NEAT1/miR-181a-5p/HMGB1 Axis Regulates Macrophage Polarization and Inflammation in Sepsis Models

Summary

This protocol integrates clinical samples, cultured macrophages, and a cecal ligation and puncture (CLP) mouse model to investigate how the NEAT1/miR-181a-5p/HMGB1 axis regulates macrophage polarization and inflammation in sepsis, providing step-by-step guidance that can be adapted to other lncRNA-mediated immune pathways.

Abstract

Sepsis is characterized by a dysregulated host immune response and remains a leading cause of mortality worldwide. Long non-coding RNA NEAT1 has been implicated in inflammatory diseases, but its specific role in macrophage polarization during sepsis has not been fully defined. Here, we systematically examine the NEAT1/miR-181a-5p/HMGB1 axis across clinical samples, cultured macrophages, and a CLP mouse model. Quantitative PCR, western blotting, dual-luciferase reporter assays, and RNA pull-down experiments are used to confirm the competitive endogenous RNA (ceRNA) interaction among NEAT1, miR-181a-5p, and HMGB1. Functional assays, including immunofluorescence, transwell migration, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, are applied to assess macrophage polarization, migration, and apoptosis. In vivo, the CLP model combined with ELISA and histopathology validates the impact of NEAT1 knockdown on cytokine profiles and organ injury. NEAT1 and HMGB1 are upregulated, whereas miR-181a-5p is downregulated, in patients with sepsis and in lipopolysaccharide-stimulated macrophages. Silencing NEAT1 promotes M2 macrophage polarization, reduces pro-inflammatory cytokines, impairs macrophage migration, and alleviates tissue damage in septic mice via the miR-181a-5p/HMGB1 axis. To our knowledge, this is the first integrated protocol to characterize the lncRNA-microRNA-HMGB1 regulatory circuit in sepsis using harmonized clinical, in vitro, and in vivo approaches. It provides a methodological framework for targeting NEAT1-related ceRNA networks as potential therapeutic strategies.

Introduction

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection¹. Despite advances in clinical management, its mortality remains high, and the molecular mechanisms underlying immune imbalance are not fully understood^2,3. Among immune regulators, long non-coding RNAs (lncRNAs) have emerged as important modulators of inflammatory signaling and macrophage function^4,5. NEAT1, a nuclear-enriched abundant transcript, is a structural component of paraspeckles and has been associated with immune and tissue-injury responses^6,7. Prior studies have linked NEAT1 to intestinal inflammation, ischemia/reperfusion injury, and acute respiratory distress syndrome^8,9,10.

In the context of sepsis, accumulating evidence indicates that lncRNAs regulate macrophage polarization and inflammatory signaling, thereby influencing disease progression and outcome^11,12. For example, lncRNAs KCNQ1OT1 and SNHG1 have been reported to modulate sepsis-induced myocardial injury through microRNA-mediated pathways^13,14. However, whether NEAT1 regulates macrophage polarization through the miR-181a-5p/HMGB1 axis in sepsis has not been systematically defined. To address this gap, we established a protocol that combines patient blood samples, lipopolysaccharide-stimulated RAW264.7 macrophages, and a cecal ligation and puncture mouse model to interrogate the NEAT1/miR-181a-5p/HMGB1 axis^15,16. Using quantitative PCR, western blotting, dual-luciferase reporter assays, RNA pull-down, and functional readouts of macrophage M1/M2 polarization, migration, and apoptosis, this protocol provides a reproducible experimental framework to define NEAT1 as a regulator of macrophage function in sepsis and to support the development of lncRNA-targeted strategies^17,18.

Protocol

All procedures involving human participants were approved by the Ethics Review Board of the Affiliated Hospital of Hebei University (Approval No. HDFY-LL-2021-232). Written informed consent was obtained from all participants or, when applicable, from their legal surrogates. All animal experiments were conducted in accordance with institutional guidelines and national regulations, and were approved by the same ethics committee. The approved protocol specified details of the cecal ligation and puncture (CLP) procedure, adenoviral administration, anesthetic regimen, and humane endpoints.

