Mir-199a-3p aggravates neuroinflammation in an Alzheimer’s disease transgenic mouse model by promoting M1-polarization microglia

Abstract

Background

Chronic neuroinflammation, driven by M1-polarized microglia, is a core pathological mechanism of Alzheimer’s disease (AD). Elevated expression levels of miR-199a-3p and pro-inflammatory cytokines were detected in the hippocampi of AD transgenic mice and in LPS-stimulated BV2 microglial cells. We hypothesized that miR-199a-3p exacerbates neuroinflammation by promoting M1 microglial polarization in AD progression.

Objective

To explore the role of miR-199a-3p in AD-associated neuroinflammation.

Methods

AD transgenic (APPswe/PSEN1dE9) mice and LPS-treated BV2 cells were used to assess miR-199a-3p effects in vivo and in vitro. Inflammatory cytokines and markers for microglial cell typing were detected. Transcriptome sequencing was performed on miR-199a-3p-modulated BV2 cells, and the sequencing data were cross-analyzed with public databases to predict miR-199a-3p-mediated pathways.

Results

Intracerebroventricular administration of miR-199a-3p agomir exacerbated amyloid deposition and impaired cognitive function in AD mice, and promoted microglial polarization toward the M1 phenotype. Conversely, treatment with miR-199a-3p antagomir attenuated AD pathology and suppressed M1 polarization. In LPS treated BV2 cells, miR-199a-3p mimics promoted M1 polarization, while inhibitors reversed this effect. Transcriptome analysis revealed that miR-199a-3p downregulated WDR76, subsequently suppressing cell cycle-associated pathways, IL-17 signaling, and FOXO pathways, resulting in an increase in the proportion of M1 type microglia.

Conclusion

MiR-199a-3p aggravates neuroinflammation of AD by promoting M1-polarization microglia. These findings highlight miR-199a-3p as a potential therapeutic target for AD.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12868-025-00965-5.

Introduction

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder and the leading cause of dementia in older adults [1].The core pathological features of AD includes the accumulation of amyloid β (Aβ) peptides and neurofibrillary tangles composed of hyperphosphorylated neuronal tau protein and a state of chronic neuroinflammation [2]. Significant efforts have been attempted to develop drugs for AD, but little has been achieved [3]. AD places an enormous pressure and burden on patients, families, and society [4]. A deeper understanding of AD pathogenesis is critical for developing novel interventions; however, the underlying mechanisms and key contributing factors remain incompletely elucidated. Thus, investigating the pathogenesis of AD and exploring new treatment strategies is crucial for clinical advancement.

Microglia, the resident immune cells of the central nervous system (CNS), play a key role in chronic inflammation [5–7]. Clinical PET imaging studies have demonstrated heightened microglial activation in the brains of AD patients, providing direct evidence of the involvement of microglia in AD [8]. In response to neuronal injury, microglia rapidly activate and polarize into either a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype [9]. Meanwhile, microglia polarized toward the M1 or M2 phenotypes can quickly shift change their phenotypes to adapt to the microenvironment. In the early stage, microglial activation helps to clear Aβ protein in general, however, the further secretion of interleukin (IL)-1β and IL-6 will recruit additional microglia around the amyloid plaque, the inflammatory factors secreted at a later stage will damage neuron function and aggravate the Aβ protein deposition [10]. Thus, deciphering the mechanisms governing microglial activation may offer new strategies to mitigate AD progression.

MicroRNAs (miRNAs), small noncoding RNAs, have been shown in many studies to play a key role in AD through RNA silencing and post-transcriptional regulation [11]. IIntriguingly, miR-199a-3p has been implicated in inflammation, though its role appears context-dependent. For instance, miR-199a-3p alleviates inflammatory levels in macrophages by inhibiting RUNX1 expression [12].MiR-199a-3p has also been shown to inhibit inflammation through the IKK β/NF-κB signaling pathway in renal tubular epithelial cells [13]. Xue et al. delivered miR-199a-3p by fabricating macrophage membrane coated nanoparticles and suppressed inflammation in mice with acute myocardial infarction [14]. In contrast, miR-199a-3p has also been reported to promote inflammation in macrophages [15]. Microglia are the resident macrophages of the central nervous system (CNS) [16], thus we hypothesized that miR-199a-3p similarly exerts a pro-inflammatory role in microglia.

In this study, we demonstrated that miR-199a-3p expression was significantly increased in the hippocampus of AD model mice, while in vitro and in vivo experiments confirmed that miR-199a-3p promotes microglia activation through inhibition of WDR76 gene expression, increases secretion of cellular inflammatory factors (IL-1β, iNOS), modulates neuroinflammation, and exacerbatesthe progression of AD. We believe that our findings provide new perspectives for understanding the role of miR-199a-3p in AD and are valuable for developing new therapeutic strategies.

