Identification Of GNG2 In Breast Cancer Development Based On GEO And TCGA Database

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IDENTIFICATION OF DIFFERENTIAL EXPRESSION GENE GNG2 BAESD ON GEO/TCGA DATABASEObjective: The differential expression genes(DEGs)with important research value in breast cancer were screened by bioinformatics based on the large sample databases of GEO and TCGA,which provided theoretical basis for the further discovery of new therapeutic targets and prognostic markers for breast cancer.Methods: The GSE45827,GSE50428 and GSE57297 expression profile chip were downloaded from GEO database,a total of 181 breast cancer samples and 23 normal control samples were incorporated into differential gene screening system.Meanwhile,the platform annotation file corresponding to each chip was also downloaded.According to the annotation file,the probe was converted to the gene name(if a gene name corresponds to multiple probes,the average is taken).The Sva package in R(3.5.1)was used for batch normalization after merging the original data of the three chips.The DEGs between the breast cancer patients and normal controls were extracted based on empirical Bayes t-tests provided by the R/Limma package,DEGs with |log Fold Change|?1,adjust P<0.01 were selected,and the TCGA database was used to verify the screened DEGs.Heat map of DEGs were drawn using R/Pheatmap package.GO enrichment analysis and KEGG pathway enrichment analysis of DEGs were performed using Cluster Profiler R package(cutoff of P < 0.05 and false discovery rate(FDR)< 0.05).The protein-protein interaction(PPI)network based on all DEGs was constructed using STRING online database(interaction score >0.7)and visualized by Cytoscape software.Survival analysis of core genes was performed using Kaplan–Meier plotter online tool.Results: Based on the conjoint analysis of three expression profile chips,1321 DEGs were identified,of which 624 were up-regulated and 697 were down-regulated in breast cancer.The GO terms for enrichment of DEGs,containing biological process,cell component and molecular function,were the extracellular structure organization,extracellular matrix,and cell adhesion molecular binding.KEGG enrichment analysis showed that most of the genes were enriched in PI3K-AKT signaling pathway.More importantly,GNG2,the core gene of the PPI network,appears the obvious lower expression in breast cancer and regulates most of the downregulation genes,and participates in PI3K-AKT signaling pathways.Moreover,Kaplan-Meier survival analysis showed that low GNG2 expression was associated with worse overall survival and disease-free survival in breast cancer patients.The results suggested that GNG2 might play an important role in the development of breast cancer.Conclusion: The differential expression profile of breast cancer was obtained based on the combined analysis of three chips in GEO database.The PPI network revealed that GNG2 may play an important role in the occurrence and development of breast cancer.PART ? GNG2 INHIBITS BREAST CANCER CELL PROLIFERATION IN VITRO AND IN VIVOObjective: The effect of GNG2 overexpression on the breast cancer cell proliferation were investigated in vitro and in vivo.Methods: The low expression of GNG2 in breast cancer was further confirmed in TCGA and GEPIA online databases.The MCF-7 and MDB-MA-231 cells with stable overexpression of GNG2 and GFP was screened by using Flow cytometry.The effects of GNG2 overexpression on the breast cancer cell proliferation,apoptosis and cycle were studied in vitro by flow cytometry.Cell proteins and total RNA were extracted,Q-PCR and Western blot were then used to detect the expression of cycle activating protein Cyclin D1,proliferation marker protein ki-67 and anti-apoptotic protein BCL-2.In order to inquiry the effect of GNG2 overexpression on breast cancer cell proliferation under the condition of co-culture of breast cancer cells and adipocytes,we constructed a co-culture system.The effect of GNG2 on breast cancer cell proliferation was studied by subcutaneous tumorigenesis in nude mice.Result: GNG2 was significantly downregulated in breast cancer,and could hardly be detected by Western blot.Stable MCF-7 and MDA-MB-231 cell lines overexpressing GNG2 were successfully constructed by transfection with overexpression lentivirus.In vitro experiments showed that upregulation of GNG2 markely inhibited cell proliferation,promoted cell apoptosis,and induced sub-G1 phase arrest.Q-PCR and western blot analysis revealed that cyclin D1,Ki-67 and BCL-2 were downregulated in