Journal: Frontiers in Immunology

Article Title: MiR-34a deficiency enhances nucleic acid sensing and type I IFN signaling in a mouse model of Alzheimer’s disease

doi: 10.3389/fimmu.2026.1694824

Figure Lengend Snippet: Effects of miR-34a knockdown on ISGs expression in Gardiquimod-treated microglia in vitro . Depletion of miR-34a in BV2 cells by miR-34a inhibitor (A) and expression levels of the ISGs in miR-34a knockdown BV2 cells followed by Gardiquimod treatments (B) . Workflow of primary microglia isolation from adult mouse brain (C) created with BioRender.com ; the image in step 3 was documented in our lab) and assessment of isolated primary microglia by Iba1 immunostaining (D) Scale bar 20 µm) and qPCR analysis (E) . Depletion of miR-34a in primary microglia using miR-34a inhibitor, and the subsequent expression of the ISGs following Gardiquimod treatment (F) . The levels of interferon β in the primary microglia culture medium (G) . Data was analyzed by independent T-test. (Data from three independent experiments with duplicates *P<0.05, #P<0.01, and ##P<0.001).

Article Snippet: Congenic C57BL/6-Tg (Thy1-APPSwDutIowa)BWevn/Mmjax (referred to as Tg-SwDI, Stock No: 34843-JAX) and B6(Cg)-Mir34ATM1LHE/J (referred to as miR-34a KO or miR-34a -/- , Stock No: 018279) mice were obtained from Jackson Laboratory.

Techniques: Knockdown, Expressing, In Vitro, Isolation, Immunostaining

Journal: Frontiers in Immunology

Article Title: MiR-34a deficiency enhances nucleic acid sensing and type I IFN signaling in a mouse model of Alzheimer’s disease

doi: 10.3389/fimmu.2026.1694824

Figure Lengend Snippet: Effects of miR-34a KO (miR-34a -/- ) on exploratory activity and anxiety-like behaviors in open-field. The percentages of peripheral time (A) , central time (B) , moving fast time (C) , moving slow time (D) , resting time (E) , and total travelled distances (F) in a 5-min session are shown as mean ± SD for 3 days. Data was analyzed by 2 x 2 x 3 ANOVAs with repeated measures on the day factor. (miR-34a +/+ Non-Tg: n=11, miR-34a -/- Non-Tg: n=12, miR-34a +/+ Tg-SwDI: n=9, miR34a -/- Tg-SwDI: n=10).

Article Snippet: Congenic C57BL/6-Tg (Thy1-APPSwDutIowa)BWevn/Mmjax (referred to as Tg-SwDI, Stock No: 34843-JAX) and B6(Cg)-Mir34ATM1LHE/J (referred to as miR-34a KO or miR-34a -/- , Stock No: 018279) mice were obtained from Jackson Laboratory.

Techniques: Activity Assay

Journal: Frontiers in Immunology

Article Title: MiR-34a deficiency enhances nucleic acid sensing and type I IFN signaling in a mouse model of Alzheimer’s disease

doi: 10.3389/fimmu.2026.1694824

Figure Lengend Snippet: Effects of miR-34a KO (miR-34a -/- ) on cognitive function in Morris water maze. Total path lengths (cm) per day (A) , Total escape latencies per day (s) (B) , and Percentages of time spent in the target quadrant in which the hidden platform were previously placed are shown as mean ± SD (C) . Data was analyzed by 2 x 2 x 5 ANOVAs with repeated measures on the day factor for (A, B) and analyzed by one-way ANOVA and Post-hoc LSD tests were used for (C) . (miR-34a +/+ Non-Tg: n=11, miR-34a -/- Non-Tg: n=12, miR-34a +/+ Tg-SwDI: n=9, miR34a -/- Tg-SwDI: n=10, *P < 0.05).

