Function And Mechanism Of ZNF8 On The Specific Promotion Of Lung Metastasis Of Breast Cancer

Background:Breast cancer has become the highest incidence of malignant tumors in women worldwide,which seriously threatens women's health.Metastasis is the leading cause of death in breast cancer patients.Breast cancer has a tendency to metastasize to specific organs.The most common metastatic sites are bone,lung,and liver.However,the mechanism of this specific metastasis tendency is unclear.Therefore,screening and identifying regulatory molecules for breast cancer-specific organ metastasis is of great significance for the prediction of breast cancer-specific organ metastasis and the determination of therapeutic targets.The KRAB-containing zinc finger protein family is the largest transcription factor/transcription regulator family in mammals,and plays an important role in normal development and tumorigenesis.However,the function of most KZNF family members is unknown.ZNF8 is a member of the KZNF family,and its function and molecular mechanism have not been reported.Objectives:1.To confirm the role of ZNF8 in breast cancer tumorigenesis,development and specific organ metastasis.2.To explore the regulatory mechanism of ZNF8 on TGF? signaling pathway,and confirm ZNF8 affect breast cancer tumorigenesis,development,and metastasis to lung depending on TGF? pathway.Methods:The breast cancer sample data from the TCGA database was used to analyze the correlation between ZNF8 and breast cancer.Immunohistochemistry was used to detect the expression level of ZNF8 in breast cancer and adjacent clinical samples.Western Blot and qPCR were used to measure the expression level of ZNF8 in breast epithelial cells and breast cancer cells.Lentivirus overexpression and knockout systems were used to establish ZNF8 overexpression or knockout cell lines,and the effects of ZNF8 on the cytological effects of breast cancer were tested by transwell,cell proliferation,and clony formation experiments,respectively.RNA-seq combined with KEGG pathway enrichment analysis were used to analyze the differentially expressed genes and the related signaling pathway in ZNF8 knockout cells.Co-immunoprecipitation(Co-IP)was used to verify the interaction between ZNF8 and Smads.qPCR was used to detect whether ZNF8 depends on TGF? signaling pathways to regulate lung metastasis-related target genes ANGPTL4 and NEDD9 Transwell assays were used to detect whether ZNF8 depends on the TGF? signaling pathway to affect breast cancer cell metastasis in vitro.A nude mouse metastasis model using ZNF8 knockout breast cancer cell lines was established to explore whether ZNF8 knockout specifically inhibit breast cancer metastasis to lung relying on TGF? signaling pathway.Western Blot was used to detect the effect of ZNF8 on the expression of epithelial-mesenchymal transition(EMT)-related genes of breast cancer cell lines.Results:1.The data from TCGA database showed that the amplification mutation is the major type of ZNF8 mutation in breast cancer,and mutations of ZNF8 were associated with poor disease/progression-free survival among patients with breast cancer.2.ZNF8 expression was significantly upregulated in cancer tissues compared with adjacent tissues,and significantly correlated with histological grade,HER2 status,lymph node and distant metastasis.High ZNF8 levels had significantly poorer survival than those with low ZNF8 levels3.The protein and mRNA level of ZNF8 in breast cancer cells were higher than normal breast epithelial cells,and in most cases,the expression level of ZNF8 was positively correlated with the cell malignancy.4.ZNF8 can promote the invasion and metastasis of breast cancer cells,but have no effect on cell proliferation.ZNF8 can obviously increase the size of clones,but only slightly increase the number of clones in clony formation assay.ZNF8 can specifically promote lung metastasis of breast cancer cells.5.The differentially expressed genes in ZNF8 knockout cells is associated with TGF? signaling pathway.And ZNF8 can interact with Smad2/3/46.ZNF8 depended on the TGF? signaling pathway to increase the expression of lung metastasis-related target genes such as ANGPTL4 and NEDD9,the metastasis capacity in vitro and metastasis to lung in vivo of cancer cells.7.ZNF8 promoted the expression of EMT-related genes in breast cancer cells.Conclusions and significance:In summary,ZNF8 is highly expressed in breast cancer,and its high expression was associated with a poor prognosis for patients.ZNF8 can regulate the metastasis ability of breast cancer cells in vitro and in vivo and specifically promote lung metastasis of breast cancer by regulating the TGF? signaling pathway.In addition,ZNF8 can promote the occurrence of EMT in breast cancer cells.Therefore,ZNF8 may serve not only as a diagnostic and prognostic indicator but also as a therapeutic target for breast cancer and its metastasis to lung.