Mechanism Of MiR-485-5p/NQO1 Signaling Axis Mediates Aerobic Glycolysis In Colorectal Cancer Malignant Progression

Background:Colorectal cancer(CRC)is one of the most common digestive tract tumors.Colorectal cancer has over 1.8 milion new cases occurred every year,with the mortality rate ranking second in the world,its incidence rate and mortality rate are on the rise.Relevant epidemiological studies have confirmed that not only age,genetics,intestinal homeostasis,diet structure,but also diabetes closely related to the occurrence of colorectal cancer.With the pathogenesis of colorectal cancer has gradually clarified,a large number of studies have confirmed that aerobic glycolysis played a vital role in the evolution of colorectal cancer.Therefore,intervene aerobic glycolysis pathway and target the treatment become new hotspots of research in colorectal cancer.NAD(P)H:quinone oxidoreductase 1(NQO1)is a cytosolic flavoenzyme that catalyzes the dielectronic reduction in eukaryotic cells,reduces the formation of semiquinone,a single electron reduction product of quinone and quinone analogues.Recent researches showed that NQO1 regulated tumor cells proliferation,migration and apoptosis through energy metabolism pathways such as aerobic glycolysis,glutamine catabolism and lipid metabolism,mainly involving in breast cancer,liver cancer,lung cancer and cervical cancer.In this study,miR-485-5p,targeted NQO1,is a small non-coding RNA,regulates the post transcripitional expression of multiple target genes through complementary pairing with 3'-UTR(untranslated region)of multiple target gene mRNA.Previous studies on miR-485-5p in tumor mainly focused on cell proliferation,metastasis,apoptosis and chemoradiotherapy resistance.At present,there are no reports on the relationship between miR-485-5p and NQO1,and whether miR-485-5p/NQO1 plays a role in regulating the evolution of colorectal cancer through aerobic glycolysis pathway,which needs further research.Objectives:This study aimed to investigate the biological function and related molecular mechanism of miR-485-5p/NQO1 axis in the progression of colorectal cancer:1)Databases combined with clinicopathological data to clarify the relationship between the expression of NQO1 and clinicopathological parameters of colorectal cancer patients;2)To elucidate the underlying molecular mechanism of NQO1 involved in proliferation,metastasis,anoikis and aerobic glycolysis in colorectal cancer;3)To reveal the targeted regulation relationship between miR-485-5p and NQO1,to explore the mechanism of miR-485-5p/NQO1 signal axis regulating the malignant evolution and aerobic glycolysis of colorectal cancer.Materials and Methods:1.Database and colorectal cancer tissue samples:1)The expression of NQO1 in pan-cancers including colorectal cancer was obtained from bioinformatics databases;2)The expression of NQO1 in colorectal cancer tissues was detected by immunohistochemistry,the relationship between the expression level of NQO1 and clinicopathological parameters of patients was analyzed;3)Kaplan-Meier and Cox proportional hazards model were used to calculated the relationship between NQO1 expression and prognosis.2.Experiments in vitro:1)Lentivirus transfection was used to establish stable colorectal cancer cell lines with NQO1 overexpression or knockdown;2)The effects of NQO1 expression on colorectal cancer cells proliferation was detected by MTT,colony formation assay and EdU staining;3)Wound healing,transwell and immunofluorescence assay were used to reveal the effects of NQO1 expression on colorectal cancer cells metastasis and EMT progression;4)Anoikis cells were cultured onto poly-HEMA coated dishes.The effects of NQO1 expression on colorectal cancer cells anoikis was detected by Hoechst 33342 staining,flow cytometry assay and Apoptosis/Necrosis detection kit;5)The effects of NQO1 mediated aerobic glycolysis in the progression of colorectal cancer was determined by Seahorse Bioscience to detect the oxygen consumption rate(OCR),extracellular acidification rate(ECAR),2-DG/3-BrPA rescue experiments;6)The microRNA targeting NQO1 were predicted by bioinformatics prediction and verified by dual luciferase reporter gene experiments;7)miR-485-5p mimic and inhibitor were transfected by lipofectamine 3000.The molecular mechanism of miR-485-5p/NQO 1 axis regulating