The Molecular Biomarker Roles Of Paip1 In Lung Adenocarcinoma

Background:Lung adenocarcinoma(LADC)is one of the common histological subtypes of lung cancer.It has a low five-year survival rate,due to high degree of malignancy,high frequency of recurrence and metastasis.As a result,it became as a serious threat to the health of human.Because of the complex biological characteristics of LADC,a lot of studies are focused on identifying the gene alteration in LADC development and progression.Recently,it is important to find out the target genes for cancer therapy and improve the prognosis of LADC patients.Polyadenylate-binding protein-interacting protein 1(Paipl)is a specific mammalian protein.It interacts with poly adenylate binding protein(PABP)and eukaryotic translation initiation factor 4A(eIF4A),and is involved in the initiation of translation and protein biosynthesis.Scotto L et al.have found that the expression of Paipl was increased during the development of cervical cancer[1].Piao J et al.have shown that the expression of Paip1 was related to histological grade and clinical stage,and its overexpression indicated the poor prognosis of the breast cancer patients[2].Additionally,Wu S has reported that silencing of Paip1 expression could significantly inhibited the proliferation and migration of hepatoma cells[3].These findings suggest that the high expression of Paip1 plays a key role in the development of various types of tumors.However,the roles of Paipl in LADC have not yet been reported.Objectives:Using immunohistochemistry(IHC)staining to determine the expression of Paip1 in LADC,and analyze the relationship between the expression of Paip1 and the clinicopathological features of LADC patients.In addition,using western blot,colony-formation and wound healing assays to explore the effects of Paip1 on LADC cells behavior in vitro and elucidate the possible molecular mechanism.Materials and methods:1.Bioinformatics and tissue specimen analysis:The mRNA expression levels of Paip1 in LADC clinical samples were obtained from Oncomine database.A total of 58 LADC patients with strict follow-up and 60 adjacent normal lung tissues were selected for IHC staining of Paip1.The correlation between Paipl expression and the clinicopathological features,overall survival(OS)rates of LADC patients were analyzed.2.Experiments in vitro:The protein expression levels of Paip1 were detected by western blot in LADC cell lines,including A549,H1299,H1975 and H1944.Then,the cells with high expression of Paip1 were selected for subsequent experiments.Si-RNA-mediated transduction was performed to knockdown of Paipl expression in LADC cells.Then,the greatest effective sequence of Paip1 siRNA was demonstrated by detecting the Paip1 expression in siRNA-transfected A549 and H1299 cells using western blot analysis and the sequence was selected for subsequent in vitro experiments.The cell proliferation viability was measured using MTT,colony formation and EdU assay.The migration ability was determined using wound healing and transwell assay.Meanwhile,the expression of EMT biomarkers and transcriptional factors were detected using immunofluorescence(IF)staining and western blot analysis in Paipl silenced LADC cells.Results:1.Paipl is overexpressed in LADC tissues and correlated with poor prognosis of LADC patients:Oncomine database analysis showed that the mRNA expression levels of Paipl in LADC tissues were higher than that those of normal lung tissues(P<0.01).IHC staining showed that the positive rate and strongly positive rate of Paipl protein expression were 77.6%(45/58)and 46.6%(27/58)in LADC tissues,which were remarkably higher than adjacent normal lung tissues(P<0.01).The overexpression of Paip1 was significantly correlated with histological grade and clinical stage(P<0.01).Regard to survival,Kaplan-Meier analysis showed that patients with high Paip1 expression exhibited a lower rate of overall survival than that of low Paip1 expression(P<0.01).2.Knockdown of Paipl inhibits the proliferation and migration of LADC cells:MTT,colony formation and EdU assay showed that knockdown of Paipl expression could significantly decreased proliferation and colony formation ability of A549 and H1299 cells(P<0.01),In addition,wound healing and transwell assay showed that knockdown of Paipl expression significantly inhibited the migration ability of A549 and H1299 cells(P<0.01).3.Knockdown of Paipl inhibits EMT progression in LADC cells:The results of IF and western blot showed that knockdown of Paip 1 up-regulated the expression of epithelial marker(E-cadherin)and down-regulated the expression of mesenchymal marker(Vimentin)and transcriptional factors(Snail and Slug)(P<0.01).Conclusions:1.Paipl expression is frequently up-regulated in LADC,and its overexpression correlates with poor prognosis in LADC patients.2.Paipl overexpression promotes proliferation and EMT-related migration of LADC cells.