IGF2BP2 Maintains Mesenchymal Phenotype,Stemness And Chemoresistance In Breast Cancer Cells

Objective:Breast cancer is a major health concern for women worldwide and is the most common malignant tumours in women.According to the latest data from the Global Cancer Study 2023,the incidence of breast cancer is approximately 31%,ranking 1st in the incidence of malignant tumours in women,and the mortality rate of breast cancer is approximately 15%,ranking 2nd in the mortality rate of malignant tumours in women.Studies have shown that Epithelial to Mesenchymal Transition(EMT)and stem cell-like properties(Stemness)of tumour cells are important mechanisms for metastasis,chemoresistance and recurrence of malignant tumours.Therefore,it is important to identify the mechanisms regulating EMT,stemness and drug resistance in breast cancer cells in order to improve breast cancer treatment,prolong the survival of breast cancer patients and improve their quality of life.Previous studies have shown that IGF2BP2 is involved in the development and progression of cancers including colorectal cancer,oesophageal adenocarcinoma,glioma,hepatocellular carcinoma,lung cancer,pancreatic cancer and many other diseases.IGF2BP2 acts as an m RNA binding protein that increases genomic stability,complexity,specificity and multifunctionality.Additional studies have shown that IGF2BP2 functions as a tumour promoter and that its amplification and overexpression are associated with tumour progression.Importantly,high levels of IGF2BP2 m RNA were associated with short survival,suggesting that IGF2BP2 has potential prognostic value in breast cancer.At this stage,studies have demonstrated that IGF2BP2 is involved in the development of some tumours.However,little has been reported on the regulatory mechanisms of IGF2BP2 in breast cancer cells.The aim of this study is to investigate the important role of IGF2BP2 in breast cancer and to clarify the regulatory mechanisms of IGF2BP2 on the mesenchymal phenotype,migration,invasion,metastasis,stemness and chemoresistance of breast cancer cells,so as to provide new therapeutic directions and precise therapeutic targets for breast cancer patients.Methods:1.The relationship between IGF2BP2 and mesenchymal-like phenotype-related indicators and stem cell characteristic-related indicators in breast cancer tumour tissues was analysed by CCLE database and GSE12777 dataset in the GEO database.Correlation between IGF2BP2 expression levels and prognosis through Kaplan-Meier Plotter website.2.Detection of IGF2BP2 expression in clinical breast cancer tissue samples: analysis of IGF2BP2 protein expression in 25 breast cancer tissue samples and 15 paraneoplastic tissue samples by immunohistochemistry.3.Three breast cancer cell lines,two mesenchymal-like breast cancer cell lines(MDA-MB-231 and Hs 578T)and one epithelial-like breast cancer cell line(MCF7)were selected for the experiment.The expression of IGF2BP2 was significantly higher in the mesenchymal-like breast cancer cell line than in the epithelial-like breast cancer cell line.Establishment of stable and transient cell lines: Using sh IGF2BP2 lentiviral vector(sh IGF2BP2-0,sh IGF2BP2-1 and sh IGF2BP2-2)stable transfer of breast cancer cells MDA-MB-231 and Hs 578 T,respectively.Western blot assay and Real-time PCR assay were used to verify the transfection efficiency.IGF2BP2 overexpression(oe IGF2BP2)plasmid was used to transiently transfect breast cancer cells MCF7,and the transfection efficiency was verified by Western blot assay and Real-time PCR assay.4.To explore the effect of IGF2BP2 on the mesenchymal phenotype of breast cancer cells: Real-time PCR was used to detect the expression of VIM,FN1 and ACTA-2 in MDA-MB-231 cells and Hs 578 T cells;immunofluorescence was used to detect the