Screening Of Downstream Genes Regulated By HDAC5-LSD1 Axis In Breast Cancer Cells

Objective Histone lysine specific demethylase 1(LSD1)can specifically catalyze the demethylation of H3K4Me1 and H3K4Me2 in the gene promoter region,thereby inhibiting gene transcription.Previous study by us found that HDAC5 and LSD1 synergistically promote the malignant proliferation,migration and invasion of breast cancer cells in human breast cancer cells.This study aims to further identify the downstream target genes co-regulated by HDAC5-LSD1 axis,and make foundation for following breast cancer research.Methods(1)HDAC inhibitors were used to treat MDA-MB-231 and MDA-MB-468 cells.The effects of HDAC inhibitors on LSD1 and USP28 m RNA levels and protein levels were analyzed by q PCR and western blot.(2)HDAC5 or LSD1 Knock-out MCF-7 cells were used to analyze the downstream pathways and genes co-regulated by HDAC5 and LSD1 with RNA-Seq.(3)Tn5 and p A-Tn5 transposases were prepared by prokaryotic expression and chitin column affinity chromatography.(4)ATAC-Seq library were constructed by PCR.Results(1)Besides sulforaphane,couples of HDAC inhibitors can up-regulate m RNA levels of HDAC5 in MDA-MB-231 cells,but all these inhibitors have no significant effects on m RNA levels of LSD1 and USP28.(2)HDAC inhibitors can up-regulate HDAC5 and LSD1 protein expression in breast cancer cell lines without significantly affecting USP28 protein levels.(3)HDAC5 and LSD1 co-regulated multiple pathways and genes in MCF-7 cells.(4)Successfully prepared Tn5 and p A-Tn5 transposases and established ATAC-Seq library.Conclusion(1)Multiple histone deacetylase inhibitors can up-regulate the expression of HDAC5 in MDA-MB-231 cells;(2)HDAC5 and LSD1 coordinately regulate the expression of multiple signaling pathways and genes in breast cancer MCF-7 cells;(3)Successfully prepared transposases Tn5 and p A-Tn5 for ATAC-Seq and CUT & Tag,which laid the foundation for the subsequent ATAC-Seq and CUT & Tag experiments.