Study On The Mechanism Of SOX5 To Maintain The Malignant Phenotype Of Non-small Cell Lung Cancer

Background and objective: Lung cancer is the malignant tumor with the highest morbidity and mortality,and non-small cell lung cancer(NSCLC)accounts for the most proportion.The malignant phenotypes of tumor cell,such as self-renewal,quick proliferation,high migration and invasion capabilities,are the underlying causes for the local recurrence and distant metastasis of NSCLC.Therefore,in-depth study of the maintenance mechanism of NSCLC malignant phenotype,in order to clarify the occurrence and development mechanism of NSCLC,to find a specific therapeutic target is of great significance.Recent studies had found that SOX2 can interact with YAP,a key effector molecule in Hippo signaling pathway,to promote the progression of malignancy.Sex determining reion Y-box 5(SOX5),the same member of the SOX supergene family,had also been found aberrantly expressed in many kinds of tumors and can promote the proliferation,invasion,migration,and other malignant phenotypes of tumor,but the expression and function of SOX5 in NSCLC progression is still unclear.So the aim of this study is to explore the expression of SOX5 in NSCLC and its roles in the maintainance of malignant phenotype and the potential molecular mechanism.Methods: The relationship between the expression of SOX5 and the prognosis of NSCLC patients was analyzed by GEO database,qRT-PCR and Western blot were performed to detect the expression of SOX5 in human bronchial epithelial cells(HBE)and three kinds of NSCLC cell lines(A549,H1299,H1975),A549 and H1975 tumorspheres and its monolayer cells.Then,A549 and H1975 cells were infected with lentivirus to build stable down-and up-regulated SOX5 cell lines,and the infection efficiency were verified by qRT-PCR and Western blot.Moreover,the colony formation assay,CCK-8 assay,tumorsphere formation assay,transwell invasion assay,wound healing assay and immunofluorescence invadopodia staining were used to explore the effects of SOX5 on the proliferation,innvasion and migration abilities of A549 and H1975 cells.Western blot was performed to explore the effect of SOX5 on the expression of stemness markers OCT4,NANOG,SOX2 and invasion,migration related proteins MMP2,VIMENTIN and SLUG.In vivo,we injected A549 cells with stable silencing of SOX5 subcutaneously into the right flanks of nude mouse to form xenograft,The xenograft tumor volume and weight were measured and the expression of SOX5,YAP1,SOX2 and MMP2 were determined by immunohistochemistry(IHC).The tumorsphere formation assay,transwell invasion assay,wound healing assay and immunofluorescence invadopodia staining were performed to explore the influence of YAP1 on NSCLC malignant phenotype.The effect of down-regulated YAP1 on the expression of stemness markers OCT4,NANOG,SOX2 and invasion and migration related proteins MMP2,VIMENTIN and SLUG were performed with western blot.On the mechanism,Co-Immunoprecipitation(Co-IP),cell immunofluorescence and the nuclear and cytoplasmic extracts assay were performed to explore the interaction protein with SOX5.Moreover,overexpressing of SOX5 was executed in the knockdown YAP1-A549 cells,and whether SOX5 can rescue the fuction of silcencing YAP1 was observed.Results: 1.We found that the highly expression of SOX5 suggests poor prognosis in NSCLC patients by GEO database,qRT-PCR and Western blot showed SOX5 highly expressed in three kinds of NSCLC cells but at a lower level in HBE,and the expression in tumorspheres is higher than in monolayer cells.2.After infected with lentiviral,qRT-PCR and Western blot showed the expression of SOX5 decreased after silencing of SOX5,while increased after overexpressed SOX5 in both A549 and H1975 cells,the knockdown and overexpression of SOX5 stable cell lines were successfully constructed 3.The colony formation assay and CCK-8 assay showed that knockdown of SOX5 decreased the volume and number of colony and attenuated the proliferation ability,while up-regulated SOX5 increased the volume and number of colony and promoted the proliferation ability.4.The tumorsphere formation assay showed that down-regulated SOX5 decreased the volume and number of tumorspheres,while up-regulated SOX5 increased the volume and number of tumorspheres.5.Transwell invasion assay,wound healing assay and immunofluorescence invadopodia staining showed that down-regulated SOX5 decreased the number of invasioned cells on the transwell chamber,slowed down the rate of wound healing and decreased the formation of invadopodia,while up-regulated SOX5 increased the number of invasioned cells on the transwell chamber,accelerated the rate of wound healing,and increased the formation of invadopodia.6.Western blot showed that down-regulated SOX5 decreased the expression of stemness markers OCT4,NANOG,SOX2,and invasion and migration related proteins MMP2,VIMENTIN and SLUG,while up-regulated SOX5 increased the expression of those proteins.7.In vivo,knockdown of SOX5 slowed down the growth of xenograft,and IHC showed that knockdown of SOX5 decreased the expression of YAP1,SOX2 and MMP2.8.After knockdown of YAP1 in NSCLC cells,the tumorsphere formation ability of NSCLC was decreased,the number of invasioned cells on the transwell chamber was decreased,the rate of wound healing and the formation of invadopodia were decreased,Western blot showed that down-regulated YAP1 decreased the expression of stemness markers OCT4,NANOG,SOX2,and invasion and migration related proteins MMP2,VIMENTIN and SLUG.9.SOX5 and YAP1 were both higher expressed in tumorspheres than in monolayer cells.Western blot showed that YAP1 can synchronous change with the down or up-regulated SOX5.And in turn,knockdown of YAP1 also decreased the expression of SOX5.10.Co-IP showed that SOX5 interacted with YAP1 to form a complex in NSCLC cells.Immunofluorescence assay showed that SOX5 and YAP1 preferentially localized in the nuclei of NSCLC cells,and the nuclear and cytoplasmic extracts assay demonstrated that the overexpression of SOX5 promoted the translocation of YAP1 into the nucleus.While overexpressed SOX5 in shYAP1-A549 cell partialy rescued the functions of silencing YAP1.Conclusion: 1.SOX5 highly expressed in NSCLC cells and can promote the proliferation,self-renewal,invasion and migration of NSCLC cells.2.Knockdown YAP1 can decrease the self-renewal,invasion and migration abilities of NSCLC cells.3.SOX5 can interact with YAP1 to promote NSCLC progression.