MCF-7 and MDB-MA-231 cells overexpressing GNG2.In addition,GNG2 overexpression could also significantly inhibit the proliferation of breast cancer cells induced by adipokine.Furthermore,in vivo experimental results further confirmed that GNG2 can inhibit the proliferation of breast cancer cells.Conclusion: Both in vivo and in vitro experiments showed that GNG2 inhibited breast cancer cells proliferation.Meanwhile,GNG2 had a significant inhibitory effect on the proliferation of breast cancer cells induced by adipokine.PART ? POTENTIAL REGULATORY MECHANISM OF GNG2 IN BREAST CANCER CELL PROLIFERATIONObjective: To explore the specific mechanism of GNG2 inhibiting breast cancer cell proliferation.Methods: The effects of GNG2 on the participating signaling pathways were determined by Western blot and KEGG pathway analysis.The key downstream molecules of GNG2 was screened by Q-PCR to clarify the positive regulation of GNG2 on MRAS.MRAS inhibitory plasmids was transfected in the MCF-7 and MDB-MA-231 cells with stable overexpression of GNG2 and GFP to determine whether GNG2 was MRAS-dependent in tumor inhibition.Result: The GSEA analysis results illuminated that GNG2 high expression group was enriched in apoptosis,natural killer cell mediated cytotoxicity,antigen processing and presentation,whereas GNG2 low expression group was enriched in DNA replication,oxidative phosphorylation,biosynthesis of unsaturated fatty acids,spliceosome and base excision repair.KEGG analysis indicated that GNG2 may inhibit cell proliferation through RAS-ERK and RAS-PI3K-AKT pathway.Western blot results confirmed that GNG2 indeed inhibited the phosphorylation of ERK and AKT.There are four subproteins in the RAS family,including HRAS KRAS MRAS NRAS.It was found that GNG2 could only affect the expression of MRAS through Q-PCR screening rather than NRAS KRAS HRAS,and Western blot also further proved that GNG2 overexpression could induce the expression of MRAS.However,the regulatory effect of GNG2 on ERK and AKT activity was ceased after inhibiting the expression of MRAS,suggesting that the regulatory effect of GNG2 on AKT and ERK activity was MRAS-dependent.Conclusion: Bioinformatics analysis suggested that GNG2 may inhibit cell proliferation in multiple ways.The results of the assay confirmed that GNG2 inhibited the activity of AKT and ERK in a MRAS-dependent manner.PART ? ASSOCIATION WITH GNG2 EXPRESSION AND CLINICOPATHOLOGIC VARIABLESObjective: In order to further evaluate the correlation between GNG2 and clinicopathologic variables downloaded from TCGA in breast cancer patients.Methods: The gene expression raw data of breast cancer and corresponding clinical information were downloaded from TCGA official website(https://portal.gdc.cancer.gov/).For the m RNA expression data,Perl was used to combine the m RNA expression values of all genes in 1230 samples into a matrix,and the expression values of single gene GNG2 in all samples were extracted.The samples were then divided into two groups with high and low expression according to the median value of GNG2 expression values.For the clinical data,the clinical information such as the survival time,ID,age,TNM stage,ER state,PR state,HER2 state,etc.was extracted,and the missing value was deleted.The R(3.5.1)software was used to conduct logistic regression analysis and Cox analysis for the extracted clinical data and the expression values of GNG2,and the corresponding 95% confidence intervals and P values were recorded and sorted into tables.To evaluate the diagnostic value of GNG2 in breast cancer patients,we used SPSS software to draw a receiver operating characteristic(ROC)curve.Result: a total of 1109 breast cancer specimens and 121 normal specimens were downloaded from TCGA.Logistic regression analysis revealed that low expression of GNG2 was significantly correlated with high stage(OR = 0.616 for stage II vs.I,P = 0.004),tumor proliferation(OR = 0.584 for > 2cm vs.? 2cm;P < 0.001),tumor status(OR = 0.091;P < 0.001)and age(OR = 0.681;P < 0.001).Univariate and multivariate Cox regression analysis showed that low expression of GNG2 was significantly correlated with poor overall survival(P < 0.05).In addition,age also showed a weak correlation with overall survival of breast cancer(OR close to 1;P < 0.05).The ROC curve analysis indicated that GNG2 might be a novel diagnostic biomarker for breast cancer(AUC= 0.887;P < 0.001).Conclusion: Low expression of GNG2 could independently indicate poor prognosis for breast cancer patients,and GNG2 might be a novel diagnostic biomarker for breast cancer.