Article Snippet: Congenic C57BL/6-Tg (Thy1-APPSwDutIowa)BWevn/Mmjax (referred to as Tg-SwDI, Stock No: 34843-JAX) and B6(Cg)-Mir34ATM1LHE/J (referred to as miR-34a KO or miR-34a -/- , Stock No: 018279) mice were obtained from Jackson Laboratory.

Techniques:

Journal: Frontiers in Immunology

Article Title: MiR-34a deficiency enhances nucleic acid sensing and type I IFN signaling in a mouse model of Alzheimer’s disease

doi: 10.3389/fimmu.2026.1694824

Figure Lengend Snippet: Effects of miR-34a KO (miR-34a -/- ) on Aβ load and APP processing in the brain. Detection of Aβ deposits by immunohistochemistry with anti-Aβ antibody, 6E10 in miR-34a +/+ (A) and miR-34a -/- (B) Tg-SwDI mice. Scale bars 200 µm. Average percentages of areas showing Aβ immunereactivity measured by morphometry in the polymorph layer of the dentate gyrus (PoDG) are shown (C) . The levels of soluble (D) and insoluble (E) Aβ40 and Aβ42 in the cerebral cortex were measured by ELISA. Data are shown as mean ± SD. The ratios of insoluble Aβ42 to Aβ40 in the cerebral cortex (F) . Effects of miR-34a KO on levels of IFITM3 (G, H) in lysates of the cerebral cortex were analyzed by western blotting using anti-ITIFM3 antibody (G) , and bar graph represents the results of densitometric analysis of IFITM3 (H) after normalizing with GAPDH levels. Data was analyzed by independent T-test. (A-F) miR-34a +/+ Tg-SwDI: n=9, miR34a -/- Tg-SwDI: n=10, *P < 0.05, #P < 0.01 and ##P < 0.001; G and H: n=4/group, *P < 0.05).

Article Snippet: Congenic C57BL/6-Tg (Thy1-APPSwDutIowa)BWevn/Mmjax (referred to as Tg-SwDI, Stock No: 34843-JAX) and B6(Cg)-Mir34ATM1LHE/J (referred to as miR-34a KO or miR-34a -/- , Stock No: 018279) mice were obtained from Jackson Laboratory.

Techniques: Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: MiR-34a deficiency enhances nucleic acid sensing and type I IFN signaling in a mouse model of Alzheimer’s disease

doi: 10.3389/fimmu.2026.1694824

Figure Lengend Snippet: Effect of miR-34a KO on microglia activation by IBA1 staining. Brain sections were subjected to immunofluorescence staining using anti-IBA1 antibody (green) and DAPI staining (blue). The dentate gyrus from miR-34a +/+ (A) and miR-34a -/- (B) Tg-SwDI mice are shown. Scale bars 50 µm. The total number of microglia/35 µm thick section (C) and the ratio of microglial cell body to cell size (D) in the dentate gyrus were determined. Data was analyzed by independent T-test. (miR-34a +/+ Tg-SwDI: n=9, miR34a -/- Tg-SwDI: n=10, ##P < 0.001).

Article Snippet: Congenic C57BL/6-Tg (Thy1-APPSwDutIowa)BWevn/Mmjax (referred to as Tg-SwDI, Stock No: 34843-JAX) and B6(Cg)-Mir34ATM1LHE/J (referred to as miR-34a KO or miR-34a -/- , Stock No: 018279) mice were obtained from Jackson Laboratory.

