| Background/Objective Colorectal cancer(CRC)is the third most common malignant tumor and one of the leading causes of cancer-related death worldwide.One of the major issues is tumor metastasis.Therefore,identifying novel and potential biomarkers for CRC metastasis is of great importance.With the rapid development of big data,weighted gene co-expression network analysis(WGCNA)has emerged as a novel holistic approach for microarray analysis,which could identify the correlation between gene modules and clinical features,so as to identify hub genes.In this study,we aimed to identify hub genes associated with metastasis of CRC by performing WGCNA,then futher analyze the results through experiments.Methods The microarray data from Gene Expression Omnibus(GEO)database,GSE41258,was used to construct a co-expression network and identify gene modules related to clinical features of CRC.Furthermore,protein–protein interaction(PPI)network analysis was also performed to screen hub nodes.Screening of differentially expressed genes(DEGs)was used to identify highly expressed genes in CRC.To validate the hub genes,an independent dataset from GEO database,GSE17536,was used for survival analysis.Gene ontology(GO)and gene set enrichment analysis(GSEA)were performed to explore potential function of hub genes.To validate the expression of ITGBL1,immunohistochemistry(IHC),quantitative real-time PCR(q RT-PCR)and western blotting were performed in CRC tissues and cell lines.Subsequently,ITGBL1 was silenced by RNA interfering.The effect of ITGBL1 were evaluated through colony formation,cell proliferation,cell apoptosis,cell cycle,cell migration and invasion assays.Besides,ITGBL1 was further overexpressed by plasmid transfection to evaluate the effect on CRC cell migration,invasion and epithelialmesenchymal transformation(EMT),as well as the influence on key molecules in the TGF?/Smad signaling pathway.Finally,to explore potential micro RNA(mi RNA)targeting ITGBL1,we searched on bioinformatics databases,then q RT-PCR and western blotting were performed to validate the correlation between mi RNA and ITGBL1.Results A total of 16 modules were screened and the turquoise module was identified to be significant(r=-0.2,P=0.009).Therefore,we screened hub genes in the turquoise module and identified ITGBL1 as a hub gene highly expressed in CRC which was also associated with overall survival and relapse-free survival of CRC patients.Then,IHC showed that ITGBL1 was associated with tumor size(P=0.017)and distant metastasis(P=0.046)of CRC patients.q RT-PCR and western blotting showed ITGBL1 was upregulated in CRC tissues compared with adjacent tissues(3.057±0.421 vs.1.483±0.211,P<0.05).In addition,ITGBL1 were up-regulated in HCT116,HT29 and DLD1 CRC cell lines(P<0.05).Our results demonstrated that ITGBL1 promoted the abilities of colony formation and proliferation of CRC cells,as well as migration,invasion and EMT(P<0.05).Besides,overexpression of ITGBL1 promoted the phosphorylation of Smad2 and Smad3.Finally,we found mi R-497-5p was a potential mi RNA targeting ITGBL1 directly.Our results showed that mi R-497-5p was down-regulated in CRC tissue(2.709±0.356 vs.5.080±0.222,P<0.05)and negatively correlated with ITGBL1 expression(r=-0.53,P<0.05).Furthermore,overexpression of mi R-497-5p inhibited the expression of ITGBL1(0.6343±0.0513 vs.1.102±0.014,0.3768±0.0356 vs.1.132±0.023,P<0.05).Re-overexpression of ITGBL1 partly abolished the tumorsuppressive effect of mi R-497-5p on cell migration and invasion.Conclusion By performing WGCNA,ITGBL1 was identified as a novel gene associated with metastasis of CRC.ITGBL1 was highly expressed in CRC and promoted migration and invasion.Higher expression of ITGBL1 indicated lower survival rate of CRC patients.Our study provided a new insight into the pathogenesis of CRC and a novel potential biomarker for further research in future studies. |
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