Cell culture and NASH cell model construction

MEM basal medium (10-010-CVR, Corning) supplemented with 10% FBS (10099–141, GIBCO) and 1% P/S was utilized for culturing the human hepatocellular carcinoma cell line HepG2. The cells were incubated at 37 °C with 5% CO2. To construct the NASH cell model, HepG2 cells were exposed to a mixture of FFA comprising oleic acid and palmitic acid in a ratio of 2:1, at a concentration of 1 mmol/L, for a duration of 24 h. The steatosis HepG2 cells were subjected to treatment with varying concentrations of TEC: low concentration (25 μM), medium concentration (50 μM), and high concentration (75 μM) [10].

CCK-8 assay

HepG2 cells from each group were quantified by CCK-8 kit (Beyotime) to detect cell viability. To obtain a single-cell suspension, HepG2 cells were dissociated using Trypsin–EDTA (11668–500, Invitrogen). The cells were adjusted to a concentration of 3 × 104 cells/mL for each group. Each well of a 96-well plate was seeded with 100 μL of the cell suspension, resulting in 3000 cells/well. Each sample had 6 replicate wells, and the edge wells were filled with 100 μL of sterile water or PBS. The plate was incubated at 37 °C with 5% CO2 overnight. After the addition of 10 μL of CCK-8 to each well, the plate was incubated for 2 h. The OD value at 450 nm was quantified using the microplate reader (Infinite M1000, TECAN).

Detection of lipid oxidation

The lipid peroxidation levels in liver tissues and cells were quantitatively measured utilizing the Lipid Peroxidation MDA Assay Kit (Beyotime, S0131S). MDA (Malondialdehyde) is a natural byproduct of lipid peroxidation in living organisms. When cells from animals or plants undergo oxidative stress, lipid peroxidation occurs. Some fatty acids, after being oxidized, gradually break down into a complex series of compounds, including MDA [32]. Therefore, the level of lipid oxidation can be detected by measuring the level of MDA. Tissue and cells were lysed using cell lysis buffer (P0013). The ratio of tissue weight to lysis buffer was 10%, and for every 1 million cells, 0.1 mL of lysis buffer was utilized. The lysates were subjected to centrifugation at 10,000–12,000 g for 10 min. An appropriate amount of TBA was weighed, and TBA stock solution with a concentration of 0.37% was prepared. MDA can react with TBA (Thiobarbituric Acid) at higher temperatures and in acidic environments to form a red MDA-TBA adduct. To prepare the MDA detection working solution, 1500 μL of TBA dilution solution, 500 μL of TBA stock solution, and 30 μL of antioxidant were mixed. The solution was heated at 70 °C and vigorously vortexed to promote dissolution. Three centrifuge tubes were prepared: one with 0.1 mL of PBS, one with 0.1 mL of different concentrations of standard solutions for the standard curve, and one with 0.1 mL of the sample for measurement. Then, 0.2 mL of MDA detection working solution was added to each tube. The tubes were heated at 100 °C or in a boiling water bath for 15 min. The tubes were centrifuged at 1000 g for 10 min at room temperature. The absorbance was measured at 532 nm using the microplate reader.

Detection of Fe2+ levels

The levels of Fe2+ were quantitatively measured using the Intracellular Iron Colorimetric Assay Kit (Applygen, E1042). To prepare a mixture called Solution A, the lysis buffer was combined with a 4.5% potassium permanganate solution in a 1:1 ratio. Solution A was incubated with the test samples at 60 °C for one hour. Upon reaching room temperature, centrifugation was carried out, followed by the addition of 30 μL of the Fe2+ detection reagent. The absorbance of the samples was quantified within the range of 540–580 nm.

Detection of reactive oxygen species (ROS) levels

ROS levels in HepG2 cells were measured using the Reactive Oxygen Species Assay Kit (Solarbio, CA1410). The fluorescent probe DCFH-DA was diluted 1:1000 in serum-free culture medium to a final concentration of 10 μmol/L. An appropriate volume of diluted DCFH-DA was added to fully cover the cells. The cells were incubated at 37 °C for 30 min. Cells were washed three times and then observed and imaged using a fluorescence microscope.

