The Mechanism Study Of NQO1 Regulating Hepatocellular Carcinoma Progression Via SREBP1-mediated Lipid Metabolism

Background: As one of the most common gastrointestinal malignancies,hepatocellular carcinomais(HCC)is the fourth rank of cancers incidence in China and the second leading cause of cancer-related death worldwide.The five-year survival rate of patients is less than 20%.Due to the latent onset,most patients are already in the middle or late stage when diagnosed,which seriously affects the prognosis of patients.Surgical resection is the preferred treatment for HCC,but it is only applicable to 15%of patients in the early stage.The clinical treatment for patients in the late stage,including the combination of systemic radiotherapy and chemotherapy,however,it is prone to treatment tolerance,results in poor efficacy.It has been recognized that metabolic associated fatty liver disease(MAFLD)caused by obesity and high fat diet has become the main reason for the rising incidence of HCC.Hence,researchers have gradually paid attention to the relationship between metabolic disorders and HCC.Recent studies have found that abnormal lipid metabolism is one of the most significant metabolic changes in HCC.Activation of key enzymes in lipid metabolism pathway accelerates the rate of lipid production and decomposition,provides membrane synthesis materials and energy for cell proliferation and metastasis,thus promoting the malignant evolution of HCC.Therefore,intervention of key enzymes and regulatory factors in lipid metabolism pathway is expected to provide a reliable new direction for target therapy of HCC and improve patient prognosis.NAD(P)H quinone oxidoreductase 1(NQO1),a cytoplasmic flavoenzyme,catalyzes double electron reduction reactions in eukaryotic cells and reduces the formation of semiquinone,a single electron reduction product of quinone and its derivatives.Researchers have found that NQO1 is highly expressed in breast cancer,lung cancer,cervical cancer and other tumors,and participates in tumor proliferation,metastasis and malignant progression by regulating metabolism reprogramming.Previous studies of our group have shown that NQO1 expression is up-regulated in HCC tissues and closely related to poor prognosis of patients.Yang et al.found that NQO1 regulates glycolysis by targeting the pyruvate kinase PKLR and promotes the malignant evolution of breast cancer.However,the role and mechanism of NQO1 in tumor lipid metabolism have not been reported.Sterol regulatory element binding protein 1(SREBP1)is a key transcription factor in nuclear and regulates lipid metabolism in eukaryotic cells,which is closely related to the occurrence and development of tumors.In our study,we firstly found that NQO1 could promote triglyceride production and upregulate key enzymes expression in lipid synthesis pathway,among which SREBP1 has a prominent differential expression.However,whether NQO1 regulates the malignant progression of HCC through SREBP1-mediated lipid metabolism and its specific molecular mechanism remains to be further elucidated.Objectives: To explore the biological functions and molecular mechanisms of NQO1/SREBP1 signaling axis in the malignant evolution of HCC.(1)To clarify the effects of NQO1 on the biological functions of HCC,including proliferation,metastasis,EMT,angiogenesis and lipid anabolism;(2)To elucidate the molecular mechanism of NQO1 regulates Snail protein stability through SREBP1-mediated lipid anabolism and participates in the malignant evolution of HCC;(3)To explore the role of NQO1 and SREBP1 as molecular markers in HCC prognosis assessment.Materials and Methods: 1.Databases and IHC of HCC tissue specimens analysis:(1)The expression of NQO1 in pan-carcinoma and HCC tissues was analyzed by UALCAN,TIMER and other bioinformation databases;(2)Immunohistochemical staining(IHC)was used to detect NQO1 expression in HCC tissue microarray,and the relationship between the NQO1 expression level and clinicopathological features was analyzed;(3)Kaplan-Meier Plotter and other databases were used to analyze the relationship between NQO1 expression and survival of HCC patients.2.In vitro experiments:(1)Lentivirus transfection was used to construct stable cell lines with NQO1 knockdown and overexpression,and the transfection effects were verified by western blot and q RT-PCR;(2)MTT,colony formation,Ed U and other assays were used to detect the proliferation of HCC cells with NQO1 differential expression;(3)The effect of NQO1 on autophagy in HCC cells was detected by AO staining and western blot;(4)The effects of NQO1 on metastasis and EMT process of HCC cells were observed by wound healing,transwell,cell morphology,immunofluorescence staining and western blot;(5)The effect of NQO1 on the angiogenesis ability of HCC was determined by vascular mimicogenesis,microtubule formation,chick chorioallantoic membrane assay and HUVECs function detection;(6)The effects of NQO1 on lipid metabolism of HCC cells were detected by BODIPY493/503 staining,triglyceride and cholesterol production kit and western blot;(7)The interaction between NQO1 and SREBP1 was verified by Co IP and immunofluorescence double-labeling assays;(8)Protein synthesis inhibition CHX,proteasome inhibition MG132 and ubiquitination experiments were used to