expression of Vimentin,Fibronectin and α-SMA in MDA-MB-231 cells and Hs 578 T cells.The expression of Vimentin,Fibronectin and α-SMA in MDA-MB-231 cells and Hs 578 T cells was detected by immunofluorescence assay;the morphological changes of MCF7 cells were detected by Phalloidin staining assay.5.Exploring the effect of IGF2BP2 on migration and invasion of breast cancer cells:migration and invasion of MDA-MB-231 cells,Hs 578 T cells and MCF7 cells using scratch assay and Transwell assay.6.Exploring the effect of IGF2BP2 on the metastatic ability of breast cancer cells: in vivo assay to detect lung metastasis in MDA-MB-231 cells;HE staining assay to detect lung metastasis in MDA-MB-231 cells.7.Exploring the effect of IGF2BP2 on the stemness of breast cancer cells: CD44,p-STAT3 and STAT3 expression in MDA-MB-231 cells and Hs 578 T cells were detected by Western blot.CD44 expression in MDA-MB-231 cells and Hs 578 T cells was detected by Real-time PCR assay.Flow cytometry was used to detect a subpopulation of LGR5-positive cells in MDA-MB-231 cells and Hs 578 T cells.Sphere-forming ability of MDA-MB-231 cells and Hs 578 T cells was detected using a sphere-forming assay.Flow cytometry was used to detect a subpopulation of LGR5-positive cells in MCF7 cells.Sphere-forming ability of MCF7 cells using sphere-forming assay.8.Exploring the effect of IGF2BP2 on drug resistance in breast cancer cells: The cell viability of MDA-MB-231 cells,Hs 578 T cells and MCF7 cells was examined using the CCK8 assay.Apoptosis of MDA-MB-231 cells and Hs 578 T cells was detected by flow cytometry assay.Sphere-forming resistance assay was used to detect the sphere-forming ability of MDA-MB-231 cells,Hs 578 T cells and MCF7 cells.9.Exploring the effect of IGF2BP2 on SNAI2: The correlation between IGF2BP2 and SNAI2 was analysed by CCLE database and GSE12777 dataset in the GEO database.Western blot assay and Real-time PCR assay were used to detect SNAI2 expression in MDA-MB-231 cells and Hs 578 T cells.The binding of IGF2BP2 protein to SNAI2 m RNA in MDA-MB-231 cells and Hs 578 T cells was detected by RIP assay.SNAI2 m RNA expression in MDA-MB-231 cells and Hs 578 T cells was detected by Real-time PCR assay after treatment with actinomycin D.10.Exploring the effect of SNAI2 on drug resistance in breast cancer cells: SNAI2overexpression(oe SNAI2)plasmid was used to transiently transfect sh IGF2BP2MDA-MB-231 cells,and Western blot assay and Real-time PCR assay were used to verify the transfection efficiency.The viability of sh IGF2BP2 MDA-MB-231 cells was measured by CCK8 assay.Sphere-forming ability of sh IGF2BP2 MDA-MB-231 cells was detected by sphere-forming resistance assay.11.Exploring the effect of SNAI2 on the mesenchymal phenotype of breast cancer cells:sh IGF2BP2 MDA-MB-231 cell morphology was examined using Phalloidin staining assay.The migration of sh IGF2BP2 MDA-MB-231 cells was detected by scratch assay.Transwell assay was used to detect sh IGF2BP2 MDA-MB-231 cell invasion(24h).12.Exploring the relationship between IGF2BP2,SNAI2 and FZD7 respectively: the correlation between IGF2BP2 and FZD7 and the correlation between SNAI2 and FZD7 were analyzed by CCLE database and GSE12777 dataset in the GEO database.Western blot assay and Real-time PCR assay were used to detect the expression of FZD7 in MDA-MB-231 cells and Hs 578 T cells.Western blot assay and Real-time PCR assay were used to detect the expression of SNAI2 in sh IGF2BP2 MDA-MB-231 cells;Western blot assay and Real-time PCR assay were used to detect the expression of SNAI2 in MCF7 cells.13.Exploring the effect of FZD7 on drug resistance in breast cancer cells: FZD7overexpression(oe FZD7)plasmid was used to transiently transfect breast cancer cells sh IGF2BP2 MDA-MB-231 cells,and Western blot assay and