Materials and methods

Animals and treatments

Seven-month-old male C57BL/6J wild-type mice and APPswe/PSEN1dE9 mice (AD mice) were obtained from the Guangdong Medical Laboratory Animal Center and Guangdong Sja Biotechnology Co., Ltd., both located in Guangdong, China.The mice were housed and individually fed at the animal experimental center of Zhujiang Hospital.The animals had ad libitum access to water and were housed in a temperature-controlled environment (22 ± 2 °C) with a 12-hour light/dark cycle.All animal experiments were approved by the Ethics Committee of Zhujiang Hospital, Southern Medical University (LAEC-2021-126).After one month of adaptation to the new environment, mice were randomly assigned to four groups: AD + agomir (APPswe/PSEN1dE9 mice intracerebroventricularly injected with miR-199a-3p agomir, n = 6), AD + antagomir (APPswe/PSEN1dE9 mice intracerebroventricularly injected with miR-199a-3p antagomir, n = 6), AD + NC (APPswe/PSEN1dE9 mice intracerebroventricularly injected with miR-199a-3p negative control, n = 6), and wild + NC (wild-type C57BL/6 mice intracerebroventricularly injected with negative control, n = 6).The agomir, antagomir, and negative control of miR-199a-3p were synthesized and designed by GenePharma Co. Ltd. (Shanghai, China)After anesthetization with tribromoethanolamine (200 mg/kg, intraperitoneally), mice were placed in a stereotaxic frame in the prone position.A hole was drilled 0.3 mm posterior to the bregma, 1 mm lateral to the midline, and 2.5 mm below the skull surface.A 4 µL volume (1.67 OD) of agomir, antagomir, and vector was injected into the lateral ventricles of the mice using a Hamilton 10 µL syringe (Hamilton, Switzerland) at a rate of 0.5 µL/min for 8 min, with a TFD03-01 Split Style Laboratory Syringe Pump (Baoding Lead Fluid Technology Co., Ltd).After a 10-minute pause, the microsyringe was removed, and the scalp was sutured.

Additionally, before and after the intraventricular injection, cerebral blood flow in the main brain vessels was monitored using a laser speckle imaging system.One month after the intraventricular injection, the mice underwent a seven-day water maze test.At the end of the experiment, the mice were euthanized by cervical dislocation under deep anesthesia induced by intraperitoneal injection of tribromoethanol (200 mg/kg). Death was confirmed by absence of vital signs and reflex responses, and their brain tissue and blood samples were collected for further analysis.

Cell culture and transfection

BV2 microglia cells were obtained from iCell Bioscience Inc. (China) and cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F12 (DMEM/F12) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS).The cells were cultured in an incubator at 37 °C with 5% CO2.MiR-199a-3p mimics and siRNA were purchased from GenePharma Co. Ltd. (Shanghai, China).Cells were treated with lipopolysaccharide (LPS) (Sigma-Aldrich, USA) at a concentration of 1 mg/mL for 24 h before transfection.Transfection was performed using EndofectinTM MAX (GeneCopoeia, USA) according to the manufacturer’s instructions.After transfection, cells were collected at 24 and 48 h for real-time PCR and Western blotting assays, respectively.

Next generation sequencing

Twenty-four hours after BV2 transfection with miR-199a-3p mimics or negative control, RNA was extracted and sent for next-generation sequencing by Frasergen (Wuhan, China).The integrity and purity of the RNA were assessed and confirmed to be satisfactory.A total of 1 µg of RNA per sample was used as input for RNA library preparation.RNA sequencing libraries were generated using the MGIEasy RNA Library Prep Kit.The clustering of index-coded samples was performed on a cBot cluster generation system with the MGI2000 detector (BGI, China).All procedures were performed according to the manufacturer’s instructions.Only genes with an absolute log2 ratio ≥ 2 and an FDR significance score < 0.01 were used for subsequent analysis.Genes were compared against various protein databases using BLASTX, including the NCBI non-redundant protein database (Nr) and the Swiss-Prot database, with a cut-off E-value of 10^− 5.

Bioinformatics analysis

Genes identified as differentially expressed in the sequencing data were used for subsequent bioinformatics analyses.Bioinformatics analyses were performed using the OECloud tools at https://cloud.oebiotech.cn.Genes downstream of miRNAs were predicted using TargetScan and miRDB.Venn diagrams were used to cross-analyze multiple datasets.The GEO2R online tool was used to analyze the GSE49329 expression profile in the Gene Expression Omnibus (GEO) database.STRING was used to predict related genes of key downstream genes and to construct a protein-protein interaction (PPI) network.

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

Total RNA was extracted from the mouse hippocampus and the BV2 cell line using Trizol reagent (Tiangen Biotech, China).RNA concentration and purity were assessed using a Nanodrop spectrophotometer (BioTek Instruments, Inc., USA).The Fast All-in-One RT Kit (ES Science Biotech, China) and the PrimeScript RT Reagent Kit (Takara, Japan) were used for mRNA and miRNA reverse transcription, respectively.qRT-PCR amplification was performed on a CFX Connection Real-Time System (Bio-RAD, USA) using SYBR qPCR SuperMix Plus (Data Invention Biotech, China).All experiments were conducted according to the manufacturer’s instructions.All reactions were normalized to the internal reference genes β-actin for mRNAs and U6 for miRNAs.The relative expression of genes was analyzed using the 2^–ΔΔCt method.The primers used are listed in Supplementary File 1.

Western blotting

Total protein from cells or hippocampal tissues was extracted using precooled RIPA lysis buffer (Fdbio Science, China) at 4℃.The protein concentration of each sample was determined using the BCA protein assay kit (Fdbio Science, China).Protein aliquots were boiled, resolved on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes (Fdbio Science, China).The membrane was blocked with skim milk at room temperature for 1 h.The PVDF membranes were incubated overnight at 4 °C with IL-1β (1:1000, Affinity, China), iNOS (1:1000, Wanleibio, China), Arg-1 (1:1000, Wanleibio, China), and β-actin antibody (1:10,000, Abmart, China).After washing three times with TBST for 5 min each, the membrane was incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:10,000, Fdbio Science, China) or HRP-labeled goat anti-mouse IgG at room temperature for 1 h.The FDbio-Dura ECL kit (Fdbio Science, China) was used to detect the protein, and imaging was performed using the Alliance Q9 & Alliance Q9 Chroma (UVITEC, UK).β-actin was used as an internal control to calculate the relative expression of the target protein using ImageJ software.

