The Role Of Super-enhancer-related Inhibitor THZ2 In The Chemoresistance Of Osteosarcoma

Purpose: Chemotherapy is necessary for osteosarcoma treatment,however,chronic treatment has not significantly improved the 5-year survival rate of patients due to the emergence of chemoresistance.In this study,we will explore the role of a novel epigenetic element,the superenhancer(SE),in primary and acquired chemoresistant osteosarcoma cells,and investigate the effect of SE-related inhibitor THZ2 on cisplatin efficacy to find more rational strategies for osteosarcoma chemotherapy.Methods: We screened primary and acquired chemoresistant osteosarcoma cells,detected the expression of SE-regulated genes and genes related to SE function in the cell lines by q RT-PCR and WB.The binding of proteins to H3K27ac(a SE-associated marker)was detected by CO-IP."Compusyn" software calculated the combination index of THZ2 and cisplatin.The drug effects on apoptosis were detected by flow cytometry.Cell phenotyping assays determine the interference of drugs on cell function.Results: The SE-regulated gene Sox9 was upregulated in both primary and acquired chemoresistant osteosarcoma cells,and prolonged exposure of sensitive osteosarcoma cells to cisplatin increased SOX9 expression.Prediction of SOX9 target genes revealed that its downstream genes play important roles in cancer transcriptional dysregulation and cell proliferation.MED1 is one of the essential proteins for SE to perform gene regulation functions.The results showed that although the expressions of MED1 and p-MED1 in chemoresistant cells were lower than those in sensitive cells,the ratio of p-MED1/MED1 was significantly higher in chemoresistant cells.THZ2 inhibited SOX9 expression and p-MED1/MED1 ratio in chemoresistant cells.The combination index values of cisplatin and THZ2 combination in sensitive osteosarcoma cell lines were mostly in the antagonistic region,while most of them were in the synergistic region in the acquired chemoresistant cell lines,and in the primary drug-resistant cell line,they were all in the synergistic region.THZ2 partially downregulated cisplatin-induced increases in SOX9 and p-MED1/MED1 ratios in chemoresistant cells.Conclusion: We found upregulation of SOX9 and p-MED1/MED1 in primary and acquired osteosarcoma chemoresistant cell lines,and THZ2 repressed these phenomena and tended to synergize with cisplatin in resistant cell lines.These findings may help promote rational treatment of osteosarcoma and improve the efficacy of chemotherapy.