Patient sample collection

Patients with sepsis were prospectively recruited at the Affiliated Hospital of Hebei University between February 2022 and July 2024. A total of 50 patients met the inclusion criteria, including 26 males and 24 females, aged 24-69 years (mean 52.4 ± 10.2 years). Sepsis was diagnosed according to Sepsis-3 criteria, requiring suspected or confirmed infection plus an increase in Sequential Organ Failure Assessment (SOFA) score by ≥ 2 points. For each patient, the primary infection source (such as pulmonary, intra-abdominal, urinary tract, or bloodstream infection) and disease severity (sepsis or septic shock) were recorded, and the distribution of SOFA scores was summarized to characterize disease burden. As a control group, 39 healthy individuals without clinical evidence of infection or systemic inflammatory disease were enrolled (20 males and 19 females, aged 26-70 years, mean 50.7 ± 11.0 years), and there were no significant differences in age or sex between groups.

Peripheral venous blood was collected from patients with sepsis before initiation of sepsis-specific treatment whenever feasible. Blood (typically 5-10 mL) was drawn into ethylenediaminetetraacetic acid (EDTA)-coated tubes and gently inverted several times to mix the anticoagulant. Samples were processed within approximately 1 h of collection. Plasma was obtained by centrifugation at 1,500 × g for 10 min at room temperature, aliquoted into labeled microtubes, and stored at -80 °C for subsequent cytokine measurements. For RNA and protein extraction, peripheral blood mononuclear cells (PBMCs) were isolated from a portion of the same samples using a density-gradient separation medium according to the manufacturer's instructions. The cells were then washed twice in PBS, counted, and stored as cell pellets at -80 °C.

Cell culture and transfection

RAW264.7 murine macrophage cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator at 37 °C with 5% CO₂. Cells were passaged with a cell scraper before reaching over-confluence to preserve a stable phenotype.

For transfection, RAW264.7 cells were seeded at 5 × 10⁵ cells per well in 6-well plates in 2 mL of complete medium approximately 24 h before reagent exposure. When cultures reached 70-90% confluence, nucleic acids (such as NEAT1 or HMGB1 expression plasmids, short-hairpin constructs, miR-181a-5p mimics or inhibitors, and their corresponding controls) were complexed with a lipid-based transfection reagent in serum-free medium according to the manufacturer's instructions. Typically, 2-3 µg of plasmid DNA or 50-100 pmol of small RNA were mixed with the recommended volume of reagent, allowed to form complexes at room temperature for 10-15 min, and then added dropwise to cells in antibiotic-free medium. After 4-6 h, the medium was replaced with fresh complete medium, and cells were incubated for a further 24-48 h before downstream assays.

Induction of macrophage polarization

To induce inflammatory macrophage polarization, RAW264.7 cells were allowed to adhere in 6-well plates for at least 4 h in complete medium. The medium was then replaced with DMEM containing 100 ng/mL lipopolysaccharide (LPS), and cells were incubated for 6 h at 37 °C with 5% CO₂. After stimulation, cells were immediately processed for RNA and protein extraction, immunofluorescence staining, migration assays, or apoptosis analysis, depending on the experimental design.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from cultured cells, PBMCs, or mouse tissues using a phenol-guanidinium-based monophasic reagent according to standard procedures. In brief, samples were lysed in 1 mL of reagent per 10⁶ cells or 50-100 mg of tissue, incubated for 5 min at room temperature, and then mixed vigorously with chloroform (0.2 mL per 1 mL of reagent). After incubation for 2-3 min, lysates were centrifuged at 12,000 × g for 15 min at 4 °C to separate the phases. The aqueous phase was transferred to a new tube, and RNA was precipitated with an equal volume of isopropanol. The mixture was then incubated for approximately 10 min and centrifuged again at 12,000 × g for 10 min at 4 °C. The resulting pellet was washed with 75% ethanol, centrifuged at 7,500 × g for 5 min at 4 °C, briefly air-dried, and dissolved in RNase-free water. RNA concentration and purity were evaluated using UV spectrophotometry, and samples with an A₂₆₀/A₂₈₀ ratio of approximately 1.8-2.0 were used for downstream analyses. When necessary, integrity was verified by agarose gel electrophoresis.

