MTA2 Triggered R-Loop Increases Stemness In Hepatocellular Carcinoma

MTA2 is an important molecule to promote the progression of malignant tumors,such as hepatocellular carcinoma,but its molecular mechanism has not been studied extensively.According to the data mining and experimental verification of HCC in The Cancer Genome Atlas(TCGA),it is found that MTA2 was highly expressed in HCC.The high expression level of MTA2 was related to short overall survival and disease-free survival,and the expression of MTA2 increases with the malignant progression of HCC.Bioinformatics analysis and in vitro and in vivo experimental verification show that MTA2 can drive the stemness of HCC,which is different from classical molecular mechanisms involving the nucleosome remodeling and deacetylation(Nu RD)transcriptional inhibitory complex and promoting the epithelial-mesenchymal transition and proliferation of tumors.Further data analysis and biochemical experimental studies found that,unlike CD133~-HCC cells with low stemness,MTA2can recruit CHD4 and HDAC2 in CD133~+HCC cells with stemness features to form an incomplete NURD complex and induce the formation of R-Loop during transcription.The R-Loop formed in the open reading frame of genes can pause the transcription of the genes.Using high-throughput sequencing and bioinformatics analysis,it was found that among the genes with both MTA2 and R-Loop regulatory signals inside the open reading frame,BDH1 is most significantly related to the prognosis of HCC.BDH1,which is a key enzyme for the metabolic process of ketone bodies,regulates the chemical transformation of the ketone bodies?-hydroxybutyric acid(?HB)and acetoacetic acid.?HB is the most abundant ketone body in mammals,which can be taken up by tissues in need of energy and converted into energy during starvation.At the same time,?-hydroxybutyric acid is also a starvation-related signal molecule,which can regulate gene expression by protein?-hydroxybutyrylation(Kbhb).The reduction of BDH1 leads to the accumulation of?HB,which allows superfluous?HB to produce a cascade effect on the opening of chromatin in the stemness-related genes through the?-hydroxybutyrylation of lysines in several proteins,especially histone H3K9bhb.Using the specimens from 340 cases with HCC,it was verified by immunostaining and in situ proximity ligation assay that R-Loop,Kbhb and H3K9bhb induced by MTA2 are correlated with the progression and prognosis of HCC,and could be potential prognostic markers and drug targets for HCC.This work reveals the important roles of the R-Loop,ketone metabolism,and acylation in the process of MTA2 promoting the stemness and further confirms that tumor progression is closely related to the microenvironment and metabolism.