Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: NRDC is a direct target of miR-136-3p in human myotubes. Skeletal muscle NRDC mRNA is responsive to training and inactivity. (A) Tissue mRNA expression of NRDC from the Human Protein Atlas database showing enriched expression of NRDC in human skeletal muscle. (B) The miR-136-3p target site in the NRDC gene is highly conserved in mammals. (C) Luciferase activity in HEK293 cells co-transfected the NRDC 3’UTR and miR-136-3p with or without anti-miR136-3p inhibitors. miR-136-3p transfection downregulates NRDC (D) mRNA and (E) representative image of protein abundance in human myotubes. (F) Publicly available data ( GSE14413 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 6 weeks of endurance training ( n = 8). (G) Publicly available data ( GSE120862 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 2 months of aerobic training ( n = 10). (H) Publicly available data ( GSE14901 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 14 days of immobilization ( n = 24). * p < 0.05, ** p < 0.005. GSE = gene set enrichment; HEK293 = human embryonic kidney; miR = microRNA; NC = negative control; NRDC = nardilysin convertase; nTPM = normalized transcripts per million; si NRDC = small interfering RNA of NRDC ; UTR = untranslated region.

Article Snippet: MiR-136-3p was labeled with Cy3 using Silencer small interfering RNA (siRNA) Labeling Kit with Cy3 Dye (Thermo Fisher Scientific) and loaded into exosome-enriched EVs with Exo-Fect siRNA/miRNA Transfection Reagent (System Biosciences, Palo Alto, CA, USA).

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Quantitative Proteomics, Negative Control, Small Interfering RNA

Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: Cellular metabolism in human myotubes after miR-136-3p transfection or NRDC silencing. Mitochondrial respiration in miR-136-3p-transfected or NRDC- silenced human myotubes was monitored using the Mitochondrial Stress Test. (A) OCR and (B) ECAR were measured using the Seahorse XFe24 Extracellular Flux Analyzer. The trace shows representative data. (C) Quantification of the mitochondrial respiration data for basal respiration, maximal respiration, ATP production, and spare respiratory capacity obtained from 3 independent experiments. Human myotubes were transfected with miR-136-3p or siRNA against NRDC before determination of (D) uptake of radiolabeled glucose, (E) rates of radiolabeled glucose oxidation, (F) conversion of radiolabeled glucose into glycogen, (G) rate of radiolabeled palmitic acid oxidation, (H) protein synthesis as assessed by incorporation of puromycin, and (I) lactate release into the media. Results are expressed as mean ± standard error of the mean. * p < 0.05, ** p < 0.005 vs. control cells. ECAR = extracellular acidification rate; FCCP = carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; miR = microRNA; NC = negative control; NRDC = nardilysin convertase; ns = no significance; OCR = oxygen consumption rate; OigoA = oligomycin A; Rot/AA = rotenone and antimycin A; si NRDC = small interfering RNA of NRDC; siRNA = small interfering RNA; scr = negative control for small interfering RNA.

Article Snippet: MiR-136-3p was labeled with Cy3 using Silencer small interfering RNA (siRNA) Labeling Kit with Cy3 Dye (Thermo Fisher Scientific) and loaded into exosome-enriched EVs with Exo-Fect siRNA/miRNA Transfection Reagent (System Biosciences, Palo Alto, CA, USA).

Techniques: Transfection, Control, Negative Control, Small Interfering RNA

Journal: Non-coding RNA Research

Article Title: CircEif3c/miR-96–5p/PHF20L1/MEOX2 axis in perivascular preadipocyte exosomes mediates fibroblast dysfunction and vascular remodeling