Western blot (WB)

The HepG2 cells were centrifuged at 800 g for five minutes at 4 °C. Five times the volume of RIPA lysis buffer (Thermo) was added to the cells. The mixture was placed on ice for 10 min and vortexed for 30 s every five minutes. The lysate was centrifuged at 12,000 g for 10 min at 4 °C. SDS-PAGE gel electrophoresis was performed, and subsequently, the proteins were transferred onto a PVDF membrane. The PVDF membrane was then blocked with TBST solution containing 5% skim milk for three hours. Primary antibodies against GPX4 (Abcam, ab125066, 1:5000 dilution), ACSL4 (Abcam, ab155282, 1:50000 dilution), GAPDH (Proteintech, 60004-1-Ig, 1:10000 dilution), HDAC1 (Proteintech, 10197-1-AP, 1:10000 dilution), H3K18la (Jingjie PTM Biolab, PTM-1406RM, 1:1000 dilution), H3K18ac (Proteintech, 39129, 1:5000 dilution), Histone H3 (Abcam, ab8896, 1:15000 dilution), ATF3(Abcam, ab254268, 1:1000 dilution), ATF4(Abcam, ab270980, 1:1000 dilution), CHAC1(Proteintech, 15207-1-AP, 1:1000 dilution) were added and incubated at 4 °C overnight. The secondary antibodies Goat Anti-Rabbit IgG H&L(HRP) (Abcam, ab6721, 1:20,000 dilution), Goat Anti-Rabbit IgG H&L(HRP) (Beyotime, A0208, 1:1000 dilution), or Goat Anti-Mouse IgG H&L(HRP) (Beyotime, A0216, 1:1000 dilution) were added and incubated for two hours. An ECL substrate (Thermo) was utilized for chemiluminescent detection, and the images were acquired with a chemiluminescence imaging system.

Oil red O staining

Lipid content in HepG2 cells was examined using Oil Red O staining. Fresh staining solution, Oil Red O Staining Solution (SANGON BIOTECH, E607319), was prepared by mixing ddH2O and the staining solution at a ratio of 6:4. The cells were fixed with 4% Paraformaldehyde Fix Solution (SANGON BIOTECH, E672002) at room temperature for 30 min. After two washes, the cells were treated with a freshly prepared staining solution. Then the cells were stained at room temperature for 60 min. After three washes, the stained cells were observed and photographed with a microscope.

Construction of NASH mouse model

Male C57BL/6 mice (8 weeks old) were purchased from SPF Biotech (BEIJING). There were two animal experiments conducted. In the first experiment, a random allocation was employed to divide the mice into five groups (n = 5). NC group: Mice were fed a standard diet for 18 weeks. NASH group: Mice were fed a composite high-fat diet (88.3% regular diet + 10% lard + 1.5% cholesterol + 0.2% sodium cholate) for 18 weeks. NASH + TEC group: After 10 weeks on the high-fat diet, mice were orally gavaged with three different concentrations of TEC (25 uM, 50 uM, 75 uM and 1.5 mL/100 g) [10] once daily for eight consecutive weeks. The second animal experiment consisted of four groups (n = 5). NC group and NASH group: These two groups were treated the same as before. NASH + TEC group: The concentration of TEC was 75 μM. NASH + TEC + tRF-31R9J antagomir/NC group: NASH mice were orally gavaged with TEC and subsequently injected with tRF-31R9J antagomir (5 μg/mouse in 1.5 mL saline) via the tail vein twice a week for 8 weeks. Upon completion of the experiment, the animals were humanely euthanized by administering an overdose of CO2, and confirmation of death was obtained through cervical dislocation. Liver tissues were immediately subjected to pathological examination and biochemical analysis.

This study was approved and supervised by the Animal Care and Ethics Committee of Guizhou Medical University Affiliated Hospital. All animal handling procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

RNA extraction from steatosis HepG2 was performed utilizing TRIzol™ (Invitrogen). A reverse transcription kit (Thermo, K1622) was utilized to perform the reverse transcription of RNA. The program was configured with the following parameters: 42 °C for 6 min, followed by 70 °C for 5 min. The obtained cDNA was further subjected to real-time PCR amplification using the 2 × Master Mix kit (Roche) in a real-time PCR machine (Applied Biosystems Inc, ABI Q6). U6 was employed as an endogenous control for normalization. The primers utilized in this study were obtained from SANGON BIOTECH and the primer sequences are provided in Table S1. The 2−ΔΔCT method was employed to calculate the relative expression of the genes.

Cell transfection

Cell spreading was performed in 6-well plates with 5 × 105 HepG2 cells per well. The cells were cultured in antibiotic-free medium (RC-003, STEMERY). Lipofectamine™ 2000 transfection reagent (Invitrogen), tsRNA-19724 mimics, tsRNA-19724 inhibitor, NC mimics, and NC inhibitor were diluted in OPTI-MEM (CORNING) and incubated for five minutes. The diluted RNA and transfection reagent were incubated at room temperature for 15 min before being added to the cells. The medium was replaced with antibiotic-free medium after six hours for subsequent experiments. The sequences of transfection fragments are provided in Table S2.

H&E staining

Liver tissues were fixed in 4% paraformaldehyde to prepare pathological sections. Each sample was embedded in paraffin and cut into sections that were 4 μm thick. The sections were immersed in hematoxylin staining solution for 10 min, followed by eosin staining for three minutes. Subsequently, dehydration and mounting were performed. The sections were observed using the microscope to evaluate lipid degeneration.

Immunofluorescence (IF)

For cellular immunofluorescence, the HepG2 cells were seeded in a 12-well plate with 5 × 10^4 cells per well. HepG2 cells were transfected with tRF-31R9J mimics and HDAC1 interfering fragments utilizing lipo2000 (Invitrogen). After six hours, each group was treated with FFA. To fix the cells, 300 μL of 4% paraformaldehyde was added and incubated for 30 min. Then, 300 μL of 0.5% Triton X-100 was added for permeabilization for 15 min. Subsequently, 300 μL of 3% BSA was added for blocking for 30 min. For each well, 500 μL of primary antibody against H3K18la (Jingjie PTM Biolab, PTM-1406RM, 1:100 dilution) and H3K18ac (Proteintech, 39129, 1:500 dilution) was added and incubated overnight at 4 °C. Then, 250 μL of secondary antibody Alexa Fluor 488-labeled Goat Anti-Rabbit IgG (H + L) (Abcam, ab150077, 1:250 dilution) was added and incubated in the dark for one hour. Subsequently, 300 μL of DAPI was added for dark staining for 10 min. The images were observed using a fluorescence microscope.

For tissue immunofluorescence, liver tissue sections were fixed with 4% paraformaldehyde. The sections were incubated with primary antibodies against ACSL4 (Abcam, ab155282, 1:200 dilution) and GPX4 (Abcam, ab125066, 1:150 dilution) at 37 °C for one hour. The sections were incubated with the secondary antibody, Alexa Fluor 488-labeled Goat Anti-Rabbit IgG (H + L) (Abcam, ab150077, diluted 1:250). The fluorescence signals were visualized using the fluorescence microscope.

Detection of triglyceride (TG) content

The TG Content Assay Kit (Enzyme-linked Biotechnology, ml076637) was employed for the detection of TG content in liver tissue. Approximately 0.1 g of tissue was weighed and combined with 1 mL of reagent 1. After homogenizing on ice, the mixture was centrifuged at 8000 g, 4 °C for 10 min. Reagent 2 and reagent 3 were added to the supernatant. The mixture was incubated for 20 min, and the absorbance was measured at 505 nm wavelength with a plate reader (ThermoFisher, FC).

Total cholesterol (TC) content detection

The measurement of TC content in liver tissue was performed using the TC Content Assay Kit (Solarbio, BC1980). The working solution was prepared by mixing reagent 1, reagent 2, and reagent 3 in a ratio of 3 mL:20 μL:3 μL, respectively. Approximately 0.1 g of tissue was weighed and added to 1 mL of extraction solution, followed by homogenization on ice. The mixture was then centrifuged at 10,000 g, 4 °C for 10 min. The working solution was added to the supernatant, and left to incubate for 15 min. The absorbance was measured at 500 nm wavelength with the plate reader (ThermoFisher, FC).

RNA pull-down

HePG2 cells were lysed using 100 μL of cell lysis buffer to obtain a protein concentration of 3 μg/μL. After centrifugation, the supernatant was collected, and 50 μL of streptavidin-coated magnetic beads were added. Then, 50 pmol of labeled negative control probe/target probe was added separately, and the mixture was incubated for 15–30 min. A total of 100 μL of 1 × Protein-RNA Binding Buffer was added to the magnetic beads and mixed. The RNA–protein binding system mixture was prepared and added to the magnetic beads. The mixture was incubated at 4 °C for 30–60 min. The 1 × wash buffer was added for washing and eluting the RNA-bound protein complex. Then, 20 μL of the pull-down complex obtained from the experiment was loaded onto a 10% SDS-PAGE gel. After electrophoresis, silver staining was carried out according to the instructions of the silver staining kit (Beyotime, P0017S).

IF/Fluorescence in situ hybridization (FISH)

Co-localization of HDAC1 and tRF-31R9J was performed using the IF/FISH dual staining method. According to the previously described method, HDAC1 localization was performed using a primary antibody against HDAC1 (Proteintech, 10197-1-AP) and the secondary antibody Cy3-labeled Goat Anti-Rabbit IgG (H + L) (Beyotime, A0516, 1:300 dilution). The tRF-31R9J was localized using the FISH kit (Bersinbio). HepG2 cells were fixed with 4% paraformaldehyde. Subsequently, the cells were hybridized with a specific probe for tRF-31R9J and counterstained with DAPI. Photographs of the samples were taken using the fluorescence microscope.

RNA immunoprecipitation (RIP)

The HDAC1-knockdown HepG2 cells were lysed and the lysates were aspirated into nuclease-free centrifuge tubes. To a new tube, 50 μL of magnetic beads and either 5 μg of HDAC1 antibody or 1 μg of IgG antibody were added and incubated at room temperature with rotation. The cell lysates were then centrifuged at 14,000 rpm for 10 min at 4 °C. A total of 100 μL of the supernatant was added to the magnetic bead-antibody tube and incubated overnight at 4 °C with rotation. RNA was extracted using the Trizol method and purified overnight using column chromatography.

Transcriptome sequencing

The steatosis hepatocarcinoma cells treated with TEC were divided into tRF-31R9J knockdown group and control group for transcriptome sequencing. TRIzol™ reagent (Invitrogen) was employed for the extraction of total RNA. mRNA was enriched and cDNA libraries were constructed. The libraries were then sequenced using Illumina novaseq6000. The raw data underwent quality control procedures to acquire clean reads. The clean reads were mapped to the reference genome sequence. RPKM (Reads Per Kilo Million) was used for normalization. Differential gene expression analysis was performed using the DESeq algorithm. In the screening process, genes with Log2FC greater than 1 or less than −1, and FDR less than 0.05 were considered as differentially expressed genes (DEGs).

The selected DEGs were annotated with pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg) to obtain all the pathways in which the DEGs are involved.

Chromatin immunoprecipitation sequencing (ChIP-seq) data processing

The ChIP-seq data of H3K18la from the GSE208727 dataset in the GEO database were analyzed. The filtered clean reads were mapped to the reference genome of the corresponding species using the bowtie2 software. The alignment results were visualized. The macs2 software was used for peak calling and peak annotation analysis to identify regions of histone lactylation and the distribution of peaks on gene elements. The MAnorm2 software was used for differential analysis and annotation of histone modifications. In the screening process, peaks with a M.value greater than 1 or less than −1, and p-value less than 0.05 were considered as differentially modified peaks.

ChIP-qPCR

Approximately 5 × 106 HepG2 cells were fixed in 1% formaldehyde. The cells were sonicated to fragment the DNA into 300 bp fragments. H3K18la antibody (PTMBIO, PTM-1406RM) and H3K18ac antibody (Proteintech, 39,129) were incubated with pre-treated Protein A/G magnetic beads, respectively. IgG was employed as a negative control. ChIP DNA was extracted and qPCR analysis was performed to detect the expression of ATF3, ATF4, and CHAC1. The primer sequences are provided in Table S1. All qPCR reactions were performed on a real-time fluorescence quantitative PCR instrument (ABI Q6, Applied Biosystems Inc., USA).

Statistical analysis

All experiments were conducted with a minimum of three biological replicates. The results are presented as mean ± standard deviation (SD). Statistical analysis was performed utilizing GraphPad Prism 8. For comparisons between two groups, a t test was employed; while, one-way analysis of variance (ANOVA) followed by Tukey's test was used for comparisons among multiple groups. A p value less than 0.05 was considered statistically significant.