The Molecular Biomarker Roles Of Overexpression Paip1 In Gastric Cancer

Background:Gastric cancer(GC)is a malignancy with high incidence and mortality in the world.In China,the incidence and mortality of GC rank among the top five in the total incidence and mortality of cancer.Current studies have shown that the occurrence and development of GC is closely related to the abnormal expression and amplification of many tumor-related genes.With the development of molecular targeted therapy,more and more targeted drugs have been used in the treatment of GC.These drugs can effectively inhibit proliferation and angiogenesis of GC,which in turn improve the prognosis of patients.However,invasion and metastasis are still the main causes of death in GC patients.Therefore,a key of improvement of the prognosis and life quality for patients is searching for early diagnostic markers and potential therapeutic targets of invasion and metastasis of GC.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),involved in the initiation of translation and protein biosynthesis.At present,many studies have confirmed the abnormal expression of Paipl in various cancer tissues and closely related to the clinical stage and histological differentiation of tumors.Guan et al.found that silencing of Paipl significantly inhibited the proliferation and migration of pancreatic carcinoma cells.These findings suggested that the high expression of Paipl plays a key role in the development of tumors.However,the role of Paipl in GC has not been reported yet.Objectives:To evaluate the expression of Paipl protein in GC,and analyze the relationship with clinicopathological and prognosis of GC patients.To investigate the effects of Paipl on the biological function of GC cells and elucidate its possible molecular mechanism.Materials and methods:The expression of Paipl mRNA and protein in GC and normal tissues were detected by immunohistochemical staining,UALCAN and Oncomine database analysis.The relationship between the expression of Paipl,prognosis and clinicopathological features of GC patients was evaluated by statistical methods.Western blot analysis was used to detect the different expression of Paipl protein in human gastric cell line GES1 and four different human GC cell lines(BGC823,SGC7901,MGC803,AGS);the localization of Paipl in GC cells was detected by immunofluorescence staining;Transfect Paipl specific small interfering RNA(siRNA)(Paipl siRNA),and use western blotting to detect changes in Paipl protein level after siRNA transfection.The silencing effects of Paipl on the proliferation and migration of SGC7901 and MGC803 cells were observed by using EdU(5-ethynyl-2’-deoxyuridine)technique of labeling,cloning,cell scratching and transwell assay.Flow cytometry was used to detect the changes of cell cycle in GC cells transfected with si-Paip1,and Western blot was used to detect the expression of cyclin marker:cyclin D1.Western blot was used to detect the expression of EMT-related markers after Paipl knockdown.Results:1.Paipl was highly expressed in GC tissues and was closely related to the prognosis of GC patients:IHC staining showed that the positive and strong positive rate of Paipl were 85.6%(77/90)and 41.1%(37/90)in GC tissues,which were significantly higher than the adjacent gastric tissue(36.7%and 6.7%)(P<0.05).UALCAN and Oncomine database analysis showed that the mRNA of Paipl in the different GC tissues were significantly higher than that of the adjacent gastric tissue(P<0.01).Paipl protein was mainly localized in cytoplasm by IF assay.Further statistics analysis showed that over-expression of Paipl was closely related to the clinical grade,lymphatic invasion and age of patients with GC.In addition,the survival time of patients with high expression of Paipl was significantly shorter than that of patients with low expression of Paip1,and multivariate analysis showed that Paipl expression and age were independent risk factors affecting the prognosis of GC patients.2.Silencing Paipl inhibited the proliferation of GC cells and cell cycle progression:Paip1 protein was significantly higher in four different human GC cell lines(BGC823,SGC7901,MGC803,AGS)than human gastric cell line GES1 cells,and two GC cell lines(SGC7901 and MGC803)were selected for subsequent experiments.The expression of Paipl in SGC7901 and MGC803 cells was significantly decreased by Western blotting after Paipl siRNA transfection(P<0.05).Cloning formation experiment and EdU experiment showed that the ability of proliferation was significantly lower than control group when Paipl knockdown(P<0.05).Flow cytometry showed the cell cycle of SGC7901 and MGC803 cells was mainly blocked in G1/S phase after Paipl knockdown(P<0.05),and the expression level of cyclinD1,the cell cycle-associated protein was down-regulated after silencing(P<0.05).3.Silencing Paipl inhibited GC cells migration and EMT progression:Wound healing and Transwell assays showed that the migration of GC cells(SGC7901 and MGC803)were significantly inhibited after silencing Paip1;Western blot analysis showed that after silencing Paip1,the expression of mesenchymal markers Vimentin and Snail were down-regulated,and the expression of epithelial marker E-cadherin was up-regulated(P<0.05).Conclusions:(1)Paip1 expression was frequently up-regulated in GC and correlated with poor prognosis of GC patients.(2)Silencing of Paipl inhibited the proliferation of GC cells by blocking in G1/S phase.(3)Paipl knockdown inhibited the migration of GC cells via EMT pathway.