| Background:Cervical cancer is one of the common gynecological malignancy,and is the fourth rank mortality of female malignancies,seriously endangers women's health.Targeted drugs targeting such as vascular endothelial growth factor(VEGF)and programmed cell death protein 1(PD-1)immunosuppressant represent the current targeted therapy and immunotherapy for cervical cancer,have great risks in treating cancer,and off-target conditions.There are also shortages in common usage of the immuno-agents.Therefore,seeking and identifying effective biomarkers other than VEGF and PD-1 is of great value for diagnosis and treatment on cervical cancer patients.Aerobic glycolysis,also known as the Warburg effect,is characterized as an increase in cell glucose uptake,over activated glycolysis,and an increase in lactate production in metabolites.Recent studies argued that tumor cells shut down mitochondrial oxidation phosphorylation functions and rely on glycolysis mainly for energy purchase,in both hypoxia and aerobic conditions.Aerobic glycolysis is widely recognized as a common metabolic model in tumor,and some drugs,targeting aerobic glycolysis to inhibit tumor progression have already been set into clinical trial and applications.Associated coding genes involved in the procedure of glycolytic regulations also became the hotspot of conquering cancer.NAD(P)H quinone oxidoreductase 1(NQO1)is a flavoenzyme,widely distributed in various organs of the human body.As a multifunctional catalyzing quinone,NQO1 plays important roles in reducing toxicity,and balancing the redox reactions in exogenous quinones.Mutations in the C609T allele of NQO1 lead to an instability of the tumor suppressor p53 and increased a risk of tumorigenesis.Recent study showed that NQO1 plays key roles in glycolysis,glutamine catabolism and fatty acid metabolism regulation to promote cancer progression.Our previous study showed that,NQO1 was highly expressed in cervical cancer tissues and was associated with poor prognosis.However,there are few reports discussed about the biological processes of NQO1 aberrant expression and its related mechanisms in cervical cancer progression.The transcription of Hypoxia-inducible factor 1?(HIF1?)increased under hypoxia conditions is often highly expressed in rapid proliferating cells and anaerobic tumor cells.In cancer cells,HIF1? high expression promote the expression and activity of glycolysis related proteins,which enhances the aerobic glycolysis of tumor cells.The homeobox gene SIX 1 plays a key role in embryonic development and is highly expressed in cervical cancer.Recent studies have shown that Sixl promotes glycolysis of breast cancer in hypoxia environment,though studies have reported the combination of NQO1 and HIF1?,yet it remains for further discussion whether the two are involved in the regulation of aerobic glycolysis in cervical cancer.Objective:To clarify and demonstrate the molecular mechanisms of NQO1 in the aerobic glycolysis and malignant progression regulation in cervical cancer.(1)To clarify the role of NQO1 the malignant progression of cervical cancer,and the role to serve as a molecular marker;(2)To discover the role of NQO1 in the proliferation,metastasis,aerobic glycolysis and other biological processes of cervical cancer cells,and to clarify its molecular mechanisms;(3)To reveal the regulatory relationship between NQO1 and HIF1?,and further demonstrate the molecular mechanisms of NQO1 in aerobic glycolysis and malignant progression regulation of cervical cancer.Materials and Methods:(1)Correlation analysis of cervical cancer tissue samples and database:1)Pan-cancerous analysis of NQO1 expression analysis in cervical cancer tissues and cells were performed by GEPIA,Human Protein Atlas,cBioPortal and CCLE biological databases;2)The expression of NQO1 in cervical cancer tissues and cells was detected by immunohistochemical(IHC)staining and Western blot assay,the relationship between the high expression of NQO1 and clinicopathological parameters of cervical cancer patients was analyzed.