Techniques: Activation Assay, Staining, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: MiR-34a deficiency enhances nucleic acid sensing and type I IFN signaling in a mouse model of Alzheimer’s disease

doi: 10.3389/fimmu.2026.1694824

Figure Lengend Snippet: Effects of miR-34a KO on expression levels of homeostatic and DAM genes. Expression levels (Heatmap) of marker genes for three different microglial stages (homeostatic, stage 1 DAM, and stage 2 DAM) are shown as fold changes miR-34a -/- to miR-34a +/+ Tg-SwDI mice, based on bulk RNAseq of the hippocampus (A) . (n=5/group, *Q < 0.05, ##Q < 0.001) Sequence alignment of the mature miR-34a with CSF1R 3’ untranslated region (3’UTR). The seed sequences and target mRNA are highlighted in red (B) . Effects of miR-34a KO on levels of CSF1R in lysates of the cerebral cortex were analyzed by western blotting using CSF1R antibody (C) and bar graph represents the results of densitometric analysis of CSF1R after normalizing with GAPDH levels. Data was analyzed by independent T-test. (n=4/group, ##P < 0.001).

Article Snippet: Congenic C57BL/6-Tg (Thy1-APPSwDutIowa)BWevn/Mmjax (referred to as Tg-SwDI, Stock No: 34843-JAX) and B6(Cg)-Mir34ATM1LHE/J (referred to as miR-34a KO or miR-34a -/- , Stock No: 018279) mice were obtained from Jackson Laboratory.

Techniques: Expressing, Marker, RNA sequencing, Sequencing, Western Blot

Journal: Frontiers in Immunology

Article Title: MiR-34a deficiency enhances nucleic acid sensing and type I IFN signaling in a mouse model of Alzheimer’s disease

doi: 10.3389/fimmu.2026.1694824

Figure Lengend Snippet: Effects of miR-34a KO on hippocampal transcriptome in Tg-SwDI mice. Bubble plot of GO enrichment analysis by Enrichr (A) . The y-axis indicates the annotated terms of GO biological process in order of adjusted P value (-log10 (FDR)) and x-axis indicates combined score that represents a comprehensive metric combining the p-value and z-score. The bubble sizes represent the number of genes enriched in a specific GO term. The bubble color represents the adjusted P value. Volcano plot of DEGs between miR-34a -/- and miR-34a +/+ Tg-SwDI mice (B) . Red and blue dots represent upregulated and downregulated genes, respectively, and gray dots represent genes with no significant difference. The gray horizontal line represents 0.05 FDR and the gray vertical line indicates the log2FC threshold of 0.1. (n=5/group).

Article Snippet: Congenic C57BL/6-Tg (Thy1-APPSwDutIowa)BWevn/Mmjax (referred to as Tg-SwDI, Stock No: 34843-JAX) and B6(Cg)-Mir34ATM1LHE/J (referred to as miR-34a KO or miR-34a -/- , Stock No: 018279) mice were obtained from Jackson Laboratory.

Techniques:

Journal: ACS Omega

Article Title: Aptamer-Driven Nanozyme-SERS Bifunctional Sensors with Enrichment Separation Effect for Ultrasensitive Detection of Liver-Fibrosis-Related Biomarkers

doi: 10.1021/acsomega.5c12530

Figure Lengend Snippet: Correlation analysis of target miRNAs with disease. (A) Volcano plot of differentially expressed miRNAs in liver samples from three groups of CCl 4 -induced liver fibrosis mice and three groups of corn-oil-treated normal mice. (B) Intersection genes between liver fibrosis differentially expressed genes and mmu-miR-34a-5p target genes. (C) GO enrichment analysis bubble chart. (D) KEGG enrichment analysis Sankey bubble chart.

Article Snippet: Sstreptavidin-modified Fe 3 O 4 nanoparticles (400 nm) and nucleotides mentioned in , including miR-34a, single-base mismatch sequence (MT1), triple-base mismatch sequence (MT3), random sequence, nucleic acid aptamer sequence (cDNA), and nucleic acid aptamer complementary sequence (H 1 ), were all procured from Sangon Biotech (Shanghai) Co., Ltd. (China).

