Targeting NAT10 inhibits hepatocarcinogenesis via ac4C-mediated SMAD3 mRNA stability

To investigate the regulatory role of NAT10 in ac4C RNA modification, we generated NAT10-knockdown SK-Hep-1 cell lines using short hairpin RNA (shRNA) technology, with shNC (non-targeting control) serving as a negative control. The efficiency of NAT10 knockdown was confirmed through quantitative PCR (qPCR) and Western blot (WB) analyses. Total RNA was subsequently extracted from the cells, and the RNA samples were treated with sodium borohydride to facilitate RedaC:T-seq for ac4C profiling. Sequencing analysis revealed that NAT10 knockdown significantly reduced the levels of ac4C in SK-Hep-1 cells, suggesting a critical role for NAT10 in regulating this post-transcriptional modification.