doi: 10.1016/j.ncrna.2026.01.006

Figure Lengend Snippet: Characterization of cultured cells and the impact of high glucose on PVPAC function. (A and D) Immunofluorescence and (B and C) flow cytometry (FCM) identification of PVPACs/AFs. Characteristic surface markers of PVPACs and AFs were analyzed by FCM, with blue curves indicating isotype controls and red curves representing test samples. PVPACs and AFs exhibited positive expression of CD34, Pref1, Sca1, vimentin, and α-actin, respectively, scale bar = 50 μm. (E–J) Normal glucose (NG, 5.5 nmol/L) and high glucose (HG, 30 nmol/L) had different effects on the biological functions of PVPACs. (E) Glucose uptake assay. Quantitative determination of 2-[3H] deoxyglucose (2-DOG) uptake by PVPAC cells under stimulation of glucose at different concentrations. HG effectively stimulated glucose uptake in PVPAC, while it had no obvious change in NG group. n (the number of experiments) = 6 (F) EDU experiment, scale bar = 100 μm. (G) CCK8 assays. n (the number of experiments) = 3 (H) Crystal violet staining; Scale bar = 200 μm. (I) Scratch migration assays. Scale bar = 200 μm. (J) Adipogenic induction and differentiation assessed by Oil Red O staining, scale bar = 200 μm. Data are shown as mean ± SD from three independent experiments. n (the number of experiments) = 3. Statistical significance was determined by one-way ANOVA with Dunnett's post-hoc test; vs. the control (NG) group, ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; n.s, no significance.

Article Snippet: After 48 h of co-incubation with AFs using control mimic, siR-control, Exo-(siR-)circEif3c mimic, Exo-(siR-)miR-96–5p mimic, Exosome (PVPAC-Exo), GW4689, and Exo-(siR-) pAd-MEOX2, respectively, the results exhibited that PVPAC-Exo-circEif3c mimic, siR-miR-96–5p mimic, and siR-pAd-MEOX2 significantly stimulated the migration and proliferation of AFs.

Techniques: Cell Culture, Immunofluorescence, Flow Cytometry, Expressing, Staining, Migration, Control

Journal: Non-coding RNA Research

Article Title: CircEif3c/miR-96–5p/PHF20L1/MEOX2 axis in perivascular preadipocyte exosomes mediates fibroblast dysfunction and vascular remodeling

doi: 10.1016/j.ncrna.2026.01.006

Figure Lengend Snippet: PVPAC/AF co-culture model confirms that PVPAC-derived exosomes mediated intercellular communication. (A) Schematic diagram of primary PVPAC/AF cells culture with subsequent exosome isolation. (B) PVPAC/AF cells co-culture model. (B1) Schematic of the transwell-based co-culture setup. (B2) Representative TEM micrograph showing exosome morphology, scale bar = 100 nm. (B3) NTA-derived size distribution and concentration profiles of isolated exosomes. (B4) Crystal violet assay assessing cell proliferation under different glucose conditions, scale bar = 200 μm. (B5 and B6) Quantitative histograms corresponding to (B3) and (B4), respectively. Data are compared across mono-vs. co-culture systems under normal (NG) or high glucose (HG). vs NG + AF group, ∗P < 0.05, ∗∗P < 0.01. (C) Confocal microscopy tracking exosome uptake. Scale bar = 50 μm. (C1) PKH67-labeled PVPAC-derived exosomes (green) enriched in PVPAC cytoplasm. (C2) PKH67-labeled AF-derived exosomes abundant within AF cytoplasm. (C3) Time-course imaging displayed PVPAC-Exo accumulation in AFs, peaking at 4 h. (D) Quantification of migration and proliferation capacities in AFs after 24-h treatment with PVPAC-Exo (1 × 10 6 particles/mL), using PBS as a vehicle control, scale bar = 200 μm. (E) Impact of NG, HG, and GW4869 on exosome biology, scale bar = 100 nm. (E1) Morphology assessed by TEM. (E2) Proliferation measured via crystal violet. (E3) Western blot quantification of vimentin and exosomal markers (CD63, TSG101) in AFs. (F) RT-PCR analysis of circEif3c and miR-96–5p in AFs and PVPACs after 24 h NG vs. HG. HG induced highest circEif3c and lowest miR-96–5p expression in PVPACs. (G–K) Systematic comparison of exosomal protein signatures across culture modalities. (G1)Single-cell culture. (G2)Dual-cell co-culture. (G3) Co-culture pre-loaded with 1 × 10 6 /mL PVPAC-Exo. (H–K) Bar graphs present mean ± SD. n (the number of experiments) = 3; one-way ANOVA with Dunnett's post-test. ∗vs. respective NG group: ∗P < 0.05, ∗∗P < 0.01; vs. respective HG group: #P < 0.05, ##P < 0.01.