the proliferation and migration of colorectal cancer cells through aerobic glycolysis were detected by colony formation assay,transwell assay and aerobic glycolysis related metabolic kits;8)Bioinformatics database was uesd for clarifying the expression of miR-485-5p in colorectal cancer and its relationship with the prognosis of patients.3.Experiments in vivo:1)The nude mouse subcutaneous xenograft model was used to investigate the effects of NQO1 on colorectal cancer cell growth in vivo;2)Metastasis lung colonization model was established by inoculating colorectal cancer cells to validate the effects of NQO1 on the formation of lung metastasis in vivo;3)Immunohistochemical staining and Western Blot assays were used to compare the expression of Ki67,EMT and glycometabolism related markers in different tumor tissues.3)Results:Part 1.NQO1 is a prognostic biomarker for colorectal cancer patients:1)Based on the data from databases,NQO1 was significantly up-regulated in a variety of tumors including colorectal cancer.2)In tissue microarrays,the expression of NQO1 in colorectal cancer tissues was signficantly higher than that in adjacent normal tissues.Moreover,clinicopathological parameters analysis showed that the expression level of NQO1 was closely associated with the clinical stage and lymph node metastasis;Kaplan-Meier analysis demonstrated that the patients with high expresssion of NQO1 had a lower overall survival;Cox regression model analysis revealed that high expresssion of NQO1 was an independent prognostic factor for survival in colorectal cancer.Part 2.NQO1 regulates the malignant biological process of colorectal cancer:1)We found that NQO1 overexpression promoted cell viability,colony formation and DNA replication ability by MTT,colony formation assay and EdU staining in colorectal cancer cells;Nude mouse xenograft tumor model experiment revealed that NQO1 promoted the growth of tumor.2)Wound healing and transwell assays showed that NQO1 overexpression obviously promoted the horizontal and vertical migration capacity of the colorectal cancer cells;Western Blot and immunofluorescence assay showed that NQO1 affected the expression of EMT related markers;HE staining showed that NQO1 promoted the formation of pulmonary metastatic nodules.3)Bioinformatics databases analysis revealed that NQO1 interacted with apoptosis related markers;Hoechst 33342 staining showed that NQO1 overexpression inhibited apoptosis;Flow cytometry implied that NQO1 overexpression promoted anoikis-resistant of colorectal cancer cells;Western Blot showed that NQO1 overexpression induced down-regulation of pro-apoptotic protein Bax and cl-Caspase 3/8,and up-regulation of anti-apoptotic protein Bcl-2.The results of Apoptosis/Necrosis detection kit were consistent.4)Seahorse flux analyser proved that NQO1 overexpression elevated ECAR and reduced OCR of colorectal cancer cells.Western Blot analysis showed that NQO1 overexpression promoted the protein expression of glucose transporter and glycolytic enzymes;Moreover,2-DG and 3-BrPA could reverse the ability of NQO1 promote the proliferation and metastasis of colorectal cancer cells.Part 3.miR-485-5p/NQO1 signal axis regulates colorectal cancer progression through aerobic glycolysis:1)Bioinformatics prediction software uncovered that miR-485-5p targeted NQO1 gene,and inhibited NQO1 mRNA and protein expression.2)Dual luciferase reporter gene experiment confirmed that miR-485-5p targetd the 3'-UTR site of NQO1 and inhibited NQO1 mRNA and protein expression.3)NQO1 can reverse miR-485-5p inhibit aerobic glycolysis in colorectal cancer by measuring the glycolysis phenotype(glucose uptake,lactate production and ATP level);NQO1 can reverse the malignant biological behavior of colorectal cancer cells inhibited by miR-485-5p through the rescue experiments of proliferation and metastasis.4)ENCORI and Kaplan-Meier plotter databases showed that the expression of miR-485-5p was significantly down-regulated in colorectal cancer than in adjacent non-tumor tissues.The patients with low expression of miR-485-5p exhibited shorter survival time.Conclusions:NQO1 was high-expressed in colorectal cancer tissues and predicts patients' poor prognosis.miR-485-5p/NQO1 signaling axis mediates aerobic glycolysis to regulate the proliferation,metastasis and apoptosis of colorectal cancer.