detect the effect of NQO1 on the stability of p53 protein;(9)The binding site of p53 and SREBP1 was detected by luciferase reporter gene assay;(10)The mechanism of NQO1 regulates the proliferation and metastasis of HCC via SREBP1 was explored by rescue experiments using si RNA,western blot,etc;(11)The influence of NQO1/SREBP1 on Snail protein stability was determined by correlation analysis in databases,q RT-PCR,Co IP and ubiquitination experiments;(12)si RNA and rescue experiments were used to verify that NQO1/SREBP1 regulates the malignant evolution of HCC by Snail;(13)The coexpression of NQO1 and SREBP1 in HCC and its correlation with patients survival were analyzed by databases and IHC.3.In vivo experiments:(1)The xenograft tumor of nude mice was established to verify the effect of NQO1 on the proliferation ability of HCC;(2)Lung metastasis model was established by tail vein injection to determine the effect of NOQ1 on HCC metastasis ability;(3)IHC was used to detect the effects of NQO1 on the expressions of Ki67,EMT and lipid metabolism related markers in tumor tissues.Results: 1.High expression of NQO1 predicted poor prognosis of HCC patients: The results of databases and IHC showed that NQO1 expression was upregulated in HCC tissues.Clinicopathological features analysis revealed that the high expression of NQO1 was correlated with tumor size and pathological grade.Kaplanmeier Plotter databases analysis showed that the overall survival of patients with high expression of NQO1 was closely related to obesity.2.NQO1 promoted the malignant progression of HCC:(1)NQO1 promoted proliferation of HCC cells and inhibited autophagy: MTT,colony formation and other experiments showed that high expression of NQO1 promoted the proliferation activity,clonal formation ability and DNA replication ability of HCC cells.AO staining showed that autophagosomes decreased after high expression of NQO1,and western blot found that NQO1 downregulated the expression of autophagy related markers Beclin1,P62 and LC3 B,suggesting that NQO1 inhibited autophagy.The xenograft tumor model showed that high expression of NQO1 promoted tumorigenesis,and vice versa.(2)NQO1promoted the migration,invasion and EMT process of HCC cells: Wound healing and Transwell experiments showed that high expression of NQO1 enhances the horizontal and vertical migration abilities of HCC cells.Morphological observation revealed that the cells with high expression of NQO1 underwent mesenchymal transformation,in addition,immunofluorescence and western blot experiments suggested that NQO1 affects the expression of EMT-related markers.Lung metastasis model showed that NQO1 high expression increases the number of lung metastatic nodules,and vice versa.(3)NQO1 promoted angiogenesis of HCC cells: It was found that NQO1 promotes angiogenesis and microtubule formation by angiogenesis mimicy,microtubule formation and CAM assays.Western blot showed that NQO1 promotes the expression of angiogenesis related markers MMP family and VEGF.HUVECs function assays showed that the culture medium supernatant of NQO1 high expression cells promotes the proliferation and migration of HUVECs,and vice versa.(4)NQO1promoted HCC cells lipid anabolism: The results of BODIPY493/503 staining,triglyceride and cholesterol levels detection showed that NQO1 promotes the neutral lipid production ability of HCC cells,and western blot showed that NQO1 promotes the expression of lipid anabolism related markers.3.The molecular mechanism of NQO1/p53/SREBP1/Snail in the progression of HCC:(1)NQO1 interacted with SREBP1 through p53 and promoted the malignant progression of HCC:Bioinformatics analysis combined with Co IP and immunofluorescence co-location assays found that NQO1 interacts with SREBP1.Ubiquitination experiments suggested that NQO1 regulates the stability of p53 protein.The binding site of p53 to the 3’UTR promoter of SREBP1 sequences was verified by luciferase reporter gene assay.MTT,wound healing and Transwell rescue assays showed that NQO1/p53 promotes the proliferation,metastasis and EMT process of HCC cells through SREBP1 mediated lipid anabolism.(2)NQO1/p53/SREBP1 plays a carcinogenic role by regulating the stability of Snail protein through ubiquitination: Co IP assay verified the interaction between NQO1,SREBP1 and Snail.By adding CHX,MG132 and ubiquitination experiments,it was found that NQO1/SREBP1 regulated Snail protein stability through ubiquitination.MTT,wound healing,Transwell and other rescue experiments showed that Snail involved in NQO1/SREBP1 promoting the proliferation and metastasis of HCC cells.4.NQO1 and SREBP1 were biomarkers for HCC prognosis evaluation and target therapy: Bioinformatics analysis combined with IHC showed that compared with non-tumor adjacent tissues,NQO1 and SREBP1 were highly expressed in HCC tissues and closely related to shorten survival of patients.NQO1 and SREBP1 could synergistically suggest poor prognosis of patients.Conclusions: NQO1 promoted the proliferation,metastasis,angiogenesis and lipid anabolism of HCC cells,and NQO1 regulated Snail protein stability through SREBP1-mediated lipid anabolism,thus promoting the malignant evolution of HCC.NQO1 and SREBP1 could serve as effective biomarkers for the target therapy and prognosis evaluation of HCC patients.