Journal: International Journal of Molecular Medicine

Article Title: miR-223-3p promotes microglial lactylation and M1 polarization via the FBXW7/Notch1/Hes1/SIRT1 axis

doi: 10.3892/ijmm.2026.5849

Figure Lengend Snippet: miR-223-3p is dysregulated in patients with AD. (A and B) Expression of miR-223-3p in (A) parietal lobe tissues and (B) prefrontal cortex tissues of patients with AD compared with controls. (C and D) miR-223-3p levels in both (C) serum and (D) CSF samples from patients with AD relative to those from controls in the GEO dataset. (E and F) miR-223-3p levels in both (E) serum and (F) CSF samples from patients with AD relative to those from controls according to the ADNI dataset. (G) ROC curve of miR-223-3p in parietal lobe tissues for AD from GEO dataset. (H) ROC curve of miR-223-3p in prefrontal cortex tissues for AD from GEO dataset. (I) ROC curves of miR-223-3p in serum for AD from GEO dataset. (J) ROC curves of miR-223-3p in CSF for AD from GEO dataset. (K) ROC curves of miR-223-3p in serum for AD from ADNI dataset. (L) ROC curves of miR-223-3p in CSF for AD from ADNI dataset. * P<0.05, ** P<0.01 and *** P<0.001. miR, microRNA; AD, Alzheimer's disease; CSF, cerebrospinal fluid; GEO, Gene Expression Omnibus; ADNI, AD Neuroimaging Initiative; ROC, receiver operating characteristic; AUC, area under the curve; CI, confidence interval.

Article Snippet: Synthetic miR-223-3p mimics and inhibitors were purchased from Shanghai GeneChem Co., Ltd. To upregulate miR-223-3p expression, 2×10 5 cells were transfected with synthetic miR-223-3p mimics (final concentration: 50 nM), while a scrambled sequence served as the negative control (NC, 50 nM).

Techniques: Expressing, Gene Expression

Journal: International Journal of Molecular Medicine

Article Title: miR-223-3p promotes microglial lactylation and M1 polarization via the FBXW7/Notch1/Hes1/SIRT1 axis

doi: 10.3892/ijmm.2026.5849

Figure Lengend Snippet: miR-223-3p promotes microglial M1 polarization. (A) RT-qPCR analysis of miR-223-3p expression. (B) RT-qPCR analysis of M1/M2 polarization marker genes in microglia. (C) Western blot detection of iNOS and Arg-1 protein levels. (D) Flow cytometric analysis of ROS levels. (E) Heatmap of genes differentially expressed between the high- and low-miR-223-3p groups. (F) KEGG pathway enrichment analysis of differentially expressed genes. (G) Gene Set Enrichment Analysis of glycolysis-associated pathways in miR-223-3p-regulated microglia. ** P<0.01 and *** P<0.001. miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; iNOS, inducible nitric oxide synthase; ROS, reactive oxygen species; KEGG, Kyoto Encyclopedia of Genes and Genomes; NC, negative control.

Article Snippet: Synthetic miR-223-3p mimics and inhibitors were purchased from Shanghai GeneChem Co., Ltd. To upregulate miR-223-3p expression, 2×10 5 cells were transfected with synthetic miR-223-3p mimics (final concentration: 50 nM), while a scrambled sequence served as the negative control (NC, 50 nM).

Techniques: Quantitative RT-PCR, Expressing, Marker, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: miR-223-3p promotes microglial lactylation and M1 polarization via the FBXW7/Notch1/Hes1/SIRT1 axis

doi: 10.3892/ijmm.2026.5849

Figure Lengend Snippet: miR-223-3p promotes microglial M1 polarization through the regulation of lactylation. (A) Heatmap of metabolite profiles across three experimental groups. (B-E) LC-MS/MS quantification of (B) lactate, (C) pyruvate, (D) NADP + and (E) NADPH levels. (F and G) ECAR assays measuring basal and compensatory glycolysis. (H and I) OCR assays assessing mitochondrial respiration. (J) Lactate content assay assessing intracellular lactate level. (K) Western blot analysis of global lactylation levels. (L) Western blot analysis of lactylation levels following treatment with glycolytic inhibitors oxamate (10 mM; 24 h) and 2-DG (10 mM; 24 h). (M) Reverse transcription-quantitative PCR analysis of microglial polarization markers. (N and O) Flow cytometric analysis of ROS levels. * P<0.05, ** P<0.01 and *** P<0.001. miR, microRNA; ECAR, extracellular acidification rate; OCR, oxygen consumption rate; 2-DG, 2-deoxy-D-glucose; ROS, reactive oxygen species; NC, negative control.

Article Snippet: Synthetic miR-223-3p mimics and inhibitors were purchased from Shanghai GeneChem Co., Ltd. To upregulate miR-223-3p expression, 2×10 5 cells were transfected with synthetic miR-223-3p mimics (final concentration: 50 nM), while a scrambled sequence served as the negative control (NC, 50 nM).

