Function And Mechanism Of BZW2 In Lung Adenocarcinoma

Chapter Ⅰ BZW2:biomarker associated with lung adenocarcinoma prognosis,tumor immune microenvironment and DNA repair damageBackground:Lung cancer is the leading cause of cancer-related death worldwide,among which lung adenocarcinoma(LUAD)is its most dominant pathological type.Despite the long-term survival benefit of targeted therapy,immunotherapy and other means for patients with advanced LUAD,the 5-year overall survival rate is still less than 20%.Due to the difficulty in early diagnosis and treatment of LUAD patients,it is crucial to find new biomarkers or therapeutic targets for LUAD.Basic leucine zipper and W2 domain containing protein 2(BZW2),an important member of the basic leucine zipper(bZIP)superfamily,is involved in translation initiation.BZW2 is overexpressed in various solid tumors and involved in tumor progression,but the function and mechanism of BZW2 in LUAD are largely unexplored.Objective:The study aimed to explore the expression characteristics,relation with clinical prognosis,epigenetic and immune infiltration characteristics of BZW2 in LUAD via bioinformatics;and analyze the correlation between BZW2 with DNA damage repair and construct a relevant prognostic risk model.Methods:1.TCGA,UCSC,the Human Protein Atlas,CancerSCEM,and CCLE were used to analyze the expression of BZW2 in pan-cancer and/or LUAD on tissue,single-cell,and cell line level.The subcellular localization of BZW2 was obtained from COMPARTMENTS.2.The correlation of BZW2 expression with clinical stage and lymph node stage was analyzed by UALCAN;Survival analysis was performed using Logrank test,univariate and multivariate Cox regression;and Nomogram was constructed.3.The mutational landscape,methylation levels of BZW2 were analyzed via cBioPortal and UALCAN.4.Functional enrichment analysis was performed using Gene Set Enrichment Analysis(GSEA),Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.5.Calculated the Pearson correlations between BZW2 expression and the ESTIMATE score,XCELL immune score;and immune regulatory genes expression,including MHC,chemokines,chemokines receptors,immune stimulatory and inhibitory genes.6.BZW2 related differentially expressed genes(DEGs)were filtered by the limma R package,and a prognostic risk score model for DNA damage repair related DEGs was developed using lasso algorithm.Kaplan-Meier curves,ROC curves,and expression prognostic heatmaps were plotted;the Pearson correlations were calculated between risk scores and expression levels of inhibitory immune checkpoints.Results:1.Compared with normal tissues,BZW2 was significantly overexpressed in LUAD and pan-cancer;BZW2 was expressed at high levels in LUAD cell lines,including H1975 and A549;BZW2 was mainly localized in the cytosol.2.High BZW2 expression was associated with worse clinical stage and lymph node stage in LUAD patients(P<0.05);Logrank test showed that LUAD patients in BZW2 high expression group had shorter overall survival(OS)(HR=1.563,95%CI:1.169-2.090,P=0.003);Multivariate Cox regression analysis identified BZW2 as a prognosis related biomarker(HR=1.441,95%CI:1.073-1.935,P=0.015);and Nomogram was built.3.BZW2 was localized at 7p21,1.Amplifications and mutations were the most predominant forms of genomic alterations,DNA methylation levels were lower in the BZW2 high expression group,and TP53,TTC,MUC16,and CSMD3 were the most frequently mutated genes.4.Enrichment analysis revealed that BZW2 was involved in RNA transport,cell cycle,chemokine signaling pathway,DNA replication,mismatch repair,nucleotide excision repair and other biological processes.GSEA analysis of BZW2 expression were positively correlated with the unfolded protein response,MYC_TARGETS_V2,G2M checkpoint,E2F targets,mTORC1 signaling,DNA repair,MYC_TARGETS_V1 and mitotic spindles.5.The expression levels of BZW2 were significantly negatively correlated with the stromal,immune and ESTIMATE scores in LUAD(P<0.05).BZW2 level was significantly negatively correlated with the infiltration proportion of CD8+T cells(P=2.40e-07),NKT cells(P=1.41e-02),B cells(P=13.92e-11),while positively related with Myeloid-Derived Suppressor Cell(MDSC,P<0.0001),M2 type tumor-associated macrophage(TAMs,P<0.01).BZW2 was negatively correlated with the expression levels of immunoregulatory genes,including MHC,chemokines,chemokines receptors and immune checkpoint inhibitors.6.The limma algorithm selected 258 significantly upregulated and 282 downregulated genes,and a DNA damage repair related risk model including four genes NUDT1,EXO1,NEIL3 and BZW2 was constructed using lasso Cox regression:Risk score=0.05629360*NEIL3+0.068783973*NUDT1+0.12760316*EXO1+0.058865386*BZW2.Survival analysis of TCGA training set(GSE50081 validation set)showed significant differences between high and low risk groups(HR=1.60,95%CI:1.19-2.15,P=1.6e-3;HR=1.70,95%CI:1.07-2.71,P=0.02).ROC curves showed AUC values of 0.65,0.62,and 0.64(0.70,0.61,and 0.59)at 1,3,and 5 years,respectively.The risk score was positively associated with expression of inhibitory immune checkpoints such as PD1,PDL1,PDL2,and LAG3.Conclusions:1.BZW2 was highly expressed in LUAD,which may serve as a biomarker associated with LUAD prognosis.2.BZW2 negatively regulated anti-tumor immune response.3.BZW2 was involved in regulating cell cycle and DNA damage repair,and associated with mTOR and c-MYC signaling pathway.Chapter Ⅱ The role and mechanism of BZW2 in regulating biological behavior of lung adenocarcinomaObjective:The study aimed to explore the role and functional mechanism of BZW2 in lung adenocarcinoma(LUAD)progression