(2)In vitro experiments:1)Cancer cell lines with overexpressed and knockdown of NQO1 were constructed by lentivirus transfection;2)The effect of NQO1 on the proliferation viability of cervical cancer cells was determined by MTT and colony formation assay.3)Flow cytometry and Western blot assay were used to detect the effect and regulation of NQO1 on apoptosis of cervical cancer cells;4)The effect of NQO1 on metastasis and invasion of cervical cancer cells was determined by wound healing assay and Transwell assay.5)The regulation of NQO1 on EMT progression of cervical cancer cells were detected by IHC and Western blot assay.6)The regulatory role of NQO1 in the glycolysis of cervical cancer was determined by certain kit and Seahorse Metabolite Analyzer system.7)Use glycolysis inhibitors to inhibit or use glucose to stimulate the glycolysis of cervical cancer cells to further clarify the regulatory role of NQO1 in aerobic glycolysis of cervical cancer cells;8)Cobalt chloride(CoCl2),and ubiquitination related experiments were used to detect the regulatory role of NQO1 on HIF1? protein ubiquitination;9)HIF1?/SIX1 in aerobic glycolysis of cervical cancer and its regulatory mechanisms were detected by small interfering RNA(siRNA)transfection.(3)In vivo experiment:the subcutaneous tumor-bearing models and tail vein injection metastasis models were established using nude mice to observe the effects of NQO1 on the proliferation and metastasis of cervical cancer cells.Results:Section ?:NQO1 was highly expressed in cervical cancer tissues and correlated with malignant progression of cervical cancer:1)The bioinformatics analysis and IHC staining assay showed that NQO1 was highly expressed in cervical cancer tissues and cells.2)NQO1 is highly expressed in cervical cancer tissues and is associated with the proliferation and metastasis of cervical cancer.3)MTT,colony formation assay we showed that NQO1 promoted the proliferation of cervical cancer cells.4)NQO1 overexpression inhibited the apoptosis rate of cervical cancer cells,demonstrated by Western blot and flow cytometry.5)NQO1 overexpression promote the migration and invasion ability of cervical cancer cells through wound healing and Transwell assays.6)NQO1 overexpression promote the epithelial-mesenchymal transition(EMT)of cervical cancer cells,analyzed by using Western blot and IF staining of EMT associated biomarkers.Section ?:The role of NQO1 in cervical cancer glycolysis:1)By analyzing the database,NQO1 biological function and related pathway enrichment analysis,showed that NQO1 plays key roles in metabolites production regulation.Western blot showed that NQO1 involved in glycolysis protein expression regulation,implicated the aerobic glycolysis of cervical cancer cells,and promote the proliferation and metastasis of cervical cancer cells.2)NQO1 reduced the generation of ROS in cervical cancer cells and inhibit the activation of AMPK related signaling pathway,determined by using Western blot analysis.Section.?:NQO1 regulates the aerobic glycolysis of cervical cancer through HIFla/SIX1:1)HIFla was knocked down by siRNA,and HIF1? generation was induced by CoCl2 treatment.Glycolysis related metabolites and phenotypes such as proliferation and metastasis of cervical cancer cells were performed.NQO1 regulates the aerobic glycolysis and malignant progressions of cervical cancer cells through HIF1?.2)Co-IP assay and IF co-localization staining assay showed that NQO1 bound to HIF1?.By using MG 132 proteasome inhibitor,and CHX protein synthesis inhibitor and ubiquitination analyzation,NQO1 showed an inhibition on HIF1? ubiquitination by proteasome,and increased HIF1? protein stability.3)IHC staining showed that SIX1 was highly expressed in cervical cancer tissues.Knock down SIX1 effectively reduced the level of aerobic glycolysis and malignant progressions of cervical cancer cells.NQO1 regulates the level of aerobic glycolysis and malignant progressions of cervical cancer cells through HIF1?/SIX1 regulation.Conclusion:NQO1 and SIX1 are highly expressed in cancer tissues and are correlated with the progression of cervical cancer.NQO1 promotes aerobic glycolysis,proliferation,and metastasis by inhibiting AMPK signaling and HIF1?/SIX1 expression enhancement. |
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