Article Snippet: After 48 h of co-incubation with AFs using control mimic, siR-control, Exo-(siR-)circEif3c mimic, Exo-(siR-)miR-96–5p mimic, Exosome (PVPAC-Exo), GW4689, and Exo-(siR-) pAd-MEOX2, respectively, the results exhibited that PVPAC-Exo-circEif3c mimic, siR-miR-96–5p mimic, and siR-pAd-MEOX2 significantly stimulated the migration and proliferation of AFs.

Techniques: Co-Culture Assay, Derivative Assay, Isolation, Concentration Assay, Crystal Violet Assay, Confocal Microscopy, Labeling, Imaging, Migration, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Comparison, Single Cell

Journal: Non-coding RNA Research

Article Title: CircEif3c/miR-96–5p/PHF20L1/MEOX2 axis in perivascular preadipocyte exosomes mediates fibroblast dysfunction and vascular remodeling

doi: 10.1016/j.ncrna.2026.01.006

Figure Lengend Snippet: Verification of the circEif3c and miR-96-5p interaction. (A – C) Identification of differentially expressed exosomal circRNAs (Exo-circRNAs). All the exosomal circRNAs in the culture medium following a co-culture model of AFs/PVPACs cells stimulated by HG (30 nM) for 24 h. The differential Exo-circRNA was analyzed by RNA microarray and displayed by volcano plot (A) and Heatmap (B). (C) A flowchart depicting the screening strategy. By integrating bioinformatics data from PVPAC-Exo, AF-Exo, and circBase, the top three highly expressed circRNAs were selected. (D) Selection of the optimal circRNA candidate. After the initial screening, the three candidate circRNAs were further narrowed down to the optimal one. Following 24 h of HG stimulation, qRT-PCR in PAPVCs revealed that circEif3c was expressed at significantly higher levels than the other two candidates. (E and F) Validation of circular structure of circEif3c . (E) Divergent primer design and Sanger sequencing validation for circEif3c amplification. (F)The primer of exosomal circEif3c was identified and verified by RT-PCR. Agarose gel analysis of circEif3c PCR products amplified with divergent versus convergent primers. (G) Stability assessment of circEif3c. Actinomycin D assay for circEif3c and Eif3c expression. (H) Efficiency of the circEif3c probe . A circRNA pull-down assay in AFs transfected with OE-circEif3c or OE-control plasmid confirmed the specific enrichment efficiency of the circEif3c probe, with a control probe as a negative control. (I – K) Screening of candidate miRNAs. Differential miRNA expression in exosomes was analyzed by microarray and displayed as a volcano plot (I) and a heatmap (J). (K) A flowchart showing the selection of miR-96–5p, miR-15a-5p, and miR-322–5p from the intersection of miRNA microarray data, TargetScan, and Circbank. (L) circRNA pull-down assay for miRNA interaction. In AFs transfected with OE-miR-96–5p mimic or control plasmid, the circEif3c probe exhibited significant enrichment of miR-96–5p, confirming their interaction. (M) AGO2-RIP assay. RIP analysis using an anti-Ago2 antibody in AF exosomes from cells overexpressing circEif3c or miR-96–5p demonstrated significant co-enrichment of both circEif3c and miR-96–5p with Ago2, indicating their incorporation into the RISC complex. (N) Luciferase reporter assay. Bioinformatic prediction identified putative miR-96–5p binding sites on circEif3c. Luciferase assay confirmed a direct interaction, as the miR-96–5p mimic suppressed the activity of the wild-type (WT) but not the mutant (Mut) circEif3c reporter. (O) miR-96-5p enrichment by circEif3c probe. The circRNA pull-down assay further confirmed the direct binding between the circEif3c probe and miR-96–5p. (P) Immunofluorescence colocalization. Cy5-labeled circEif3c and Cy3-labeled miR-96–5p plasmids showed clear colocalization in AFs 24 h post-transfection, providing visual evidence of their interaction, scale bars = 30 μm. n (the number of experiments) = 3, ∗P < 0.05; ∗∗P < 0.01.