Flow cytometry

Prior to antibody staining, cell receptors were blocked with anti-murine CD16/CD32 Fc receptor (Elabscience Biotechnology Co., Ltd, China) at 4 °C for 10 min.After blocking, BV2 cells were double-stained with PerCP-conjugated anti-murine CD40 (Elabscience Biotechnology Co., Ltd, China) and PE-conjugated anti-murine CD206 (Elabscience Biotechnology Co., Ltd, China) for 20 min.Cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter Biotechnology, USA).Ten thousand events were recorded, with M1-type microglia identified by positive expression of CD40 and M2-type microglia identified by positive expression of CD206.Furthermore, flow cytometry analysis of microglia in mouse hippocampal tissue was performed according to the following protocol.After euthanasia, part of the hippocampal tissue was dissected and placed in a 6-well plate.The tissue was homogenized using a filter head, and 2 mL of PBS was added to prepare a cell suspension by passing the solution through a 40 μm cell strainer. Antibodies against CD206 (Biolegend, USA), CD86 (Biolegend, USA), CD11b (Tonbo, USA), and CD45 (Tonbo, USA) were then added and incubated overnight in the dark.Among these, CD45 + and CD11b + were used as criteria to identify microglial cells in the brain. CD206 + and CD86- microglia were classified as M2 microglia, while CD206- and CD86 + microglia were classified as M1 microglia. Cells that were CD206- and CD86- were considered unactivated microglia.The results were analyzed using CytExpert software (Beckman Coulter Biotechnology, USA).

Elisa

BV2 cell culture medium was collected 48 h post-transfection, centrifuged at 3000 RPM for 20 min, and the supernatant was collected.After anesthesia was induced with tribromoethanolamine (200 mg/kg, intraperitoneally), orbital blood samples were collected via enucleation.The blood was allowed to clot at room temperature for 20 min, then centrifuged at 3000 RPM for 25 min.The serum (supernatant) was carefully collected for IL-1β (RUIXIN Biotech, China) ELISA analysis.IL-1β concentrations were expressed as pg/mL in the supernatant.Plates were read using an enzyme marker (Biotek ELX808, USA) at a wavelength of 450 nm.

Immunohistochemistry

Mice were anesthetized intraperitoneally with tribromoethanol (200 mg/kg) and transcardially perfused with 40 mL of 0.9% saline solution (pre-cooled to 4 °C) before brain removal.One hemisphere was placed in 4% paraformaldehyde at 4 °C for 24 h for paraffin embedding.Briefly, brain sections were incubated with 3% H2O2 in methanol, blocked with 10% goat serum, and incubated overnight at 4 °C with anti-beta amyloid 1–42 antibody (1:120, ab2539; Abcam).After washing, the sections were incubated with goat anti-rabbit biotinylated secondary antibody (1:200; Abcam).After rinsing with PBS, streptavidin-labeled peroxidase was added and incubated for 30 min.Following another rinse, freshly prepared 3,3′-diaminobenzidine (DAB) solution was added, and the reaction was allowed to develop.The sections were stained with hematoxylin and differentiated by immersion in 1% hydrochloric acid in alcohol.The sections were then washed in ammonia and stained blue, followed by rinsing with water.Images were captured using the PANNORAMIC MIDI II– Legacy (3DHISTECH Ltd., Hungary).

Morris water maze

Mice in each group underwent a spatial navigation test 28 days post-modeling.A round pool (120 cm in diameter, 55 cm in height) filled with non-toxic white water-based tempura paint (22 ± 1 °C) was divided into four quadrants, each with equal distances on the rim.An 8-cm target platform was placed in the center of the SW quadrant. It was above the water surface on the first day and submerged 1 cm below the surface from the second to the sixth day.The platform remained in the same position throughout the learning trials.A video tracking system (Shanghai Jiliang Software Technology Co., Ltd., China) was used to monitor and record the swimming activity of the mice during the test.Each test allowed each mouse 60 s to find the platform, and mice that successfully located the platform were allowed to rest on it for 10 s.If a mouse failed to find the platform, it was gently guided to it and allowed an additional 25 s to facilitate spatial memory formation.Each mouse was trained four times daily at 30-minute intervals.A probe test was conducted on the seventh day, when the platform was removed.Each mouse was placed in the water in the quadrant furthest from the one containing the platform used on days 1–6, and allowed to navigate freely for 60 s.

Monitoring changes in cerebral blood flow

The Laser Speckle Imaging System (LSCI, Wuhan SIM Opto-technology Co., China) was used to monitor changes in cerebral blood flow (CBF) in the brains of mice.The system consisted of an Olympus ZS61 microscope, a continuous wavelength (λ = 785 nm) laser source, a camera, and a computer.Before lateral ventricle injection, brain blood flow in mice was dynamically measured using speckle signals (image exposure time: T = 20 ms), and changes in blood flow in the anterior and posterior fontanels were recorded.Cerebral blood flow was re-measured using the LSCI system 14 days after lateral ventricle injection.Due to variations in cerebral vessels among mice, the anterior and posterior fontanels were used as markers to calculate the average blood flow in these two regions, which was taken as the average value of cerebral blood flow in the mice.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9.0 software (GraphPad).Data were presented as mean ± standard deviation.No corrections for multiple comparisons were applied, as statistical analyses were limited to pairwise comparisons between two experimental groups using unpaired t-tests.A P-value of < 0.05 was considered statistically significant.

Results

MiR-199a-3p was increased in the hippocampal tissues of AD mice and LPS-stimulated microglia

To determine whether miR-199a-3p expression is altered in AD, we performed qRT-PCR to measure miR-199a-3p levels in hippocampal tissue from 7-month-old APPswe/PSEN1dE9 transgenic AD mice and age-matched wild-type C57BL/6J mice. MiR-199a-3p levels were significantly higher in the AD mice than in wild-type controls(Fig. 1A). The observed upregulation of miR-199a-3p in AD mice suggests that it may contribute to AD pathogenesis. Given that neuroinflammation is a core pathological mechanism in AD [1] and that miR-199a-3p might influence disease progression by modulating the inflammatory state, we next assessed miR-199a-3p expression in an in vitro model of microglial activation. Mouse microglia cells(BV2) were stimulated with lipopolysaccharide (LPS) to mimic an inflammatory environment. We found that miR-199a-3p expression was significantly increased in LPS-stimulated microglia compared to untreated cells(Fig. 1B). These results demonstrated that miR-199a-3p was significantly upregulated in both the hippocampal tissues of AD mice and in LPS-stimulated microglia.

Fig. 1.

MiR-199a-3p is up-regulated in AD hippocampus tissues and in mouse microglia treated with LPS. A qRT-PCR analysis of miR-199a-3p expression in hippocampus tissues of APPswe/PSEN1dE9 and C57BL/6J mice. B qRT-PCR analysis of miR-199a-3p expression in BV2 treated with 1 mg/mL LPS for 24 h. *p < 0.05, **p < 0.01

MiR-199a-3p promoted the progression of AD in a mouse model through intracerebroventricular injection

We hypothesized that miR-199a-3p may function as a pro-inflammatory factor in microglia and thus promote the progression of AD. To test this hypothesis, we modulated the expression of miR-199a-3p in the hippocampal region of AD mice by intracerebroventricular injection of antagomir, agomir, or negative control. Figure 2A illustrates the entire treatment protocol in mice. Changes in the cerebral blood flow were monitored before and after lateral ventricle injection using laser speckle imaging. We divided the changes in cerebral blood flow after injection by the value of cerebral blood flow before injection and defined this value as the change ratio of blood flow. Mice injected with the antagomir exhibited a significant increase in cerebral blood flow, whereas those injected with the agomir showed a significant decrease (Fig. 2B–D). Moreover, wild-type mice displayed significantly higher cerebral blood flow compared to AD mice (Fig. 2C). These results suggest that inhibiting miR-199a-3p expression improves cerebral perfusion and may alleviate hypoperfusion and hypoxia in the brain of mice. To further evaluate the functional impact of miR-199a-3p, we conducted a Morris water maze test one month after injection to assess spatial learning and memory. Mice treated with miR-199a-3p agomir exhibited longer escape latencies from the second day of the learning phase, indicating impaired spatial learning (Fig. 2E). As expected, AD model mice demonstrated poorer performance than age-matched wild-type controls during both the training and probe phases (Fig. 2F–H). In the probe trial, mice injected with the antagomir displayed increased platform crossings and longer time spent in the target quadrant, without any difference in swimming speed, compared to the negative control group. In contrast, mice injected with the agomir performed significantly worse (Fig. 2G–I). To investigate the effect of miR-199a-3p on Aβ deposition, brain tissue sections were analyzed by immunohistochemistry. Mice receiving miR-199a-3p agomir displayed increased amyloid plaque accumulation in the hippocampus, whereas antagomir treatment mitigated Aβ deposition in AD mice (Fig. 2J, K). These findings indicate that miR-199a-3p exacerbates pathological features of AD, including impaired cerebral blood flow, cognitive deficits, and amyloid deposition, while its inhibition may confer neuroprotective effects.

Fig. 2.

MiR-199a-3p promotes the progression of AD in mouse models through intracerebroventricular injection. A Schematic diagram showing the study design to explore the role of miR-199a-3p in AD. B Representative images of the cerebral blood flow of mice before and after intracerebroventricular injection. C Bar plot of cerebral blood flow from mice before and after injection. D The bar graph of the change ratio of the cerebral blood flow of mice before and after injection. Ratio = (Post-injection CBF (14 days) − Pre-injection CBF) ÷ Pre-injection CBF. E–I The miR-199a-3p negative control, agomir and antagomir were injected into the hippocampus of AD mice and the miR-199a-3p negative control was injected into the hippocampus of wild-type mice twice. One month later, the mice were subjected to the Morris water maze test. E Representative images and thermal trajectory map of traces to the hidden platform on day 7 of the Morris water maze. F Latencies to the hidden platform in the Morris water maze in the learning stage were recorded (n = 6 for each group, repeated measures student test. G Times of mice crossing the platform area. H Stay times of mice in the target quadrant, and I the swimming speed on day 7 were analyzed (n = 6 for each group). J, K The expression of amyloid protein in the hippocampus was detected by immunohistochemical staining. Scale bars, 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

MiR-199a-3p promoted M1 polarization of microglia in the hippocampus

Previous studies have commonly used CD11b⁺ and CD45⁺ as markers to identify microglia, with CD40, CD86, IL-1β, and iNOS serving as indicators of M1 polarization, while CD206 and Arg-1 are considered markers of M2 microglia. The M1/M2 microglial ratio is approximately reflected by the proportion of CD86⁺/CD206⁻ cells to CD86⁻/CD206⁺ cells, or by the expression ratio of iNOS to Arg-1.

