The Role And Mechanism Of RNA Acetyltransferase NAT10 In Regulating Vascular Neointima Formation

Part-I: The Role of RNA Acetyltransferase NAT10 in Regulating Vascular Neointima Formation Background: Percutaneous coronary intervention is currently the preferred treatment for patients with coronary atherosclerotic heart disease,but the occurrence of postoperative restenosis is a significant problem that challenged clinical treatment.The underlying mechanisms of vascular neointima formation remain unclear,but studies have reported that RNA modification plays a crucial role in the progression of vascular neointima formation.Recently,some research indicated that N-acetyltransferase 10(NAT10)could impact m RNA stability and translation efficiency in an m RNA N4-acetylcytidine(ac4c)way.Nevertheless,there is no study about NAT10 in cardiovascular disease,even in vascular neointima formation.Objective: We used in vivo and in vitro experiments to explore the role of NAT10 in vascular neointima formation.Methods:1 To characterize NAT10 expression and m RNA ac4 c modification in vascular neointima formation,we quantified NAT10 by Western Blot and m RNA ac4 c modification by Dot blot in SD rats receiving balloon injury in the carotid artery.2 To characterize NAT10 expression and localization in VSMCs,we quantified NAT10 by Western Blot and NAT10 localization by immunofluorescence staining in rat primary VSMCs treating platelet-derived growth factor.3 To clarify the regulatory role of NAT10 in vascular neointima formation.NAT10 knockdown and overexpression adenovirus were constructed respectively and performed in the rat carotid artery balloon injury model.The effectiveness of the intervention was verified by Western Blotting.Hematoxylin-eosin staining was used to observe the alterations of vascular neointima formation and Ki67 immunofluorescence staining to detect the difference in cell proliferation.4 To clarify the regulatory role of NAT10 in VSMCs proliferation,migration,and apoptosis.We achieved in vitro knockdown or overexpression of NAT10 by transfecting si RNA or plasmids into rat VSMCs.CCK-8 and PH3 immunofluorescence staining experiments were used to detect rat VSMCs proliferation changes;Scratch test and Transwell assay were used to detect rat VSMCs migration capacity;Tunel staining and cell flow cytometry were used to detect rat VSMCs apoptosis ability.Results:1 NAT10 and m RNA ac4 c modification were significantly increased in the tissue of rat carotid artery vascular neointima formation.NAT10 also increased significantly and localized in the nucleus in the rat primary VSMCs treating platelet-derived growth factor.2 Knockdown of rat carotid vascular NAT10 expression suppressed the vascular neointima formation,reduced cell proliferation,and promoted cell apoptosis.3 Overexpression of rat carotid vascular NAT10 expression promoted vascular neointima formation and promoted cell proliferation.4 Knockdown of NAT10 expression in rat VSMCs could inhibit cell proliferation and migration,promote cell apoptosis,and block cell cycle progress.5 Overexpression of NAT10 could promote cell proliferation and migration.Conclusion:After vascular injury,the expression of NAT10 protein in vascular media VSMCs increased,regulating the proliferation,migration,and apoptosis of VSMCs to affect the development of vascular neointima formation.Part-II: NAT10 affects m RNA stability and translation efficiency through ac4 c modification to regulate VSMCs proliferation,migration,and apoptosis.Background: RNA modification plays an essential role in the progression of cardiovascular disease,and the newly discovered m RNA ac4 c modification can regulate m RNA stability and translation efficiency.The role of m RNA ac4 c modification in physiological and pathological processes is rarely studied,even in cardiovascular disease.Objective: We used in vitro experiments to investigate whether NAT10 affects m RNA stability and translation efficiency through m RNA ac4 c modification,regulating the vascular neointima formation.Methods: 1 To clarify the regulatory role of NAT10 was in an m RNA ac4c-dependent way,the acetylase domain of NAT10 was mutated and was transfected to rat VSMCs.CCK-8 assay was used to detect rat VSMCs proliferation changes.2 To clarify that NAT10 affects m RNA stability and translation efficiency and then regulates the proliferation and migration of VSMCs.Transcriptome,RNA acetylation immunoprecipitation,RNA metabolism,and ribosome imprinting highthroughput sequencing were used after NAT10 knockdown in VSMCs.Results: 1 After mutation of the NAT10 acetylase domain,the effect of NAT10 on promoting the proliferation of VSMCs was disappeared.2 Transcriptome sequencing indicated that differential gene expression after NAT10 knockdown was highly enriched in the regulation of cell proliferation,cell death,and other biological processes.3 Acetylated RNA immunoprecipitation(ac RIP)sequencing suggested that differential ac4 c modification after NAT10 knockdown was highly enriched in the regulation of cell proliferation,migration,and other biological processes.4 Combined analyses of ac RIP sequencing and RNA metabolism sequencing,indicating that NAT10 affects m RNA stability through m RNA ac4 c modification and regulates the proliferation,migration,and other biological processes.5 Combined analyses of ac RIP sequencing and ribosome imprinting sequencing,revealing that NAT10 affects m RNA translation efficiency through m RNA ac4 c modification and regulates the proliferation,migration,and other biological processes.Conclusion:NAT10 regulates downstream gene stability and translation efficiency through m RNA ac4 c modification and plays a regulatory role in the proliferation,migration,and apoptosis of VSMCs.