Real-time quantitative reverse transcription PCR (RT-qPCR)

RNAs were isolated from AML12 cells or liver tissues using Triquick reagent (Solarbio, R1100, Beijing, China), and subsequently were reverse transcribed to cDNA (Solarbio, RP1100, Beijing, China). qPCR with 2×SYBR Green PCR Mastermix (Solarbio, SR1110, Beijing, China) was used to detect relative mRNA expression, normalized to Gapdh expression. Primer sequences are provided in Supplementary Table 1.

Oil red O staining and FFA quantification

After AML12 cells were incubated with 150 µmol/L PA for 48 h, the cells were first washed with PBS and fixed with 4% paraformaldehyde, followed by multiple washes with water. Next, the cells were then washed with water for 3 times, incubated with diluted Oil red O (Beyotime, C0157S, Shanghai, China) for 10 min, and were washed with water again. The photographs were taken with microscope (Leica DM IL LED Fluo, Wetzlar, Germany). The liver tissue slices were added to diluted Oil red O, and incubated for 20 min, decolorized with 70% alcohol, and then washed with water again. Next, the liver slices were further stained using hematoxylin. The cellular FFA content were detected with FFA content assay kit (Solarbio, BC0590, Beijing, China).

Immunoblotting

The AML12 cells or liver tissue protein samples were extracted with ProteoPrep^® total protein isolation kit (Sigma, MO, USA), and the content was detected using the BCA quantitative analysis kit (Sigma, Shanghai, China). Subsequently, approximate 100 µg protein was separated by SDS-PAGE (70 V, 30 min then 120 V, 90 min) and transferred (300 mA, 60 min) and transferred to PVDF membranes (Millipore, MA, USA). The membrane was then blocked with 5% non-fat milk for 2 h, and incubated in diluted primary antibody including NAT10 (13365-1-AP), SREBP-1 (14088-1-AP), ACC1 (21923-1-AP), FASN (10624-2-AP) and β-actin (66009-1-Ig) [all provided by Proteintech, Wuhan, China], SCAP (Abcam, ab308060, MA, USA) and Cd36 (Abcam, ab255331, MA, USA). The next day, the membranes were incubated with HRP-labeled secondary antibodies for 1 h, and after washed for 3 times, chemiluminescence reaction was performed by using BeyoECL plus kit (Beyotime, P0018S, Beijing, China). Images were captured using the GelDoc XR Biorad system (Bio-Rad, CA, USA) for further analysis.

mRNA ac4C dot blot

The mRNA was isolated from about 10 µg of total RNA using Dynabeads™ mRNA purification kit (Invitrogen, CA, USA), and mRNA was denatured at 95 °C for 5 min, and was placed at ice immediately for 1 min. Next, the mRNA was added to PVDF membranes (Millipore, MA, USA), the membrane was cross-linked for 3 times at ultraviolet 254 nm, and subsequently were blocked with 5% non-fat milk containing 0.1% PBST. The membrane was incubated with anti-ac4C antibody (Abcam, ab252215, MA, USA) overnight at 4 °C. The membrane was washed for 3 times and incubated using an anti-rabbit IgG-HRP (Sigma, MO, USA) for 1.5 h at room temperature, and then washed for 3 times. The membrane was incubated with ECL (Beyotime, P0018S, Shanghai, China) and 0.02% methylene blue (Solarbio, M8030, Beijing, China) was used to stain membrane. After that, membrane was washed with RNase-free water (Solarbio, R1600, Beijing, China) for 1.5 h, and the membrane was photographed with ChemiDoc imaging system (Bio-Rad, CA, USA).

Hepatocyte-targeted mouse adeno-associated virus (AAV) shRNA-NAT10 plasmid construction

The overexpressed NAT10 (OE-NAT10, NM_153126. 4), OE-Srebf1, OE-Scap and overexpressed negative control plasmid (OE-NC), siRNA NAT10 (siRNA-NAT10) and the siRNA negative control plasmid (siRNA-NC) was constructed by Shanghai Sanggong Biotechnology Co., Ltd, China. Hepatocyte-targeted AAV-shRNA-NAT10 and AAV-shRNA-NC plasmids were designed and constructed by Shanghai Gene Chemistry Co., Ltd, China. The NAT10 target sequence was 5’-GCTGGATTTGTTCCTGTCTAT-3’, and element sequences was pAAV-ApoE/hAATp-EGFP-MIR155(MCS)-SV40 PolyA. The cells were seeded in six-well palate and when cells growth to 80–90% confluence and then were transfected by 50nM siRNA-NAT10 and siRNA-NC.

