NAT10 promotes synovial aggression by increasing the stability and translation of N4-acetylated PTX3 mRNA in rheumatoid arthritis.

Recent studies indicate that N-acetyltransferase 10 (NAT10)-mediated ac4C modification plays unique roles in tumour metastasis and immune infiltration. This study aimed to uncover the role of NAT10-mediated ac4C in fibroblast-like synoviocytes (FLSs) functions and synovial immune cell infiltration in rheumatoid arthritis (RA). FLSs were obtained from active established patients with RA. Protein expression was determined by western blotting or immunohistochemistry or multiplexed immunohistochemistry. Cell migration was measured using a Boyden chamber. ac4C-RIP-seq combined with RNA-seq was performed to identify potential targets of NAT10. RNA immunoprecipitation was used to validate the interaction between protein and mRNA. NAT10 haploinsufficiency, inhibitor remodelin or intra-articular Adv-NAT10 was used to suppress arthritis in mice with delayed-type hypersensitivity arthritis (DYHA) and collagen II-induced arthritis (CIA) and rats with CIA. We found elevated levels of NAT10 and ac4C in FLSs and synovium from patients with RA. NAT10 knockdown or specific inhibitor treatment reduced the migration and invasion of RA FLSs. Increased NAT10 level in the synovium was positively correlated with synovial infiltration of multiple types of immune cells. NAT10 inhibition in vivo attenuated the severity of arthritis in mice with CIA and DTHA, and rats with CIA. Mechanistically, we explored that NAT10 regulated RA FLS functions by promoting stability and translation efficiency of N4-acetylated PTX3 mRNA. PTX3 also regulated RA FLS aggression and is associated with synovial immune cell infiltration. Our findings uncover the important roles of NAT10-mediated ac4C modification in promoting rheumatoid synovial aggression and inflammation, indicating that NAT10 may be a potential target for the treatment of RA, even other dysregulated FLSs-associated disorders.

Liu D ，Kuang Y ，Chen S ，Li R ，Su F ，Zhang S ，Qiu Q ，Lin S ，Shen C ，Liu Y ，Liang L ，Wang J ，Xu H ，Xiao Y ... -  《-》

Eukaryotic translation initiation factor 6-mediated ribosome biogenesis promotes synovial aggression and inflammation by increasing the translation of SP1 in rheumatoid arthritis.

Fibroblast-like synoviocytes (FLSs) play critical roles in synovial inflammation and aggression in rheumatoid arthritis (RA). Here, we explored the role of eukaryotic translation initiation factor 6 (eIF6) in regulating the biological behaviors of FLSs from patients with RA. FLSs were isolated from the synovial tissues of RA patients. Gene expression was assessed via RT-qPCR, and protein expression was evaluated via Western blotting or immunohistochemistry. Proliferation and nascent peptide synthesis were evaluated via EdU incorporation and HPG labeling, respectively. Cell migration and invasion were observed via Transwell assays. Polysome profiling was conducted to analyze the distribution of ribosomes and combined mRNAs. The in vivo effect of eIF6 inhibition was evaluated in a collagen-induced arthritis (CIA) rat model. We found that eIF6 expression was elevated in FLSs and synovial tissues from RA patients compared to those from healthy controls and osteoarthritis patients. Knockdown of eIF6 inhibited the migration, invasion, inflammation, and proliferation of FLSs from patients with RA. Mechanistically, eIF6 knockdown downregulated ribosome biogenesis in FLSs from with RA, leading to a decrease in the proportion of polysome-associated specificity protein 1 (SP1) mRNA and a subsequent reduction in the translation initiation efficiency of SP1 mRNA. Thus, eIF6 controls SP1 expression through translation-mediated mechanisms. Interestingly, intra-articular eIF6 siRNA treatment attenuated symptoms and histological manifestations in CIA rats. Our findings suggest that an increase in synovial eIF6 might contribute to rheumatoid synovial inflammation and aggression and that targeting eIF6 may have therapeutic potential in RA patients.

Shen C ，Peng C ，Zhang S ，Li R ，Liu S ，Kuang Y ，Su F ，Liu Y ，Liang L ，Xiao Y ，Xu H ... -  《-》

ALKBH5-Mediated RNA m(6) A Methylation Regulates the Migration, Invasion, and Proliferation of Rheumatoid Fibroblast-Like Synoviocytes.

Fibroblast-like synoviocytes (FLSs) are critical for promoting joint damage in rheumatoid arthritis (RA). N6 -methyladenosine (m6 A) modification plays key roles in various diseases, but its role in the pathogenesis of RA is largely unknown. Here, we investigate increased demethylase ALKBH5 promotion of proliferation, migration, and invasion of RA FLSs via regulating JARID2 expression. ALKBH5 expression in FLSs was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. 5-ethynyl-2'-deoxyuridine, scratch wound healing, and transwell assays were implemented to determine the role of ALKBH5 on RA FLS proliferation, mobility, and migration. Then, m6 A sequencing combined with RNA sequencing was performed to identify the potential targets of ALKBH5. RNA immunoprecipitation and RNA pulldown were then used to validate the interaction between the protein and messenger RNA (mRNA). Collagen-induced arthritis (CIA) and delayed-type hypersensitivity arthritis (DTHA) models were further established to assess the therapeutic potency of ALKBH5 in vivo. We demonstrated that ALKBH5 expression was increased in FLSs and synovium from RA. Functionally, ALKBH5 knockdown inhibited the proliferation, migration, and invasion of RA FLSs, whereas overexpression of ALKBH5 displayed the opposite effect. Mechanistically, ALKBH5 mediated m6 A modification in the JARID2 mRNA and enhanced its mRNA stability in cooperation with IGF2BP3. Intriguingly, the severity of arthritis was attenuated in mice with DTHA and ALKBH5 knockout or rats with CIA and intra-articular injection of ALKBH5 short hairpin RNA. Our findings suggest that ALKBH5-mediated m6 A modification is crucial for synovial hyperplasia and invasion in RA. ALKBH5 might be a potential therapeutic target for RA and even for dysregulated fibroblasts in a wide range of diseases.

