SKAP1 Regulates Gastric Cancer Proliferation,Invasion And Migration Through JAK1/PI3K/AKT Axis

Background and Objectives:Gastric cancer(GC)is the fourth leading cause of cancer death worldwide,accounting for 7.7% of all cancer deaths,following lung,colorectal and liver cancer.Early diagnosis of GC is challenging due to the lack of specific symptoms and signs,resulting in a low detection rate for early-stage disease.Patients with advanced GC often miss the opportunity for surgery,and current treatment methods are limited.Therefore,identifying new biomarkers for early detection,targeted therapy,and prognosis prediction is urgent.Studies have shown that SKAP1 is up-regulated in various tumors,including colon cancer,malignant mycosis fungoides,and breast cancer,promoting proliferation and invasion and is associated with poor prognosis.However,the role and mechanism of SKAP1 in GC remain unclear.This study aims to investigate the expression of SKAP1 in GC tissues and adjacent normal tissues,analyze its correlation with clinicopathological parameters and prognosis,and to further explore the expression of SKAP1 in GC cells and its potential function and molecular mechanism in cell proliferation,migration,and invasion.Materials and Methods:Ⅰ.Analysis of SKAP1 expression,prognosis,and bioinformatics in GC tissues1.Analysis of SKAP1 expression in GC:(i)Using the TIMER database,we analyzed the expression of SKAP1 in various tumor tissues and their corresponding normal tissues.(ii)We analyzed the expression difference of SKAP1 in GC tissues and normal gastric epithelial tissues using sequencing data from TCGA and GEO databases.Furthermore,we collected 21 pairs of GC and adjacent tissues from a hospital and detected the expression of SKAP1 in GC and adjacent tissues via q RT-PCR and Western Blot.2.Correlation between SKAP1 and clinicopathological parameters of GC patients and prognosis analysis:(i)We analyzed the correlation between SKAP1 expression and clinicopathological parameters of GC patients using the TCGA-STAD dataset.(ii)The Kaplan-Meier Plotter database was used to analyze the relationship between SKAP1 expression and overall survival(OS),post-progression survival(PPS),and progression-free survival(PFS)of GC patients.3.Prediction of the possible mechanism of SKAP1 in regulating GC progression via bioinformatics: We performed Gene Set Enrichment Analysis(GSEA)with data from SKAP1 high and low expression groups to predict the molecular mechanism of SKAP1 in regulating GC progression.Ⅱ.SKAP1 regulates the malignant biological behavior of GC cells through JAK1/PI3K/AKT signaling axis1.Effects of SKAP1 on biological behaviors of GC cells:(i)GC cells MKN45 and HGC27 were transfected with si RNA-SKAP1,and the silencing efficiency was detected by Western Blot and q RT-PCR.(ii)MTS,Ed U and colony formation assays were used to detect the changes in cell proliferation ability after silencing SKAP1.(iii)The changes of invasion and migration ability of GC cells after silencing SKAP1 were analyzed by Transwell invasion and migration assay.(iv)The relationship between apoptosis and SKAP1 expression was detected by flow cytometry.2.SKAP1 regulates JAK1/PI3K/AKT signaling axis in GC cells: We evaluated changes in key genes from pathways enriched in the SKAP1-overexpressing group using q RT-PCR and assessed the expression levels of JAK1,P-JAK1,PI3 K,P-PI3 K,AKT,and P-AKT in GC cells after silencing SKAP1 using Western Blot analysis.3.Effect of PI3 K agonist on the biological behavior regulated by SKAP1 in GC cells: On the basis of silencing SKAP1,(i)Western Blot was used to detected the change in the expression levels of JAK1,PI3 K,AKT and their phosphorylated proteins after the application of PI3 K agonist 740 Y-P.(ii)The change of cell proliferation ability after the application of PI3 K agonist 740 Y-P was detected by MTS,Ed U and cloning formation assays.(iii)Transwell invasion and migration assays were used to analyze the changes in the invasion and migration capacity of GC cells after the application of PI3 K agonist 740 Y-P.Results:Ⅰ.Expression,prognosis and bioinformatic analysis of SKAP1 in GC1.SKAP1 is highly expressed in gastric cancer tissues:(i)SKAP1 is highly expressed in a variety of tumors,especially gastrointestinal tumors such as esophageal cancer,gastric cancer,colon cancer,and rectal cancer.Analysis of TCGA and GEO data showed that the expression of SKAP1 in GC tissues was significantly higher than in adjacent tissues.(ii)We collected 21 pairs of GC tissues and adjacent tissues and found that the expression of SKAP1 was significantly higher in GC tissues using q RT-PCR and Western Blot.2.The relationship between SKAP1 and clinicopathological parameters of GC patients and prognosis analysis:(i)High expression of SKAP1 was significantly correlated with T stage and Grade of GC patients(P < 0.05).(ii)Kaplan-Meier Plotter database analysis showed that GC patients with high SKAP1 expression had significantly shorter OS,PPS,and PFS than those with low expression(P < 0.05).Thus,high expression of SKAP1 is a poor prognostic factor for GC patients.3.The possible mechanism of SKAP1 regulating gastric cancer progression predicted by bioinformatics: GSEA enrichment analysis showed significant enrichment of the JAK-STAT pathway,PI3K-AKT pathway,and TNF-α pathway in the SKAP1 high-expression group.Moreover,SKAP1 overexpression was associated with proliferation-related biological processes(such as MYC target genes,E2 F target genes,G2 M cell cycle checkpoint,spindle formation,DNA repair,etc.)and inflammatory response.Ⅱ.SKAP1 regulates the malignant biological behavior of GC cells through JAK1/PI3K/AKT signaling axis1.Effects of silencing SKAP1 on biological behaviors of GC cells:(i)The results of WB and q RT-PCR showed that the application of si RNA-SKAP1 efficiently silenced SKAP1 expression in GC cells.(ii)Silencing SKAP1 decreased cell proliferation,invasion,and migration ability according to MTS,Ed U,and colony formation assays,and transwell invasion and migration assay.However,SKAP1 had no significant effect on GC cell apoptosis,as detected by flow cytometry.2.SKAP1 regulates JAK1/PI3K/AKT signaling axis in GC cells:(i)q RT-PCR showed that silencing SKAP1 significantly decreased JAK1 m RNA levels.(ii)WB showed that SKAP1 knockdown reduced protein levels of JAK1,P-JAK1,P-PI3 K,and P-AKT in GC cells.3.Effect of PI3 K agonist on the biological behavior regulated by SKAP1 in GC cells:(i)WB analysis showed that adding PI3 K agonist 740 Y-P increased P-PI3 K and P-AKT protein levels compared with the SKAP1 silencing group without affecting JAK1,P-JAK1,and PI3 K levels.(ii)MTS,Ed U,and colony formation assays showed that the proliferation ability of GC cells was significantly increased after adding PI3 K agonist 740 Y-P compared with the SKAP1 silencing group.(iii)Transwell invasion and migration assay showed that adding PI3 K agonist 740 Y-P also significantly increased the invasion and migration ability of GC cells compared with the SKAP1 silencing group.Conclusion:1.SKAP1 is highly expressed in GC tissues and associated with poor prognosis.2.High expression of SKAP1 promotes the proliferation,invasion,and migration of MKN45 and HGC27 GC cells but has no significant correlation with cell apoptosis.3.SKAP1 may promote the proliferation,invasion and migration of GC cells by activating JAK1/PI3K/AKT signaling axis,thus promoting the progression of GC.