Cell transfection

When AML12 cells were grown in approximately 80% confluency, the cells were transfected with OE-NAT10, OE-Srebf1, OE-Scap, OE-NC, siRNA-NAT10 and siRNA-NC with Lipo 5000 (Solarbio, L3200, Beijing, China) according to operation manual. After 48 h, the cells were treated with or without 150 µmol/L PA.

ac4C-specific RNA immunoprecipitation (RIP) PCR (acRIP-PCR) and NAT10-RIP-PCR

After AML12 cells transfected with siRNA-NAT10 or siRNA-NC for 48 h, the total RNA was isolated and mRNA was purified using a mRNA purification kit (Invitrogen, CA, USA). The acRIP (Millipore, 17–700, MA, USA) was performed. Briefly, the mRNA was incubated with anti-ac4C antibody (Abcam, ab252215, MA, USA) and anti-IgG antibody (Abcam, ab172730, MA, USA) using Protein G magnetic beads. The ac4C modified mRNA was collected and further were amplified using RT-qPCR. NAT10-RIP-PCR method was performed according to acRIP-PCR. The Srebf1 and Scap primers were designed according to ac4C modification sites predicted by PACES software (http://www.rnanut.net/paces/). The primer sequences are listed in Supplementary Table 1.

RNA decay assay

After AML12 cells were transfected with siRNA-NAT10 and siRNA-NC for 48 h, the two group of cells were harvested after 5 µg/mL Actinomycin D (Selleck, S8964, TX, USA) treatment for 0, 2, 4, 6 h. The total RNA was extracted and reverse transcribed, and then the Srebf1 and Scap expression were amplified using qPCR.

Dual-luciferase reporter assay

According to Srebf1 and Scap mRNA ac4C motif site predicted by PACES software, the pGL3-basic-Srebf1-wild type (Srebf1-WT), pGL3-basic-Scap-wild type (Scap-WT), pGL3-basic-Srebf1-mutation (Srebf1-MUT, mutation sequence: CTGCTGGCAAGTCTC) and pGL3-basic-Scap-mutation (Scap-MUT, mutation sequence: CGACCGAATACCCGA) plasmid were constructed. Approximately 1 µg plasmid was transfected into HEK293T and evaluation of firefly luciferase and renilla luciferase was carried out using a dual-luciferase reporter assay kit (Solarbio, D0011, Beijing, China).

In vivo study

To explore NAT10 and mRNA ac4C expression in liver tissue following a high-fat diet in mice, 6-weeks-old male mice were fed by chow diet (‌D12450J)‌ (n=5) and high-fat diet (D12492), all feed were provided by Research Diets, NJ, USA (n=5) for 12 weeks, the liver tissue were collected at 12th week. In addition, for AAV mediated NAT10 hepatocyte-targeted silencing experiment, 6-weeks-old male C57BL/6J mice were injected with hepatocyte-targeted AAV-shRNA-NAT10 (4×10¹¹v.g, n=5) or AAV-shRNA-NC (4×10¹¹v.g, n=5) through tail vein injection, and then subsequently were fed with a high-fat diet for 12 weeks. Additionally, two group of mice were injected again using same dose at 4th week. Furthermore, for Remodelin gavage experiment, male 6-weeks-old C57BL/6J mice were gavage with DMSO (n=5) and Remodelin (100 mg/kg/day, n=5), respectively, and two groups also were fed with high-fat diet for 12 weeks. All animal procedures were conducted in accordance with ethical guidelines approved by the Animal Ethics Committee of The Third Xiangya Hospital of Central South University, Changsha, China, (Approval NO: 2023-S027).