Article Snippet: After 48 h of co-incubation with AFs using control mimic, siR-control, Exo-(siR-)circEif3c mimic, Exo-(siR-)miR-96–5p mimic, Exosome (PVPAC-Exo), GW4689, and Exo-(siR-) pAd-MEOX2, respectively, the results exhibited that PVPAC-Exo-circEif3c mimic, siR-miR-96–5p mimic, and siR-pAd-MEOX2 significantly stimulated the migration and proliferation of AFs.

Techniques: Co-Culture Assay, Microarray, Selection, Quantitative RT-PCR, Biomarker Discovery, Sequencing, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing, Pull Down Assay, Transfection, Control, Plasmid Preparation, Negative Control, Luciferase, Reporter Assay, Binding Assay, Activity Assay, Mutagenesis, Immunofluorescence, Labeling

Journal: Non-coding RNA Research

Article Title: CircEif3c/miR-96–5p/PHF20L1/MEOX2 axis in perivascular preadipocyte exosomes mediates fibroblast dysfunction and vascular remodeling

doi: 10.1016/j.ncrna.2026.01.006

Figure Lengend Snippet: Role of PVPAC-Exo-circEif3c in regulating AF biological functions and its potential mechanism. PVPAC-derived exosomal circEif3c (Exo-circEif3c) promoted AFs migration and proliferation, whereas silencing exosomal circEif3c suppresses these processes. (A) Time-course analysis of circEif3c expression in AFs after Exo-circEif3c treatment (0, 6, and 12 h; 0 h as control). (B) Stable silencing efficiency and specificity of circEif3c in AFs; Exo-siR-control served as the control. (C and D) Effects of PVPAC-Exo-siR- circEif3c-1 and -2 on AF migration and proliferation assessed by wound healing and proliferation assays. Scratch closure percentage and migrated cell numbers were quantified using ImageJ and GraphPad Prism 9.5, scale bar = 150 μm. (E) and (F) FCM analysis of AF proliferation and apoptosis following treatment with PVPAC-Exo-circEif3c, Exo-miR-96–5p, and Ad-MEOX2 interaction. (G) Western blot analysis of vimentin, PHF20L1, and MEOX2 expression in AFs under high glucose and circEif3c modulation. (H) Effects of Exo-circEif3c on the expression of vimentin, PHF20L1, MEOX2, and LC3 in AFs. GAPDH was used as a loading control. All data above are presented as mean ± SD from three independent experiments. vs. the control group, ∗P < 0.05, ∗∗P < 0.01(one-way ANOVA with Dunnett's post-hoc test), n (the number of experiments) = 3.

Article Snippet: After 48 h of co-incubation with AFs using control mimic, siR-control, Exo-(siR-)circEif3c mimic, Exo-(siR-)miR-96–5p mimic, Exosome (PVPAC-Exo), GW4689, and Exo-(siR-) pAd-MEOX2, respectively, the results exhibited that PVPAC-Exo-circEif3c mimic, siR-miR-96–5p mimic, and siR-pAd-MEOX2 significantly stimulated the migration and proliferation of AFs.

Techniques: Derivative Assay, Migration, Expressing, Control, Western Blot

Journal: Non-coding RNA Research

Article Title: CircEif3c/miR-96–5p/PHF20L1/MEOX2 axis in perivascular preadipocyte exosomes mediates fibroblast dysfunction and vascular remodeling