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: miR-223-3p promotes microglial lactylation and M1 polarization via the FBXW7/Notch1/Hes1/SIRT1 axis

doi: 10.3892/ijmm.2026.5849

Figure Lengend Snippet: miR-223-3p promotes microglial lactylation and M1 polarization by suppressing SIRT1 expression. (A) RT-qPCR analysis of SIRT1 mRNA expression. (B) Western blot analysis of SIRT1 protein levels. (C) Western blot analysis of lactylation levels following treatment with SIRT1 activator SRT1720 (5 μ M; 24 h). (D) Western blot analysis of lactylation levels following treatment with SIRT1 inhibitor EX-527 (10 μ M; 24 h). (E and F) RT-qPCR and Western blot validation of SIRT1 overexpression in rescue experiments. (G) Western blot detection of global lactylation levels. (H) RT-qPCR analysis of microglial polarization markers. (I) Western blot analysis of iNOS and Arg-1 protein expression. (J and K) Flow cytometric analysis of ROS levels. * P<0.05, ** P<0.01 and *** P<0.001. miR, microRNA; SIRT1, sirtuin 1; RT-qPCR, reverse transcription-quantitative PCR; iNOS, inducible nitric oxide synthase; ROS, reactive oxygen species; NC, negative control; OE, overexpression.

Article Snippet: Synthetic miR-223-3p mimics and inhibitors were purchased from Shanghai GeneChem Co., Ltd. To upregulate miR-223-3p expression, 2×10 5 cells were transfected with synthetic miR-223-3p mimics (final concentration: 50 nM), while a scrambled sequence served as the negative control (NC, 50 nM).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Biomarker Discovery, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: miR-223-3p promotes microglial lactylation and M1 polarization via the FBXW7/Notch1/Hes1/SIRT1 axis

doi: 10.3892/ijmm.2026.5849

Figure Lengend Snippet: miR-223-3p suppresses SIRT1 transcription via the Notch1/Hes1 signaling pathway, thereby promoting microglial lactylation and M1 polarization. (A) RT-qPCR analysis of Notch1 and Hes1 mRNA levels. (B) Western blot analysis of NICD1 and Hes1 protein expression. (C) Western blot analysis of SIRT1 expression following silencing Hes1. (D) Western blot detection of global lactylation levels. (E) RT-qPCR analysis of microglial polarization markers. (F and G) RT-qPCR and western blot detection of SIRT1 following treatment with Notch1 inhibitor DAPT (20 nM; 24 h). (H) Western blot detection of global lactylation levels. (I) RT-qPCR analysis of microglial polarization markers. (J) Western blot analysis of iNOS and Arg-1 protein expression. (K) Flow cytometric analysis of ROS levels. * P<0.05, ** P<0.01 and *** P<0.001. miR, microRNA; SIRT1, sirtuin 1; RT-qPCR, reverse transcription-quantitative PCR; iNOS, inducible nitric oxide synthase; ROS, reactive oxygen species; NC, negative control; ns, not significant.

Article Snippet: Synthetic miR-223-3p mimics and inhibitors were purchased from Shanghai GeneChem Co., Ltd. To upregulate miR-223-3p expression, 2×10 5 cells were transfected with synthetic miR-223-3p mimics (final concentration: 50 nM), while a scrambled sequence served as the negative control (NC, 50 nM).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: miR-223-3p promotes microglial lactylation and M1 polarization via the FBXW7/Notch1/Hes1/SIRT1 axis

doi: 10.3892/ijmm.2026.5849

Figure Lengend Snippet: miR-223-3p targets FBXW7 to reduce Notch1 ubiquitination and degradation. (A) Venn diagram showing the overlap of predicted miR-223-3p targets from three databases. (B) Reverse transcription-quantitative PCR analysis of downstream target mRNA expression. (C) Western blot detection of FBXW7 protein expression. (D) Predicted binding site between miR-223-3p and the 3' untranslated region of FBXW7 . (E) Dual-luciferase reporter assay confirming the direct interaction between miR-223-3p and FBXW7 . (F) Effects of the proteasome inhibitor MG132 and the lysosomal inhibitor CQ on NICD1 protein expression in BV2 cells with altered miR-223-3p expression. (G) CHX chase assay showing the dynamics of NICD1 protein degradation in BV2 cells. (H and I) Co-immunoprecipitation analysis of the interaction between FBXW7 and NICD1. (J) Effect of FBXW7 overexpression on NICD1 ubiquitination in 293T cells. (K) Western blot analysis of FBXW7-mediated regulation of NICD1 expression and ubiquitination in microglia. ** P<0.01 and *** P<0.001. miR, microRNA; CHX, cycloheximide; NC, negative control; ns, not significant.

Article Snippet: Synthetic miR-223-3p mimics and inhibitors were purchased from Shanghai GeneChem Co., Ltd. To upregulate miR-223-3p expression, 2×10 5 cells were transfected with synthetic miR-223-3p mimics (final concentration: 50 nM), while a scrambled sequence served as the negative control (NC, 50 nM).

Techniques: Ubiquitin Proteomics, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Binding Assay, Luciferase, Reporter Assay, Immunoprecipitation, Over Expression, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: miR-223-3p promotes microglial lactylation and M1 polarization via the FBXW7/Notch1/Hes1/SIRT1 axis

doi: 10.3892/ijmm.2026.5849

Figure Lengend Snippet: Schematic representation of the proposed mechanism of miR-223-3p in microglial M1 polarization. miR, microRNA; SIRT1, sirtuin 1.

Article Snippet: Synthetic miR-223-3p mimics and inhibitors were purchased from Shanghai GeneChem Co., Ltd. To upregulate miR-223-3p expression, 2×10 5 cells were transfected with synthetic miR-223-3p mimics (final concentration: 50 nM), while a scrambled sequence served as the negative control (NC, 50 nM).

Techniques: