ac4C acetylation regulates mRNA stability and translation efficiency in osteosarcoma
Zhang Wen-jie, Jia Gao, Lei Fan, Juan Wang, Bin He, Yunhua Wang, Xiaotong Zhang, Hui Mao
Abstract
Objective N4-acetylcytidine (ac4C) acetylation can promote target gene expression through improved mRNA stability. To explore the role of ac4C acetylation in osteosarcoma, U2OS and MG63 cell lines were treated with the N -acetyltransferase 10 (NAT10) inhibitor Remodelin. Reverse transcription–polymerase chain reaction (RT-PCR) and Western blot were used to test the gene and protein expression efficiency. Methods The proliferation rate of osteosarcoma cells was measured by a cell counting kit-8 (CCK8) assay. The cell cycle and apoptosis were analyzed by flow cytometry. The invasiveness of osteosarcoma cells was detected by a transwell invasion assay. The ac4C acetylation of target genes was screened by acetylated RNA immunoprecipitation and sequencing (acRIP-seq). Results We found that when osteosarcoma cells were treated with Remodelin at the optimal concentration, their NAT10 expression and the cell proliferation was inhibited, the cells in the G1 phase increased ( P < 0.05) but those in the S phase decreased, the apoptotic cells in the early and late stages increased, and the cells invasiveness decreased ( P < 0.05). Conclusions The farnesyltransferase subunit beta gene ( FNTB ) was identified by acRIP-seq as one of the target genes of ac4C acetylation and was further verified by RT-PCR and Western blot analyses. Remodelin was demonstrated to reduce the stability and protein translation efficiency of target gene mRNA in osteosarcoma cells. In conclusion, inhibition of ac4C acetylation in osteosarcoma can block proliferation and metastasis as well as promote apoptosis and cell cycle arrest. Ac4C acetylation contributes to the stability and protein translation efficiency of the downstream target gene mRNA.