Identification Of TAF7 In Metastatic Triple-negative Breast Cancers And Primary Mechanism Study On Its Regulating Cancer Cell Metastasis

Breast cancer is mainly divided into four types:Luminal A,Luminal B,HER2~+and triple-negative.Among them,triple-negative breast cancer(TNBC)patients have poor prognosis,and the main reason leading to the deterioration of TNBC is the metastasis of cancer cell.Single-cell RNA sequencing(sc RNA-seq)can detect the gene expression level of each cell in a sample,analyze the activity of signaling pathways in cells,and accurately analyze cell life activities.This study intends to conduct sc RNA-seq on TNBC clinical tissue samples that have already metastasized,and deeply mine the data,screen out the molecules that cause TNBC metastasis,and further explore the mechanism of its regulation of cancer cell metastasis.Firstly,scRNA-seq was performed on the collected tissue samples of TNBC patients with metastases,and gene expression data of 13 098 cells were obtained.Through dimensionality reduction clustering,extraction of cell marker genes to identify cell population types,and analysis based on chromosome copy number inference,13 cell clusters identified,namely 5 breast cancer cell clusters,as well as breast epithelial cell cluster,epidermal cell cluster,CD8~+T cell cluster,fibroblast cluster,macrophage cluster,monocyte cluster,endothelial cell cluster,and neutrophil cluster.By comparing the gene expression levels of gene sets related to cell cycle and epithelial mesenchymal transition(EMT)in these five cancer cell populations,and combining the results of gene set variation analysis(GSVA),it was found that cancer cells cluster 6#has the strongest proliferation and metastasis ability,and the most active EMT signaling pathway and G2/M checkpoint signaling pathway,indicating that cancer cell cluster 6#has the highest degree of malignancy and may play an important role in the proliferation and metastasis of cancer cells in this sample.By constructing the gene regulatory network of cancer cell group 6#,multiple transcription factors including TATA box–binding protein–associated factor 7(TAF7)were screened.By analyzing the expression level of TAF7 in each cell population,it was found that the expression level of TAF7 in cancer cell cluster was significantly higher than that in normal breast epithelial cell cluster.In addition,by analyzing the clinical information and gene expression data of TNBC patients in the TCGA database,it was found that the high expression of TAF7 was significantly associated with the poor prognosis of TNBC patients.Secondly,in order to determine the expression level of TAF7 in TNBC,this study further collected tumor tissue samples from 3 patients with metastatic TNBC.Immunohistochemical detection found that the expression level of TAF7 in tumor tissue was significantly higher than that in adjacent normal tissue.In addition,the data of TNBC patients in the TCGA database were also analyzed and it was found that the expression level of TAF7 in tumor tissues was significantly higher than that in normal tissues.Meanwhile,three other groups of TNBC sample scRNA-seq datasets were analyzed,and the results showed that TAF7 expression was significantly higher in cancer cell clusters than in normal breast epithelial cell cluster.These results further verified that TAF7 was highly expressed in TNBC.Finally,in order to explore the biological functions of TAF7 in TNBC,this study analyzed the biological functions related to TAF7 using the sequencing results of cancer cell cluster in the metastatic TNBC samples in this case.GO enrichment analysis of differentially expressed genes showed that TAF7 was significantly associated with cancer cell proliferation and metastasis;GSVA analysis showed that EMT signaling pathway and G2/M checkpoint signaling pathway were highly active in cancer cell cluster with high TAF7 expression.The above results suggest that TAF7 may be an important molecule regulating cancer cell proliferation and metastasis in metastatic TNBC.Then,using the metastatic TNBC cell lines MDA-MB-231 and LM2,it was found that TAF7 was highly expressed in both MDA-MB-231 and LM2 cells by q RT-PCR and Western blotting experiments.Using si RNA and sh RNA interference technology,after knocking down the expression of TAF7 in cells,it was found that the proliferation and migration ability of cells were decreased,and the expression level of proliferating cell nuclear antigen(PCNA)was decreased,the expression of vimentin was decreased,and the expression level of E-cadherin increased.The above results indicate that TAF7 may promote cancer cell proliferation by up-regulating PCNA;promote cancer cell metastasis by promoting cancer cell EMT process.In summary,in this study,scRNA-seq and data mining of tissue samples from TNBC patients with metastases found that TAF7 can promote cancer cell proliferation and metastasis.In metastatic TNBC cell lines MDA-MB-231 and LM2,the decreased expression of TAF7 was found to inhibit the proliferation of cancer cells by down-regulating PCNA,and inhibiting the EMT process of cells by down-regulating Vimentin and up-regulating E-cadherin,thereby inhibiting TNBC migration and infiltration.This study lays a foundation for further revealing the metastasis mechanism of TNBC.