Research On The Mechanism Of HDAC5 Regulating MRNA M~6A Modification In Breast Cancer Cells

Research Background Breast cancer is the malignant tumor with the highest incidence of women in my country and even in the world,and it is also the main cause of female cancer deaths except lung cancer.HDAC5 and m RNA m~6A play a key role in breast cancer.Objective This project aims to study the role and mechanism of HDAC5 in the regulation of m~6A methylation in breast cancer cells.Methods The TCGA database and immunohistochemistry were firstly used to analyze the correlation between the expression of METTL14,Spi-1 and HDAC5 in breast cancer tissues and the relationship with the malignant degree of breast cancer;then verifing the activity of the METTL14 and Spi-1 gene promoter in MCF-7 cells by dual luciferase reporter gene when knockouting HDAC5 gene.Real-time quantitative PCR and western blot were used to further prove that the protein and m RNA expression levels of METTL14 and Spi-1were regulated by HDAC5 in the HDAC5 knockout MCF-7 cell lines,while silencing or overexpressing the expression of Spi-1.The level of m RNA m~6A modification was detected by LC-MS/MS in human breast cancer cells MCF-7 and mouse breast cancer cells 4T-1 with HDAC5 gene knockout.Finally we observed HDAC5 inhibitor LMK-235 had an effect on the proliferation and migration of 4T-1 cells in vivo using 4T-1 cell tumor-bearing mode.Results HDAC5 and METTL14 were highly expressed and their expressions were positively regulated,while Spi-1 was lowly expressed and were negatively related to the expression of the former two in breast cancer tissues.HDAC5 could regulate the activity of the promoter regions of METTL14 and Spi-1 genes.At the same time,HDAC5 can up-regulate the protein and m RNA expression levels of METTL14 in MCF-7 cells by inhibiting the expression of Spi-1.HDAC5 gene knockout resulted in down-regulation of the overall methylation level of m RNA m~6A in MCF-7 cells and 4T-1 cells.HDAC5 inhibitor LMK-235 treatment significantly inhibited the proliferation of 4T-1 cells in mice.Conclusion This study shows that HDAC5 can up-regulate the expression of METTL14by inhibiting Spi-1,and thereby maintain the level of m RNA m~6A modification in breast cancer cells.The proliferation and metastasis of breast cancer cells could be inhibited by HDAC5.It suggests that histone deacetylation modification and m~6A methylation modification are related to the epigenetic regulation of the occurrence and development of breast cancer.