Techniques:

Journal: ACS Omega

Article Title: Aptamer-Driven Nanozyme-SERS Bifunctional Sensors with Enrichment Separation Effect for Ultrasensitive Detection of Liver-Fibrosis-Related Biomarkers

doi: 10.1021/acsomega.5c12530

Figure Lengend Snippet: Performance evaluation of nanozyme SERS sensors: (A) SERS spectra at different time intervals and (B) two-dimensional intensity heatmap. (C) SERS spectra and local method plots of nanozyme SERS sensors from different batches. (D) SERS spectra of nanozyme SERS sensors stored for different days and a line chart of signal intensity at 1602 cm –1 . (E) Scatter plot of 20 randomly selected positions and (F) 3D surface heatmap with projections. (G) Bar chart demonstrating nanozyme SERS sensor selectivity for miR-34a. (H) Effect of small-molecule interferences in simulated serum on miR-34a detection. (I) The effect of mismatched miRNA and increased serum component concentrations on miR-34a detection.

Article Snippet: Sstreptavidin-modified Fe 3 O 4 nanoparticles (400 nm) and nucleotides mentioned in , including miR-34a, single-base mismatch sequence (MT1), triple-base mismatch sequence (MT3), random sequence, nucleic acid aptamer sequence (cDNA), and nucleic acid aptamer complementary sequence (H 1 ), were all procured from Sangon Biotech (Shanghai) Co., Ltd. (China).

Techniques:

Journal: ACS Omega

Article Title: Aptamer-Driven Nanozyme-SERS Bifunctional Sensors with Enrichment Separation Effect for Ultrasensitive Detection of Liver-Fibrosis-Related Biomarkers

doi: 10.1021/acsomega.5c12530

Figure Lengend Snippet: SERS spectra of miR-34a at different concentrations in serum (A) and a heatmap at 1602 cm –1 (B). Linear equation fitted between the characteristic peak intensity at 1602 cm –1 and the logarithm of the miR-34a concentration (C).

Article Snippet: Sstreptavidin-modified Fe 3 O 4 nanoparticles (400 nm) and nucleotides mentioned in , including miR-34a, single-base mismatch sequence (MT1), triple-base mismatch sequence (MT3), random sequence, nucleic acid aptamer sequence (cDNA), and nucleic acid aptamer complementary sequence (H 1 ), were all procured from Sangon Biotech (Shanghai) Co., Ltd. (China).

Techniques: Concentration Assay

Journal: ACS Omega

Article Title: Aptamer-Driven Nanozyme-SERS Bifunctional Sensors with Enrichment Separation Effect for Ultrasensitive Detection of Liver-Fibrosis-Related Biomarkers

doi: 10.1021/acsomega.5c12530

Figure Lengend Snippet: Detection of miR-34a in a mouse model of liver fibrosis. (A) Average SERS spectra from serum samples of mice with different degrees of liver fibrosis. (B) Quantitative differentiation of SERS intensity at 1602 cm –1 in liver tissue sections from mice with varying fibrosis stages via t test.

Article Snippet: Sstreptavidin-modified Fe 3 O 4 nanoparticles (400 nm) and nucleotides mentioned in , including miR-34a, single-base mismatch sequence (MT1), triple-base mismatch sequence (MT3), random sequence, nucleic acid aptamer sequence (cDNA), and nucleic acid aptamer complementary sequence (H 1 ), were all procured from Sangon Biotech (Shanghai) Co., Ltd. (China).

Techniques:

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: miR-34a-5p was selected as the key miRNA in the differential regulation of autophagy in testicular somatic cells by fluoride. (A) Network toxicological analysis results of miRNAs related to fluoride-induced male reproductive injury. (B) HAMDB database analysis results of autophagy-related candidate miRNAs. (C and D) qRT-PCR revealed autophagy-related candidate miRNA expression changes in (C) TM3 and (D) TM4 cells after fluoride treatment. (E) FISH of miR-34a-5p in the testis of control and fluoride-treated mice (dashed areas indicate Leydig cells, and white arrows indicate Sertoli cells). All values in the figure are means ± SEM. n = 3. * P < 0.05, ** P < 0.01.

