Mechanism Of NQO1/CPT1A Regulating Pancreatic Adenocarcinoma Evolution Through Fatty Acid Oxidation

Background:Pancreatic adenocarcinoma(PAAD)is one of the quite normal malignant tumors of digestive system.With the increase of new cases and deaths from PAAD worldwide,the 5-year survival rate is as low as about 10%.At present,the treatment regimen is still mainly surgery and chemotherapy,but the treatment effect is little,the survival rate and prognosis of patients were not greatly improve.Therefore,it is necessary to explore the molecular mechanism of PAAD from a more in-depth perspective and provide new possibilities for the diagnosis and treatment of PAAD.Energy metabolism reprogramming has been recognized to play an important role in the occurrence and development of malignant tumors.Fatty acid oxidation(FAO),as an emerging research focus,exists in the malignant evolution of most cancers.Carnitine palmitoyl-Teansferase 1A(CPT1A),a member of the Carnitine system family,is a key rate-limiting enzyme of the FAO process,which plays a key role in cancer progression through regulating the FAO process in cancer cells.NADPH quinone oxidoreductase 1(NAD(P)H: quinone oxidoreductase 1,NQO1)is a soluble flavinase ubiquitous in mammals.As a superoxide reductase,NADPH oxidoreductase participates in the binding and regulation of m RNA translation,interacts with proteins and degrades proteasome.Many years of studies have found that NQO1 regulates the evolution of various malignant tumors,such as breast cancer,carcinoma of lung and hepatocellular carcinoma,through a variety of energy metabolism pathways.Our research group has previously found that the high expression of NQO1 in PAAD is associated with poor prognosis,but the biological process and molecular mechanism of NQO1 in PAAD has not been further explored.Whether NQO1 participates in the evolution of PAAD through FAO and its relationship with key enzymes in the process of FAO has not been reported,which requires further investigation and analysis.Objective: To further explore the effects of NQO1 on the biological functions of PAAD cells,and initially clarify the roles and molecular mechanisms of NQO1 and CPT1 A,a key FAO enzyme,in regulating FAO and its evolution in PAAD.Materials and methods: 1.Correlation analysis between NQO1 and PAAD:1)The expression of NQO1 in PAAD was verified by exploring GEPIA,TIMER2.0,ENCORI,UCSC Xena,UALCAN and Oncomine databases;2)Survival analysis was performed using Oncolnc,HPA,GEPIA and Kaplan-Meier databases;2.In vitro experiments: 1)Construction of stable differentially expressed NQO1 cell lines;2)Effects of MTT,plate cloning and Ed U on proliferation of PAAD cells;3)Transwell and scratch assay were used to determine the effect of NQO1 on the metastasis of PAAD cells;4)Immunofluorescence and protein imprinting assay were used to detect the effect of NQO1 on EMT process of PAAD cells;5)The specific molecular mechanism of NQO1 in PAAD was predicted by enrichment analysis of STRING database and Linked Omics database;6)The regulation of NQO1 on FAO in PAAD was determined by FAO kit,ATP kit and protein imprinting assay;7)ETX was used to determine the molecular mechanism of NQO1 in regulating FAO in PAAD by MTT,plate cloning,cell scratch and protein imprinting experiments;8)Ch IPBase,GEPIA and TIMER databases were used to explore the correlation between NQO1 and CPT1 A,immunohistochemical staining was used to verify the expression of CPT1 A in PAAD tissues,and the interaction between NQO1 and CPT1 A was detected by immunofluorescence male localization and Co IP assay;9)The regulation of NQO1 in the ubiquitination degradation of CPT1 A protein was detected by ubiquitination technique;10)The role and regulatory mechanism of NQO1 and CPT1 A in fatty acid oxidation,proliferation and metastasis of PAAD were investigated by lentivirus transfection,plate cloning,cell laceration,Transwell and western blot;3.In vivo experiments: 1)A tumor bearing model was established in nude mice to explore the effect of differentially expressed of NQO1 on the proliferation of PAAD;2)The expression changes of NQO1,proliferation markers and FAO markers in subcutaneous tumor-bearing tissues of different groups of mice were compared by immunohistochemical staining.Results: 1.The expression of NQO1 is up-regulated in PAAD tissues and is associated with poor prognosis: A database search showed that NQO1 was highly expressed in PAAD tissues,and survival analysis in the database showed that overexpression of NQO1 predicts poor prognosis in patients;2.NQO1 overexpression is associated with lymph node metastasis in patients with PAAD:HPA database and immunohistochemistry experiments showed that NQO1 protein was significantly higher in PAAD tissues than in normal paracancer tissues,it was mainly located in the nucleus with a few expression in the cytoplasm yet;The expression of NQO1 was only correlated with lymphatic metastasis(P < 0.05);3.NQO1 promotes the proliferation of PAAD: MTT,plate cloning,Ed U,subcutaneous implantation of nude mice and immunohistochemical staining were used to detect Ki67 protein expression in subcutaneous tumor tissues of mice,it was found that NQO1 overexpression can promote the proliferation of PAAD cells in vivo and in vitro,and vice versa;4.NQO1 promoted the migration,invasion and EMT process: Cell scratch and Transwell experiments showed that NQO1 overexpression promoted the migration and invasion of PAAD cells;Immunoblotting,immunohistochemical staining and immunofluorescence of EMT-related proteins showed that NQO1 regulated the EMT process of PAAD cells;5.NQO1 plays a carcinogenic role in the progression of PAAD through FAO: The correlation between NQO1 and the processes related to FAO and key enzymes of FAO was found through GO enrichment analysis and exploration database;The results of FAO and ATP kit showed that the content of FAO and ATP in PAAD cells increased significantly when NQO1 was overexpressed;The expression levels of FAO-related markers in PAAD cells were also up-regulated when NQO1 was overexpressed;After the addition of FAO inhibitors,MTT,plate cloning,cell scratches and western blot experiments showed that the inhibitors reversed the promotion effect of NQO1 overexpression on PAAD cells;6.NQO1/CPT1 A is involved in the evolution of PAAD: Mining database showed that NQO1 was positively correlated with the rate-limiting enzyme CPT1 A in FAO,and CPT1 A was highly expressed in a variety of cancer tissues.Immunofluorescence co-location assay showed that NQO1/CPT1 A was co-located in the nucleus and cytoplasm;Further Co IP experiments showed that NQO1 had an interaction with CPT1A;Ubiquitination experiments showed that NQO1 increased the stability of CPT1 A by inhibiting the ubiquitination of CPT1 A by proteasome.After CPT1 A was silenced in PAAD cell lines with NQO1 overexpression,plate cloning,Ed U,Transwell,cell blot and western blot confirmed that down-regulation of CPT1 A expression partially inhibited the promotion of NQO1 overexpression on proliferation,migration and EMT process of pancreatic cancer cells.Conclusion: NQO1 is highly expressed in PAAD,which is closely related to the malignant evolution of PAAD.Moreover,NQO1/CPT1 A interacts with PAAD and plays an important role in PAAD cell proliferation and metastasis by activating the FAO pathway.