| Background:Breast cancer is one of the most common malignancies in the female population,with the second highest mortality rate after lung cancer,according to cancer statistics for 2022.10%-20%of breast cancers are negative for human epidermal growth factor receptor 2(HER2),estrogen receptor(ER)and progesterone receptor(PR)expression,known as triple-negative breast cancer(TNBC).In recent years,both the incidence and mortality of TNBC are increasing gradually,and the patients with TNBC tend to be younger and younger.TNBC is highly invasive and heterogeneous.Patients with TNBC have a tendency for earlier recurrence,earlier metastasis,more reduced overall survival(OS)and poorer prognosis compared with other breast cancer patients.Surgery and chemotherapy are still the main treatment methods for TNBC.As the specific pathogenesis of TNBC and its related influencing factors have not been fully clarified,most therapeutic measures for TNBC are not effective.Only a few individualized treatments have been shown to improve the prognosis and outcome of TNBC patients.Therefore,we need to learn more about the mechanisms of the development of the TNBC,as well as to identify new biomarkers for the diagnosis and prognosis of TNBC patients.Objectives:1.In this study,we screened G protein-coupled receptor 34(GPR34),a key gene of TNBC in TCGA database by bioinformatics techniques and analyzed the expression of GPR34 in TNBC tissues and paired paracancerous tissues.To investigate the expression level of GPR34 in TNBC based on immunohistochemistry staining,and to explore the relationship between GPR34 expression levels,clinicopathological parameters and prognosis of TNBC patients.2.To explore whether GPR34 could be used as a therapeutic target of TNBC and a biomarker to evaluate the prognosis of the disease,laying a foundation for the mechanism research of TNBC and seeking potential targeted therapy.Methods:1.In this study,RNA sequencing data of 126 TNBC patients and clinical data of 116 TNBC patients were obtained in TCGA.2.Using R software,TNBC differentially expressed genes(DEGs)were screened based on the results of relevant survival analysis of tumor microenvironment,stromal cell and immune cell differential analysis.First,we used STRING to construct a protein-protein interaction network(PPI)for DEGs and screened the core genes for TNBC with a confidence level higher than 0.400;then we performed both survival analysis and clinical correlation analysis on DEGs to identify prognosis-related genes in TNBC(p<0.05).GPR34 was finally screened as a key gene in TNBC after the intersection of the above two methods by Venn analysis.3.To verify the relationship between GPR34 and survival of TNBC patients in TCGA,to speculate the pathway of GPR34 using GSEA analysis and explore the relationship between GPR34 expression and the level of immune cell infiltration.4.95 TNBC patients who received surgical treatment in the Department of breast surgery,Qilu Hospital of Shandong university from June 1st,2011 to October 31st,2017 were collected.The expression of GPR34 protein was detected by immunohistochemical staining(IHC)in the tumor tissues of TNBC and paraffin sections of TNBC specimens.The relationship between GPR34 protein expression level and the clinicopathological characteristics of TNBC patients were analyzed by SPSS 26.0.Finally,according to the follow-up data,we used Kaplan-Meier method to plot the OS curve.Results:1.Among the 135 tumor microenvironment-related DEGs,133 up-regulated genes and 2 down-regulated genes were screened in TNBC based on the criteria of false discovery rate FDR<0.05 and |log fold-change|>1.0,and GPR34 belonged to upregulated expressed genes.2.We screened 72 core genes for TNBC by constructing protein interaction networks for DEGs and screened 2 prognosis-related genes by performing the survival analysis and clinical correlation analysis.Finally,GPR34 was identified as a key gene for TNBC.3.In TCGA,we found that GPR34 mRNA was highly expressed in TNBC patients,and patients with high GPR34 mRNA expression had relatively shorter OS(p<0.05).4.GSEA analysis showed that GPR34 was enriched in various signaling pathways such as T-cell receptor signaling pathway,B-cell receptor signaling pathway,chemokine signaling pathway and JAK-STAT signaling pathway.Immune cells infiltration analysis showed that memory B cells,macrophage M2,resting mast cells and neutrophils were related to the expression of GPR34.5.Immunohistochemical staining showed that GPR34 protein was highly expressed in TNBC tissues(p<0.05).Patients with high GPR34 protein expression had a shorter OS(p<0.05).6.GPR34 expression was significantly different between TNBC patients in T1,T2 stages and those in T3,T4 stages(p<0.001).There was no significant correlation between GPR34 expression and age,histological grade,presence of lymph node metastasis and TNM stage of TNBC patients(p>0.05)Conclusion:1.The expression level of GPR34 protein in TNBC was significantly higher than that in peripheral normal breast tissue,suggesting that it may be associated with the development of TNBC.2.In TNBC,patients with high GPR34 expression have reduced survival and poor prognosis,and we speculated that it could be used as a biomarker to assess prognosis,and may become a potential future therapeutic target for the treatment of TNBC. |
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