Article Snippet: Sections (4 μm) of testicular tissue digested with proteinase K for 15 min were used with FAM-labeled miR-34a-5p or Cy3-labeled Rest probes (Servicebio; Table ).

Techniques: Quantitative RT-PCR, Expressing, Control

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: Fluoride differentially regulates miR-34a-5p to modulate autophagy in testicular somatic cells. (A to F) Testicular somatic cells were treated with lentiviruses expressing miR-34a-5p/miR-NC and Inhibitor-miR-34a-5p/Inhibitor-NC. (A and D) Relative expression of miR-34a-5p. (B, C, E, and F) Relative expression of LC3B and p62 proteins. (G to I) miR-34a-5p overexpression rescued fluoride-treated TM3 cells. (G) Relative expression of miR-34a-5p. (H and I) Relative expression of LC3B and p62 proteins. (J to L) miR-34a-5p knockdown rescued fluoride-treated TM4 cells. (J) Relative expression of miR-34a-5p. (K and L) Relative expression of LC3B and p62 proteins. All values in the figure are means ± SEM. n = 3. * P < 0.05, ** P < 0.01 versus control, miR-NC, or Inhibitor-NC; # P < 0.05, ## P < 0.01 versus 0.25 mM NaF.

Article Snippet: Sections (4 μm) of testicular tissue digested with proteinase K for 15 min were used with FAM-labeled miR-34a-5p or Cy3-labeled Rest probes (Servicebio; Table ).

Techniques: Expressing, Over Expression, Knockdown, Control

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: miR-34a-5p targets REST in testicular somatic cells exposed to fluoride. (A) Expression changes of p62 mRNA in TM3 and TM4 cells after fluoride treatment and overexpression or knockdown of miR-34a-5p. (B) Prediction results of miR-34a-5p target genes by miRWalk combined with ChEA3 database. (C and F) Differential expression of the top 15 predicted target genes after fluoride treatment in (C) TM3 and (F) TM4 cells. (D, E, G, and H) Relative mRNA expression of Elf1 , Foxo1 , Foxp1 , Klf10 , Mef2a , Rest , Smad5 , and Yy1 . (I) Schematic showing the targeted binding sites of miR-34a-5p to FOXO1 and REST 3′ UTR. (J and K) The histogram shows the relative fluorescence intensity of each group. (L) FISH of miR-34a-5p and Rest mRNA in testicular tissue from control mice (white arrow indicates the positive area within the Sertoli cells or Leydig cells). (M and N) Differential expression of REST protein in testicular somatic cells after overexpression or knockdown of miR-34a-5p. All values in the figure are means ± SEM. n = 3. * P < 0.05, ** P < 0.01.

Article Snippet: Sections (4 μm) of testicular tissue digested with proteinase K for 15 min were used with FAM-labeled miR-34a-5p or Cy3-labeled Rest probes (Servicebio; Table ).

Techniques: Expressing, Over Expression, Knockdown, Quantitative Proteomics, Binding Assay, Fluorescence, Control

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: miR-34a-5p targets REST to regulate autophagy in testicular somatic cells. (A to F) TM3 and TM4 cells were treated with lentivirus expressing OE-REST, si-REST, OE-NC, or si-NC. (A and D) Relative Rest mRNA expression. (B, C, E, and F) Relative LC3B, p62, and REST protein expression. (G to K) REST overexpression rescued TM3 and TM4 cells overexpressing miR-34a-5p. (G and H) Relative miR-34a-5p, Rest , and p62 mRNA expression. (I to K) Relative REST, LC3B, and p62 protein levels. All values in the figure are means ± SEM. n = 3. ns indicates not significant; * P < 0.05, ** P < 0.01 versus control; # P < 0.05, ## P < 0.01 versus miR-34a-5p.

Article Snippet: Sections (4 μm) of testicular tissue digested with proteinase K for 15 min were used with FAM-labeled miR-34a-5p or Cy3-labeled Rest probes (Servicebio; Table ).

