| Background and objective Worldwide,lung cancer is one of the most common cancers in humans,a leading cause of cancer-related death in men and women.The discovery of a potent gene regulating tumorigenesis and drug resistance is of high clinical importance.STIL promotes cell proliferation in prostate cancer,Pancreatic cancer and gastric cancer.Knock down STIL induced cell cycle arrest in G2/M phase and cell apoptosis of gastric cancer(GC)cells.Transcriptome analysis indicated that STIL STILencing modulated many gene expression,particularly for downregulating the IGF-1/PI3K/AKT pathway.High expression of STIL m RNA was associated with shorter disease-free survival time in colorectal cancer(CRC)patients,and STIL regulates beta-catenin levels through p-AKT in colorectal cancer,independent of Shh pathway.Knockdown of STIL inhibited cell proliferation of prostate cancer and induced cell apoptosis,and promotes tumor growth through MAPK/ERK,PI3K/Akt and AMPK pathways in prostate cancer.STIL regulates beta-catenin levels through p-AKT,independent of Shh pathway.Wnt/β-catenin alterations are prominent in human malignancies.In non-small cell lung cancer(NSCLC),β-catenin and APC mutations are uncommon,but Wnt signaling is important in NSCLC cell lines,and Wnt inhibition reduces proliferation.Wnt pathway activation correlates with poor survival in human lung adenocarcinoma,pancreatic ductal adenocarcinoma and mesothelioma.Human LUAD,in particular metastasis,is frequently associated with increased expression of Wnt pathway-activating genes and down regulation of negative regulators of the pathway.SCL/TAL1 interrupting locus(STIL)is associated with the progression of several tumors;however,the biological role and underlying molecular mechanism of STIL in lung cancer remain poorly understood,and particularly the association between the biological role of STIL and Wnt/β-catenin pathway is unclear.Therefore,our objective was to investigate the biological role and the underlying molecular mechanism of STIL in lung cancer and to identify whether STIL promotes or inhibits cell proliferation and invasion through Wnt/β-catenin pathway.Methods To identify Differentially expressed genes(DEGs)in lung cancer patients,we first analyzed publicly available m RNA Expression profiling data by array of four independent datasets obtained from GEO database that included932 samples,using R language and Limma package in Rstudio software,and next performed univariate Cox proportional-hazards regression analysis to assess prognostic value of the above DEGs across all lung patients within GSE30219 dataset and analyze the association between these differentially expressed genes expression and overall survival time(or poor prognosis)by integrating survival time,status data and m RNA expression data.Additionally,the multivariate Cox proportional-hazards regression analysis was conducted to further determine which genes predict poor prognosis independent of age,gender and the TNM stage etc.and could be independent poor prognostic biomarkers in patients with lung cancer.Subsequently,to further explore and validate the relevance between Top 25 independent poor prognostic genes obtained from multivariate Cox regression analysis and overall survival time,Kaplan-Meier survival analysis was conducted across all lung cancer patients of Kaplan-Meier plot(https://kmplot.com/analysis)and the log-rank test was used to assess significance.To explore the functional role of STIL in cell proliferation and invasion in lung cancer cells,we transfected STIL si RNA or p EGFP-C1-STIL to lung cancer cells to knockdown or overexpress STIL,and then tested cell proliferation and invasion using cck-8 and transwell invasion experimental methods,respectively.To investigate whether STIL promotes cell proliferation and invasion in lung cancer cells through activating Wnt/β-catenin pathway,we first transfected p EGFP-C1-STIL to lung cancer cells followed by treatment of Wnt/β-catenin pathway inhibitor LGK-974,and then detected cell proliferation and invasion by using cck-8 and transwell invasion methods,respectively.To further investigate whether STIL activates Wnt/β-catenin pathway and this activation can be rescued by Wnt/β-catenin pathway inhibitor LGK-974,we next performed western blot to test protein expression level of β-catenin,WNT7 B and Wnt10 B that are described as critical molecular of Wnt/β-catenin pathway.Statistical analyses were conducted in R(www.R-project.org)or Graph Pad Prism7.0,and all survival analyses of GEO Database were