High Expression Of CELSR3 Promoted Proliferation And Invasion In Lung Adenocarcinoma: Via Bioinformatics Analysis

Part One Screening of CELSR family in lung adenocarcinoma,based on integrated bioinformatics analysisObjective:To investigate the value of CELSR3 in LUAD,based on integrated bioinformatics analysis.Methods:1.Oncomine database is an online cancer microarray database that aimed at facilitating discovery from genome-wide expression analyses.The expression levels of CELSR m RNA（log₂-transformed）in lung cancer tissues and normal tissues were searched using Oncomine database,and the transcription levels of CELSRs in all lung cancer tissues were determined for statistical comparison.To obtain the important CELSR probes,we set the following thresholds:P-value<0.05,fold change>1.5,and gene ranks in the top 10%.2.UALCAN is an interactive web resource that provides comprehensive cancer transcriptome date for in-depth analysis of The Cancer Genome Atlas（TCGA）gene expression and clinical data from 31 types of cancer.Using UALCAN,we also analyzed the relative CELSR3 expression in LUAD across tumors and corresponding normal tissues,in addition to different tumor subgroups.3.The GEPIA is an interactive website based on TCGA and GTEx-based project,where RNA sequencing expression in tumors and corresponding normal tissues can be analyzed.We used GEPIA to analyze the correlation of TCGA expression data and the Spearman method to determine the correlation coefficient between two genes.4.The prognostic value of the CELSRs m RNA expression was assessed by the Kaplan-Meier Plotter an online database containing gene expression data and survival information for lung cancer patients.Using this database,we analyzed the overall survival（OS）and first progression（FP）of patients with LUAD.The patient samples were divided at the median expression into two groups with either high expression or low expression,and evaluated using the Kaplan-Meier survival plot[P-value<0.05,false detection rate（FDR）<0.05).The risk ratio（HR）had a 95%confidence interval and log-rank p-value.5.The Linked Omics database is a website used to analyze 32 TCGA cancer-related dataset.We used the Link Finder module of Linked Omics to study the differentially expressed genes related to CELSR3 in the TCGA LUAD（n=515）.Pearson correlation coefficient was used for statistical analysis of the results,and Link Finder was used to establish a statistical diagram of a single gene.Functional Enrichment Analysis was applied using the Web-based Gene Se T Analysis Toolkit（Web Gestalt）.The data in the Link Finder results were signed and sequenced,and we used gene set enrichment analysis（GSEA）for gene ontology（GO）analysis,KEGG,and network analysis.We used the Molecular Signatures Database（MSig DB）to analyze the network（FDR<0.05;and 500 simulations were performed）.6.TIMER is a comprehensive resource for the systematic analysis of immune infiltrates across 32 tumor types.TIMER employs a previously published statistical method to infer the abundance of tumor-infiltrating immune cells（TIICs）from gene expression profiles.We analyzed the expression of CELSR3 in different types of lung cancer and the correlation of CELSR3 expression with infiltration of immune cell,including B cells,CD8+T cells,CD4+T cells,macrophages,neutrophils and dendritic cells（DCs）,through gene patterns.Moreover,we studid the correlations between CELSR3expression and genetic markers in TIICs through related modules.7.The TISIDB is a web-portal that combines 4176 records from 2530publications and reports on 988 genes associated with antitumor immunity.We can explore the function of genes of interest and their role in tumor immune interactions by analyzing the TISIDB database and through high-throughput data analysis and literature mining.On the X-axis,CELSR3is used as the gene symbol,and on the Y-axis,the TIICs-related gene marker is used as the gene symbol.We used Log₂RSEM to determine the gene expression level.Results:1.Expression of CELSR3 m RNA is higher in lung adenocarcinoma than in normal lung tissue.2.CELSR3 m RNA expression was statistically significant in sex,age,N stage,M stage and T stage in lung adenocarcinoma.3.Patients with high expression of CELSR3 m RNA in lung adenocarcinoma have a poor prognosis.4.Analysis of GO and KEGG pathways of genes co-expressed with CELSR3 in lung adenocarcinoma showed that CELSR3 was related to cell cycle,DNA replication,and cell adhesion.5.ATM/PLK,CDK1,CDK2,CHEK1 and CHEK2 associated with CELSR3 in lung adenocarcinoma;mi RNAs included mi R-517,mi R-503,mi R-370,mi R-331,mi R-197,mi R-125,and mi R-412;The transcription factors were E2F1 and E2F4.6.CELSR3 m RNA expression was correlated with CD8+T cell infiltration in lung adenocarcinoma cells.7.CELSR3 m RNA expression was correlated with the CCL17/CCR4axis.Conclusions:1.CELSR3 m RNA is highly expressed in lung adenocarcinoma and is associated