Real-time PCR assay were used to verify the transfection efficiency.The viability of sh IGF2BP2 MDA-MB-231 cells was measured by CCK8 assay.Sphere-forming resistance assay was used to detect the sphere-forming ability of sh IGF2BP2 MDA-MB-231 cells.14.Exploring the effect of FZD7 on the mesenchymal phenotype of breast cancer cells:sh IGF2BP2 MDA-MB-231 cell morphology was examined using Phalloidin staining assay.The migration of sh IGF2BP2 MDA-MB-231 cells was detected using a scratch assay.Detection of sh IGF2BP2 MDA-MB-231 cell invasion using Transwell assay(24h).Results:1.Bioinformatics analysis results In breast cancer cell lines,IGF2BP2 was positively correlated with mesenchymal phenotype-related indicators VIM,FN1,SNAI2 and ZEB1,and with stemness-related indicators CD44,FZD7,ITGA6 and IL-6.Breast cancer patients with high IGF2BP2 expression had shorter overall survival,distant metastasis-free survival and post-progression survival.2.IGF2BP2 was differentially expressed in breast cancer tissues and paraneoplastic tissues The expression of IGF2BP2 protein in 25 breast cancer tissues and 15 paraneoplastic tissues was analysed by immunohistochemistry.The results showed that 23(92%)breast cancer tissues were positive for IGF2BP2 expression and 2(13%)paraneoplastic tissue was positive for IGF2BP2 expression.3.IGF2BP2 maintains the mesenchymal phenotype of breast cancer cells IGF2BP2 knockdown MDA-MB-231 cells and Hs 578 T cells showed a morphological transformation towards the epithelial state and decreased expression of Vimentin,Fibronectin and α-SMA.IGF2BP2 overexpressing MCF7 cells showed a morphological transformation towards the mesenchymal state cells and increased expression of Vimentin.4.IGF2BP2 promotes breast cancer cell migration and invasion IGF2BP2 knockdown of MDA-MB-231 cells and Hs 578 T cells resulted in diminished cell migration and invasion,while IGF2BP2 overexpression of MCF7 cells resulted in enhanced cell migration and invasion.5.IGF2BP2 promotes breast cancer cell metastasis In vivo analysis of the effect of IGF2BP2 on lung metastasis in breast cancer cells showed that IGF2BP2 knockdown of MDA-MB-231 cells significantly reduced the metastatic foci on the surface of the lung.HE staining further confirmed that IGF2BP2 knockdown of MDA-MB-231 cells had a reduced ability to metastasize in the lung.6.IGF2BP2 induced stemness in breast cancer cells IGF2BP2 knockdown decreased CD44,p-STAT3 and STAT3 protein levels in MDA-MB-231 cells and Hs 578 T cells.IGF2BP2 knockdown decreased CD44 m RNA levels in both MDA-MB-231 cells and Hs 578 T cells.Flow cytometry revealed a significant decrease in the subpopulation of IGF2BP2 knockdown MDA-MB-231 cells and LGR5 positive cells in Hs 578 T cells.Sphere-forming assay IGF2BP2 knockdown MDA-MB-231 cells and Hs 578 T cells showed a significant decrease in sphere-forming rate.Flow cytometry revealed a significant increase in the subpopulation of LGR5-positive cells in IGF2BP2 overexpressing MCF7 cells.Sphere-forming assays revealed a significant increase in the sphere-forming rate of IGF2BP2 overexpressing MCF7 cells.7.IGF2BP2 confers chemoresistance to breast cancer cells CCK8 assays showed that IGF2BP2 knockdown MDA-MB-231 cells and Hs 578 T cells were more sensitive to the chemotherapeutic agent Adriamycin(ADR).Flow cytometry assays showed an increase in apoptosis of IGF2BP2 knockdown MDA-MB-231 cells and Hs 578 T cells following the effects of ADR.Sphere-forming resistance assays showed that IGF2BP2 knockdown MDA-MB-231 cells and Hs 578 T cells were significantly less sphere-forming after ADR,and CCK8 assays showed that IGF2BP2 overexpressing MCF7 cells were even less sensitive to chemotherapeutic ADR.The sphere-forming resistance assay showed that the sphere-forming ability of IGF2BP2 