ARHGAP21 Inhibits Epithelial-Mesenchymal Transition By Inactivated WNT Signaling Pathway In Non-Small Cell Lung Cancer

Background and purposeRhoGTPases family is one of the key factors regulating the cytoskeleton.However,the RhoGTPases family proteins are also regulated by various factors.Rho GTPase activating protein 21(ARHGAP21)is one of those factors and is reported to be involved in cell adhesion,migration,Golgi regulation,and cell trafficking.However,ARHGAP21 has not been reported in non-small cell lung cancer.This study aims to clarify the expression and function of ARHGAP21 in non-small cell lung cancer and provide a new target for the diagnosis and treatment of non-small cell lung cancer.Method1.To explore the expression of ARHGAP21 in non-small cell lung cancer(1)The expression of ARHGAP21 in cancer and paracancer tissues was analyzed based on TCGA and CPTAC databases.(2)The expression of ARHGAP21 in cancer and paracancer tissues was detected by immunohistochemistry.(3)Western blot was used to detect the expression of ARHGAP21 in non-small cell lung cancer cell lines and immortalized normal bronchial epithelial cells.2.To explore the relationship between ARHGAP21 expression and prognosis of patients with non-small cell lung cancer(1)The relationship between ARHGAP21 expression and prognosis of patients was analyzed based on Kmplotter lung cancer survival database.(2)The relationship between ARHGAP21 expression in tissue chips and prognosis was analyzed according to immunohistochemical scores.3.To explore the effect of ARHGAP21 on the intracellular and extracellular migration ability of non-small cell lung cancer cells(1)ARHGAP21 knockdown cell line and knock-down efficiency verification.(2)The effect of ARHGAP21 on cell migration in vitro was verified by Transwell assay.(3)The effect of ARHGAP21 on cell migration in vitro was verified by scratch healing experiment.(4)The effect of ARHGAP21 on cell metastasis in vivo was verified by lung metastasis model in nude mice.4.Bioinformatics predicts the mechanism of action of ARHGAP21(1)To search for molecules related to ARHGAP21 expression based on GEPIA and TCGA data.(2)KEGG and String databases were used to analyze the possible cellular functional processes and signaling pathways involved in ARHGAP21.5.The mechanism of action of ARHGAP21 was verified by cell biology experiments(1)The effect of ARHGAP21 on the mRNA level of the complex molecule was verified by qPCR.(2)The effect of ARHGAP21 on the molecular protein level of the complex was verified by Western blot.(3)The ubiquitination level test confirmed that ARHGAP21 promoted the ubiquitination degradation of β-catenin by regulating the expression of complex molecules.Results1.The analysis of TCGA,CPTAC database,clinical samples and 7 cell lines showed that the expression of ARHGAP21 in NSCLC tissues was downregulated compared with normal tissues.2.Immunohistochemical score of tissue chip combined with clinical parameter analysis showed that low expression of ARHGAP21 was associated with poor prognosis.3.Cell biological function experiments showed that knockdown of ARHGAP21 could promote the migration of non-small cell lung cancer cells in vitro and in vivo.4.Bioinformatics analysis showed that APC was highly correlated with ARHGAP21 expression.And ArhGAP21-related proteins are widely involved in WNT signaling pathway.5.Molecular mechanisms suggest that down-regulation of ARHGAP21 promotes EMT process in non-small cell lung cancer cells through activation of WNT signaling pathway,thus promoting tumor progressionConclusionARHGAP21 is downregulated in non-small cell lung cancer and is an un-protective factor for prognosis.Downregulation of ARHGAP21 leads to activation of WNT signaling pathway and promotes metastasis of non-small cell lung cancer cells.