The Mechanisms Of The Interaction Of ZEB1-mediated LUCAT1 Transcription In Regulating Breast Cancer Stem Cells Properties

Objective:Female breast cancer is currently the highest incidence of malignant tumor in the world.These are malignant tumors with diverse phenotypes containing massive heterogeneous information,genetically or epigenetically.Breast cancer stem cells(BCSCs),a small cluster of cells in breast cancer,are suggested as the driving factor of cancer development,self-renewal,and heterogeneity;and the root of chemotherapy resistance,radiotherapy resistance,and recurrence and metastasis.The mode of treatment targeting BCSCs presents a broader exploration space and heavier clinical value.Therefore,finding specific stem cell indicators and investigation on their biological characteristics and molecular mechanisms are of practical significance.The transcription factor zinc finger E-box binding homeobox 1(ZEB1)is an important member of the ZEB family of transcription factors,which plays an important role in regulating cell differentiation and tissue specificity.ZEB1 can promote the malignant phenotype of tumors by regulating the epithelial-to-mesenchymal transition(EMT)of malignant tumors,showing ectopic expression in multiple cancer types.Also,it regulates cell plasticity,playing a key role in multiple steps of cancer development.This study analyzed the expression of ZEB1 in breast cancer tissues and its correlation with clinicopathology and prognosis.Moreover,we clarified the biological characteristics of ZEB1 in promoting BCSCs properties and the molecular mechanism independent of EMT progress.This new regulatory network of BCSCs provides a theoretical basis to explore specific targets for breast cancer stem cell targeted therapy,also as additional evidence for ZEB1 impacting malignant tumors through EMT-independent process.Methods:1.BCSCs induction and stemness identification:MCF-7 and T47D breast cancer cell-lines were induced into MCF-7 CSCs and T47D CSCs with serum-free medium under suspension culture.We observed the change of cell morphology and performed q RT-PCR,western blot,flow cytometry,agarose clone formation assay,CCK-8 assay and Transwell assay to identify BCSCs properties of inducted MCF-7 CSCs and T47D CSCs.2.Bioinformatics-based analysis:We extracted RNA-Seq data from TCGA database and analyzed the differential expression of ZEB1 in CD44⁺CD24^-/low and non-CD44⁺CD24^-/low breast cancer patients and its correlation with stemness indicators.3.The relation of ZEB1 expression with clinical significance in breast cancer specimens:The expression of ZEB1(immunohistochemistry)and LUCAT1(in situ hybridization)was detected in 110 specimens of breast cancer patients.Pearson chi-square test and logistic regression correlation were used to analyze clinicopathological factors with ZEB1 expression.Kaplan-Meier survival analysis was conducted to assess the correlation between ZEB1 expression and the prognosis of breast cancer patients.4.Cytology experiment:Plasmid transfection was performed to construct cell models with different expressions of ZEB1,LUCAT1 and mi R-5582-3p.We detected the proliferation ability of breast cancer cells and the self-renewal ability of breast cancer stem cells by cloning assay and sphere-formation assay respectively.QRT-PCR and Western blot were conducted to detect the m RNA and protein levels of stem markers such as OCT4,NANOG and SOX2.The expression of stemness markers CD24 and CD44 on cell surface was detected by flow cytometry.5.Bioinformatics-based target gene prediction and molecular mechanism experiments:We used JASPAR,mi Randa and Target Scan databases to predict target genes;dual luciferase reporter gene experiments to verify the specific binding between ZEB1 and LUCAT1 promoters,mi R-5582-3p and ZEB1;the direct binding of ZEB1 to the LUCAT1promoter region was confirmed by chromatin immunoprecipitation;RNA pull down experiment further proved that ZEB1 directly binds to mi R-5582-3p by specific sequences.Results:1.Induction and stemness identification of breast cancer stem cells:After cell culture and induction,the morphology of MCF-7 and T47D changed significantly into mammospheres.Compared to the parental cells,the proportion of CD44⁺/CD24^-cells increased in MCF-7 CSCs and T47D CSCs(p<0.0001).MRNA and protein levels of stem cell markers,such as OCT4,NANOG and SOX2 increased(p<0.05).Clone formation ability was significantly enhanced after induction(p<0.01).Also,MCF-7 and T47D mammospheres appeared stronger migration capabilities(p<0.01),and obvious paclitaxel resistance.2.ZEB1 expression is associated with lymph node metastasis(p<0.001),HER2positive expression(p<0.001),Ki-67 status(p=0.014),histological grade(p=0.042)and TNM staging(p<0.001)in breast cancer specimens.Patients with higher ZEB1expression had shorter overall survival(HR=2.37,p=0.002)and disease-free survival(HR=1.910,p=0.038).3.TCGA analysis indicated that ZEB1 is highly expressed in CD44⁺CD24^-/low breast cancer patients(p<0.05),and it is positively correlated with the expression of stemness-related Hedgehog pathway(p<0.0001).4.ZEB1 promotes the properties of BCSCs:When ZEB1 overexpressed in MCF-7and T47D,the m RNA and protein expression of OCT4,NANOG and SOX2 were increased(p<0.01),and the ratio of CD44⁺CD24^-/low cell subgroup increased as well(p<0.0001).Enhanced proliferation and colony forming ability(p<0.05)was found.Depletion of ZEB1 in MCF-7 CSCs and T47D CSCs decreased the m RNA and protein expression of OCT4,NANOG and SOX2(p<0.01).The proportion of CD44⁺/CD24^-/lowcell subset shrunk(p<0.0001)and the self-renewal ability of MCF-7 CSCs and T47D CSCs was found decreased with ZEB1 knockdown(p<0.01).5.ZEB1 binds to the LUCAT1 promoter and mediates LUCAT1 transcription to regulate BCSCs properties.LUCAT1 is transcriptionally activated by ZEB1 and completely binds with mi R-5582-3p,endowing the activity of ZEB1,forming ZEB1/LUCAT1/mi R-5582-3p feedback loop to regulate BCSCs properties.Conclusion:1.ZEB1 has the biological function of regulating properties of BCSCs.The high expression of ZEB1 in breast cancer indicates a poor prognosis.2.ZEB1 mediates the transcription of long non-coding RNA LUCAT1 to regulate BCSCs properties.After LUCAT1 compete binding with mi R-5582-3p,the repression of ZEB1 is released and contributes to ZEB1/LUCAT1/mi R-5582-3p feedback loop.