline hydroxylase,P4HA2 plays essential roles in regulating protein folding and stability in mammalian cells.P4HA2 upregulation has been reported to be associated with occurrence of many tumors;however,the mechanism underlying P4HA2 abnormal expression and its function in TGF-β-mediated EMT and metastasis of NSCLC cell is still unclear.This study aims to explore the transcriptional mechanism by which TGF-β/SMAD.signaling regulates P4HA2 expression,and to reveal the function of P4HA2 in relation to TGF-β-induced EMT and Akt signaling,so as to provide new insights and theoretical basis for the mechanism exploration and clinical diagnosis in NSCLC.Methods:(1)The relationship between the expression of P4HA2 and the prognosis of NSCLC patients was analyzed based on TCGA database.(2)With the use of real-time qPCR,the expression of P4HA2 in tissues was determined,and the expression in NSCLC was compared with the paired-adjacent samples.(3)Gene set enrichment analysis(GSEA)based on the TCGA database were used to analyze the signaling pathways related to P4HA2.(4)A dual-fluorescent reporter vector containing P4HA2 promoter region was constructed and transfected into cells.In the setting of TGF-β stimulation,the activity of the constructs in the cell lines with SMAD3 and SMAD4 knocked out was detected.(5)ChIP assay was used to verify the binding effect of SMAD3/SMAD4 complex with the promoter region of P4HA2 gene.(6)Cell migration and invasion were analyzed by transwell assay.(7)Western blot was used to detect proteins expression.Results:(1)The survival rate of NSCLC with high expression of P4HA2 is significantly lower than that of the patients with low P4HA2 expression.(2)P4HA2 is highly expressed in tissues of NSCLCs as compared with the paired-adjacent samples.(3)The TGF-β signaling pathway is positively correlated with the expression of P4HA2,and TGF-β1 promotes P4HA2 expression.(4)After SMAD3 and SMAD4 were knocked out,the TGF-β1-induced pGL3 activity of P4HA2 promoter is inhibited.(5)The enrichment of SMAD3 and SMAD4 to P4HA2 promoter is observed to be increased following TGF-βtreatment.(6)Overexpression of P4HA2 promotes EMT,migration and invasion of NSCLC cells.(7)P4HA2 activates the PI3K-Akt signaling pathway in NSCLC cells.Conclusion:The present study reveals that the proline hydroxylase,P4HA2,is highly expressed in NSCLC tissues,and high-expression of P4HA2 is related to the poor prognosis of NSCLC patients.TGF-β activates the transcriptional activity of SMAD3/SMAD4 complex,which in turn accelerates the expression of P4HA2.P4HA2.promotes cell EMT and activates the PI3K-Akt signaling pathway in NSCLC cells.