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: MiR-136-3p content in cell culture medium and uptake of extracellular miR-136-3p into cultured myotubes. MiRNA content in (A) human myotubes and (B) human pancreatic islets culture media. Results were first normalized using RNU1A1 and then presented in relation to miR-23a-3p content for n = 4 different donors for myotubes cultures and n = 4 donors for human islets. Left panel (C) shows bright-field image of cultured human myotubes and right panel (C) shows a representative fluorescence image of cultured human myotubes with cells exposed to human serum-derived EVs loaded with Cy3-miR-136-3p. Cy3 fluorescence (red) is detected in the whole cytoplasm of the human myotubes. (D) Representative fluorescence image of human myotubes exposed to HEK293 culture medium with EVs loaded with Cy3-miR-136-3p (red). (E) Representative image of human myotubes exposed to EVs loaded with TexasRed-labeled with a control RNA (orange). Nuclear Hoechst staining is shown in blue. Scale bar = 100 μm. EVs = extracellular vesicles; HEK293 = human embryonic kidney; miR = microRNA; Rel = relative; RNU1A1 = U1 small nuclear RNA.

Article Snippet: Human embryonic kidney (HEK293) cells were obtained from American Type Culture Collection (ATCC) and cultured in high-glucose (4.5 g/L) Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% (vol/vol) FBS.

Techniques: Cell Culture, Fluorescence, Derivative Assay, Labeling, Control, Staining

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: Human embryonic kidney (HEK293) cells were obtained from American Type Culture Collection (ATCC) and cultured in high-glucose (4.5 g/L) Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% (vol/vol) FBS.

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

Journal: Molecular Therapy. Nucleic Acids

Article Title: microRNA-422a promotes HIV replication and innate immune evasion by targeting MECP2

doi: 10.1016/j.omtn.2026.102844

Figure Lengend Snippet: miR-422a directly targets MECP2 (A) Schematic diagram showing the predicted miR-422a target sites within the MECP2 3′UTR. The predicted target seed sites and their corresponding mutated sequences are highlighted in red. (B) HEK-293T cells were cotransfected with MECP2 3′UTR luciferase reporter vector (wild-type or mutant versions), pRL-TK, and miR-422a mimic or negative control (NC) for 24 h. Cells were then harvested for luciferase analysis. Relative luciferase activity is shown. (C) Unstimulated or stimulated primary CD4+ T cells were transfected with miR-422a mimic or NC for 72 h. Cells were then collected to detect MECP2 mRNA levels by RT-qPCR. (D) Stimulated primary CD4+ T cells were transfected with the indicated concentrations of miR-422a mimic or NC for 72 h. Cells were then collected to detect MECP2 protein levels by western blot. β-actin was used as the endogenous control. (E) Relative quantification of MECP2 protein from the western blot in (D). (F) MECP2 protein expression was analyzed by western blot following the establishment of stable cell lines. (G) MECP2 KO-Jurkat or scramble-Jurkat cells were infected with HIV-1 NL4-3 . The percentage of Gag-positive cells was detected by flow cytometry at the indicated time point. (H and I) Stimulated primary CD4+ T cells were nucleofected with Cas9-RNP targeting MECP2 for 24 h, then infected with HIV-1 NL4-3 for 6 days. Cells and supernatants were collected for Gag detection and p24 measurements by flow cytometry (H) and ELISA (I), respectively. (J) MECP2 KO-Jurkat or scramble-Jurkat cells were transfected with miR-422a mimic or its NC for 24 h, then infected with HIV-1 NL4-3 (or left uninfected). Six days after infection, cells were collected, fixed, and permeabilized, then stained with RD-fluorescent Gag antibody to detect Gag expression by flow cytometry. The percentage of Gag-positive cells was determined by flow cytometry. Each dot represents data from one donor. Data are representative of the results of three independent experiments ( n = 3 biologically independent samples, mean ± SEM). Statistical significance was analyzed by unpaired or paired student t tests. p ≤ 0.05 [∗], p ≤ 0.01 [∗∗], p ≤ 0.001 [∗∗∗], p ≤ 0.0001 [∗∗∗∗].

Article Snippet: HEK-293T cells were obtained from American Type Culture Collection (ATCC) (#CRL-3216) and cultured in DMEM medium (Thermo Fisher Scientific, #11-965-092) supplemented with 10% FBS and 1% penicillin-streptomycin (Thermo Fisher Scientific, #15140122).

Techniques: Luciferase, Plasmid Preparation, Mutagenesis, Negative Control, Activity Assay, Transfection, Quantitative RT-PCR, Western Blot, Control, Quantitative Proteomics, Expressing, Stable Transfection, Infection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: microRNA-422a promotes HIV replication and innate immune evasion by targeting MECP2

doi: 10.1016/j.omtn.2026.102844

Figure Lengend Snippet: miR-422a directly targets MECP2 (A) Schematic diagram showing the predicted miR-422a target sites within the MECP2 3′UTR. The predicted target seed sites and their corresponding mutated sequences are highlighted in red. (B) HEK-293T cells were cotransfected with MECP2 3′UTR luciferase reporter vector (wild-type or mutant versions), pRL-TK, and miR-422a mimic or negative control (NC) for 24 h. Cells were then harvested for luciferase analysis. Relative luciferase activity is shown. (C) Unstimulated or stimulated primary CD4+ T cells were transfected with miR-422a mimic or NC for 72 h. Cells were then collected to detect MECP2 mRNA levels by RT-qPCR. (D) Stimulated primary CD4+ T cells were transfected with the indicated concentrations of miR-422a mimic or NC for 72 h. Cells were then collected to detect MECP2 protein levels by western blot. β-actin was used as the endogenous control. (E) Relative quantification of MECP2 protein from the western blot in (D). (F) MECP2 protein expression was analyzed by western blot following the establishment of stable cell lines. (G) MECP2 KO-Jurkat or scramble-Jurkat cells were infected with HIV-1 NL4-3 . The percentage of Gag-positive cells was detected by flow cytometry at the indicated time point. (H and I) Stimulated primary CD4+ T cells were nucleofected with Cas9-RNP targeting MECP2 for 24 h, then infected with HIV-1 NL4-3 for 6 days. Cells and supernatants were collected for Gag detection and p24 measurements by flow cytometry (H) and ELISA (I), respectively. (J) MECP2 KO-Jurkat or scramble-Jurkat cells were transfected with miR-422a mimic or its NC for 24 h, then infected with HIV-1 NL4-3 (or left uninfected). Six days after infection, cells were collected, fixed, and permeabilized, then stained with RD-fluorescent Gag antibody to detect Gag expression by flow cytometry. The percentage of Gag-positive cells was determined by flow cytometry. Each dot represents data from one donor. Data are representative of the results of three independent experiments ( n = 3 biologically independent samples, mean ± SEM). Statistical significance was analyzed by unpaired or paired student t tests. p ≤ 0.05 [∗], p ≤ 0.01 [∗∗], p ≤ 0.001 [∗∗∗], p ≤ 0.0001 [∗∗∗∗].

Article Snippet: HIV-1 NL4-3 viral stocks were generated by transfection of proviral DNA (BEI, #ARP-114) into HEK-293T cells via polyethylenimine (PEI, Polysciences, #26913-06-4) as described previously.

Techniques: Luciferase, Plasmid Preparation, Mutagenesis, Negative Control, Activity Assay, Transfection, Quantitative RT-PCR, Western Blot, Control, Quantitative Proteomics, Expressing, Stable Transfection, Infection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining