| Backgrounds: Breast cancer is the most common malignant tumor in women,accounting for 30% of new cancer cases in women,and about 2.26 million new breast cancer patients were diagnosed in 2020,which is the leading cause of cancer death in women.The formation of breast cancer changes the structure or regulation of cell oncogenes under the influence of external factors,leading to uncontrolled cell growth and differentiation,and malignant transformation.On the other hand,the tumor microenvironment also affects the progression of breast cancer.Metastasis is the cause of tumor recurrence in most cases,and breast cancer patients with distant metastases have a poor prognosis,with a five-year survival rate of less than 30%.Therefore,an in-depth understanding of the underlying mechanisms of the occurrence and progression of breast cancer metastases has significant clinical significance.Zinc,in the form of zinc finger proteins,is often involved in gene expression in vivo,maintenance of the structure of proteins and nucleic acids,intracellular molecular transport,and immunity.Because the zinc finger protein was originally discovered to look like a finger,many people thought that finger-like proteins should be called zinc finger proteins,but this definition has been expanded by the discovery of similar proteins.Zinc finger proteins generally refer to all proteins that need to bind zinc in order to function properly.Zinc finger proteins do not have a fixed structure and function,they generally play a role in regulating gene expression and stabilizing structure.Zinc finger protein 746(ZNF746)was originally identified as a substrate for the E3 ubiquitin ligase Parkin and is associated with Parkinson’s disease.There is growing evidence that abnormal expression of ZNF746 is associated with cancer progression.ZNF746 may promote colorectal cancer progression by increasing the stability of c-Myc.Down-regulation of ZNF746 inhibited the invasion and epithelial-mesenchymal transformation of NSCLC cells.Melatonin inhibits bladder tumorigenesis by down-regulating ZNF746.Based on the above experimental results,we speculate that ZNF746 as a tumor promoting gene can promote cancer progression by influencing transcription factors and tumor cells.However,it is unclear whether ZNF746 plays a role in breast cancer.The Notch signaling pathway has been clearly defined in cell proliferation and differentiation.One of the key features of the Notch signaling pathway is the direct link between extracellular signaling and transcriptional output,without the need for extended protein chains as many other signaling pathways do.In many biological processes,including morphological changes during cancer onset and progression,there is a strong correlation between Notch mediated gene expression activity and cell morphological regulation.The Notch signaling pathway controls cell proliferation,fate,differentiation,and cell death by touching short distance signals between nearby cells.Fibroblasts are an important component of stromal tumor growth and have also been shown to be affected by Notch regulation.In recent years,more and more studies have shown that abnormal activation of Notch signaling pathway is associated with tumorigenesis,including breast cancer.Notch signaling is modulated in tumors by relationships between different segments of the IL-6/STAT3 pathway.The initial medical evidence supporting Notch signaling control was via IL-6,in which breast cancer cells induce Jagged1 autocrine.Notch promotes breast cancer progression through tumor-initiating cell maintenance,tumor cell fate assignment,proliferation,survival,and motility.In addition,Notch is considered to be a decisive mechanism regulating various paracrine and paracrine communications in the tumor microenvironment(TME).The Notch signaling pathway is activated by interactions between ligands and receptors in neighboring cells.Notch expression is regulated by hypoxia and inflammatory cytokines(IL-1,IL6,etc.)and activated by ligand binding.It contains four Notch receptors,namely notch-1,Notch-2,Notch-3 and Notch-4.And five related ligands,Jagged-1,Jagged-2 and Dll-1(Delta-like-1),Dll-3(Delta-like-3)and Dll-4(Delta-like-4);These protein molecules are membrane-bound,conduct paracrine signaling,and are present in arteries rather than veins.Jagged1 is one of the major ligands in the Notch signaling pathway.When Jagged1 binds to the Notch receptor,the Notch intracellular domain(NICD)will be released in the cytoplasm and transferred to the nucleus,then the downstream target genes will be transcribed and activated.Such as HES1.It has been found that the variation of Jagged1 is related to the occurrence and development of tumors.Meanwhile,Notch signaling pathway also plays an important role in determining the cell fate of macrophages,especially in differentiation into tumor-associated macrophage(TAM).TAM is an important component of breast tumor inflammatory infiltration.TAM may play a role in preventing or promoting tumors depending on the polarization of macrophages.Macrophages mainly differentiate into M1-like and M2-like phenotypes in tumors.M1-like macrophages represent typically activated macrophages,and M2-like macrophages represent alternately activated macrophages.TAM of M2-like macrophages is closely related to tumor growth and metastasis.Macrophages exhibit transcriptome signals associated with the Notch signaling pathway.Activation of Jagged1/Notch signaling pathway affects differentiation and polarization of macrophages.Previous studies have shown that GATA1 promotes the proliferation and metastasis of ovarian cancer through Jagged1/Notch signaling pathway,HOXA-AS2 accelerates the proliferation and migration of cervical cancer cells by activating Notch pathway,and PKMYT1 enhances the cell proliferation and tumor formation of non-small cell lung cancer by activating Notch signaling pathway.In this study,we investigated the effects of ZNF746 overexpression or silencing on the proliferation,migration,and invasion of breast cancer cells,as well as the effects of conditioned media from ZNF746 overexpression or silencing tumor cells on the polarization of M2 macrophages.In addition,we explored the role of Jagged1/Notch