Clinical sample collection

Liver tissue samples were obtained from patients who underwent Liver resection surgery for benign Liver diseases, such as liver hemangioma, at the Second Hospital of Shandong University. The samples consisted of 12 cases of MASH Liver samples and 12 cases of normal liver samples. These samples were collected with prior informed consent from the patients and approved by the Ethics Committee of the Second Hospital of Shandong University (KYLL-2023-413). For each patient, a surgical resection was performed, and the liver tissue samples were immediately collected and stored in liquid nitrogen until further analysis. All MASH samples were histologically confirmed with a Steatosis, Activity, Fibrosis (SAF) score ≥ 4, defined as steatosis ≥ 1, activity ≥ 2, and fibrosis ≥ 1. The European Association for the Study of the Liver (EASL), the European Association for the Study of Diabetes (EASD), and the European Association for the Study of Obesity (EASO) jointly issued clinical practice guidelines for NAFLD, recommending the use of the Steatosis, Activity, Fibrosis (SAF) scoring system for grading the severity of NASH. The SAF scoring system comprises: (1) Steatosis (S 0–3); (2) Activity (A 0–4), calculated as the sum of lobular inflammation (0–2) and ballooning degeneration (0–2); (3) Fibrosis (F 0–4), resulting in a total score ranging from 0 to 11.Within the SAF scoring system, the concurrent histological presence of steatosis, lobular inflammation, and ballooning degeneration is sufficient for a diagnosis of NASH. The SAF scoring system is primarily used for clinical diagnosis and evaluation. All MASH samples were diagnosed by experienced pathologists based on histopathological examination.

Transcriptome sequencing

Transcriptome sequencing was performed on 3 MASH Liver samples and 3 normal liver samples. The samples were processed for RNA extraction using a commercial kit following the manufacturer’s instructions (Tiangen, Beijing, China). The RNA samples were then subjected to library preparation using the Illumina TruSeq Stranded Total RNA Library Prep Kit. The raw sequencing data were preprocessed to remove low-quality reads, adapter sequences, and contaminated reads. The clean reads were then aligned to the human reference genome using a splice-aware aligner. The quantification of gene expression levels and identification of novel transcripts were performed using established bioinformatics tools. Differential gene expression analysis was conducted using a suitable statistical framework. Genes with a log fold change (logFC) > 2 and a P-value < 0.05 were considered significantly differentially expressed. Based on these criteria, a total of 746 differentially expressed lncRNAs were identified between the MASH liver samples and normal liver samples.

RT-qPCR detection

Total RNA was extracted from the liver tissue samples or cells using an RNA extraction kit (Qiagen, Germany) according to the manufacturer’s instructions. The quantity and quality of the isolated RNA were assessed using a NanoDrop spectrophotometer and Agilent Bioanalyzer. For cDNA synthesis, 1 µg of total RNA was reverse transcribed into cDNA using a reverse transcription kit (Vazyme, China). RT-qPCR was performed using a SYBR Green master mix and specific primers on a real-time PCR system. The primers used for RT-qPCR were designed based on the sequences of the target mRNAs and Linc01271. The sequences of the primers used in this study are provided in Supplementary Table S1. The relative expression levels of the target lncRNAs and mRNAs were calculated using the 2^-ΔΔCt method, with normalization to an internal control gene such as β-actin.

Cell culture

The THLE-2 cell line was used in this study and was cultured according to standard protocols. THLE-2 was purchased from American Type Culture Collection (Manassas, VA, USA). The cells were maintained in Dulbecco’s Modification of Eagle’s Medium (DMEM, Gibco, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified atmosphere with 5% CO2 at 37 °C. The culture medium was refreshed every 2–3 days, and the cells were passaged upon reaching 70–80% confluence using trypsin-EDTA. The cells were routinely checked for mycoplasma contamination to ensure the quality of the cell culture.

Primary hepatic stellate cells (HSCs) were isolated from human liver samples and cultured on uncoated plastic dishes in RPMI-1640 medium supplemented with 8% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 2 mM L-glutamine at 37 °C under 5% CO₂. To prevent spontaneous activation, cells were maintained in low-serum conditions (≤ 8% FBS) and passaged no more than twice within 7 days; experiments were conducted within 72 h post-isolation. Cell identity was confirmed by vitamin A autofluorescence (365 nm excitation) and quiescent status was validated by < 10% α-SMA positivity via immunofluorescence.

Primary human Kupffer cells were isolated from human liver samples using a modified collagenase digestion protocol. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin-streptomycin, and 2 mM L-glutamine at 37 °C under 5% CO₂ for 4 h, followed by medium change to remove non-adherent cells (primarily endothelial cells).To maintain functional quiescence, KCs were cultured in ≤ 10% FBS and used within 48 h post-isolation without passaging.