Hematoxylin and eosin (H&E) staining

The liver tissues were fixed with 4% paraformaldehyde for overnight at 4 °C, and the liver tissue were paraffin imbedded and cut into 5µM sections. After deparaffinized and rehydrated, the specimens were stained with H&E staining kit (Solarbio, G1120, Beijing, China). Microscopic images were captured using a Leica DM IL LED Fluo microscope (Wetzlar, Germany).

Biochemical analysis

The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity were assayed by corresponding assay kits (BC1555 and BC1565, Solarbio, Beijing, China) along with the triglyceride (TG) and cholesterol (TC) assay kits (BC0625 and BC1985, Solarbio, Beijing, China). 100 mg of liver tissue were added 1mL of a mixture containing n-heptane and isopropyl alcohol at a volumetric ratio of 1:1, and were centrifuged at 10,000 rmp at 4. The supernatant was used to detect liver TG content.

Glucose tolerance test (GTT) and insulin tolerance test (ITT)

For AAV mediated NAT10 hepatocytes targeted silencing experiment and Remodelin gavage experiment, after 12 weeks, mice were subjected to a fasting period of 12 h, the mice were then intraperitoneally injected with 1.5 g/kg of glucose and their blood glucose levels were monitored at various time points −0, 30, 60, 90, and 120 min post-injection. In addition, for ITT, after mice were fasted for 12 h, they were injected with 1 IU/kg of insulin (Novo Nordisk, Copenhagen, Denmark), the blood glucose levels were measured at the same intervals as the GTT experiment. Area under the curve (AUC) for GTT and ITT were calculated by Graphpad Prism 10 software.

NAT10 radically induces AML12 cell lipogenesis

To further explore the role of NAT10 on lipogenesis in vitro, NAT10 was overexpressed or silenced in AML12 cells for 48 h, and were treated with 150µmol/L PA, and Oil red O staining results showed that NAT10 significantly promoted lipid accumulation in AML12 cells, and conversely, silencing of NAT10 strongly inhibited lipogenesis in AML12 cells (Fig. 2A). The cellular content of FFA was greatly enhanced or reduced in NAT10 overexpressed or silenced in AML12 cells respectively after 150µmol/L PA treatment (Fig. 2B). Additionally, some critical molecules of hepatocyte lipogenesis, such as, Srebf1, acetyl-CoA carboxylase alpha (Acaca), and Fasn mRNA and protein, including Cd36, were also positively regulated by NAT10, especially Srebf1 mRNA and precursor and mature SREBP-1 were increased or decreased even without PA intervention (Fig. 2C-F). Furthermore, knockdown of NAT10 suppressed mRNA ac4C modification, which was revealed by using ac4C dot blot (Fig. 2G). In addition, Oil red O staining displayed that a specific inhibitor of NAT10, Remodelin, strongly repressed AML12 cell lipogenesis (Fig. 2H), decreased cellular FFA contents (Fig. 2I), and inhibited Srebf1, Acaca, Fasn mRNA and protein, including Cd36 expression (Fig. 2J-K). The results demonstrated that NAT10 notably promoted lipogenesis in AML12 cells.

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Fig. 2

NAT10 improves AML12 cells lipogenesis and mRNA ac4C modification. (A) Overexpressing or silencing of NAT10 promoted or inhibited AML12 cell lipid droplets form (Oil red O staining, scale bar =50 μm), (B) cellular FFA content, (C) Srebf1, (D) Acaca, (E) Fasn mRNA and (F) protein including Cd36 expression after AML12 were intervened with 150µmol/L PA. (G) Knockdown of NAT10 decreased mRNA ac4C modification in AML12 cells. (H) 20µmol/L Remodelin inhibited AML12 cellular lipid droplet forms (Oil red O staining, scale bar =50 μm), (I) cellular FFA content, (J) Srebf1, Acaca and Fasn mRNA and (K) protein including Cd36 expression. Data were represented as mean±SEM. *P<0.05, **P<0.01, ***P<0.001

