NQO1/Snail Axis Promotes Glioma Progression Via PI3K/AKT/mTOR Signaling Pathway

Background:Glioma is the most common primary malignant tumor in adult nervous system,with high morbidity and majority.At present,surgical resection followed by radiotherapy and adjuvant chemotherapy is the standard treatment strategy for glioma,but characterized by rapid tumor invasive growth,the recommended therapy often is incapable of resecting the tumor entirely and leads to tumor recurrence and dismal prognosis.Therefore,it is urgent to explore new effective therapeutic strategies and potential targets for glioma.NAD(P)H:quinone oxidoreductase(NQO1)has been studied extensively as an important component of the cellular antioxidant defense program because of its ability to catalyze the two electron reduction of quinines to hydroquinones,thus preventing the formation of radicals and reactive oxygen species due to one-electron reduction of cytotoxic quinines and the autooxidation of semi-quinones.NQO1 also functions as an important role in detoxification during metabolism.It has been reported that polymorphism of NQO1 gene C609T site could lead to reduce or complete loss of NQO1 enzyme activity,thus reducing NQO1 functions of detoxification and maintaining cell stability,and increasing the risk of tumor occurrence.Recently,many studies showed that NQO1 is up-regulated in a number of solid tumors,such as breast cancer,gastric cancer,and ovarian cancer,and high NQO1 expression has.been correlated with a significant increase in cell proliferative activity as well as drug resistance.The previous research of our group found that NQO1 inhibitor?-rapaquinone may suppress invasion and migration of gastric and breast cancer cells by inhibiting EMT process Via P13K/AKT/mTOR signaling pathway.Meanwhile,some studies have shown that PI3K/Akt/mTOR signaling pathway is clo sely related to the malignant progression of glioma.Snail,as one of the regulator of EMT markers,is upregulated in variety of tumors and correlates with tumor invasion and metastasis.It has reported that knockdown the expression of Snail could inhibit the proliferation,migration and invasion of glioma cells.However,whetherNQO1 affects glioma metastasis and EMT process,and whether this regulation is related to Snailremains unclear.Objectives:The present study aimed to investigate the biological function and molecular mechanism of NQO1/Snail signal axis in the malignant progression of glioma:(1)To evaluate the correlation between NQO1 expression and clinicopathological characteristics,further clarify the value of NQO1 as a prognostic biomarker in glioma.(2)To explore the effects and mechanism of NQO1 silence or overexpression on biological functions of glioma cells,including proliferation,migration,and invasion.(3)To detect the correlation between NQO1 and Snail,and the potential molecular mechanisms of NQO1/Snail signaling axis in glioma malignant progression.Materials and Methods:1.Bioinformatic analysis and glioma tissues:(1)The Oncomine database,Human Protein Atlas database,GEPIA database were used to explore the expression level of NQO1 mRNA in glioma tissues.(2)Immunohistochemistry was performed to detect the protein expression level of NQO1 in glioma patients and the correlation between NQO1 expression of tumors and patients' clinicopathological characteristics were statistically analyzed.(3)GEPIA database was used to explore the correlation between NQO1 mRNA expression and prognosis in glioma.Survival curves obtained using the Kaplan-Meier method.2.In vitro experiments:(1)Established stable silence or overexpress NQO1 expression glioma cells lines using lentivirus infection.(2)MTT assay,colony formation assay,and EdU staining were performed to assess the effects of NQO1 expression levels on proliferation of glioma cells.(3)Wound healing assay and Transwell assay were performed to characterize the effects of NQO1 expression levels on migration and invasion of glioma cells.(4)To clarify the molecular mechanism of NQO1 promotingthe malignant progression of glioma through PI3K/AKT/mTOR signaling pathway,colony formation assay,EdU staining assay and Transwell assay were performed following LY294002 and Rapamycin treatment.(5)The LinkedOmics database,cBioportal database,and ChIPBaseV2.0 database were used to predict the target genes of NQO1 and enrichment analysis was performed.(6)Immunofluorescence assay was used to detect the co-localization of NQO1 and Snail.(7)To explore the molecular mechanism of NQO1/Snail signaling axis on proliferation,migration and invasion of glioma cells,the colony formation assay, wound healing assay and Transwell assay were performed using small interfering RNA(siRNA)specific transfected for Snail in NQO1 stable overexpression cell lines of glioma.3.In vivo experiments:(1)The subcutaneous xenograft model of nude mice was established to compare the effect of differential expression of NQO1 on tumor growth.(2)Immunohistochemistry staining was performed to compare the expression levels of Ki67 and EMT related markers in different tumor tissues group.(3)The lung metastasis tumor model of nude mouse was established by injection of glioma cells with different expression of NQOl into the tail vein to compare the formation of lung metastasis.Results:1:.High expression of NQO1 in glioma tissues was correlated with patients'poor prognosis.(1)Bioinformatics analysis showed that mRNA expression of NQO1 was increased in glioma tissues compared with the adjacent normal tissues.(2)Immunohistochemical staining showed that NOQ1 was highly expressed in glioma tissues compared with adjacent non-cancerous tissues,especially in high pathologicalgrade and histologic infiltration tissues.TCGA analysis results showed that patients with high NQO1 expression had significantly shorter tumor-free survival than those with low NQO1 expression,but insignificantly with overall survival in glioma patients.2.NQO1 plays roles in glioma cells proliferation migration and EMT pro gression regulation via PI3K/AKT/mTORpathway.(1)The results of MTT assay,colony formation assay and EdU staining assay showed that cell proliferation and colony formation were inhibited when the expression of NQO1 was suppressed in glioma cells;oppositely,overexpression of NQO1 promoted the proliferation and colony formation ability.(2)The results of wound healing assay and Transwell assay indicated that overexpression of NQO1 promoted cell invasion and migration in glioma cells.Instead,when the expression of NQO1 was suppressed,the cell invasion and migration were significantly reduced.(3)Bioinformatic analysis showed that NQO1 was co-expressed with EMT markers.Furthermore,both western blot assay and immunofluorescence analysis showed that NQO1 regulate the protein expression levels of EMT-related markers.(4)Western blot analysis showed that NQO1 regulated the expression levels of P13K/AKT/mTOR signaling pathway-related proteins.The results of MTT,colony formation,EdU and Transwell assay indicated that LY294002 and Rapamycin reserved the promotive effect of NQO1 overexpression on cell proliferation,colony formation,DNA duplication,migration and invasion in,glioma cells.Both Western blot and immunofluorescence analysis demonstrated that effect of NQO1 overexpression on the p13K/AKT/mTOR pathway was partially reversed by LY294002 and Rapamycin.3.NQO1 regulates the malignant progression of glioma through Snail.The results of bioinformatic analysis confirmed thatSnail expression was positively correlated with NQO1 expression in glioma.Based on colony formation and Transwell assay,we found that knocking down Snail expression level could reverse the promotion effect of NQO1 overexpression on proliferation,migration,invasion ability of glioma cells and EMT process,and activate PI3K/AKT/mTOR signaling pathway.Conclusions:NQO1 was high-expressioned in glioma and correlated with patients' poor prognosis.NQO1/Snail axis played a crucial role in glioma malignant evolution via PI3K/AKT/mTOR signaling pathway.