Journal: Diseases

Article Title: Impact of miR-181a on SIRT1 Expression and Senescence in Hutchinson–Gilford Progeria Syndrome

doi: 10.3390/diseases13080245

Figure Lengend Snippet: Interconnection between autophagy and inflammatory pathways. Under high-nutrient conditions, the mechanistic target of rapamycin (mTOR) is activated, leading to the inhibition of the autophagic pathway, which involves the initiation, elongation, maturation, and fusion of the autophagosome [ , , ]. Inflammation signaling typically results in the activation of NF-κB, which can occur through canonical or non-canonical mechanisms. The canonical activation of NF-κB is initiated by the binding of growth factors to tumor necrosis factor (TNF) or toll-like receptors (TLRs), which activate TNF receptor-associated factor (TRAF) proteins. This leads to the activation of the IKK complex, degradation of IKB, and translocation of the NF-ĸB1-RelA complex into the nucleus to activate proinflammatory genes. In contrast, the non-canonical pathway is triggered by signals from TNF receptors that activate the NF-ĸB-inducing kinase, leading to IKK activation [ , , , ]. TLR4 signaling also induces the JAK/STAT pathway, another critical component of inflammatory signaling [ , , ]. Autophagy and inflammation are connected through the DNA damage response. DNA damage activates the cytosolic DNA sensors cGAS and STING, which, in conjunction with ATM, can stimulate both inflammatory responses and autophagic vesicle formation, as well as the central cell-cycle regulator p53 [ , , , ]. Proteins analyzed by miRNA interactions are illustrated in green.

Article Snippet: Cultures were treated in the starving medium with 10 ng/ml transforming growth factor β1 (TGFβ1, HZ-1011, Proteintech, Rosemont, IL, USA) for 96 h and collected for RNA isolation or fixed for immunofluorescence staining. miR-181a-5p mimic (Thermo Fisher, miRVanaTM miR-181a-5p mimic, 4464066) and miR-181a-5p inhibitor (Thermo Fisher, miRVanaTM miR-181a-5p inhibitor, 4464084) were transfected in a concentration of 25 μM for 6 and 9 days and 10 nM for 6 and 9 days, respectively, at a cell density of 30–50%.

Techniques: Inhibition, Activation Assay, Binding Assay, Translocation Assay

Journal: Diseases

Article Title: Impact of miR-181a on SIRT1 Expression and Senescence in Hutchinson–Gilford Progeria Syndrome

doi: 10.3390/diseases13080245

Figure Lengend Snippet: Identification and profiling of miRNAs regulating autophagy and inflammation pathways in control and HGPS. ( A ) Literature search procedure. Proteins from both autophagy and inflammation pathways were examined for miRNA regulation, and overlapping miRNAs were analyzed using RT-qPCR. ( B ) RT-qPCR analysis. miRNAs identified through text mining were analyzed in normal and HGPS fibroblasts during replicative senescence. Control fibroblasts (GMO1651c, GMO1652c, GMO3349c, GMO1582B) and HGPS fibroblasts (HGADFN127, HGADFN188, HGADFN003) were compared at passages with a replicative senescence under 5% (young) and over 20% (old) intragroup, as well as same-aged groups. Relative expression levels were normalized to U6 small nuclear 1 (RNU-6). Graphs show mean values ± standard deviation. Statistical significance was calculated using ANOVA with Tukey’s post hoc test (* p < 0.05, ** p < 0.01, (control: n = 4, HGPS: n = 3)). Non-significant changes are not indicated.

Article Snippet: Cultures were treated in the starving medium with 10 ng/ml transforming growth factor β1 (TGFβ1, HZ-1011, Proteintech, Rosemont, IL, USA) for 96 h and collected for RNA isolation or fixed for immunofluorescence staining. miR-181a-5p mimic (Thermo Fisher, miRVanaTM miR-181a-5p mimic, 4464066) and miR-181a-5p inhibitor (Thermo Fisher, miRVanaTM miR-181a-5p inhibitor, 4464084) were transfected in a concentration of 25 μM for 6 and 9 days and 10 nM for 6 and 9 days, respectively, at a cell density of 30–50%.

Techniques: Control, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: Diseases

Article Title: Impact of miR-181a on SIRT1 Expression and Senescence in Hutchinson–Gilford Progeria Syndrome

doi: 10.3390/diseases13080245

Figure Lengend Snippet: Elevated TGFβ1 levels in HGPS promote upregulation of miR-181a-5p expression. ( A ) TGFβ1 mRNA expression level in three control (GMO5565, GMO5757c, HGFDFN369) and HGPS (HGADFN127, HGADFN003, HGADFN164) fibroblast cell strains. Comparisons were made between the young and old stages intragroup as well as same-aged stages. Expression was normalized to GAPDH. ( B ) Representative immunofluorescence staining of the control cell line 5757c P18 with α-SMA following TGFβ1 treatment. Scale bar = 50 µm ( C ) Quantification of immunofluorescence signal in control (GMO5757c P18) and HGPS (HGADFN003 P12) cell strains. Comparisons were made between treated and non-treated samples. ( D ) Western blot of α-SMA and GAPDH as a loading control in control (GMO5757c P18) and HGPS (HGADN271 P11) cell strains. ( E ) Relative miR-181a-5p levels following TGFß1 treatment in three control (HGFDFN369, GMO5757c, GMO3349c) and HGPS (HGADFN271, HGADFN003, HGADFN164) cell strains with a senescence index <5%. Expression was normalized to U6 small nuclear 1 (RNU-6). Comparisons were made between treated and non-treated samples. ( A , C , E ) Graphs show mean values ± standard deviation. Statistical significance was calculated using ANOVA with Tukey’s post hoc test (* p < 0.05, ** p < 0.01, **** p < 0.0001 ( n = 3)).

Article Snippet: Cultures were treated in the starving medium with 10 ng/ml transforming growth factor β1 (TGFβ1, HZ-1011, Proteintech, Rosemont, IL, USA) for 96 h and collected for RNA isolation or fixed for immunofluorescence staining. miR-181a-5p mimic (Thermo Fisher, miRVanaTM miR-181a-5p mimic, 4464066) and miR-181a-5p inhibitor (Thermo Fisher, miRVanaTM miR-181a-5p inhibitor, 4464084) were transfected in a concentration of 25 μM for 6 and 9 days and 10 nM for 6 and 9 days, respectively, at a cell density of 30–50%.

Techniques: Expressing, Control, Immunofluorescence, Staining, Western Blot, Standard Deviation