Mechanism Of ZNF692 Recruitment Of PRMT1-mediated IL7R Expression Upregulation In JAK3/STAT3 Pathway Activation And Breast Cancer Cell Proliferation And Metastasis

ObjectiveThe aim of this study was to clarify the expression pattern,prognostic value,and biological functions of zinc finger protein 692(ZNF692)in breast cancer,to investigate the molecular mechanisms by which ZNF692 regulates downstream gene transcription and activation of related pathways to affect proliferation and metastasis.Methods1.The mRNA expression levels of ZNF692 in breast cancer tissues were analyzed using The Cancer Genome Atlas(TCGA)database,Gene Expression Omnibus(GEO)database and 16 paired breast cancer patient tissues;analysis of ZNF692 protein expression levels in breast cancer tissues and different breast cancer cells using tissue microarrays and western blot;to elucidate the correlation between the expression level of ZNF692 and different clinicopathological features and its prognostic value.2.Overexpression of ZNF692 in MDA-MB-231 and knockdown of ZNF692 in MCF7 and UACC812,and the effects of ZNF692 on breast cancer cell proliferation,migration and invasion were assessed in vitro by CCK8,clone formation,transwell migration and transwell invasion assays;construction of a breast cancer lung metastasis model to assess the effect of ZNF692 on the ability of breast cancer cells to metastasize in the lung.3.Identification of potential interacting proteins of ZNF692 using immunoprecipitation coupled with liquid chromatography/tandem mass spectrometry,and the interaction of ZNF692 with PRMT1 was verified by forward and reverse exogenous and endogenous immunoprecipitation(co-IP)experiments.4.Combined transcriptome sequencing and CUT&Tag(Cleavage under targets&tagmentaion)sequencing of ZNF692 and PRMT1 to establish strict screening criteria(upregulated transcript levels and log2FC>2 after overexpression of ZNF692;score>200 in CUT&Tag-seq;down-regulated transcript levels and log2FC<-2 after knockdown of PRMT1)to obtain a downstream target gene map co-regulated by ZNF692 and PRMT1;To verify the effects of ZNF692 and PRMT1 on IL7R mRNA and protein expression levels by RT-qPCR and western blot;to explore the effects of ZNF692 and IL7R promoter binding sites and PRMT1 enzyme inactivation mutants on IL7R promoter activity by dual luciferase reporter gene assay;to verify whether ZNF692 and PRMT1 promote the enrichment of histone H4R3me2a modifications in the IL7R promoter region by CUT&Tag-qPCR.5.Exploring whether ZNF692 and PRMT1 can activate the JAK3/STAT3 signaling pathway through upregulation of IL7R expression by western blot,immunofluorescence,and RT-qPCR.6.Validation of the effect of IL7R on ZNF692-mediated proliferation and metastasis of breast cancer cells by CCK8 and transwell.Results1.ZNF692 is upregulated in both mRNA and protein expression levels in breast cancer cells and tissues(P<0.01);patients with ZNF692 high expression breast cancer have a shorter overall survival time(HR=2.67,P=0.044).2.Overexpression of ZNF692 significantly enhanced proliferation,migration,and invasion of MDA-MB-231 cells,and knockdown of ZNF692 significantly inhibited proliferation,migration,and invasion of MCF7 and UACC812 cells(P<0.05);Overexpression of ZNF692 promotes the formation of lung metastases in nude mice in a tail vein model(P<0.05).3.ZNF692 interacts with PRMT1,and this interaction is independent of genomic DNA.4.ZNF692 and PRMT1 have 34 significant co-regulatory genes;among them,ZNF692 has the most significant effect on IL7R mRNA levels;ZNF692 binds to the GGCCCA motif of the IL7R promoter through its 433-510 region;overexpression of ZNF692 promotes IL7R mRNA and protein expression levels,while knockdown of PRMT1 results in IL7R mRNA and protein levels were partially neutralized by knockdown of PRMT1,and this effect was dependent on the methyltransferase activity of PRMT1;overexpression of ZNF692 resulted in a significant increase in enrichment of H4R3me2a modifications on the IL7R promoter(P<0.01),the enrichment of IL7R promoter H4R3me2a modification was significantly reduced again after knockdown of PRMT1(P<0.01).5.JAK3/STAT3 phosphorylation levels were significantly increased after overexpression of ZNF692 while knocking down IL7R or PRMT1,and JAK3/STAT3 phosphorylation levels were reduced;Increased STAT3 phosphorylation fluorescence intensity after overexpression of ZNF692 and decreased STAT3 phosphorylation fluorescence intensity level after knockdown of ZNF692;Overexpression of ZNF692 significantly upregulated the expression of JAK/STAT downstream proliferative and metastasis-related genes(cyclin D1,cmyc,BCL2,BCL2L1,MMP9,Vimentin),while knockdown suppressed the expression of these genes.6.Knockdown of IL7R inhibited the enhancement of ZNF692-mediated proliferation and metastasis of breast cancer cells;conversely,overexpression of IL7R partially restored the diminished proliferation and metastasis of breast cancer cells caused by knockdown of ZNF692.ConclusionZNF692 interacts with PRMT1 and co-binds to the IL7R promoter;ZNF692 promotes IL7R transcription and expression dependent on PRMT1 and its mediated modification of H4R3me2a,and IL7R further activates the JAK3/STAT3 signaling pathway,thereby promoting proliferation and metastasis of breast cancer cells.