doi: 10.1016/j.ncrna.2026.01.006

Figure Lengend Snippet: The regulatory mchanisms of miR-96-5p in AF biology. (A) Time-course analysis of miR-96–5p expression in AFs treated with PVPAC-Exo at 0, 6, 12, and 24 h. (B) and (C) AFs were transfected with miR-96–5p mimic and NC mimic for 24 h. Then, the migration ability of AFs through migration experiments (B) and EDU assay (C) were evaluated using Image J and GraphPad Prism 9. vs. control mimic, ∗P < 0.05, ∗∗P < 0.01, n (the number of experiments) = 3, scale bar = 150 μm. (D) Correlation analysis between extracellular ncRNA levels in culture supernatant and intracellular expression of PHF20L1 and MEOX2 by RT-qPCR. (E) Bioinformatic prediction identifying PHF20L1 and MEOX2 as potential targets of miR-96–5p. (F) Predicted miR-96–5p binding sites in the 3′UTR of PHF20L1. (G) Luciferase reporter assay displayed that miR-96–5p mimic significantly reduced luciferase activity in the PHF20L1-WT group, but not in the PHF20L1-Mut group (vs. PHF20L1-Mut, ∗∗ P < 0.01), n (the number of experiments) = 3. (H) Bioinformatics analysis indicated the miR-96–5p binding sites in the 3′UTR of MEOX2. (I) Western blot exhibited no significant change in MEOX2 protein levels upon miR-96–5p overexpression. (J) Interaction network among miR-96–5p, circEif3c, PHF20L1, and MEOX2, constructed using GEPIA, ENCORI, miRNet, NDEx, and Cytoscape. (K) Western blot analysis of PHF20L1 and MEOX2 expression in AFs transfected with control-exosome, OE-exosomes, miR-comtrol mimic, miR-96–5p mimic, and UNC1215, respectively. vs. control-exosome, miR-comtrol mimic, ∗ P < 0.05, ∗∗ P < 0.01, n (the number of experiments) = 3. (L) Predicted protein–protein interaction interface between PHF20L1 and MEOX2 using Zdock 3.0.2 and PyMOL 2.5.5. (M) Co-IP experiments confirmed an interaction between PHF20L1 and MEOX2.

Article Snippet: After 48 h of co-incubation with AFs using control mimic, siR-control, Exo-(siR-)circEif3c mimic, Exo-(siR-)miR-96–5p mimic, Exosome (PVPAC-Exo), GW4689, and Exo-(siR-) pAd-MEOX2, respectively, the results exhibited that PVPAC-Exo-circEif3c mimic, siR-miR-96–5p mimic, and siR-pAd-MEOX2 significantly stimulated the migration and proliferation of AFs.

Techniques: Expressing, Transfection, Migration, EdU Assay, Control, Quantitative RT-PCR, Binding Assay, Luciferase, Reporter Assay, Activity Assay, Western Blot, Over Expression, Construct, Co-Immunoprecipitation Assay

Journal: Non-coding RNA Research

Article Title: CircEif3c/miR-96–5p/PHF20L1/MEOX2 axis in perivascular preadipocyte exosomes mediates fibroblast dysfunction and vascular remodeling

doi: 10.1016/j.ncrna.2026.01.006

Figure Lengend Snippet: CircEif3c modulates AF proliferation and migration via the miR-96-5p/PHF20L 1 /MEOX2 axis. (A–C) Cell migration and proliferation assays. AFs were transfected for 24 h with Ad-GFP, siR-circEif3c, miR-96–5p mimic, or siR-MEOX2. Migration (A) and proliferation (B) were quantified (C). (D–F) AFs were co-incubated for 48 h with control mimic, Exo-(siR-)circEif3c mimic, Exo-(siR-)miR-96–5p mimic, PVPAC-exosome (Exo-control), GW4869, or Exo-siR-pAd-MEOX2. Migration (D) and proliferation (E) were assessed (F), scale bar = 150 μm. (G) Cellular fluorescence immunolocalization. nuclei (DAPI, blue), circEif3c (Cy5, red), miR-96–5p (Cy3, orange-yellow), MEOX2 (GFP, green).Scale bar = 30 μm. The above data were presented as mean ± SD. vs. Ad-GFP group, ∗ P < 0.05, ∗∗ P < 0.01, n (the number of experiments) = 3.