Complementary DNA (cDNA) was synthesized from total RNA using a commercially available reverse transcription kit, following the manufacturer's instructions. For miR-181a-5p, gene-specific or stem-loop primers were employed as recommended. Quantitative real-time PCR was performed using a real-time PCR system and a suitable master mix (such as SYBR Green- or probe-based chemistry) in a total reaction volume of 20 µL containing cDNA template and 0.2-0.4 µM of each primer. Typical cycling conditions included an initial denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s, with melt-curve analysis when appropriate to confirm amplicon specificity. NEAT1 and HMGB1 expression levels were normalized to GAPDH, and miR-181a-5p expression was normalized to U6 small nuclear RNA. Relative expression was calculated using the 2-ΔΔCt method and expressed as fold change compared with control groups. Primer sequences and amplicon sizes were compiled in a separate spreadsheet for submission as a Supplementary Table 1.

Western blotting

For western blotting, cells were washed twice with ice-cold PBS and lysed in an appropriate radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors. Lysates were incubated on ice for 15-30 min with occasional gentle mixing and centrifuged at 12,000 × g for 15 min at 4 °C to remove cellular debris. The supernatants were collected, and protein concentrations were determined using a bicinchoninic acid (BCA) assay.

Equal amounts of protein (typically 40 µg per lane) were mixed with loading buffer, boiled at 95 °C for 5-10 min to denature proteins, and separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels of appropriate acrylamide concentration (for example, 10-12% for HMGB1). Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes using a wet or semi-dry transfer system under conditions recommended for the molecular weight range of interest. Membranes were blocked with 5% non-fat milk or bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies against HMGB1 (for example, 1:500 dilution) and GAPDH (for example, 1:1,000 dilution) diluted in blocking buffer. After three washes in TBST, membranes were incubated with suitable horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Following additional washes, protein bands were visualized using an enhanced chemiluminescence substrate and captured with a digital imaging system. Band intensities were quantified using image analysis software, HMGB1 signals were normalized to GAPDH, and relative expression was compared across groups.

Dual-Luciferase reporter assay

Wild-type and mutant fragments of NEAT1 and the HMGB1 3′-untranslated region (3′-UTR) containing the predicted miR-181a-5p binding sites were cloned into a luciferase reporter vector¹⁹. All constructs were verified by Sanger sequencing to confirm the insertion and mutation sites²⁰. RAW264.7 cells were seeded in 24-well plates and allowed to adhere overnight. Cells were then co-transfected with reporter plasmids and either miR-181a-5p inhibitors, mimics, or corresponding negative controls using a lipid-based transfection reagent, as described for the general transfection procedure.

After approximately 24-48 h of incubation, cells were washed with PBS, lysed in an appropriate passive lysis buffer (for example, 20 µL per well), and gently agitated to ensure complete lysis. Luciferase activity was measured using a dual-luciferase assay kit and a luminometer, following the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla luciferase activity to control for transfection efficiency, and the relative luciferase activity of each group was compared with the corresponding control.

Transwell migration assay

Macrophage migration was evaluated using transwell chambers pre-coated with an extracellular matrix gel suitable for cell invasion assays. The gel was diluted 1:6 in serum-free medium on ice, and 50 µL of the diluted solution was added to the upper surface of each insert. The plate was incubated at 37 °C for approximately 4 h to allow the gel to solidify. Transfected or treated RAW264.7 cells were harvested, resuspended in serum-free medium at 1 × 10⁶ cells/mL, and 100 µL of the cell suspension (1 × 10⁵ cells) was added to the upper chamber. The lower chamber was filled with 600 µL of complete medium containing 10% fetal bovine serum as a chemoattractant.

Cells were incubated at 37 °C with 5% CO₂ for 24 h. At the end of incubation, non-migrated cells remaining on the upper surface of the membrane were gently removed with a cotton swab. Migrated cells adherent to the lower membrane surface were fixed with 95% ethanol for about 20 min at room temperature and then stained with 0.5% crystal violet solution for 10-15 min. Excess dye was removed by washing with PBS, and membranes were air-dried. Migrated cells were examined under a light microscope, and cells in several randomly selected fields per insert were counted in a blinded manner to quantify migratory capacity.

TUNEL apoptosis assay

For apoptosis detection, RAW264.7 cells were cultured on glass coverslips and subjected to the indicated treatments or transfections. Cells were fixed with 4% paraformaldehyde for 30-60 min at room temperature and rinsed with PBS. Endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide for 20 min, followed by PBS washes. A TUNEL assay kit was used, and the labeling mixture was applied to cover the cells. Samples were incubated at 37 °C for approximately 60 min in a humidified chamber, and the reaction was stopped according to the kit instructions. After incubation with streptavidin-HRP or an equivalent detection reagent for 30 min at 37 °C, nuclei were visualized with a chromogenic substrate such as 3,3′-diaminobenzidine (DAB) until brown-stained apoptotic nuclei became evident. Slides were counterstained with hematoxylin, dehydrated through graded ethanol, cleared in xylene, and mounted with a permanent medium. Apoptotic cells and total nuclei were observed under a light microscope, and the apoptotic index was calculated as the percentage of TUNEL-positive cells.

Immunofluorescence

For immunofluorescence, paraffin-embedded tissue sections were dewaxed in xylene and rehydrated through a graded ethanol series to water. Antigen retrieval was performed using an appropriate buffer (such as citrate buffer, pH 6.0) and heat-induced epitope retrieval by microwave or pressure cooker, according to antibody recommendations. After cooling and rinsing, sections were blocked with 5% normal serum in PBS for 1 h at 37 °C to reduce nonspecific binding. Tissues were then incubated overnight at 4 °C with primary antibodies against markers of macrophage polarization and inflammatory cytokines, including CD206, TNF-α, IL-1β, IL-6, inducible nitric oxide synthase (iNOS), IL-4, IL-10, and Arg-1 (commonly at 1:500 dilution), diluted in blocking buffer.

The following day, sections were washed in PBS and incubated with appropriate fluorophore-conjugated secondary antibodies for 1 h at 37 °C in the dark. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. After final washes, sections were mounted with an anti-fade mounting medium and examined under a fluorescence microscope using suitable filter sets. Images were acquired with consistent exposure settings across groups, and scale bars (for example, 20 µm) and magnifications were indicated in all figure panels and legends.

CLP-induced sepsis mouse model

Male C57BL/6 mice (24-32 g) were acclimatized for at least 1 week before surgery and then randomly assigned to Sham, CLP, CLP plus negative control short hairpin, or CLP plus NEAT1 short hairpin groups (n = 6 per group). Mice were anesthetized by inhalation of ether in a closed chamber for approximately 1-3 min until loss of the righting reflex ether or another institutional-approved anesthetic at a dose and exposure duration that induced loss of the righting reflex while maintaining adequate respiratory function. The abdominal hair was shaved, and the skin was disinfected with an appropriate antiseptic solution. A midline laparotomy of approximately 1-2 cm was performed to expose the cecum with its adjoining intestine.

For CLP, the cecum was gently exteriorized and ligated with a sterile suture at approximately two-thirds of its length, taking care not to obstruct the ileocecal valve or compromise mesenteric blood flow. The ligated cecum was punctured twice with a sterile needle of the gauge specified in the approved protocol (for example, 21 G), and a small amount of fecal material was gently extruded to ensure patency of the puncture sites. The cecum was then returned to the peritoneal cavity, and the abdominal wall and skin were closed in layers with sutures. Sham-operated mice underwent the same laparotomy and cecum exposure without ligation or puncture.

To achieve in vivo knockdown of NEAT1, an adenoviral vector encoding shNEAT1 and a corresponding negative control vector were prepared at a defined titer (for example, 1 × 10⁹ plaque-forming units/mL). A fixed volume of viral suspension (such as 100 µL per mouse) was administered via the tail vein at a specified time relative to CLP (for example, 1 h after surgery), as described in the experimental design and approved by the ethics committee. After surgery, mice received standard postoperative care, including subcutaneous fluid resuscitation if indicated, and were monitored for clinical signs of sepsis and distress. At the predetermined time point, animals were euthanized humanely by overdose inhalation of ether in a closed chamber, using approximately 5-10 mL of ether to ensure rapid cessation of respiration, followed by confirmation of death, blood was collected for cytokine analysis, and organs were harvested for histopathology, immunofluorescence, and molecular assays.