Western blot analysis revealed that, compared to mice injected with a negative control, those receiving miR-199a-3p agomir exhibited elevated expression of IL-1β and iNOS and reduced Arg-1 levels in hippocampal tissues. In contrast, mice injected with miR-199a-3p antagomir showed increased Arg-1 expression and decreased levels of IL-1β and iNOS (Fig. 3A–D). The iNOS to Arg-1 ratio further supported enhanced M1 polarization following agomir treatment and its attenuation with antagomir administration. (Fig. 3E). The content of IL-1β was also detected in the serum of mice by the ELISA, which showed that injection of agomir could aggravate the increase in inflammation, whereas antagomir administration attenuated this elevation (Fig. 3F). Flow cytometry was then used to evaluate the types of microglia present in mouse hippocampal tissue. The findings also support the above conclusion that in mice injected with antagomir, the proportion of M1 microglia (CD86⁺CD206⁻) decreased, while the proportion of unactivated microglia (CD86⁻CD206⁻) increased (Fig. 3G–I).

Fig. 3.

MiR-199a-3p promoted M1 polarization of microglia in the hippocampal region. A Western blotting analysis of iNOS, IL-1β and Arg-1 protein levels in hippocampal tissue of experimental wild type and AD mice. B–D Relative expression levels of each protein. E The proportion of M1/M2 microglia is approximately equal to the ratio of iNOS to Arg-1. F Detection of IL-1β in mouse serum by ELISA after inhibition and overexpression of miR-199a-3p. G After agomir or antagomir treatment, mice hippocampal tissue was made into a cell suspension, and stained for flow cytometry analysis. The indicated gates correspond to resting microglia (CD11b+/CD45+), M1 microglia (CD86+/CD206-), and M2 microglia (CD86-/CD206+). H, I Percentage of CD86 + cells and CD86-CD206 cells to the whole. The proportion of unactivated microglia is approximately equal to the CD86-CD206- rate

MiR-199a-3p promoted M1 polarization in BV2

As indicated above, miR-199a-3p expression was upregulated in BV2 cells following stimulation with LPS. Following antagomir injection in AD mice, the proportion of M1 microglia decreased, and inflammation was alleviated compared to the AD + NC group. Based on these findings, we speculated that miR-199a-3p may have the capacity to drive microglial polarization. To test this hypothesis, BV2 cells were transfected with miR-199a-3p mimics, inhibitors, or negative control sequences. As expected, transfection with miR-199a-3p mimics significantly increased miR-199a-3p expression, while transfection with inhibitors markedly reduced its levels compared to the control (Fig. 4A). BV2 cells were pretreated with LPS, followed by transfection with either miR-199a-3p mimics or inhibitors. Western blot analysis showed that miR-199a-3p mimics promoted M1 differentiation and increased the expression of inflammatory markers IL-1β and iNOS (Fig. 4B–F). In contrast, inhibition of miR-199a-3p expression led to a shift toward M2 polarization, with reduced IL-1β and iNOS levels and increased Arg-1 expression (Fig. 4B–F). To further verify the polarization orientation of microglia, microglial markers CD40 and CD206 were analyzed by flow cytometry to evaluate the phenotypic status of M1 and M2 (Fig. 4G). The results showed that miR-199a-3p mimics could increase the proportion of M1 cells in microglia, while miR-199a-3p inhibitors could increase the proportion of M2 cells in microglia (Fig. 4H–J). Altogether these data suggested that miR-199a-3p could promote M1 polarization of microglia.

Fig. 4.

MiR-199a-3p promoted M1 polarization in BV2. A BV2 were transfected with miR-199a-3p inhibitor or negative control or mimic. QRT-PCR was used to evaluate miR-199a-3p levels in BV2 24 h after transfection. B Western blotting analysis of iNOS, IL-1β and Arg-1 protein levels in BV2 after miR-199a–3p inhibition and overexpression. C–E Relative expression levels of each protein. F The proportion of M1/M2 microglia is approximately equal to the ratio of iNOS to Arg-1. G Transfected with the miR-199a-3p inhibitor, negative control, or mimics simulated with or without LPS, BV2 was harvested to stain for flow cytometry analysis. Gates correspond to resting microglia (CD11b+/CD45+), M1 type microglia (CD40+/CD206-) and M2 type microglia (CD40-/CD206+). H, I Proportion of CD40 + and CD206 + cells of the whole sample. J The proportion of M1/M2 microglia is approximately equal to the ratio of CD40+/CD206- to CD40-/CD206+

Bioinformatics analysis of miR-199a-3p in BV2

We transfected miR-199a-3p mimics into BV2 and performed a transcriptome analysis of transfected cell samples to identify possible targets of miR-199a-3p and the pathways involved. Principal component analysis (PCA) results showed that the difference between the experimental group and the control group was large and the difference within the group was within an acceptable range, suggesting that the biological duplication of the samples was adequate and that the experimental group was properly set up (Fig. 5A). A total of 499 differentially expressed genes were identified, of which 289 genes were significantly up-regulated and 210 genes were significantly down-regulated. Figure 5B shows the differently expressed mRNA ranked by fold change (FC), including both upregulated and downregulated genes. Table 1 shows the 7 main mRNAs that were most significantly up- or down-regulated in the miR-199a-3p high expression group compared to the controls. To validate the transcriptome analysis data, two significantly upregulated genes (IL1f9 and Mmp13) and two significantly downregulated genes (Kif11 and Npcd) were randomly selected from Table 1 for the verification of qRT-PCR, as shown in Fig. 5C. The results show that after transfection of miR-199a-3p mimics into BV2, the expression of Kif11 and Npcd was significantly decreased and the expression of IL1f9 and Mmp13 was significantly increased compared to the control group (p < 0.01).