Construction of cell lipotoxicity model

The cell lipotoxicity model was established using THLE-2 cells by treating them with a mixture of palmitic acid (PA) and oleic acid (OA). PA and OA were prepared as 100 mM stock solutions. The PA stock solution was mixed with 100 mM oleic acid (OA) in a volume of PA/OA = 1:2. This mixture was then conjugated to fatty acid-free bovine serum albumin (BSA) to create a PA/OA-BSA complex. THLE-2 cells were seeded in culture plates or dishes and allowed to adhere overnight. Subsequently, the culture medium was replaced with serum-free medium containing the PA/OA-BSA complex. The cells were then incubated for 6–12 h to induce lipotoxicity.

Construction of MASLD and MASH models in C57BL/C mice

C57BL/C mice were randomly divided into experimental groups and housed under standard laboratory conditions. To establish the MASLD model, mice were fed a high-fat diet (HFD) or a high-fat, high-cholesterol (HFHC) for 12 weeks and MASH model for 24 weeks. HFHC (52% kcal fat and 1.25% kcal cholesterol) and HFD (60% kcal fat) were purchased from Dyets Biotechnology Co., Ltd. At the end of the respective feeding periods, mice were humanely euthanized to minimize suffering. Euthanasia was performed using carbon dioxide (CO2) inhalation, followed by cervical dislocation to ensure death. These methods are widely accepted for providing a rapid and painless procedure, in accordance with guidelines from the American Veterinary Medical Association (AVMA) for euthanasia. The livers were promptly harvested for further analysis. Oil Red O staining was performed on the liver sections to assess hepatic lipid accumulation. Immunohistochemistry staining was performed on the liver sections to examine specific proteins of interest.

Fluorescence in situ hybridization (FISH) assay

THLE-2 cells were treated with 4% paraformaldehyde for 20 min and rinsed with PBS. Probes specific to the Linc01271 RNA sequence were designed using bioinformatics tools and synthesized by Tsingke (Tsingke, Beijing, China). The probes were labeled with FITC, for visualization under a fluorescence microscope. The fixed cells were permeabilized using 0.5% Triton-X. Following permeabilization, cells were incubated with the labeled Linc01271 probes in a hybridization buffer containing appropriate blocking reagents. After FISH hybridization, cells were washed and counterstained with DAPI. The stained cells were then imaged using a fluorescence microscope.

ShRNA transfection

Cells were cultured and plated, and shRNA sequences specifically targeting Linc01271 (shLinc01271) were designed and selected. A non-targeting shRNA (shNC) sequence served as the negative control. Transfection complexes were prepared using LipofectamineTM 3000 (Invitrogen) and added to the cells as described before [36]. After 24 h, the efficiency of gene knockdown was validated using RT-qPCR.

Oil red O staining

Cells or tissue samples were prepared for Oil Red O staining according to standard protocols [37]. For cell samples, cells were cultured and plated onto six-well plate. For tissue samples, tissue sections were obtained through paraffin embedding. Cell or tissue sections were fixed using 4% paraformaldehyde and permeabilization. The fixed and permeabilized cell or tissue samples were incubated with an Oil Red O working solution. The working solution was prepared by diluting Oil Red O stock solution with distilled water, according to the manufacturer’s instructions. The samples were washed and counterstained using DAPI.

Quantification of triglyceride and total cholesterol contents

Intracellular total triglyceride (TG) and total cholesterol (TC) contents were quantified with a triglyceride assay kit (ab65336; Abcam, Cambridge, MA, USA) and a total cholesterol assay kit (ab65359; Abcam) according to the manufacturer’s instructions. Lipids in the Liver were extracted and measured. Briefly, approximately 100 mg Liver was homogenized with 3 ml T-PER (Pierce, Rockford, IL). 100 µl tissue extracts were added to 1.6 ml CHCl3: MeOH (2:1). 200 µl 0.6% NaCl was added and the solutions were centrifuged. The organic layer was removed and dried, and the resulting pellet was dissolved in 100 µl phosphate-buffered saline. Triglyceride and cholesterol concentrations were then measured by using assay kits.

Western blot

After different treatments, cells were lysed in RIPA buffer (Beyotime, Haimen, China) supplemented with protease inhibitors and phosphatase inhibitors. The concentration of protein extracts was determined using the BCA method. The protein (20 µg) was then separated on 12% SDS-polyacrylamide gels, and the blots were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA). After blocking with 5% skimmed milk, the membranes were then incubated overnight with primary antibodies at 4 °C and for 1 h with secondary antibodies at room temperature. Information about the antibodies used is provided in Table S2. After rinsing, the blots were visualized using the ECL method (Thermo Scientific), and the protein signals were analyzed using the ImageJ software.