Statistical analysis

Statistical analysis was performed using SPSS version 23.0 or an equivalent statistical software package. Data are presented as mean ± standard deviation (SD) unless otherwise stated. For comparisons between two groups, an unpaired Student's t-test was used when data were normally distributed with homogeneous variances. For comparisons among three or more groups, one-way analysis of variance (ANOVA) was applied, followed by appropriate post hoc tests for multiple comparisons when a significant overall effect was detected. A two-sided p value < 0.05 was considered statistically significant. The meaning of statistical symbols (for example, p < 0.05, *p < 0.01) was defined in the figure legends, and the number of biological replicates is indicated in the corresponding text or legends.

Results

Detection of NEAT1/miR-181a-5p/HMGB1 expression and interactions in sepsis

In serum from patients with sepsis, NEAT1 and HMGB1 levels were significantly higher than in healthy controls, whereas miR-181a-5p was markedly reduced. Similar expression patterns were observed in PBMCs and in RAW264.7 macrophages stimulated with lipopolysaccharide, indicating that this axis is consistently altered in sepsis and sepsis-like inflammatory conditions (Figure 1A

Discussion

Sepsis is characterized by profound and often long-lasting immune dysregulation, in which phases of hyperinflammation and immunosuppression coexist and jointly contribute to organ failure and poor outcomes^21,22. Accumulating evidence indicates that long non-coding RNAs (lncRNAs) participate in these processes by modulating inflammatory signaling and macrophage polarization^23,24. For example, lncRNA-MAAMT ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adenoviral Vector (shNEAT1)	10692-V5	Vigene Biosciences	Adenovirus used for knocking down NEAT1 expression in vivo and in vitro.
BCA Protein Assay Kit	23227	Thermo Fisher	Kit used to quantify protein concentration in cell lysates.
Dual-Luciferase Reporter Assay Kit	E1910	Promega	Kit used for dual-luciferase reporter assays to measure miR-181a-5p interaction.
ELISA Kits (Cytokines)	N/A	R&D Systems	Kits used for measuring cytokine levels in serum and tissue samples.
Fetal Bovine Serum (FBS)	16000-044	Gibco	Supplement used in culture media for cell growth.
GAPDH Antibody	2118S	Cell Signaling Technology	Primary antibody against GAPDH used as loading control in western blot.
Hematoxylin and Eosin Staining Kit	N/A	Thermo Fisher	Kit used for histological staining of tissue sections for pathological analysis.
Immunofluorescence Antibodies (CD206)	14693-1-AP	Proteintech	Antibody used for detecting M2 macrophage marker CD206 in tissue sections.
Lipopolysaccharide (LPS)	L2630	Sigma-Aldrich	LPS used to induce inflammatory macrophage polarization in RAW264.7 cells.
miR-181a-5p Mimic	N/A	N/A	Synthetic miR-181a-5p mimic used to modulate miR-181a-5p levels.
NEAT1 Expression Plasmid	N/A	N/A	Plasmid used for overexpression of NEAT1 in RAW264.7 macrophages.
RAW264.7 Macrophage Cells	TIB-71	ATCC	Murine macrophage cell line used for in vitro experiments.
RIPA Lysis Buffer	89901	Thermo Fisher	Buffer used for lysing cells to extract total protein.
RPMI-1640 Medium	11875-093	Gibco	Culture medium for RAW264.7 macrophages.
SYBR Green Master Mix	4385612	Thermo Fisher	Reagent used for quantitative real-time PCR analysis.
Transwell Migration Assay Chambers	3422	Corning	Chambers used for transwell migration assays to assess macrophage migration.
TUNEL Apoptosis Assay Kit	ab66110	Abcam	Kit used for detecting apoptosis in RAW264.7 cells by TUNEL staining.
Western Blotting Antibodies (HMGB1)	ab18256	Abcam	Primary antibody against HMGB1 used in western blotting.