conducted using the survival package in R.P < 0.05 was considered as statistically significant.Results we first performed differentially expressed genes(DEGs)analysis of lung cancer tissues and matched adjacent non-tumor lung(NTL)tissues or normal lung tissues by using R language and Limma package in Rstudio.To identify differentially expressed genes(DEGs),using our criteria of p< 0.05 and Log FC > 1 or <-1,we identified 1487 genes in GSE31210 dataset,1641 genes in GSE30219 dataset,1453 genes in GSE19188 and 1071 genes in GSE151101,showing altered RNA expression between tumors and non-tumor lung.For overlap of 4 datasets,We identified 425 genes were deferentially expressed between lung tumor tissue and non-tumor lung tissue(P<0.05).Of these,127 genes were significantly upregulated,while 298 genes were significantly downregulated.Kaplan – Meier survival analysis revealed that overexpression of Top 25 independent poor prognostic genes in human lung cancer correlated with poor survival,higher expression of Top 25 genes including STIL in patients with lung cancer had shorter overall survival time.STIL overexpression predicts poor prognosis(HR1.92,logrank P = 7.1e-06).Consistently,univariable Cox proportional hazards regression analysis showed that increased expression level of STIL in patients with lung cancer is a poor prognostic indicator(HR 1.56,Pvalue 5.51E-12).Multivariable Cox proportional hazards regression analysis indicated that STIL overexpression is significantly associated with poor prognosis in lung cancer patients,critically,for overall survival,it’s an independent poor prognostic biomarker in lung cancer(HR 1.41,Pvalue 1.25E-05).RT-PCR analysis also revealed that the expression level of STIL m RNA significantly increased in lung cancer cells,compared to normal lung cells,and consistently,western blot results showed that higher expression level of STIL protein was detected in lung cancers.Moreover,by performing functional assays such as CCK-8 assay and transwell experiments,we demonstrated knock down of STIL by si RNA targeting STIL gene suppressed cell proliferation and invasion in lung cancer cells.Importantly,we found that STIL overexpression promotes cell proliferation and invasion in H1299 cells,and this proliferation and invasion induced by STIL overexpression was rescued by Wnt/β-catenin pathway inhibitor LGK-974.Overexpression of STIL by transfecting p EGFP-C1-STIL to lung cancer cells resulted in increased expression of β-catenin,WNT7 B and Wnt10 B.Westen blot results indicated that STIL overexpression resulted in increased expression level of β-catenin,WNT7 Band Wnt10B described as critical molecular of Wnt/β-catenin pathway.Critically,STIL-induced activation of β-catenin,WNT7 B and Wnt10 B can be rescued by Wnt/β-catenin pathway inhibitor LGK-974.These data suggested that STIL-induced cell proliferation and invasion in lung cancer are associated with Wnt/β-catenin pathway.Conclusions Using our criteria of p< 0.05 and Log FC > 1 or <-1,We identified425 genes were differentially expressed.Of these,127 genes were significantly upregulated,while 298 genes were significantly downregulated.STIL m RNA expression in lung cancer samples significantly upregulated compared to corresponding adjacent non-malignant of lung cancer patients or normal lung tissue samples without lung cancer(P<0.05).Our Differentially expressed genes(DEGs)analysis result indicated that STIL m RNA expression in lung cancer samples significantly upregulated compared to corresponding adjacent non-malignant of lung cancer patients or normal lung.tissue samples without lung cancer(P<0.05).Moreover,knock down of STIL by si RNA targeting STIL gene suppressed cell proliferation and migration of lung cancer,while STIL overexpression promotes cell proliferation and invasion in lung cancer cells.STIL-induced cell proliferation and invasion in lung cancer cells were rescued by Wnt/β-catenin pathway inhibitor LGK-974.Critically,STIL-mediated upregulation or activation of β-catenin,WNT7 Band Wnt10B that are described as critical molecular were rescued by Wnt/β-catenin pathway inhibitor LGK-974.Therefore,Our study suggests that therapeutic suppression of STIL might be advantageous due to targeting multiple aspects of lung cancer progression,and it may suggest that modulation of STIL and Wnt/β-catenin pathway inhibitor could be a successful approach to prevent progression to lung cancer,and strategies might translate into effective anti-cancer therapies. |
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