signaling in ZNF746-mediated breast cancer cells.Methods: We used UALCAN online database evaluate ZNF746 expression(http://ualcan.path.uab.edu/analysis.html)and used bioinformatics analysis the relationship between expression of ZNF746 and overall survival in patients with breast cancer.We collected 40 pairs of breast cancer tissues and adjacent tissues,and detected the expression of ZNF746 by Western Blotting(WB)and real-time fluorescence quantitative PCR(RT-q PCR).The expression of ZNF746 in normal breast epithelial cells MCF-10 A and breast cancer cell lines(MDA-MB-231,MDA-MB-468,BT-549,MDA-MB-415 and T-47D)was detected by WB and RT-q PCR.MDA-MB-468 and MDA-MB-415 cells stably silenced or overexpressed with ZNF746 were produced by lentiviral infection and purinomycin selection.The silencing or overexpression efficiency of ZNF746 was detected by RT-q PCR and WB.The effect of ZNF746 on the proliferation,migration and invasion of breast cancer cells was evaluated by MTT assay,colony formation assay,flow cytometry and cell migration and invasion assay.GSEA enrichment was used to analyze the correlation between ZNF746 and Jagged1/Notch pathway in the breast cancer public database.WB analysis was performed to determine the effect of ZNF746 on Jagged1/Notch signaling pathway.WB was used to evaluate the expression of Jagged1,NICD,HEY1 and HES1 in tumor cells.Immunohistochemical staining of ZNF746,CD163 and HES1 was performed in 95 human breast cancer tissues,and clinical characteristics of these breast cancer patients were collected.The effect of ZNF746 on the polarization of M2 macrophages was determined by cell coculture,RT-q PCR and flow cytometry.The silencing of Jagged1 was confirmed by transfection of Jagged1 specific si RNAs into breast cancer cells.The effect of ZNF746 on the proliferation,migration and invasion of breast cancer cells was evaluated by MTT assay,colony formation assay,flow cytometry and cell migration and invasion assay.GSEA enrichment was used to analyze the correlation between ZNF746 and Jagged1/Notch pathway in the breast cancer public database.WB analysis was performed to determine the effect of ZNF746 on Jagged1/Notch signaling pathway.WB was used to evaluate the expression of Jagged1,NICD,HEY1 and HES1 in tumor cells.Immunohistochemical staining of ZNF746,CD163 and HES1 was performed in 95 human breast cancer tissues,and clinical characteristics of these breast cancer patients were collected.The effect of ZNF746 on the polarization of M2 macrophages was determined by cell coculture,RT-q PCR and flow cytometry.The silencing of Jagged1 was confirmed by transfection of Jagged1 specific si RNAs into breast cancer cells.Results: We found that ZNF746 was significantly up-regulated in breast cancer tissues in the TCGA database,and the Overall Survival(OS)of breast cancer patients with high expression of ZNF746 was significantly lower than that of patients with low expression of ZNF746.ZNF746 is highly expressed in breast cancer tissues and cells compared to paracancer tissues.Overexpression of ZNF746 promoted the proliferation,migration and invasion of breast cancer cells,while ZNF746 knockout breast cancer cells showed the opposite effect.In order to explore the influence of ZNF746 on downstream pathways,we applied GSEA enrichment analysis to ZNF746 related pathways in breast cancer public database,and we found that Jagged1/Notch signaling pathway was correlated with ZNF746.Moreover,in correlation analysis,the expression of ZNF746 was positively correlated with NICD,a key gene in Jagged1/Notch pathway.We overexpressed or silenced ZNF746 and evaluated the expressions of Jagged1,NICD,HEY1 and HES1 associated with Notch signaling pathway in tumor cells using Western blot.The results showed that their expression levels were increased in cells after ZNF746 was up-regulated,but decreased after ZNF746 knockout,suggesting that the Jagged1/Notch signaling pathway was activated by ZNF746 up-regulation.We further performed immunohistochemical staining of ZNF746,CD163 and HES1 in 95 human breast cancer tissues and collected clinical features of the patients.The results showed that there were more HES1+ cells and CD163+(M2 macrophage marker)cells in breast cancer tissues with high expression of ZNF746.These results suggest that ZNF746 may be involved in the polarization of breast cancer M2 macrophages through Jagged1/Notch signaling pathway.In addition,ZNF746 enhances the production of C-C motif chemokine ligand 2(CCL2)and colony stimulating factor 1(CSF1).The conditioned medium from ZNF746 overexpressed cancer cells promoted the M2 polarization of macrophages,while the conditioned medium from ZNF746 silenced cancer cells inhibited the M2 polarization of macrophages,as evidenced by increased and decreased proportions of CD206+ and CD163+ cells,respectively.MTT results showed that ZNF746 induced cell viability was inhibited after transfection with Jagged1 si RNA.The silencing of Jagged1 reversed the increase in ZNF746-mediated cell migration and invasion.Elevated expressions of CCL2 and CSF1 induced by ZNF746 were inhibited after Jagged1 knockout.In addition,compared with ZNF746 overexpressed cells cultured in conditioned medium,ZNF746 overexpressed MDA-MB-468 cells cultured in conditioned medium transfected with Jagged1 si RNA,THP-1 cells cultured in conditioned medium transfected with ZNF746 overexpressed MDA-MB-468 cells,The expression of CD206 and CD163 and the percentage of CD163 and CD206 positive cells were decreased.Finally,we found that silencing ZNF746 significantly reduced the growth rate and tumor size in a xenograft model of nude mice.Overexpression of ZNF746 significantly increased the growth rate and tumor size.Conclusions: This study revealed the pro-tumor role of ZNF746 in breast cancer and demonstrated that ZNF746 activates the Jagged1/Notch signaling pathway and that ZNF746 promotes M2-like polarization of tumor-associated macrophages.ZNF746 promotes M2-like polarization of tumor-associated macrophages through Jagged1/Notch signaling to promote breast cancer progression,suggesting that ZNF746 may be a promising therapeutic target for breast cancer. |
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