| Background:Breast cancer is the most prevalent malignant tumor worldwide,which seriously endangers women’s health.Although the 5-year overall survival(OS)is more than 90%in patients with breast cancer,the 5-year OS has not been improved in patients with advanced metastatic breast cancer,and metastasis is still the main cause of death in metastatic breast cancer patients.Zinc Finger Protein 384(ZNF384),a Cys2His2 zinc finger protein transcription factor,participates in transcriptional regulation of a variety of extracellular matrix genes.ZNF384 has been reported to be increased in multiple types of human cancers and function as an oncogene.However,the role and mechanism of ZNF384 in breast cancer metastasis remains unknown.Objectives:We aimed to determine the expression levels of ZNF384 in breast cancer and its clinical value as a biomarker in prognostic prediction for patients with breast cancer.We further determined the effect of ZNF384 on biological behavior of breast cancer cells both in vitro and in vivo,and elucidate the molecular mechanism of ZNF384 in regulating breast cancer metastasis.Our study will provide clues and theoretical basis for the development of new targets for breast cancer therapy.Methods: 1.The expression level of ZNF384 in invasive breast cancer tissues(n = 236)and the paired adjacent normal breast tissues(n = 20)was examined by immunohistochemistry,and the differences of ZNF384 expression level between breast cancer and normal breast tissues was determined by the paired rank sum test.chi-square test was used to analyze the relationship between various clinicopathological variables and ZNF384 expression levels.The Kaplan-Meier analysis was used to determine the outcome in breast cancer patients with different ZNF384 expression.Univariate and multivariate survival analysis was performed by Cox proportional hazards regression model.The ZNF384 protein expression in breast cancer cell lines and normal breast epithelial cell line was determined by western blot.2.Lentiviral infection was used to generate the stable ZNF384-overexpressed or-depleted breast cancer cell lines,as well as the control cell lines.We performed CCK-8 and plate colony formation assays to determine the cell proliferation in vitro.Furthermore,we performed wound healing and transwell assays to determine the cell migration and invasion in vitro.The effect of ZNF384 on breast cancer growth and metastasis in vivo was evaluated by small animal live imaging after inoculation of mice with fat pads or tail vein injections.3.The effect of ZNF384 on the expression of epithelial-mesenchymal transition(EMT)markers and EMT-related transcriptional factors,and the effect of mi R-485-5p on ZNF384 expression was determined by Reverse Transcription quantitative Polymerase Chain Reaction(RT-q PCR)and western blot assays.RT-q PCR was also used to determine the effect of ZEB1 on mi R-485-5p expression.4.Chromatin Immunoprecipitation(Ch IP)analysis was used to determine the binding of ZNF384 to ZEB1 promoter and the binding of ZEB1 to mi R-485 promoter.The dual-luciferase analysis was used to determine the effect of ZNF384 on ZEB1 promoter activity,ZEB1 on mi R-485 promoter activity and mi R-485-5p on ZNF384 3’-UTR activity.Results: 1.ZNF384 was overexpressed in breast cancer tissues compared to the paired adjacent normal breast tissues.The expression level of ZNF384 was also elevated in breast cancer cell lines compared to the normal breast epithelial cell line MCF10 A.The expression level of ZNF384 in breast cancer was positively associated with tumor progression,including grade and number of metastatic lymph nodes,but negatively related to the estrogen receptor(ER)status.Although the ZNF384 high breast cancer patients had a significantly poorer prognosis compared to the ZNF384 low breast cancer patients,the ZNF384 expression level could not be used as an independent prognostic predictor in patients with breast cancer.2.Ectopic expression of ZNF384 promoted cell migration and invasion,but did not affect cell proliferation in T47 D and ZR-75-30 breast cancer cell lines in vitro.Depletion of ZNF384 suppressed cell migration and invasion,but did not affect cell proliferation in MDA-MB-231 breast cancer cell line in vitro.Depletion of ZNF384 inhibited tumor growth and metastasis in vivo.3.We observed that ZNF384-overexpressed T47 D cells exhibited a fibroblast-like and spindle-shaped morphology,whereas the control cells retained their cobblestone-like morphology.In addition,ZNF-depleted MDA-MB-231 cells exhibited a cobblestone-like morphology,whereas the control cells retained their fibroblast-like and spindle-shaped morphology.ZNF384-overexpressed T47 D cells exhibited significant upregulation of the mesenchymal markers N-cadherin and Vimentin,and EMT-related transcriptional factor ZEB1,Twist1,Snail and Slug,but dramatic downregulation of epithelial marker E-cadherin.ZNF384-depleted MDA-MB-231 cells exhibited significant downregulation of the mesenchymal markers N-cadherin and Vimentin,and EMT-related transcriptional factor Twist1,Snail,Slug and ZEB1,but dramatic upregulation of epithelial marker E-cadherin.4.ZNF384 could bind to ZEB1 promoter region and transactivated ZEB1 expression.mi R-485-5p could bind to ZNF384 3’-UTR and down-regulated its expression.ZEB1 could bind to mi R-485 promoter region and transcriptionally suppressed mi R-485-5p expression.ZNF384 down-regulated mi R-485-5p expression through up-regulating ZEB1 expression.Conclusions: Our study demonstrated that ZNF384 is highly expressed in breast cancer and associated with poor prognosis in patient with breast cancer.ZNF384 promotes breast cancer metastasis by inducing the EMT phenotype both in vitro and in vivo.Mechanically,ZNF384 transactivates ZEB1 expression and ZEB1 transcriptionally represses mi R-485-5p expression.In addition,mi R-485-5p inhibits ZNF384 expression by binding its 3’-UTR,suggesting that ZNF384-ZEB1-mi R-485 negative feedback loop regulates breast cancer metastasis.This study reveals the role of ZNF384 in breast cancer metastasis and its potential molecular mechanism,providing clues and theoretical basis for the development of therapeutic targets and prognostic predictive markers for patients with breast cancer. |
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