Research On Anti-tumor Effect And Mechanism Of 4.1N In Non-small Cell Lung Cancer

Background: Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer death worldwide.NSCLC(Non-small cell lung cancer)accounts for 85% of the total amount of lung cancer and is difficult to diagnose and treat in the early stage.NSCLC is divided into three main histological types: lung adenocarcinoma(LUAD),lung squamous cell carcinoma(LUSC)and large-cell lung carcinoma(LCLC).The 5-year survival rate for early NSCLC patients was approximately30%,and the 1-year survival rate for advanced NSCLC was only approximately 10%.Conventional clinical diagnosis methods still cannot detect NSCLC micrometastases.Due to the poor effect of traditional surgery,the drug resistance of radiotherapy and chemotherapy,and the limitations of targeted drugs and immunotherapy,overall survival rates of NSCLC patients are still very low.We still need to find new molecular targets for etiology-based diagnosis,treatment and prognosis in NSCLC,which are important for improving the efficacy of NSCLC treamtment and patient survival.Scaffolding protein 4.1N is encoded by EPB41L1 gene.4.1N has three conserved domains: FERM(4.1-ezrin-radixin-moesin),SAB(spectrin-actin-binding domain)and CTD(c-terminal domain).In between the three domains,there are three non-conservative U(unique)domains: The U1 in the N-terminal extension region;the U2 between the FERM and SAB;the U3 between SAB and CTD.4.1N is a neuron-enriched 4.1 family member and its research was focused in the nervous system before.In recent years,the anti-tumor role of 4.1N has been revealed in some cancers such as epithelial ovarian cancer,breast cancer,etc.However,the role of 4.1N in NSCLC is still elusive.Objectives: By exploring expression level,biological effect and underlying molecular mechanism of 4.1N in NSCLC,this research aims to find new molecular targets for diagnosis,treatment and prognosis of NSCLC based on 4.1N,and provided a new strategy for diagnosis and treatment of NSCLC patients.Methods: Western blot were used to detect protein expression level of 4.1N in NSCLC cell lines.4.1N expression level was analyzed by immunocytochemistry in NSCLC specimens.The correlation between4.1N expression level and clinical features was analysed.Wound healing assay,MTT assay,transwell assay and xenografts nude mice assay were performed to investigate the biological effect of 4.1N in vitro and vivo.Bioinformatics,immunoprecipitation,LC-MS/MS analysis,co-immunoprecipitation and GST pull-down assay were used to search for 4.1N-interacting molecules(including the specific interacting domains).After knocking down 4.1N in 95 C and over-expressing 4.1N in95 D cells,western blot was used to search for 4.1N-mediated pathway.Bioinformatics,Meth Surv tool(https://biit.cs.ut.ee/methsurv)and targeted bisulfite sequencing(TBS-seq)were used to analyze the methylation of 4.1N gene and its prognostic relevance.NSCLC cells were treated with the methylase inhibitor 5-Aza-Cd R,and then the 4.1N expression was detected by q PCR and western blot.4.1N m RNA and mi R-454-3p expression levels were evaluated through GCTA data analysis and q PCR.Spearman analysis was used to validate the relationship between 4.1N m RNA and mi R-454-3p expressions.Bioinformatics,double luciferase reporter assay and western blot were used to validate if the mi R-454-3p binds 4.1N m RNA 3’ untranlated region(3’UTR)in NSCLC cells.Results: 4.1N expression level was relatively lower in cells(H1299and 95D)with high metastatic ability,while 4.1N expression level was relatively higher in cells(A549,HCC827 and 95C)with low metastatic ability.Positive immunocytochemistry staining of 4.1N was found in ten normal lung tissues(10/10),while negative staining of 4.1N was detected in 55%(52/99)of NSCLC specimens.Negative immunocytochemistry staining for 4.1N was significantly associated with poorly differentiated cases and high TMN stages.4.1N suppressed NSCLC cell proliferation and migration in vivo and in vitro.flotillin-1 is a noval 4.1N-interacting molecule.4.1N contacted with flotillin-1 through the FERM and U2 domains and was involved in a β-catenin/Wnt pathway.High methylation of 4.1N gene promoter was prevelant in LUAD and LUSC tumors.The methylation level of 4.1N gene promoter was high from stage I to IV in LUAD and LUSC tumors.DNA methyltransferase inhibitor 5-Aza-Cd R decreased methylation level of the 4.1N gene promoter and restored the4.1N expression at both the m RNA and protein levels.Mi R-454-3p was significantly high-expressed in NSCLC,while 4.1N m RNA was significantly high-expressed in NSCLC adjacent tissue.Abnormally high-expressed mi R-454-3p directly targeted 4.1N m RNA 3’UTR.Mi R-454-3p expression was negatively correlated with 4.1N m RNA expression in NSCLC patients.Conclusions: 1.4.1N expression level is negatively associated with NSCLC stage and the degree of malignancy.The reduction or loss of4.1N promotes the occurrence and development of NSCLC.2.4.1N interacts with a new 4.1N-binding molecule,flotillin-1,through FERM and U2 domains.4.1N is involved in a β-catenin/Wnt pathway and inhibits cell proliferation and migration of NSCLC cells.3.Hyper-methylation within the 4.1N gene promoter region is prevelant in NSCLC.High methylation of 4.1N gene was an early event in NSCLC.4.In NSCLC,mi R-454-3p was abnomally high expressed.mi R-454-3p directly targeted 4.1N m RNA 3’UTR.27 figures,22 tables and 135 references.