Study On The Role And Mechanism Of MTHFD2 In Lung Adenocarcinoma Growth And Metastasis And The Regulation Of Its Expression

Lung cancer is one of the most common malignant tumors in the world,and it leads the cause of cancer-related death.It is estimated that 5 year survival rate of lung cancer patients remains 21% even after treatment.The histology of lung cancer includes non-small cell lung cancer and small cell lung cancer.Lung adenocarcinoma is the most common subtype of non-small cell lung cancer.The current strategies for lung adenocarcinoma treatment include surgery,radiotherapy,chemotherapy and targeted therapy.Since lack of symptoms in early stage,most patients are diagnosed with advanced stages.Although some lung adenocarcinoma patients with advanced stages can benefit from chemotherapy and targeted therapy,they will eventually develop drug resistance,which implies the exsitense of unrevealed disease mechanism.Therefore,it is necessary to explore new pathogenesis of lung adenocarcinoma,explore new therapeutic targets and improve the survival time of patients.One-carbon metabolism is an important pathway of cancer metabolic reprogramming,which plays a crucial role in cancer development.In addition to providing one-carbon units for macromolecules synthesis(such as nucleotides,aminoacids,lipids),one-carbon metabolism is also involved in epigenetic modification,ATP and NADPH production in cancer.Current studies show that one-carbon metabolism contains cytoplasmic pathway and mitochondrial pathway.In recent years,one-carbon metabolism enzymes are found to be up-regulated in cancers,especially in the mitochondrial pathway.Besides,one-carbon metabolism enzymes affect cancer cell proliferation,migration,apoptosis,stem cell characteristics and other phenotypes,which are expected to become new drug development targets.In this study,we used online databases and tissue samples from lung adenocarcinoma patients to explore the expression of methylenetetrahydrofolate dehydrogenase 2(MTHFD2)in lung adenocarcinoma,and analyze the relationship between MTHFD2 expression level and clinical characteristics of lung adenocarcinoma patients.We explored the effect of MTHFD2 on cell proliferation,migration,apoptosis and sphere formation in lung adenocarcinoma cell lines.We explored the effect of MTHFD2 on the tumorigenesis and metastasis of lung adenocarcinoma in nude mice.Then we explored the role of MTHFD2 in lung adenocarcinoma oxidative stress and its effect on AKT/GSK-3β/β-catenin signaling pathway.Finally,we investigated the role of mi R-30a-3p in regulating the expression of MTHFD2.This study reveals new mechanism of lung adenocarcinoma development,analyzes the significance of MTHFD2 in patients with lung adenocarcinoma,and explores the specific mechanism of action and expression regulation mechanism of MTHFD2 in lung adenocarcinoma.We provides new clues for condition analysis and new targets for the treatment of lung adenocarcinoma.Part Ⅰ Expression and clinical significance of MTHFD2 in lung adenocarcinomaObjective: To investigate the expression of MTHFD2 in lung adenocarcinoma and analyze the relationship between MTHFD2 expression level and patients’ clinical characteristics.Methods: We analyzed the expression of one-carbon metabolism enzymes in TCGA LUAD dataset using UCSC database.We analyzed the expression of MTHFD2 in lung adenocarcinoma using Oncomine database.Immunohistochemistry(IHC)assay was performed to detect the expression of MTHFD2 in lung adenocarcinoma patients.At last,we analyzed the relationship between MTHFD2 expression level and patients’ gender,smoking history,clinical stage,prognosis.Results: 1.TCGA LUAD dataset showed one-carbon metabolism enzymes were up-regulated in lung adenocarcinoma,and MTHFD2 had the greatest difference.GSE32863(Selamat)and GSE19188(Hou)datasets from Oncomine database showed MTHFD2 was up-regulated in lung adenocarcinoma tissues compared with normal lung tissues.IHC showed the expression of MTHFD2 