Fig. 5.

Bioinformatics analysis of miR-199a-3p in BV2. A The principal component analysis of BV2 transfected with miR-199a-3p mimics or negative control. B Volcano plot filtering identified differentially expressed mRNAs in sequencing samples. C qRT-PCR analysis of IL1f9, Npcd, Kif11, and Mmp13 in sequencing samples. D The bar graph of the enrichment pathway of up-regulated mRNA by KEGG analysis. E The bubble plot of the enrichment pathway of down-regulated mRNA by GO analysis. F The bubble plot of the enrichment pathway of up-regulating mRNA by KEGG analysis. GO Gene Ontology, KEGG Kyoto Encyclopedia of Genes and Genomes

Table 1.

14 main mRNAs that were most significantly up- or down-regulated in the miR-199a-3p high expression group compared to the controls

GeneID	Log2FoldChange	Regulation	Control-Expression	Treatment-Expression	P-value
Gm10359	−21.53890045	Down	56.02290846	1.84E−05	3.57E−08
Gm12671	−21.53890045	Down	56.02290846	1.84E−05	3.57E−08
novel.231	−9.360552563	Down	124.9427204	0.190065749	5.55E−14
novel.4225	−2.220968472	Down	33.50254985	7.186207158	0.000594569
H3c14	−1.655569906	Down	96.20999639	30.53823949	0.000989537
Npcd	−1.423611716	Down	117.2754432	43.71769483	0.000281984
Kif11	−1.248338287	Down	88.14428627	37.10281803	0.005553258
Gm5537	24.63969206	Up	1.09E-05	286.1605995	5.28E−05
novel.150	3.874994427	Up	77.11480322	1131.429011	2.86E−10
IL1f9	2.962686314	Up	19.28395354	150.3327232	2.53E−08
Hist1h2br	2.6391116	Up	7.8400055	48.8391527	0.000229688
Fosl1	2.278645543	Up	43.09736563	209.1179835	1.10E−05
Mmp13	2.147566677	Up	57.9013208	256.549078	0.000952568
Cxcl2	2.02082844132809	Up	29.41631515	119.3763364	4.98E−07

To examine the possible involvement of miR-199a-3p in signaling pathways in BV2 cells, a KEGG and GO enrichment analysis was performed based on the differentially expressed mRNA. The results of the GO analysis indicated that the changes in biological processes (BP) of the up-regulate genes were significantly enriched in the cellular processes, biological regulation, regulation of biological process, metabolic processes, and response to stimulus pathways (Fig. 5D). Changes in cell component (CC) of up-regulate genes were mainly enriched in cellular anatomical entity and protein-containing complex pathways (Fig. 5D). Changes in molecular function (MF) were enriched in the catalytic activity and binding pathways. Besides, cell cycle-related pathways were enriched in GO analysis results of down-regulate genes (Fig. 5E). Among the analysis of the KEGG pathway of up-regulated mRNA, the IL-17 signaling pathway was the most enriched, followed by the C-type lectin receptor signaling pathway and the FOXO signaling pathway. Furthermore, the NF-kappa B signaling pathway, TNF signaling pathway, PI3K-Akt signaling pathway, and the HIF-1 signaling pathway associated with inflammation were also highly enriched, as shown in Fig. 5F.

MiR-199a–3p directly targeted WDR76

MiRNA is often thought to function by binding to the 3’-UTR of target mRNA and inducing its degradation [17]. To explore potential miR-199a-3p targeting genes in microglia, bioinformatics analysis was performed with a public database, including miRDB and TargetScan, and the sequencing results were obtained. Overlapping significantly down-regulated genes in the cell lines and the miR-199a-3p target genes predicted by the miRDB database and the TargetScan database were analyzed (Fig. 6A). The results showed that 5 target genes predicted by the miRDB database and the Targetscan database were significantly downregulated. Table 2 shows the expression of the overlapping genes. To reduce the error caused by the limited number of samples, these five genes were validated in a public dataset GSE49329 [18]. In this study, microarray analysis was performed on mouse microglia, simulated by LPS, which differentiated into type M1.The results showed that the expression of WDR76 was significantly down-regulated when microglia differentiated to M1, suggesting that down-regulation of WDR76 may lead to activation of microglia to M1. Table 3 shows the expression of these genes in GSE49329. Then WDR76 mRNA expression levels were validated in cells and tissues and found that WDR76 expression decreased when miR-199a-3p was upregulated in cells and increased when miR-199a-3p was downregulated in cells, as was the trend observed in tissues (Fig. 6E, F). Therefore, we considered that miR-199a-3p may induce microglia differentiation to M1 by inhibiting the expression of WDR76. The String database (https://cn.string-db.org/) was used to construct the protein interaction network for WDR76 based on the differential genes shown in the sequencing results and the differential genes enriched in the FOXO, IL-17, cell cycle and NF- kappa B signaling pathway, suggesting that WDR76 may inhibit the related inflammatory pathway of microglia by inhibiting the cell cycle pathway (Fig. 6G).

Fig. 6.

MiR-199a–3p directly targets WDR76. A Venn diagram displaying the overlapping of the down-regulated genes and mice target genes of miR-199a–3p, as predicted by Targetscan and the miRDB database. B The heatmap of expression levels of CD151, WDR76, Fam199x, Tgif2, and Lin28b in bV2 transfected with miR-199a-3p negative control or mimics. C The heatmap showing expression of CD151, WDR76, Fam199x, Tgif2, and Lin28b in integrated expression profiles of mRNA in polarized microglia following PBS or LPS stimulation. D The alignment of a putative binding site for miR-199a-3p within the 3 ‘UTR’ of WDR76 mRNA shows a high level of sequence conservation and complementarity with miR-199a–3p. E qRT-PCR analysis of WDR76 in wild type and AD mice injected with negative control miR-199a-3p, antagomir, or agomir. F qRT-PCR analysis of WDR76 in BV2 transfected with miR-199a–3p negative control, inhibitor, or mimics with or without IL-1β stimulation. G Protein interaction network for WDR76 based on differential genes shown in the sequencing results and differential genes enriched in the FOXO, IL-17, cell cycle and NF- kappa B signaling pathway. (*p < 0.05, **p < 0.01, ***p < 0.001.)

Table 2.

5 overlapping target genes predicted by the MiRDB database and the targetscan database were significantly downregulated

Gene name	Regulation	Control-expression	Treat-expression	Log2FoldChange	P-value
Lin28b	Down	85.7741732	46.59697847	−0.880306907	0.001230176
WDR76	Down	770.5290157	483.2461078	−0.673091173	3.19E−05
Tgif2	Down	244.5244106	165.0566805	−0.567016964	0.002105783
Fam199x	Down	942.5924838	645.9488276	−0.545214299	0.00318421
Cd151	Down	643.695011	449.8463684	−0.516944911	0.00151428

Table 3.

The expression of target genes in GSE49329

ID	Regulation	P-value	LPS_mean	PBS_mean	logFC
Lin28b	UP	5.05e−01	5.15175502	5.180524439	6.96e−02
WDR76	Down	5.57e−08	6.235392686	7.556395305	−1.46
Tgif2	Down	1.83e−01	6.787730699	6.879651865	−2.58e−01
Fam199x	Down	2.42e−03	7.002790126	7.416004046	−4.57e−01
Cd151	Down	1.04e−04	10.00341668	10.31574777	−6.93e−01

Discussion

This study clarified the biological role of miR-199a-3p in the development of AD. We propose an inflammatory cytokine pathway is induced by the up-regulation of miR-199a-3p expression in microglia. Specifically, upregulated miR-199a-3p promotes microglia to toward the M1 phenotype by targeting WDR76. This shift triggers activation of the IL-17 and FOXO signaling pathways and suppresses cell cycle–related pathways, thereby enhancing the secretion of inflammatory mediators and accelerating disease progression (Fig. 7). Therefore, our results suggest that miR-199a-3p may be a new potential therapeutic target for the prevention and treatment of AD.

Fig. 7.

Description of mir-199a-3p affecting AD disease by modulating the function of microglia. Mir-199a-3p inhibits the expression of WDR76 in microglia, which activates the IL-17 and FOXO pathways and inhibits the cell cycle-associated pathway. Under this action, microglia differentiate into type M1, promote the secretion of inflammatory mediators such as iNOS, IL-1β, and accelerate the deposition of amyloid protein. The accumulation of amyloid protein and the aggravation of neuroinflammation will cause further damage to nerve cells and aggravate the degenerative changes of the central nervous system

MicroRNAs are endogenous, non-coding RNAs that exert post-transcriptional regulatory effects by binding, via their seed region, to complementary sequences within the 3′ untranslated region (3′UTR) of target gene transcripts [19]. Numerous evidence shows that miRNA play an important role in neuroinflammation and AD [20]. For example, miR-146a-5p has been shown to promote oxidative stress and pyroptosis by targeting TIGAR in the hippocampus neuronal cell model of AD [21]. Overexpression of miR-135 effectively rescued AD-like synaptic and memory deficits in the AD mouse model by targeting Rock2 [22].Furthermore, stereotaxic injection of miR-331-3p and miR-9-5p antagomir into the hippocampus activated autophagy and enhanced Aβ protein elimination, which improved cognitive decline and abnormal activity of late-onset AD mice [23]. In prior research, miR-199a-3p is often found as an inhibitory factor for cancer cell proliferation and invasion, or as a suppressor of the NF-κB pathway [13, 24]. However, our findings reveal that miR-199a-3p can promote the progression of neuroinflammation and AD. The overexpression of miR-199a-3p decreased the expression of WDR76 and promoted the secretion of inflammatory factors in microglia. There were no significant differences in the expression of IKK β, the target protein of miR-199a-3p, in microglia. Therefore, to our knowledge, this is the first report to emphasize the biological importance of miR-199a-3p in promoting the progression of AD.

Sequencing of the entire exon, in a pooled sample consisting of 6965 AD patients and 13,252 healthy controls, found that WDR76 was mutated four to five times more frequently in AD patients than in healthy non-dementia controls [25]. Furthermore, WDR76 promotes the expression of GLUT1, SAD1, and FADS2 in DNA methylation and histone modification in a manner dependent on LSH and chromatin modification, thus inhibiting the iron death pathway [26]. Thus, weakening or loss of WDR76 function can promote the onset and development of AD, but there is still a lack of research on the specific role of WDR76 in AD.

