| Breast cancer is caused by internal factors and environmental factors.Understanding the molecular pathogenesis is of great significance and value for diagnosis,intervention,prognostic evaluation,and precision treatment in the future.Previously,BRWD3（bromodomain and WD repeat domain containing 3）was found statistically significant between breast cancer patients and normal controls.BRWD3 is one of the members of the bromodomain-containing protein family.The mutations or abnormal expression of the coding genes of this family members are associated with a variety of tumors,psychiatric and neurological diseases.BRWD3 is related to chronic leukemia,melanoma,head and neck cancer,and HPV-mediated cervical epithelial transformation.In this study,we intends to find clues about the role of BRWD3 in breast cancer progression,using TCGA database and bioinformatics methods.With immunohistochemical methods combined with bioinformatics results,we analyze the expression characteristics of BRWD3 in breast cancer tissues,and the relationship between BRWD3 and clinical features and its role in breast cancer progression.Part 1:Analysis of BRWD3 gene mutations in breast cancer tissuesWe analyze BRWD3 mutations in breast cancer tissues on the c Bio Portal for cancer genomeics platform and use SIFT and Polyphen-2 to assess mutation hazards.The results showed that no BRWD3 mutation was found in 11 normal breast tissues and 10 benign breast phyllodes tumors.In 13 invasive breast cancer studies,among the 7438 samples of 7271patients,34 patients were retrieved with BRWD3（NM＿153252）mutation.The hazard assessment of the mutation types was performed.20 missense mutation types were evaluated as the harmful mutations in the SIFT assessment,and 19 missense mutation types were evaluated as the harmful mutations with Polyphen-2.However,loss-of-function truncation mutations have not been rated as harmful mutations.The clinical characteristics of these BRWD3 deleterious mutation types were analyzed.Due to the lack of Ki-67 value,it can only be preliminarily estimated as luminal（4 cases）,HER2 overexpression（1 case）,triple negative（1 case）,and unknown（2 cases）.Part 2:Correlation between BRWD3 expression level and the breast cancerGrouping matrix and expression matrix were created after the molecular data and the clinical data were downloaded using GDC-client.Differential expression was conducted with edge R.The differentially expressed genes were statistically analyzed,and the first 30,000 genes,which met the two commonly used criteria|㏒2FC|>1 and p<0.05,were selected and input into the csv file.Statistical differences between the two groups were detected with independent sample T test,while statistical differences among multiple groups were detected with one-way analysis of variance.The p<0.05 was set as a statistically significant difference.The results showed that the expression level of BRWD3 in cancer tissues was higher than that in paracancerous tissue,and the difference was statistically significant（p<0.05）.Between Invasive Ductal Carcinoma（IDC）and Invasive Lobular Carcinoma（ILC）,the difference in BRWD3 expression level was statistically significant（p<0.001）.Part 3:BRWD3 expression characteristics in breast cancer tissuesWe collected 1 case of breast hyperplasia,9 cases of breast fibroadenoma,and 32 cases of breast cancer.The methods of paraffin embedding,sectioning,HE staining and immunohistochemistry were used to observe the expression characteristics of BRWD3 in breast cancer tissues.Firstly,the immunohistochemical parameters and method were optimized and standardized.Subsequently,the expression of BRWD3 was detected in the tissues.Finally,more than 3 senior pathologists reviewed the slices,diagnosed and evaluated the positive degree.The results showed that BRWD3 was positive in the breast hyperplasia,and in fibroadenoma and breast cancer tissues.It was heterogeneity of BRWD3 distribution in the cytoplasmic,membranes and nuclei.BRWD3 presents heterogeneity in the degree of cytoplasmic staining,the distribution of cell membranes and nuclei.Part 4:Role of BRWD3 in breast cancer progression To explore the role of BRWD3 in breast cancer progression,a total of 1486 differential expression genes（DEGs）of IDC and ILC genes were obtained from TCGA database.These DEGs were enriched in cell division,cytoskeleton and inflammation.The module of BRWD3was related to the formation of IDC/ILC pathology using Weighted Gene Co-Expression Network Analysis.164 genes were selected as a network from the module of BRWD3.The biological process functions of this network were enriched in the regulation of epithelial cell migration,regulation of nitrogen-containing compound metabolism,cell adhesion,and cell communication.Part 5:Correlation between BRWD3 expression and ER,PR,and HER2 statusAccording to the BRWD3 expression level in breast cancer,the samples were divided into a high-expression group and a low-expression group.The differences in the expression and status of ER,PR and HER2 proteins in these two groups were analyzed to find the relationship between BRWD3 transcription level and pathological types.A significant correlation was observed between BRWD3 transcription level and PR status,HER2 protein expression level,and HER-2/3 phosphorylated protein（HER2＿p Y1248 and HER3＿p Y1289）expression levels.No significantly difference was observed in ER status or expression level.The relationships between BRWD3 and PR,HER2 were conducted with immunohistochemical methods.The results showed a positive correlative trend between BRWD3 and PR status,and a negative correlative trend between HER2 and PR status.ConclusionIn this study,the differences are observed in the expression of BRWD3 between cancer and adjacent tissues,IDC and ILC expression of BRWD3 in cancer tissues is positively correlated with PR,and a negative correlation with HER2.BRWD3 may affect the formation of IDC and ILC,the formation of PR/HER2 related clinical features,and the overall process of breast cancer through influencing cytoskeleton assembly,cell adhesion,and cell-social connection. |
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