NAT10-mediated ac4C Srebf1 mRNA modification promotes AML12 lipogenesis

To further explore the molecular mechanism of NAT10 regulated lipogenesis, some important genes who participate in hepatocyte lipogenesis, were evaluated by PACES website prediction. Srebf1 mRNA was predicted as a target gene ac4C modified by NAT10 (score: 0.3281) (Fig. 3A). The prediction site was found located at 1939 to1953 of mRNA sequence, belonging to CDS. The motif sequence “CTGCTCCAGCGTCTC” is a characteristic ac4C motif “CXXCXXCXX” structure (Fig. 3B). To verify whether Srebf1 mRNA was ac4C modified by NAT10, after silencing NAT10, acRIP-PCR was used to detect the Srebf1 mRNA ac4C modification level. The results proved that knockdown of NAT10 downregulated Srebf1 mRNA ac4C modification level (Fig. 3C). Moreover, NAT10-RIP-PCR also confirmed that NAT10 was able to bind to Srebf1 mRNA and silencing of NAT10 decreased Srebf1 mRNA (Fig. 3D). Besides, the RNA decay assay exhibited that silencing of NAT10 significantly decreased Srebf1 mRNA half-life time (Fig. 3E). Next, to explore whether NAT10 can directly bind to Srebf1 mRNA ac4C modification site, dual-luciferase reporter assay elucidated that mutated Srebf1 mRNA decrease the combination with NAT10, which strongly supported the fact that NAT10 can bind to the motif of Srebf1 mRNA (Fig. 3F). Furthermore, overexpression or inhibition of NAT10 increased or decreased with or without PA-induced the precursor and mature SREBP-1 protein expression (Fig. 3G). The results illustrated that NAT10 induced lipogenesis in AML12 cells via ac4C modification of Srebf1 mRNA. In addition, mice CCAAT/enhancer binding protein beta (Cebpb) (score: 0.4322), Fasn (score: 0.5839) and human SREBF1 (score: 0.2318) and FASN (score: 0.6972) were predicted as possibly ac4C modified, using PACES software. However, despite high prediction score, acRIP-PCR verified that NAT10 did not affect their mRNA ac4C modification (acRIP-PCR of human SREBF1 and FASN were performed in HepG2 cells) (Fig. 3H-K). In addition, the rescue experiment showed overexpression of Srebf1 was able to partially increase FASN and Cd36 protein expression after AML12 was knockdown of NAT10 (Fig. 3L).

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Fig. 3

NAT10 ac4C modified Srebf1 mRNA in AML12 cells. (A) The NAT10 ac4C modification motif and (B) schematic diagram of Srebf1 mRNA was predicted using PACES software. (C) NAT10-mediated Srebf1 mRNA ac4C modification was verified using acRIP-PCR. (D) NAT10 bound with Srebf1 mRNA and inhibition of NAT10 decreased Srebf1 mRNA were detected by NAT10-RIP-PCR. (E) The Srebf1 mRNA stability was detected with RNA decay assay. (F) NAT10 directly bound to Srebf1 mRNA ac4C modification motif through dual-luciferase reporter assay. (G) SREBP-1 expression was verified with or without 150µmol/L PA treatment. (H) acRIP-PCR verified that mice Cebpb, (I) mice Fasn and (J) human SREBF1 and (K) FASN (HepG2 cells) did not be ac4C modified by NAT10. (L) The rescue experiment showed that FASN and Cd36 protein expression after AML12 was transfected with siRNA-NAT10 and OE-Srebf1. Data were represented as mean±SEM. **P<0.01, ***P<0.001

NAT10 ac4C modified Scap mRNA

In addition, another critical hepatocyte lipogenesis factor, mouse Scap, was also predicted as a target gene, ac4C modified by NAT10 (score: 0.2613) (Fig. 4A). The motif sequence, CGACGACGCCCCCGA, is located from 2807 to 2821 of Scap mRNA, also positioned at CDS (Fig. 4B). acRIP-PCR revealed that silencing of NAT10 inhibited Scap mRNA ac4C modification (Fig. 4C). NAT10 also was verified that it can bind to Scap mRNA and silencing of NAT10 decrease Scap mRNA via NAT10-RIP-PCR (Fig. 4D). In addition, inhibition of NAT10 attenuated Scap mRNA stability and mRNA and protein level expression (Fig. 4E-G). Furthermore, NAT10 was validated to directly bind to Scap mRNA ac4C modification through dual-luciferase reporter assay (Fig. 4H). The rescue experiment also showed that upregulated Scap was able to partially improve FASN and Cd36 protein expression after NAT10 knockdown in AML12 cells (Fig. 4I). The results indicated that NAT10 regulated Scap mRNA ac4C modification in AML12 cells.