Dual-Luciferase reporter assay

To investigate the interaction between Linc01271, RAB35 mRNA, and the candidate miRNAs, a Dual-luciferase reporter assay was employed. The 3’ untranslated regions (3’ UTRs) of Linc01271 and RAB35 mRNA, containing the predicted miRNA binding sites, were cloned into the pmirGLO luciferase reporter vector (Promega, USA) downstream of the luciferase gene (Linc01224-luci and RAB35-luci). For the assay, THLE-2 cells were co-transfected with either Linc01271-luci or RAB35-luci and the candidate miRNAs using Lipofectamine 3000 (Invitrogen, USA). A negative control group (Ctrl) was also included in the experiment to serve as a baseline for comparison. 48 h post-transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, USA). Renilla luciferase activity was used as an internal control to normalize the firefly luciferase activity.

Adeno-associated virus

Adeno-associated virus (AAV) was used to achieve liver-specific overexpression of linc001271 in mice. For liver tropism, AAV8 serotype was selected, and virus particles were packaged in HEK293T cells using standard triple-plasmid transfection. Viral particles were purified via iodixanol gradient ultracentrifugation and titrated as viral genomes (qpcr). Male C57BL/6 mice were injected via the tail vein with AAV in 200 µL of phosphate-buffered saline (PBS). The AAV titer was 1 × 1013 viral genomes (vg)/mL, and the injection dosage was 2 × 1011 vg per mouse (200 µL per injection). A single tail-vein injection was administered 4 weeks prior to the initiation of HFHC diet feeding. Control mice were injected with an empty AAV vector.

Isolation of human Kupffer cells and hepatic stellate cells

Primary Kupffer cells and hepatic stellate cells were isolated from surgically resected human liver tissues (tumor-adjacent normal regions, cold ischemia time ≤ 30 min). Tissues underwent dual-step enzymatic perfusion via the hepatic vein: (1) Pre-perfusion with calcium-free HBSS containing 0.5 mM EGTA (37 °C, 4 ml/min, 10 min); (2) Digestion with 0.02% collagenase IV + 0.45% Pronase (Kupffer Cells isolation) or 0.05% collagenase IV alone (HSCs isolation) via recirculating perfusion (20–30 min). Digested tissue was dissociated mechanically, filtered through 200-µm mesh, and treated with 10 µg/ml DNase I. Non-parenchymal cells (NPCs) were enriched by differential centrifugation (50 ×g for 3 min → discard hepatocytes; 150 ×g for 10 min → pellet NPCs). For Kupffer Cells: NPCs were purified using a discontinuous Percoll gradient (25% over 50%) with centrifugation (500 ×g, 15 min, 4 °C), collecting cells at the interface, followed by 2-h adherence in RPMI-1640/8% FBS. For HSCs: NPCs were resuspended in 11% Nycodenz (density 1.053 g/ml) and centrifuged (2,730 r/min, 15 min), collecting the intermediate layer. Kupffer Cells identity was confirmed by > 90% CD163⁺/CD68⁺ expression (flow cytometry) and phagocytosis of FITC-latex beads; HSCs were validated by > 95% vitamin A autofluorescence (365 nm excitation) and < 10% α-SMA⁺ immunofluorescence. Viability exceeded 90% (trypan blue exclusion).Hepatocytes were plated on collagen I-coated plates at 5 × 10⁴ cells/cm² in maintenance medium (William’s E Medium + 10% FBS, 1% penicillin/streptomycin, 1 µM dexamethasone, 4 µg/mL insulin, 2 mM L-glutamine). After 4-h attachment, serum-free medium was applied, and experiments commenced 24 h post-seeding.

Co-culture of THLE-2 cells and HSCs

To investigate the interaction between hepatocytes and hepatic stellate cells, an indirect co-culture system of THLE-2 and HSCs was established. Firstly, hepatic stellate cells were seeded into gelatin-precoated 24-well cell culture plates (lower chamber) at a density of 1 × 10⁴ to 5 × 10⁴ cells per well, and cultured in RPMI-1640 medium supplemented with 8% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 2 mM L-glutamine. After HSCs were fully adherent (usually 12–24 h later), Transwell inserts (pore size 0.4 μm, polycarbonate membrane) were placed into the wells. Subsequently, Vector or Linc01271-overexpressing THLE-2 cells were seeded into the upper chamber of the Transwell at a density of 1 × 10⁵ to 5 × 10⁵ cells per well, using FBS-free DMEM medium. The two types of cells were maintained in a co-culture state within the Transwell system, where communication occurred only through soluble factors, and physical contact was blocked by the semipermeable membrane. Co-culture was performed in a constant-temperature and humidity incubator at 37 °C with 5% CO₂ for 24 to 72 h, after which the cells were collected separately for subsequent analysis.

Statistical analysis

Statistical analysis was performed using SPSS version 23.0 software (IBM Corp., Armonk, NY, USA). Data were expressed as mean ± standard deviation (SD). For comparisons between two groups, the Student’s t-test was used. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was used. For the RT-qPCR data, the relative expression levels of the target genes were calculated using the 2−ΔΔCt method. A p-value < 0.05 was considered statistically significant. Statistically significant differences were indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.