and provide a new target for the treatment of LUAD.Methods:1.RT-qPCR and Western blot were used to detect the expression of BZW2 in human LUAD cell lines(NCI-H1975,PC9 and A549)and human normal bronchial epithelial cell line(BEAS-2B).2.RT-qPCR and Western blot were used to determine the expression efficiency of the transfected siRNA,the overexpression plasmids,and the knockdown lentiviral vectors.3.CCK8 cell proliferation experiments and EdU experiments were used to explore the proliferative capacity.4.Cell scratch assay,Transwell migration and invasion assay were used to explore the effects of BZW2 on the migration and invasion abilities;Western blot was used to detect the expression level of Epithelial-Mesenchymal Transition(EMT)related markers(E-cadherin,N-cadherin,slug and vimentin).5.Western blot was used to detect the expression levels of pro-apoptotic factors,including Caspase-3,cleaved Caspase-3,cleaved PARP and Bax,and anti-apoptotic factor Bcl-2 after knockdown and overexpression of BZW2;Annexin V-FITC and PI staining were performed and apoptosis was detected by flow cytometry.6.After cisplatin induced DNA damage in PC9,the BZW2 level was detected by Western blot;Immunofluorescence was used to detect the distribution of γ-H2AX after knockdown of BZW2;The TCGA-LUAD cohort was used to explore the correlation of BZW2 with homologous recombination repair(HR)related genes;RT-qPCR was used to estimate the levels of HR related proteins,including BRCA1,BRCA2 and RAD51.7.The BZW2 protein interaction network was constructed via STRING;The expression level of H2AFY after knockdown of BZW2 was detected via RT-qPCR;The correlation analysis,differential expression analysis and survival analysis of H2AFY were performed for TCGA-LUAD cohort.8.The expression levels of cell cycle related pathway proteins p53 after knockdown and overexpression of BZW2 were detected by Western Blot.9.The expression levels of total PI3K,AKT,mTOR and phosphorylated PI3K,AKT after knockdown and overexpression of BZW2 were detected by Western Blot.10.The nude mice subcutaneous tumor model was constructed using stably BZW2 knockdown(shBZW2)and control(shNC)A549 cell lines.Subcutaneous tumor weight,volume,and growth rate were collected for comparation.Subcutaneous tumors were stripped for immunohistochemical staining of BZW2 and Ki-67.Results:1.RT-qPCR and Western blot results showed that BZW2 was significantly over expressed in NCI-H1975,PC9 and A549 compared with BEAS-2B.2.RT-qPCR and Western blot results showed that the expression level of BZW2 was significantly decreased after siRNA transfection in PC9 and A549 cell lines and increased after transfection of overexpression plasmids;BZW2 expression levels in shBZW2 cell lines were significantly decreased compared with the shNC.3.CCK8 cell proliferation assay and EdU assay found the OD value and proliferation rate of PC9 and A549 cell lines were significantly decreased after knockdown of BZW2;Whereas the proliferation of PC9 was promoted when BZW2 overexpressed.4.The cell scratch assay showed a significant decrease in the wound healing percentage after knockdown of BZW2 in PC9 and A549,and the Transwell migration and invasion assay showed a significant decrease in the cell counts crossed the chamber after knockdown of BZW2;Whereas the migration and invasion abilities of PC9 were strengthened when BZW2 overexpressed;Western blot showed that the level of E-cadherin increased significantly while N-cadherin and slug decreased after knockdown of BZW2;The level of E-cadherin was decreased after overexpression of BZW2,and the expression of N-cadherin and slug was increased.5.Western blot showed increased expression of Caspase-3,cleaved Caspase-3,cleaved PARP and Bax and decreased Bcl-2 after knockdown of BZW2 in PC9 and A549;Overexpression of BZW2 in PC9 decreased the expression of Caspase-3,cleaved caspase-3 and cleaved PARP and increased Bcl-2;Flow cytometry results showed that the apoptosis rate was increased after knockdown of BZW2,but decreased when BZW2 overexpressed.6.Western blot showed that the expression level of BZW2 was significantly decreased after cisplatin induced DNA damage in PC9 cells;Immunofluorescence results found that the proportion of y-H2AX positive cells was increased after knockdown of BZW2;RT-qPCR showed that the expression levels of BRCA1,BRCA2 and RAD51 were significantly decreased when BZW2 knockdown.7.The PPI network showed that BZW2 was closely related to H2AFY;RT-qPCR showed that the expression level of H2AFY was significantly decreased after knockdown of BZW2;H2AFY was significantly highly expressed in LUAD and associated with poor prognosis of LUAD patients.8.Western blot showed that the expression levels of p53 increased after knockdown of BZW2,whereas decreased after overexpression of BZW2.9.Western blot showed that the expression levels of mTOR,phosphorylated PI3K and AKT were decreased after knockdown of BZW2,while the levels of total PI3K and AKT were not significantly different,and the activity of PI3K/AKT/mTOR pathway was increased when BZW2 overexpressed.10.The subcutaneous tumor model in nude mice showed that the subcutaneous tumor volume,weight,and growth rate of nude mice in the shBZW2 group were significantly smaller than the shNC;Immunohistochemistry showed that the expression level of Ki67 was significantly decreased in the shBZW2 group.Conclusions:1.BZW2 promoted proliferation,migration and invasion in LUAD,and regulated apoptosis,EMT and DNA damage repair.2.BZW2 may promote LUAD progression by regulating H2AFY,p53 and PI3K/AKT/mTOR pathways.