in lung adenocarcinoma tissues was higher than that in adjacent tissues.2.In lung adenocarcinoma,the expression of MTHFD2 was higher in male patients compared with female patients.Smoking patients had higher MTHFD2 expression level than non-smoking patients.Patients with stage Ⅲ lung adenocarcinoma had higher MTHFD2 expression level than patients with stage Ⅰ.Patients with high MTHFD2 expression had shorter overall survival time than patients with low MTHFD2expression.Conclusions: 1.The expression of MTHFD2 was up-regulated in lung adenocarcinoma tissues.2.In lung adenocarcinoma,high MTHFD2 expression was positively associated with male patients,smoking habit,clinical stage and poor prognosis.Part Ⅱ The effect of MTHFD2 on cell proliferation,migration and apoptosis in lung adenocarcinomaObjective: To investigate the effect of MTHFD2 on cell proliferation,migration and apoptosis in lung adenocarcinoma.Methods: Western blot was performed to detect the expression of MTHFD2 in lung adenocarcinoma cell lines.After PC-9 and H1975 cells were transfected with si NC or si MTHFD2,CCK-8,flow cytometry,low attachment culture,would healing assay,transwell assay and western blot were performed to detect the effect on proliferation,cell cycle,apoptosis,sphere formation,migration and epithelial-mesenchymal transition(EMT)markers,respectively.After PC-9 was infected with sh NC and sh MTHFD2 lentivirus,subcutaneous xenograft tumor model and tail vein metastatic tumor model were performed to detect xenograft growth and metastasis.IHC assay and TUNEL assay were performed to detect Ki67 expression and apoptosis,respectively.Results: 1.The expression of MTHFD2 in lung adenocarcinoma cell lines was higher than that in human bronchial epithelial cells(HBEC).2.Compared with si NC group in PC-9 and H1975 cells,CCK-8 showed si MTHFD2 group had reduced proliferation ability.Flow cytometry showed si MTHFD2 group had decreased proportion of S+G2/M phase cells and more apoptotic cells.Low attachment culture showed si MTHFD2 group had limited sphere formation ability.3.Compared with si NC group in H1299 and H1975 cells,would healing assay showed si MTHFD2 group had increased would width.Transwell assay showedsi MTHFD2 group had more migrated cells.Western blot showed si MTHFD2 suppressed N-cadherin and vimentin level in H1299 cell,while si MTHFD2 suppressed vimentin level and elevated E-cadherin level in H1975 cell.4.Compared with sh NC group in PC-9 cell,subcutaneous xenograft tumor model showed sh MTHFD2 group grew much slower,and xenograft weight was decreased.IHC assay and TUNEL assay showed sh MTHFD2 group xenograft had lower Ki67 expression level and more apoptotic cells.Tail vein metastatic tumor model showed sh MTHFD2 group had less metastatic nodules.Conclusions: 1.The expression of MTHFD2 was up-regulated in lung adenocarcinoma cell lines.2.MTHFD2 knockdown suppressed cell proliferation and sphere formation abilities,blocked cell cycle,and promoted cell apoptosis in lung adenocarcinoma cells.3.MTHFD2 knockdown suppressed cell migration ability and EMT in lung adenocarcinoma cells.Part Ⅲ The role of MTHFD2 in modulating redox homeostasis and AKT/GSK-3β/β-catenin signaling in lung adenocarcinomaObjective: To investigate the mechanism of MTHFD2 in lung adenocarcinoma.Methods: GSEA was performed to reveal the potential mechanism of MTHFD2 in lung adenocarcinoma.After PC-9 and H1975 cells were transfected with si NC/si MTHFD2 and vector/MTHFD2 overexpression plasmid,flow cytometry was performed to detect cellular reactive oxygen species(ROS)levels.WST-8 assay was performed to detect cellular NADPH/NADP+ ratio.After hypoxia stimulation,flow cytometry was performed to detect cellular ROS levels and cell apoptosis.Western blot was performed to detect the expression levels of p-AKT,p-ERK and nucleus β-catenin.After PC-9 and H1975 cells were transfected with si NC/si MTHFD2 + vector/MTHFD2 overexpression plasmid,together with DMSO/MK-2206 treatment,western blot was