Objective To explore the effect of tripartite motif-containing 28(TRIM28)on the proliferation and colony-forming ability of breast cancer cells and the relationship with tumor-infiltrating immune cells.Methods A total of 39 patients with breast cancer undergoing treatment in Zhengzhou Central Hospital Affiliated to Zhengzhou University from September 2020 to January 2022 were selected as the research subjects.Paraffin-embedded tumor tissue specimens and paired paracancerous tissues were collected.The expression of TRIM28 protein in these tissues was determined by immunohistochemical staining.The MCF7 cells in the logarithmic phase were divided into the si-control group and the si-TRIM28 group.The cells in the si-control group were transfected with empty plasmids,and the cells in the si-TRIM28 group were transfected with si-TRIM28 plasmids,which specifically knocked down TRIM28.The expression level of TRIM28 protein in the two groups of MCF7 cells was detected by Western blot,the cell proliferation in the two groups was detected by cell counting kit-8 assay,and the colony formation of the MCF7 cells in the two groups was detected by colony formation assay.Use CIBERSORT algorithm to analyze the difference of tumor infiltrating immune cells in breast cancer sample tissues in The Cancer Genome Atlas(TCGA)database and its correlation with TRIM28 protein.The signaling pathways between the differences in TRIM28 gene expression levels were analyzed by gene set enrichment analysis(GSEA).TRIM28-related immunomodulatory factors were obtained from the TISIDB database and analyzed by the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.Results The TR1M28 protein in breast cancer tissues was positively stained and localized in the cytoplasm,presenting as brownish-yellow fine granules.TRIM28 in paracancerous tissues was expressed either negatively or weakly positive,while the expression of TRIM28 protein in breast cancer tissues was observed with a robust positive signal.The expression level of TRIM28 protein in tumor tissues was significantly higher than that in paracancerous tissues(t=2.258,P＜0.05).The relative expression level of TRIM28 protein of MCF7 cells in the si-TRIM28 group was significantly lower than that in the si-control group(t=61.654,P＜0.005).On day 0 of cultivation,there was no significant difference in MCF7 cell survival rate between the two groups(t=-0.547,P＞0.05).On day 4 of cultivation,the cell survival rate of MCF7 cells in the si-TRIM28 group was significantly lower than that in the si-control group(t=13.920,P＜0.05).The number of colony formation in MCF7 cells in the si-TRIM28 group was significantly fewer than that in the si-control group(t=11.528,P＜0.05).There were significant differences in the counts of macrophage subpopulations,B-cell subpopulations,T-cell subpopulations,activated natural killer cells,monocytes,and eosinophils in breast cancer tissues and paracancerous tissues(P＜0.05).The expression level of TRIM28 in breast cancer tissues was positively correlated with infiltrating monocytes and regulatory T cells(r=0.150,0.100;P＜0.05)and negatively correlated with activated dendritic cells,activated CD4 memory T cells,resting CD4 memory T cells and γδ T cells(r=-0.100,-0.160,-0.099,-0.190;P＜0.05).GSEA analysis showed that TRIM28 was involved in multiple immune/cancer-related signaling pathways,including cancer pathway,T-cell receptor signaling pathway,Notch signaling pathway,and Wnt signaling pathway.Fifty-eight immunomodulatory factors related to TRIM28 gene were obtained from the TISIDB database.The KEGG pathway enrichment analysis on these immunomodulatory factors showed that CSF1R,CXCL12,CD27,CD40 and CD40LG collectively participated in the T-cell receptor signaling pathway,natural killer cell-mediated cytotoxicity and nuclear factor-κB signaling pathway.Conclusion TRIM28 protein is highly expressed in breast cancer.The proliferative and colony-forming abilities of breast cancer cells can be inhibited by knocking down TRIM28 gene.TRIM28 protein may be involved in regulating the immune microenvironment in breast cancer through multiple immune/cancer-related signaling pathways.
breast cancertripartite motif-containing 28immune microenvironmentbioinformatics
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