Techniques: Expressing, Over Expression, Control

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: Mechanism diagram of fluoride-induced reproductive injury in male mice by differential regulation of miR-34a-5p targeting REST to modulate autophagy in testicular somatic cells.

Article Snippet: Sections (4 μm) of testicular tissue digested with proteinase K for 15 min were used with FAM-labeled miR-34a-5p or Cy3-labeled Rest probes (Servicebio; Table ).

Techniques:

Journal: GeroScience

Article Title: Angiotensin II, miR-34a, and AGTRAP crosstalk in arterial smooth muscle cells

doi: 10.1007/s11357-025-02018-5

Figure Lengend Snippet: Age-associated increase of miR-34a in monkey and rat central arteries. A miR-34a positively correlated with age in the CCA of NHP 8 to 26 years of age ( n = 10) ( Y = 0.09097 x + 0.1310; Rs = 0.6727; * p < 0.05). B miR-34a expression was higher in the CCA of 30-month (old) vs 8-month (young) old rats ( n = 5 in each group). C A higher miR-34a expression was also found in the aorta of old vs young rats ( n = 13 in each group), and this difference was present both in the adventitia ( n = 4 young, n = 5 old) and in the media ( n = 5 in each group). Statistical analysis was performed using a Mann–Whitney U test (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: Experiments with mice were performed in conformity with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and in accordance with the experimental protocol approved by the Animal Welfare Organization (OPBA) of the Italian National Ministry of Health and Cogentech (824/2020-PR). miR-34a −/− (miR-34a KO) mouse line was purchased from the Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Expressing, MANN-WHITNEY

Journal: GeroScience

Article Title: Angiotensin II, miR-34a, and AGTRAP crosstalk in arterial smooth muscle cells

doi: 10.1007/s11357-025-02018-5

Figure Lengend Snippet: Angiotensin II induces miR-34a upregulation and AGTRAP downmodulation in isolated arterial rat and human aortic smooth muscle cells. A VSMC isolated from 8-month (young) and 30-month (old) old rats were treated with Ang II (100 nM for 48 h). Ang II enhanced miR-34a expression in VSMC from old rats but had no effect in VSMC from young rats ( n = 8 in each group). B HASMC ( n = 6 in each group) were treated with Ang II alone (1 µM for 24 h), or with Valsartan (1 µM for 1 h), followed by treatment with Ang II (1 µM for 24 h). Ang II increased miR-34a expression and this effect was abolished by pretreatment with Valsartan. C HASMC was treated with Ang II (1 µM for 24 h). Ang II decreased AGTRAP ( n = 6 in each group) and SIRT1 mRNA ( n = 6–7). D Representative western blot analysis of AGTRAP and SIRT1 and densitometric ratio of AGTRAP and SIRT1 normalized to tubulin protein levels ( n = 7 in each group). Ang II decreased both AGTRAP and SIRT1 protein levels. The statistical analysis was performed using the Wilcoxon t -test ( * p < 0.05; ** p < 0.01)

Article Snippet: Experiments with mice were performed in conformity with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and in accordance with the experimental protocol approved by the Animal Welfare Organization (OPBA) of the Italian National Ministry of Health and Cogentech (824/2020-PR). miR-34a −/− (miR-34a KO) mouse line was purchased from the Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Isolation, Expressing, Western Blot