WDR76 belongs to a superfamily characterized by WDR domains, featuring a β-propeller structure with seven WD40 blades. Current literature on WDR76 is sparse; however, emerging studies suggest its involvement in inhibiting liver and colorectal cancers [27, 28]. Both the public data set (GSE49329) and our sequencing results show that when the proportion of M1 in microglia increases, the expression of WDR76 decreases significantly, suggesting that WDR76 plays a key role in microglial morphological and functional changes. In our study, administration of miR-199a-3p agomir to the lateral ventricle of mice resulted in a marked reduction in WDR76 mRNA levels and a significant increase in the proportion of M1 microglia in the hippocampus, while the introduction of the antagomir produced opposite effects. Furthermore, by constructing a PPI network related to differential genes, we found that WDR76 may mediate the cell cycle pathway through Shcbp1, Prism1, and Haus5 and then influences inflammatory pathways such as the IL-17 and NF-κB pathway, thus regulating the function of microglia.

At the same time, our sequencing results also revealed significant enrichment of the IL-17 and FOXO1 pathways in differentially regulated microglia, suggesting a relationship with microglial M1/M2 polarization. Many studies have indicated that in AD, β-amyloid deposition stimulates immune cells and continuously activates the IL-17 pathway [29]. For instance, Zhang et al. found that the expression of IL-17 in the hippocampus, serum, and cerebrospinal fluid samples of rats increased significantly after injection of Aβ1–42 [30]. IL-17 has also been shown to promote autophagy and trigger the accumulation of Aβ in neurons, ultimately leading to neurodegeneration [31, 32]. In addition, co-culture of microglia with Th1/Th17 cells results in the production of various inflammatory mediators, including IL-1β, IL-6, and TNF-α [33]. Upon IL-17 stimulation, microglia exhibit increased synthesis and secretion of inflammatory factors such as IL-6, nitric oxide and other inflammatory factors [34].The FOXO transcription factor family, including DAF-16 in Caenorhabditis elegans, plays crucial roles in vascular diseases, behavioral disorders, and neuronal damage [35, 36]. Activation of FOXO can lead to neuronal damage and death. FOXO3a promotes the injury and apoptosis of microglia under oxidative stress [37]. FOXO3 interacts with calcineurin in astrocytes to promote the expression of inflammatory cytokines (TNF-α, IL-6) and increases the deposition of β-amyloid protein [38]. However, there are no studies exploring the relationship between the FOXO pathway, the IL-17 pathway, and microglial differentiation. This study provides novel insights that amyloid protein or inflammatory factors stimulate miR-199a-3p expression in microglia, thus activating the FOXO pathway and the IL-17 pathway via inhibition of WDR76 expression, then increasing the proportion of M1 microglia and further aggravating neuroinflammation in AD.

This study has several limitations. First, direct experimental evidence supporting the regulation of WDR76 by miR-199a-3p is lacking, and our conclusions are primarily based on bioinformatics predictions. Although, according to Targetscan and the miRDB database algorithms, we can consider that miR-199a-3p can specifically bind to WDR76 and reduce the expression of WDR76. Moreover, research on the role of WDR76 in AD and neuroinflammation remains limited. The present study does not provide sufficient evidence to confirm that silencing WDR76 in microglia leads to increased secretion of inflammatory factors and accelerates AD progression. Additionally, we detected significant activation of the IL-17 and FOXO pathways, but we did not further explore the role of this pathway in microglia and AD disease. These unanswered questions will serve as the foundation and direction for our future studies.

Conclusions

This study identifies miR-199a-3p as a novel regulator that promotes microglial M1 polarization and contributes to the progression of neuroinflammation in Alzheimer’s disease (AD). We demonstrate that miR-199a-3p is upregulated in both AD mouse hippocampal tissues and LPS-stimulated microglia, accompanied by increased secretion of pro-inflammatory cytokines and exacerbation of AD-related pathological features. Based on transcriptomic and bioinformatic analyses, we propose that miR-199a-3p may induce microglial differentiation toward the M1 phenotype by inhibiting the expression of WDR76. These findings provide new evidence for the role of miR-199a-3p in AD-related neuroinflammation and support its potential as a candidate target for future therapeutic strategies. However, further studies are required to experimentally validate the regulatory relationship between miR-199a-3p, WDR76, and the downstream signaling pathways in microglial function.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

Yang Li (Conceptualization; Formal Analysis; Methodology; Funding acquisition); Duobin Wu (Methodology; Project administration); Xiaoya Gao (Supervision; Project administration); Chenyang Wang (Formal analysis; Visualization; Funding acquisition; Writing - Review & Editing); Xiaolu Bu (Formal analysis; Visualization; Writing-original draft); Mengyao Cao (Formal analysis; Visualization; Writing-original draft); Yunyu Lian (Investigation); Haocong Ling (Investigation); Mo You (Investigation); Junfei Yi (Formal analysis; Methodology).All authors reviewed the manuscript.

Funding

This study was supported by the Natural Science Foundation of Guangdong Province (Grant number 2018A0303130216) and President Foundation of Zhujiang Hospital, Southern Medical University (Grant number yzjj2023qn15).

Data availability

The datasets generated during the current study are available in the NGDC repository, CRA026641.The mRNA expression data supporting the findings of this study are openly available in the NCBI Gene Expression Omnibus database under the accession code GSE49329 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE49329). The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All animal experiments were conducted with the approval of the Zhujiang Hospital of Southern Medical University Ethics Committee (LAEC-2021-126). All methods were performed in accordance with the relevant guidelines and regulations.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.