with poor prognosis.2.The CELSR3 m RNA expression was statistically significant in sex,age,N stage,M stage and T stage in lung adenocarcinoma.3.The CELSR3 m RNA expression was related to cell cycle,proliferation and invasion.4.The CELSR3 m RNA expression in lung adenocarcinoma cells is associated with CD8+T cell infiltration and may be mediated by the CCL17/CCR4 axis.Part Two Effect of CELSR3 downexpression on proliferation and apoptosis of A549 and H1975 cells and its mechanismObjective:To investigate the effect of CELSR3 on proliferation and apoptosis in LUAD and its mechanism.Methods:1.Immunohistochemistry and Western blot were used to detect the expression level of CELSR3 in tumor tissues and non-tumor tissues of lung adenocarcinoma patients.2.Western blot assays were used to examine the expression of CELSR3in the LUAD cells lines（A549 and H1975）.3.Si-CELSR3 and NC were designed and synthesized,and transfected into A549 and H1975 cells.Expression of CELSR3 in lung adenocarcinoma A549 and H1975 cells was detected by q RT-PCR.4.CCK-8 assays were performed to examine the effect of CELSR3downexpression on proliferation of the A549 and H1975 cells.5.Flow cytometry were performed to detect the effect of CELSR3downexpression on cell cycle of the A549 and H1975 cells.6.Flow cytometry were performed to detect the effect of CELSR3downexpression on apoptosis of the A549 and H1975 cells.7.Colony Formation Assays were used to determine the effect of si-CELSR3 and NC on the cell cycle of lung adenocarcinoma A549 and H1975 cells.8.Western blot assays were used to detect the effect of CELSR3downexpression on the expression of Caspase-3 and Caspase-8 proteins in A549 and H1975 cells.Results:1.Immunohistochemical results showed that the expression of CELSR3protein in lung adenocarcinoma tissues was higher than that in normal tissues.2.Western Blot was used to detect the expression level of CELSR3protein in two lung adenocarcinoma cell lines,and the results showed that the expression level of CELSR3 protein was high in A549 and H1975 cells.Therefore,A549 and H1975 cells were used as research objects in subsequent studies.3.After transfection with different c ELSR3 si RNAs,q RT-PCR analysis showed that c ELSR3 m RNA expression was decreased in both A549 and H1975,and si RNA#1 had the best effect.4.Cell proliferation assays showed that downexpression of CELSR3could significantly inhibit the proliferative ability,compared with the control vector group（P<0.05）.6.Flow cytometry showed that the percentage of apoptotic cells in the A549 and H1975 downexpression group was significantly higher than that of the control vector group（P<0.05）.7.Compared with the control vector group,Caspase-3 and Caspase-8proteins levels signifcantly decreased upon CELSR3 downexpression（P<0.05）.Conclusions:1.Si-CELSR3 transfection can effectively reduce the expression level of CELSR3 m RNA.2.CELSR3 downexpression significantly inhibit the proliferation and clone formation ability of A549 and H1975 cells,and promote the apoptosis of A549 and H1975 cells.3.Decreased the expression of Caspase-3 and Caspase-8 might be the key mechanism for regulating apoptosis of the A549 and H1975 cells.Part Three Effect of CELSR3 downexpression on migration and invasion of A549 and H1975 cells and its mechanismObjective:To investigate the effect of CELSR3 on migration and invasion in LUAD and its mechanism.Methods:1.A CELSR3 downexpression or control vector was transfected into A549 and H1975 cells,and stably transfected A549 and H1975 monoclonal cells was selected by puromycin.2.The effects of CELSR3 on the migration ability of A549 and H1975cells were investigated by wound-healing assays.3.The effects of CELSR3 on the migration ability of A549 and H1975cells were further investigated by Transwell migration assays.4.The effects of CELSR3 on the invasion ability of A549 and H1975cells were further investigated by Transwell invasion assays.5.Western blot assays were used to detect the effect of CELSR3downexpression on the expression of E-cadherin and Vimentin proteins in A549 and H1975 cells.Results:1.Wound-healing assays showed that the migration ability was markedly decreased in the A549 and H1975 cells that downexpressed CELSR3 when compared with that of the control cells（P<0.05）.2.Transwell migration assays showed that downexpressing CELSR3inhibited the migration of A549 and H1975 cells（P<0.05）.3.Transwell invasion assays showed that downexpressing CELSR3attenuated the invasiveness of A549 and H1975 cells（P<0.05）.4.E-cadherin and Vimentin proteins levels signifcantly decreased upon CELSR3 downexpression,compared with the control vector group.Conclusions:1.CELSR3 downexpression significantly inhibited the ability of migr-ation and invasion of A549 and H1975 cells.2.CELSR3 may mediate the migration and invasion ability of A549 and H1975 lung adenocarcinoma cells by regulating the EMT pathway.