overexpressing MCF7 cells was significantly enhanced after ADR.8.IGF2BP2 regulates SNAI2 m RNA stability and expression Analysis in the CCLE database and GSE12777 dataset in the GEO database showed a positive correlation between IGF2BP2 and SNAI2.IGF2BP2 knockdown was found to significantly reduce SNAI2 expression in MDA-MB-231 cells and Hs 578 T cells by Western blot assay and Real-time PCR assay.RIP assay showed that IGF2BP2 was able to bind to SNAI2 m RNA in MDA-MB-231 cells and Hs 578 T cells.Actinomycin D assay showed that IGF2BP2 knockdown increased the rate of SNAI2 m RNA degradation in MDA-MB-231 cells and Hs 578 T cells.9.SNAI2 overexpression restores chemoresistance in sh IGF2BP2 MDA-MB-231 cells SNAI2 overexpression(oe SNAI2)plasmid was used to transiently transfect sh IGF2BP2-1 MDA-MB-231,and Western blot assays and Real-time PCR experiments confirmed increased SNAI2 expression after transfection.cck8 assays showed that SNAI2 overexpressing sh IGF2BP2 MDA-MB-231 cells were chemo The sensitivity of ADR to chemotherapy was reduced.The sphere-forming resistance assay showed that the sphere-forming ability of SNAI2 overexpressing sh IGF2BP2 MDA-MB-231 cells was significantly enhanced after ADR.10.SNAI2 overexpression restored the mesenchymal phenotype of breast cancer cells Phalloidin staining assay showed that SNAI2 overexpressing sh IGF2BP2 MDA-MB-231 cells transformed from epithelial to mesenchymal morphology.Transwell assay showed that SNAI2 overexpressing sh IGF2BP2 MDA-MB-231 cells had enhanced migratory ability,and Transwell assay showed that SNAI2 overexpressing sh IGF2BP2MDA-MB-231 cells had enhanced invasive ability.11.SNAI2 regulates FZD7 expression Analysis of the CCLE database and GSE12777 dataset in the GEO database showed that IGF2BP2 and SNAI2 were positively correlated with FZD7,respectively.western blot and Real-time PCR experiments showed that IGF2BP2 knockdown decreased SNAI2 expression in MDA-MB-231 cells and Hs 578 T cells.Western blot and Real-time PCR experiments showed that SNAI2 overexpression sh IGF2BP2 MDA-MB-231 cells showed an increase in FZD7 expression.was significantly increased.12.FZD7 overexpression restores chemoresistance in sh IGF2BP2 MDA-MB-231 cells FZD7 overexpression(oe FZD7)plasmid was used to transiently transfect sh IGF2BP2-1MDA-MB-231,and Western blot assays and Real-time PCR experiments confirmed increased FZD7 expression after transfection.CCK8 assays showed that FZD7 overexpressing sh IGF2BP2-1 MDA-MB-231 cells were less sensitive to chemotherapeutic drugs ADR sensitivity was decreased.The sphere-forming resistance assay showed that the sphere-forming ability of FZD7 overexpressing sh IGF2BP2-1MDA-MB-231 cells was significantly enhanced after ADR.13.FZD7 overexpression restored the mesenchymal phenotype of sh IGF2BP2MDA-MB-231 cells Phalloidin staining assays showed that FZD7 overexpressing sh IGF2BP2 MDA-MB-231 cells transformed from epithelial to mesenchymal morphology.Cell scratching assay showed that FZD7 overexpressing sh IGF2BP2-1 MDA-MB-231 cells had enhanced migration ability.transwell assay showed that FZD7 overexpressing sh IGF2BP2-1MDA-MB-231 cells had enhanced invasion ability.Conclusions:1.IGF2BP2 is highly expressed in breast cancer and is associated with poor prognosis.2.IGF2BP2 maintains the mesenchymal phenotype of breast cancer cells and promotes migration,invasion and metastasis of breast cancer cells.3.IGF2BP2 promotes breast cancer cell stemness and chemoresistance.4.In breast cancer,IGF2BP2 acts as an m RNA binding protein to regulate SNAI2expression;SNAI2 regulates FZD7 expression and SNAI2-FZD7 is the downstream effector pathway of IGF2BP2.5.IGF2BP2-SNAI2-FZD7 pathway can be used as a target to block breast cancer metastasis and overcome chemoresistance.