BAG2 Inhibits Cell Proliferation By Reducing The Transcriptional Activity Of The Transcription Factor FOXM1

Forkhead box M1（FOXM1）is a member of the FOX family of transcription factors.It is highly expressed in various tumor cells and participates in the occurrence and development of various types of cancer.FOXM1 not only participates in tumor cell proliferation,migration,invasion,metastasis,epithelial-mesenchymal transition process,but also participates in tumor angiogenesis,cell senescence and other processes.It is the main regulator of gene expression,cell cycle mechanism and other major physiological processes.Bcl-2-associated anthanogene（BAG2）is a member of the BAG family.It is a protein that can interact with Hsc70/Hsp70 molecular chaperones.It regulates a variety of biological responses by interacting with other proteins,such as cellular emergency,apoptosis,tumor growth,cell signal transduction.BAG2 is overexpressed in different types of tumor cells and is related to the poor prognosis of tumors.In this paper,the prokaryotic expression system was used to express the PGEX-4T2-FOXM1_688-748 plasmid,and the GST-tag purification system was used to obtain the recombinant protein GST-FOXM1_688-748.Through Pulldown-combined mass spectrometry analysis technology,the potential interaction protein of FOXM1transcription activation domain was captured from MBA-MD-231 cells,and GST-Pulldown experiment confirmed that FOXM1_688-748 interacted with BAG2.With the aid of bimolecular fluorescence complementation technology and fluorescence co-localization technology,it was determined that FOXM1 and BAG2 co-localized in the nucleus.GST-Pulldown experiment and co-immunoprecipitation experiment confirmed that BAG2 and FOXM1 actually interacted.BAG2 was overexpressed in HEK293T cells,and the changes in RNA levels of FOXM1 downstream target genes（PLK1,CDC25B,CDK1）were detected by RT-PCR,and it was found that overexpression of BAG2 down-regulated the m RNA levels of FOXM1 downstream target genes.Through dual luciferase reporter gene experiment and electrophoretic mobility shift assay（EMSA）,it was found that overexpression of BAG2 reduced the transcription activity and DNA binding ability of FOXM1.The HEK293T cells overexpressing BAG2 were detected by flow cytometry,and it was found that the proportion of cells in the S phase decreased.BAG2 was overexpressed in HEK293T cells and MCF-7 cells,and the changes in its proliferation ability were detected by cell clone formation experiments.It was found that overexpression of BAG2 inhibited the proliferation ability of HEK293T cells and MCF cells.This discovery adds a new negative regulatory protein to FOXM1,which helps to design new FOXM1 inhibitors against BAG2,thereby inhibiting the transcriptional activity of FOXM1 and blocking the activation of downstream target genes of FOXM1.