NAT10 Promotes Prostate Cancer Progression By Acetylating MRNAs Of HMGA1 And KRT8

Background and purposeThe second most common malignancy in men worldwide is prostate cancer(PCa).Cancer cell epithelial-mesenchymal transition and cell cycle modification are crucial processes influencing the growth of malignant tumors and distant metastasis,respectively.Epitranscriptomics is one of the popular fields that have emerged recently to study the effects of chemical modifications carried by RNA on gene expression.N4acetylcytidine(ac4C),the first acetylated nucleoside to be discovered,is also widely present on mRNA.In mammals,NAT10 is the only known RNA acetyltransferase responsible for catalyzing the formation of ac4C in numerous messenger RNAs and influencing the translation efficiency and stability of messenger RNAs,thereby affecting a variety of biological functions in other tumors.However,it is still unclear how NAT10 affects PCa by catalyzing ac4C.Therefore,it is crucial to investigate NAT10’s biological role in PCa and its molecular mechanism.MethodsCorrelation of NAT10 expression in PCa tissues with clinicopathological parameters was compared by bioinformatics,immunohistochemistry and Western blot.In PCa cell lines,we created NAT10 knockdown and overexpression constructs and assessed the expression of ac4C.To determine the proliferation function of PCa cells in vitro,plate clone formation test and cell proliferation assay approaches are used.By using methods like Trans well migration and scratch healing assays,we were able to assess the PCa cells’ ability to migrate in vitro.We also used flow cytometry to determine the PCa cells’ cell cycle.Using a subcutaneous transplantation tumor model in naked mice and a tail vein injection tumor formation model in naked mice,respectively,the proliferation and metastatic capacity of PCa cells were evaluated in vivo.On cell lines with NAT10 knockdown,transcriptome sequencing and RNA immunoprecipitation sequencing were carried out,and the results were assessed for downstream target mRNA of NAT10 acetylation.We used the RNA Binding Protein Immunoprecipitation and actinomycin D assay to verify the effect of ac4C modification on the stability of downstream target mRNAs and transient transfection of stable transfectants using plasmids for in vitro reversion experiments of cellular functions.ResultsAs compared to paraneoplastic tissue,NAT 10 was highly expressed in the PCa tissue group.Also it positively correlated with the patient’s Gleason score,clinical stage,and pathological T-stage.Knockdown of NAT10 in PCa cell lines showed decreased ac4C levels,their proliferation and migration abilities were significantly reduced,and the cells underwent G1 phase arrest,whereas overexpression cell lines showed the opposite phenotype.In PCa cell lines with NAT10 knockdown,the epithelialmesenchymal transition was inhibited while the cell cycle was obstructed,according to Western blot data.NAT 10 knockdown led to smaller subcutaneous graft tumors and a significantly lower number and size of lung metastases,according to in vivo studies in nude mice.HMGA1 and KRT8 were screened by RNA immunoprecipitation sequencing as target genes for NAT 10 acetylated mRNA.Knockdown of NAT 10 decreased the ac4C level of mRNA of HMGA1 and KRT8 and decreased mRNA stability.Overexpression of HMGA1 and KRT8 in stable transgenic strains with knockdown of NAT10 restored cell proliferation,cell cycle progression and migration,and epithelial-mesenchymal transition,respectively.ConclusionThe level of NAT10 expression is strongly linked with PCa patients’ poor prognosis and level of malignancy.NAT10 can increase the mRNA stability of HMGA1 and KRT8 by acetylating their mRNAs and promote cell cycle progression and epithelial-mesenchymal transition,ultimately increasing the malignancy of the tumor.In conclusion,NAT10-HMGA1/KRT8 is expected to be a new target for the treatment of PCa patients.