performed to detect the expression levels of p-AKT,p-GSK and β-catenin.CCK-8 assay and transwell assay were performed to detect cell proliferation and migration,respectively.Results: 1.GSEA result showed MTHFD2 could modulate redox homeostasis in lung adenocarcinoma.In PC-9 and H1975 cells,MTHFD2 knockdown increased cellular ROS levels while MTHFD2 overexpression had no effect on cellular ROS levels.Besides,knockdown of MTHFD2 decreased cellular NADPH/NADP+ ratio.2.Hypoxia stimulation increased cellular ROS levels in PC-9 and H1975 cells,and induced more apoptotic cells in si MTHFD2 group compared to si NC group.Besides,N-acetylcysteine could partially rescue cell apoptosis.3.GSEA result showed MTHFD2 could modulate protein serine/threonine kinase activity.Western blot showed knockdown of MTHFD2 decreased the expression levels of p-AKT and nucleus β-catenin,while it had little impact on p-ERK.Further investigation results showed MTHFD2 knockdown decreased the expression levels of p-AKT,p-GSK and β-catenin,which mimicked the effect of MK-2206.MTHFD2 overexpression increased the expression levels of p-AKT,p-GSK and β-catenin,while MK-2206 erased this effect.4.CCK-8 assay and transwell assay showed MTHFD2 knockdown suppressed cell proliferation and migration in PC-9 and H1975 cells,while MTHFD2 overexpression promoted cell proliferation and migration,respectively.Meanwhile,MK-2206 erased the promotion of MTHFD2 overexpression.Conclusions: 1.MTHFD2 modulated redox homeostasis in lung adenocarcinoma.2.MTHFD2 regulated cell proliferation and migration via AKT/GSK3-β/β-catenin signaling in lung adenocarcinoma.Part Ⅳ The role of mi R-30a-3p in regulating MTHFD2 expression in lung adenocarcinomaObjective: To investigate the role of mi RNA in regulating MTHFD2 expression in lung adenocarcinoma.Methods: c Bioportal database was applied to analyze MTHFD2 gene alternative frequency.mi RWalk、mi RDB、mi Randa and Targetscan databases were applied to predict the potential mi RNAs,which could interact with MTHFD2.Star Base database was applied to analyze the expression of mi R-30a-3p and the relationship between mi R-30a-3p and MTHFD2.q PCR was performed to detect the expression level of mi R-30a-3p in HBEC and lung adenocarcinoma cell lines.q PCR and western blot were performed to detect the m RNA and protein levels in PC-9 and H1975 cells after transfection with NC mimics and mi R-30a-3p mimics.Dual luciferase reporter assay validated the interaction between mi R-30a-3p and 3’UTR of MTHFD2.After PC-9 and H1975 cells were transfected with NC mimics/mi R-30a-3p mimics + vector/MTHFD2 overexpression plasmid,CCK-8 assay and transwell assay were performed to detect cell proliferation and migration,respectively.Results: 1.c Bioportal database showed mutation + amplification frequency of MTHFD2 gene was less than 1% in lung adenocarcinoma.mi RWalk、mi RDB、mi Randa and Targetscan databases predicted 20 potential mi RNAs.Star Base database showed mi R-30a-3p was down-regulated in lung adenocarcinoma and negatively associated with MTHFD2.2.q PCR results showed the expression level of mi R-30a-3p was lower in lungadenocarcinoma cell lines than that in HBEC.Compared with NC mimics group,mi R-30a-3p mimics group had lower m RNA and protein levels of MTHFD2 in PC-9 and H1975 cells.Dual luciferase reporter assay showed that mi R-30a-3p mimics inhibited luciferase enzyme expression with MTHFD2 wild type 3’UTR,while had little impact on that with MTHFD2 mutant type 3’UTR.4.CCK-8 assay and transwell assay showed transfection with mi R-30a-3p mimics suppressed proliferation and migration of PC-9 and H1975 cells compared to NC mimics,respectively.While overexpression of MTHFD2 partially abolished the suppression of mi R-30a-3p mimics.Conclusions: 1.mi R-30a-3p was down-regulated in lung adenocarcinoma.2.mi R-30a-3p inhibited MTHFD2 expression via binding to 3’UTR of MTHFD2 in lung adenocarcinoma.3.In lung adenocarcinoma,the oncogene role of MTHFD2 was regulated by mi R-30a-3p.