Article Snippet: After 48 h of co-incubation with AFs using control mimic, siR-control, Exo-(siR-)circEif3c mimic, Exo-(siR-)miR-96–5p mimic, Exosome (PVPAC-Exo), GW4689, and Exo-(siR-) pAd-MEOX2, respectively, the results exhibited that PVPAC-Exo-circEif3c mimic, siR-miR-96–5p mimic, and siR-pAd-MEOX2 significantly stimulated the migration and proliferation of AFs.

Techniques: Migration, Transfection, Incubation, Control, Fluorescence

Journal: Non-coding RNA Research

Article Title: CircEif3c/miR-96–5p/PHF20L1/MEOX2 axis in perivascular preadipocyte exosomes mediates fibroblast dysfunction and vascular remodeling

doi: 10.1016/j.ncrna.2026.01.006

Figure Lengend Snippet: Exosomal circEif3c/miR-96-5p/PHF20L1/MEOX2 axis drives vascular remodeling in vivo. (A) Workflow: a stable PVPAC line over-expressing circEif3c supplied exosomes (Exo-Ad-circEif3c, 10 μg/mouse) that were micro-injected into perivascular adipose tissue (PVAT) surrounding the left carotid artery for 4 weeks to initiate remodeling. Subsequently, after the model was established, treatments with (Exo)-Ad-GFP, (Exo)-Ad- circEif3c, (Exo)-Ad-miR-96–5p, and (Exo)-Ad-Meox2 were administered continuously for 2 weeks, respectively. Normal saline (NS) was used as a negative control. (B) Representative H&E-stained cross-sections and concomitant ultrasonography of the common carotid artery. Black scale bars = 50 μm, yellow scale bars = 1 mm, and white scale bars = 0.1 s. (C) Immunohistochemistry. Scale bars = 20 μm. (D) Western blotting. (E) Quantification of protein levels. (F) Tissue localization of Cy5-labeled circEif3c by immunofluorescence, scale bar = 100 μm. (G) Fluorescence intensity quantification. (H) Comparative fluorescence imaging of vascular sections: (H1) Bright-field H&E vs. dark-field GFP before and after Ad-MEOX2 transfection; Scale bars = 50 μm; (H2) DM-remodeling vs MEOX2-intervention groups. Scale bars = 30 μm. (I) Whole-animal in vivo imaging of Cy5 signal. All quantitative data above are presented as mean ± SD. vs. control, ∗ P < 0.01.∗∗ P < 0.01. n (the number of animals) = 6 in each group.

Article Snippet: After 48 h of co-incubation with AFs using control mimic, siR-control, Exo-(siR-)circEif3c mimic, Exo-(siR-)miR-96–5p mimic, Exosome (PVPAC-Exo), GW4689, and Exo-(siR-) pAd-MEOX2, respectively, the results exhibited that PVPAC-Exo-circEif3c mimic, siR-miR-96–5p mimic, and siR-pAd-MEOX2 significantly stimulated the migration and proliferation of AFs.

Techniques: In Vivo, Expressing, Injection, Saline, Negative Control, Staining, Immunohistochemistry, Western Blot, Labeling, Immunofluorescence, Fluorescence, Imaging, Transfection, In Vivo Imaging, Control

Journal: Non-coding RNA Research

Article Title: CircEif3c/miR-96–5p/PHF20L1/MEOX2 axis in perivascular preadipocyte exosomes mediates fibroblast dysfunction and vascular remodeling

doi: 10.1016/j.ncrna.2026.01.006

Figure Lengend Snippet: Schematic illustration of the PVPAC-Exo mediated circEif3c/miR-96–5p/PHF20L1/MEOX2 axis regulating vascular remodeling.

Article Snippet: After 48 h of co-incubation with AFs using control mimic, siR-control, Exo-(siR-)circEif3c mimic, Exo-(siR-)miR-96–5p mimic, Exosome (PVPAC-Exo), GW4689, and Exo-(siR-) pAd-MEOX2, respectively, the results exhibited that PVPAC-Exo-circEif3c mimic, siR-miR-96–5p mimic, and siR-pAd-MEOX2 significantly stimulated the migration and proliferation of AFs.

Techniques: