The Role And Mechanism Of FOXF2 Promoting Breast Cancer Cell Proliferation

BackgroundThe FOXF2 is a DNA binding protein that can regulate the expression of many genes,and whose expression is also regulated by various genes.FOXF2 is involved in the process of embryonic development and the formation and remodeling of extracellular matrix.Depending on the preliminary research,we come to the conclusion that FOXF2 is highly express in triple negative breast cancer,and have something to do with poorly prognosis and early metastasis.Through regulating of EMT related gene,twist and foxq1,FOXF2 inhibit the metastasis of triple negative breast cancer cells.The expression of FOXF2 is also regulated by methylation,hypermethylation status of FOXF2 promoter can cause lower expression of FOXF2.Transcription factor Sp1 is thought to be a positive transcriptional regulater of FOXF2.Previous study showed that NF-κB can promote the expression of FOXF2.In this project we confirmed that NF-κB is a positive transcription regulater of FOXF2,and NF-κB being activited in triple negative breast cancer cells and so dose the expression of FOXF2,which showing a significantly positive correlation.To our surprise we found that the FOXF2 can be an auxiliary transcription factor of Rel A,and can promote the expression of Cyclin D1 consistent with Rel A,and then mediates NF-κB promote the proliferation of breast cancer cells.These findings provide a new direction for prevention and cure of breast cancer.Objective:To clarify the potential regulatory roles of transcription factor Rel A on FOXF2,and the mechanism of FOXF2 regulates the proliferation of breast cancer cells.Provide diagnostic markers and novel therapeutic targets for the diagnosis and treatment of breast cancer.Method:1.The state of NF-κB signaling pathway in Breast cancer cell lines MCF-7,T47 D,MDA-MB-231 and BT549 were inhibition or activation.The expression of FOXF2 was detected by Western blot,RT-QPCR,so further to determine the regulatory role of NF-κB on FOXF2.2.Query mapper database we found that in the promoter of FOXF2 exist multiple binding sites of the transcription factor NF-κB.Ch IP assay to detect the NF-κB binding sites in the promoter of FOXF2.In MDA-MB-231 and MCF-7 cells inhibit the NF-κB signaling pathway,or activate the NF-κB signaling pathway respectively,Ch IP assays show the binding site changes.Dual luciferase reporter assay conformed the transcriptional activity of NF-κB in FOXF2 promoter binding sites.3.Breast cancer cell lines MCF-7,T47 D,MDA-MB-231 and BT549 were used for MTT and clone formation assay,to determine FOXF2 promoting the proliferation of breast cancer cell lines.Flow cytometry experiments to determine the effect of which period of cell cycle influence the proliferation of the process.4.To explore the mechanism of FOXF2 promoting the proliferation of breast cancer cells,MCF-7 and MDA-MB-231 were overexpressed or inhibited the expression of FOXF2,to detect the expression of Cyclin D1.And then MCF-7 and MDA-MB-231 respectively activative or inhibit NF-κB signaling pathway,IP experiments was performed to detect the interaction between the Rel A and FOXF2,Ch IP assay the binding of Rel A and FOXF2 on the promoter of Cyclin D1.5.Dual luciferase reporter assay the influence of FOXF2 on NF-κB activity,and to determine the effect of FOXF2 on Rel A transcription activity.Results1.In the breast cancer cell line MCF-7,T47 D,MDA-MB-231 and BT549 respectively activative or inhibit the NF-κB signaling pathway,Western and RT-q PCR detected FOXF2 expression,found that the activation of NF-κB,rised the expression of FOXF2 and inhibition of NF-κB,FOXF2 expression decreased.2.Ch IP assay was used to detect the binding sites of NF-κB on FOXF2 promoter.In MCF-7 cells there is only one site with Rel A weak binding.Treated with TNF-α could active the NF-κB signaling pathway in MCF-7,and we found a plurality of Rel A binding sites in FOXF2 promoter;in MDA-MB-231,there appeared four Rel A binding sites,while inhibited NF-κB signaling pathway,and Ch IP assays we found Rel A binding sites almost disappeared.Dual luciferase reporter assay transcriptional activity of NF-κB on FOXF2 promoter.In MCF-7 activation of signaling pathway or overexpression of Rel A,enhances the transcriptional activity of FOXF2,and only the Reign-542/-532 play a functional role;in MDA-MB-231 inhibiting NF-κB signaling pathway,the transcriptional activity of FOXF2 reduced.The results indicated that activition of NF-κB signaling pathway regulat the transcriptional level of FOXF2.3.In breast cancer cell lines MCF-7,T47 D,MDA-MB-231 and BT549,MTT and colony formation assay found that FOXF2 is a critical mediator of NF-κB signaling pathway-induced proliferation in breast cancer cells.4.MCF-7 and MDA-MB-231 were overexpressed or interfered with FOXF2,Western and RT-QPCR were used for detecting the expression of Cyclin D1.We found that in MCF-7,FOXF2 can promote the expression of Cyclin D1 partially.In MDA-MB-231,FOXF2 inhibitation could decrease the expression of Cyclin D1.In MCF-7 and MDA-MB-231 activatived or inhibited the NF-κB signaling pathway respectively,and then interference or overexpression of FOXF2,we found that FOXF2 interference can restore the influence of NF-κB on the expression of Cyclin D1.IP experiments was performed by Rel A and HA antibodies,and we found that when NF-κB is activated Rel A and FOXF2 could interact with each other.Ch IP conformed that Rel A and FOXF2 could facilitated the expression of Cyclin D1,and Rel A transcriptional regulation of Cyclin D1 need FOXF2.5.Dual luciferase reporter system verify that FOXF2 could enhance NF-κB transcription activity.ConclusionsNF-κB could transcriptional activative the expression of FOXF2 through RELA/p65;FOXF2 combined with Rel A,which performed as a cotranscription factor of Rel A,to promotes the expression of Cyclin D1;And then promote the proliferation of breast cancer cells.