Kuang Y ，Li R ，Wang J ，Xu S ，Qiu Q ，Lin S ，Liu D ，Shen C ，Liu Y ，Xu M ，Lin W ，Zhang S ，Liang L ，Xu H ，Xiao Y ... -  《-》

Targeting N4-acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2-mediated HMGB2 mRNA translation.

N4-acetylcytidine (ac4C) represents a novel messenger RNA (mRNA) modification, and its associated acetyltransferase N-acetyltransferase 10 (NAT10) plays a crucial role in the initiation and progression of tumors by regulating mRNA functionality. However, its role in hepatocellular carcinoma (HCC) development and prognosis is largely unknown. This study aimed to elucidate the role of NAT10-mediated ac4C in HCC progression and provide a promising therapeutic approach. The ac4C levels were evaluated by dot blot and ultra-performance liquid chromatography-tandem mass spectrometry with harvested HCC tissues. The expression of NAT10 was investigated using quantitative real-time polymerase chain reaction, western blotting, and immunohistochemical staining across 91 cohorts of HCC patients. To explore the underlying mechanisms of NAT10-ac4C in HCC, we employed a comprehensive approach integrating acetylated RNA immunoprecipitation and sequencing, RNA sequencing and ribosome profiling analyses, along with RNA immunoprecipitation, RNA pull-down, mass spectrometry, and site-specific mutation analyses. The drug affinity responsive targets stability, cellular thermal shift assay, and surface plasmon resonance assays were performed to assess the specific binding of NAT10 and Panobinostat. Furthermore, the efficacy of targeting NAT10-ac4C for HCC treatment was elucidated through in vitro experiments using HCC cells and in vivo HCC mouse models. Our investigation revealed a significant increase in both the ac4C RNA level and NAT10 expression in HCC. Notably, elevated NAT10 expression was associated with poor outcomes in HCC patients. Functionally, silencing NAT10 suppressed HCC proliferation and metastasis in vitro and in vivo. Mechanistically, NAT10 stimulates the ac4C modification within the coding sequence (CDS) of high mobility group protein B2 (HMGB2), which subsequently enhances HMGB2 translation by facilitating eukaryotic elongation factor 2 (eEF2) binding to the ac4C sites on HMGB2 mRNA's CDS. Additionally, high-throughput compound library screening revealed Panobinostat as a potent inhibitor of NAT10-mediated ac4C modification. This inhibition significantly attenuated HCC growth and metastasis in both in vitro experiments using HCC cells and in vivo HCC mouse models. Our study identified a novel oncogenic epi-transcriptome axis involving NAT10-ac4C/eEF2-HMGB2, which plays a pivotal role in regulating HCC growth and metastasis. The drug Panobinostat validates the therapeutic potential of targeting this axis for HCC treatment.

Liu H ，Xu L ，Yue S ，Su H ，Chen X ，Liu Q ，Li H ，Liang H ，Chen X ，He J ，Ding Z ，Zhang B ... -  《Cancer Communications》

NAT10 promotes liver lipogenesis in mouse through N4-acetylcytidine modification of Srebf1 and Scap mRNA.

Metabolic dysfunction associated steatotic liver disease (MASLD), closely linked to excessive lipogenesis, induces chronic liver disease. MASLD often cause other metabolic diseases, such as cardiovascular disease, diabetes and obesity. However, the mechanism of N-acetyltransferase 10 (NAT10)-mediated N4-acetylcytidine (ac4C) mRNA modification in lipogenesis of MASLD has not been fully elucidated. This study investigated the role of NAT10 in lipogenesis targeting mRNA ac4C modification. The expression of NAT10 in mouse liver was assessed after a 12-week high-fat diet. In addition, the expression of NAT10 also was detected after AML12 hepatocytes cells were treated with 150 µmol/L palmitic acid (PA). The ac4C mRNA modification was performed by dot blotting. Oil red O staining and the mRNA expression of Srebf1, Acaca and Fasn were used to assess lipogenesis in AML12 cells with NAT10 overexpression or knockdown. acRIP-PCR and NAT10 RIP-PCR were used to verify the Srebf1 and Scap mRNA ac4C modification by NAT10. Furthermore, the liver lipogenesis was evaluated by AAV-mediated target knockdown of NAT10 in mouse liver and treating a specific inhibitor, Remodelin. This study revealed that NAT10 is significantly upregulated in liver lipogenesis after a 12-week high-fat diet. NAT10 and ac4C mRNA modification were also drastically increased in AML12 cells after treated with 150 µmol/L PA. Silencing of NAT10 notably inhibited the lipogenesis in AML12 cells and AAV-mediated target knockdown of NAT10 in mouse liver. The acRIP-PCR and NAT10-RIP-PCR revealed that NAT10 ac4C modified Srebf1 and Scap mRNA, the critical modulator of liver lipogenesis, to regulate liver lipogenesis. Besides, Remodelin strongly inhibited liver lipogenesis, including liver TG, serum ALT, AST, TG and TC level and glucose metabolism. NAT10 mediates ac4C modification of Srebf1 and Scap mRNA, thereby affecting lipogenesis in the liver. This study provided a new target for the treatment of MASLD.

Wang Z ，Wan X ，Khan MA ，Peng L ，Sun X ，Yi X ，Chen K ... -  《-》