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Fig. 4

NAT10 regulated Scap mRNA via ac4C modification. (A) The Scap mRNA ac4C modification site was predicted using PACES software. (B) Location of Scap mRNA ac4C motif. (C) acRIP-PCR was used to test that NAT10 ac4C modified Scap mRNA. (D) NAT10-RIP-PCR verified NAT10 protein bound with Scap mRNA and knockdown of NAT10 decreased Scap mRNA. (E) RNA decay assay detected Scap mRNA stability after silencing of NAT10. (F) Scap mRNA and (G) SCAP protein expression was verified with or without 150µmol/L PA treatment. (H) Dual-luciferase reporter assay validated NAT10 regulated Scap mRNA via its ac4C modification motif site. (I) The rescue experiment illustrated that overexpression of Scap was able to partial increase FASN and Cd36 protein expression after AML12 was knockdown by NAT10. Data were represented as mean±SEM.*P<0.05, **P<0.01, ***P<0.001

NAT10 promoted liver lipogenesis via Srebf1 and Scap mRNA ac4C modification in vivo

Liver-specific knockout of NAT10 has been shown to reduce liver steatosis in male offspring mice after maternal high-fat diet [30]. However, there is no direct evidence that NAT10 participate in liver lipogenesis via mRNA ac4C modification. To explore whether NAT10 regulate liver lipogenesis in vivo, hepatocyte-targeted AAV-shRNA-NAT10 (n=5) and AAV-shRNA-NC (n=5) were injected through tail vein of mice at 0 and 4th week, while simultaneously fed a high-fat diet for 12 weeks (Fig. 5A). The results showed that although body weight was not significantly different in two groups, liver size was smaller and lighter in weight in AAV-shRNA-NAT10 than AAV-shRNA-NC group (Fig. 5B-D). In addition, the pathological staining like H&E and Oil red O staining showed that silencing of NAT10 inhibited liver lipid droplet formation (Fig. 5E-F). Moreover, TG content of liver, serum ALT, AST, TC and TG concentration were decreased in AAV-shRNA-NAT10 (Fig. 5G-K). Besides, GTT results showed that blood glucose level in AAV-shRNA-NC was similar to AAV-shRNA-NAT10 group except for at 30 min, however, there was no difference in AUC of GTT, ITT and AUC of ITT. These results indicated normal glucose tolerance and insulin sensitivity (Fig. 5L-O). In molecular level, acRIP-PCR results revealed that silencing of NAT10 decreased Srebf1 and Scap mRNA ac4C modification (Fig. 5P, Q). In addition, knockdown of NAT10 reduced Srebf1, Scap, Acaca and Fasn mRNA and protein expression in liver (Fig. 5R, S). The results suggested that NAT10 inhibited liver lipogenesis through the downregulation of Srebf1 and Scap mRNA ac4C modification.

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Fig. 5

Silencing of NAT10 ac4C modified Srebf1 and Scap mRNA to inhibit mice liver lipogenesis. (A) The protocol of AAV-shRNA-NAT10 injection in vivo (the cartoon image was provided by www.figdraw.com). (B) The mice and liver size after mice were injected with AAV-shRNA-NAT10 and AAV-shRNA-NC and fed with high-fat diet for 12 weeks. (C) The body weight and (D) liver weight in two groups. (E) H&E (scale bar =50 μm) and (F) Oil red O staining (scale bar =25 μm) in liver. (G) The liver TG, (H) serum ALT, (I) AST, (J) TC, (K) TG, (L) GTT, (M) GTT-AUC, (N) ITT and (O) ITT-AUC in two groups. (P) Knockdown of NAT10 inhibited Srebf1 and (Q) Scap mRNA ac4C modification in liver tissue using acRIP-PCR. (R) Knockdown of NAT10 blocked Srebf1, Scap, Acaca and Fasn mRNA and (S) protein expression in liver. *P<0.05, **P<0.01, ***P<0.001

Not applicable.