The Mechanism Study Of Zinc Finger Protein 276 Activates Wnt/β-catenin Pathway Through CYP1B1 To Promote Breast Cancer Proliferation And Metastasis

ObjectiveThis study aimed to explore the biological function and molecular mechanism of zinc finger protein ZNF276 in breast cancer,and to clarify its role and significance as a transcription factor in the development of breast cancer.Methods1.The mRNA and protein expression levels of ZNF276 in breast cancer tissues and its correlation with the clinicopathological characteristics of breast cancer patients were detected by TCGA database analysis combined with tissue microarray,and the mRNA and protein expression levels of ZNF276 in MCF-7,MDA-MB-231,SK-BR-3,BT-474 and UACC-812 breast cancer cell lines were analysed by RT-qPCR and Western blot.2.Two types of breast cancer cells were used for overexpression/knockdown of ZNF276.The effect of ZNF276 on the proliferation,invasion and migration of breast cancer cells in vitro was assessed by CCK-8,clone formation assay,transwell and wound healing assay,and the effect of ZNF276 on the growth of breast tumors in vivo was evaluated by subcutaneous tumorigenesis experiments in nude mice.3.Transcriptome sequencing combined with CUT Tag to explore the potential downstream target genes of ZNF276.The dual luciferase reporter gene experiment was used to verify the transcriptional regulatory relationship of ZNF276 to the target gene promoter.The functional binding domains between ZNF276 and target gene promoters were defined by constructing truncated sequence expression plasmids.4.The associated pathway regulated by ZNF276 was explored through TOP/FOP Flash assay,immunofluorescence,RT-qPCR and Western blot,to further proved whether ZNF276 can affect the proliferation,invasion and migration of breast cancer cells in vitro by regulating target genes.5.The correlation between the expression of ZNF276,the target gene and pathway-related gene was analyzed by RT-qPCR and western blot.6.Screening of potential ZNF276 interacting proteins by immunoprecipitation and mass spectrometry,and identification of ZNF276 binding proteins by co-immunoprecipitation,TOP/FOP Flash assay and dual luciferase reporter gene assay to further demonstrate whether ZNF276 regulates target gene transcription and expression by recruiting the interacting protein.Results1.TCGA database showed that the mRNA expression of ZNF276 in breast cancer tissues was lower than that in adjacent tissues(P<0.01),and the prognosis of breast cancer patients with low ZNF276 expression was poor.Immunohistochemistry demonstrated that the protein level of ZNF276 was upregulated in breast cancer tissues(P<0.05),and the expression level was associated with the age of breast cancer patients.The mRNA and protein expression levels of ZNF276 were significantly higher in MDA-MB-231,MCF-7,BT-474 and UACC-812 breast cancer cell lines than in normal breast cells(P<0.05 or P<0.01),while the expression was down-regulated in SK-BR-3 cell lines(P<0.001).2.Overexpression of ZNF276 promoted the proliferation,invasion and migration of breast cancer cells,while ZNF276 knockdown produced the opposite effects(P<0.01 or P<0.001).Compared with the control group,the mice injected with the ZNF276 overexpression cells increased significantly in tumor volume and grew faster(P<0.05 or P<0.001).3.Transcriptome sequencing screened out 4173 differentially expressed genes which including 2194 up-regulated genes and 1979 down-regulated genes,combined with 4432 genes detected by CUT Tag,we screened 379 common genes.15 genes with relative higher fold change were screened out for RT-qPCR,in combination with previous studies,we speculate that CYP1B1 is a potential target gene of ZNF276.Dual luciferase reporter gene assay proved that CYP1B1 is a downstream target gene of ZNF276 in breast cancer cells,and ZNF276 binds to the region from-1127 bp to-921 bp of the CYP1B1 promoter through the C2H2 domain and activates transcription.4.ZNF276 could up-regulate the expression of CYP1B1,activate the Wnt/β-catenin signaling pathway,and promote β-catenin nuclear translocation,thereby promoting the proliferation,invasion and migration of breast cancer cells in vitro.5.The expressions of ZNF276,CYP1B1 and β-catenin in breast cancer tissues and cells were positively correlated(P<0.05 and r>0).6.ZNF276 promotes CYP1B1 transcription through recruitment of MAGEB2 and activates the Wnt/β-catenin signaling pathway.ConclusionZNF276 promotes proliferation,invasion and metastasis of breast cancer cells by recruiting MAGEB2 protein to regulate CYP1B1,promoting β-catenin nuclear translocation and activating the Wnt/β-catenin pathway.