Journal: GeroScience

Article Title: Angiotensin II, miR-34a, and AGTRAP crosstalk in arterial smooth muscle cells

doi: 10.1007/s11357-025-02018-5

Figure Lengend Snippet: Aging and miR-34a modulate AGTRAP expression. Experiments in ( A–B ) were performed with VSMC isolated from 8-month-old (young) and 30-month-old (old) rats. A AGTRAP mRNA expression was not modulated by aging in VSMC of young ( n = 9) and old ( n = 6) rats. B AGTRAP protein was lower in VSMC of old rats. Representative western blot analysis of AGTRAP in young and old rat VSMC and densitometric ratio of AGTRAP normalized to vinculin protein levels ( n = 5, young; n = 6, old). C–D AGTRAP expression was modulated by miR-34a and aging in vivo. Thoracic aortas of WT and miR-34a KO male mice, 2-month-old (young) and 18-month-old (old), were tested for AGTRAP expression by immunohistochemistry. C Aging was associated with markedly lower AGTRAP protein expression in both WT and miR-34a KO mice. AGTRAP was higher in young miR-34a KO mice compared to the WT control. In old mice, AGTRAP exhibited a trend toward higher expression in miR-34a KO vs WT, but the difference was not statistically significant. The percentage of positive area per AGTRAP was calculated by determining the ratio of positive AGTRAP area to total aortic cross-sectional area in one section per animal and multiplying by 100. Each circle or square in the graph represents data from a single mouse (WT young, n = 8; WT old, n = 4; miR-34a KO, young, n = 9; miR-34a KO, old, n = 5). D Representative images (one mouse per group) of the quantification shown in ( C ) of an aortic section field stained for AGTRAP (brown signal) with a specific antibody and counterstained with hematoxylin (purple signal). Calibration bar applied to all panels = 20 µm. E AGTRAP is a direct target of miR-34a. HEK293 were transfected with firefly luciferase constructs containing the Human 3′UTR-AGTRAP wild-type (wt) or mutated in miR-34a seed sequence (mut). The panel shows a schematic representation of miR-34a binding sites. The wt and mut seed sequences are indicated in bold, whereas mut nucleotides are indicated in small letters in red. The seed sequence nucleotides indicated are specific for the 3′UTR of Human AGTRAP ENST00000376627.2. F These constructs were co-transfected with a plasmid encoding either miR-34a or miR-scramble (miR-scr) sequences as control. Values were normalized according to Renilla luciferase activity ( n = 6 in each group). The overexpression of miR-34a decreased luciferase activity of 3′UTR-AGTRAP wt but had no effect on 3′UTR-AGTRAP mut. In ( A ), ( B ), and ( C ) the statistical analysis was performed using Mann–Whitney U test. In ( F ), statistical analysis was performed using Wilcoxon t -test ( *p<0.05 ; ** p < 0.01; *** p < 0.001)

Article Snippet: Experiments with mice were performed in conformity with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and in accordance with the experimental protocol approved by the Animal Welfare Organization (OPBA) of the Italian National Ministry of Health and Cogentech (824/2020-PR). miR-34a −/− (miR-34a KO) mouse line was purchased from the Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Expressing, Isolation, Western Blot, In Vivo, Immunohistochemistry, Control, Staining, Transfection, Luciferase, Construct, Sequencing, Binding Assay, Plasmid Preparation, Activity Assay, Over Expression, MANN-WHITNEY

Journal: GeroScience

Article Title: Angiotensin II, miR-34a, and AGTRAP crosstalk in arterial smooth muscle cells

doi: 10.1007/s11357-025-02018-5

Figure Lengend Snippet: Effect of miR-34a overexpression and AGTRAP knockdown on miR-34a targets in isolated HASMC. A–C HASMC were infected for 48 h either with a lentivirus encoding miR-34a or with a control virus (miR-scr); miR-34a ( n = 8 in each group), AGTRAP ( n = 9 in each group), and SIRT1 ( n = 8 in each group). miR-34a and mRNA expression were quantified by RT-qPCR. A miR-34a increased, and B AGTRAP and SIRT1 mRNA levels were lower in miR-34a overexpressing cells compared to scramble control. C Representative western blot analysis and densitometric ratio of AGTRAP and SIRT1, normalized to β-actin protein levels ( n = 6 in each group), demonstrated a significant decrease of AGTRAP and SIRT1 protein in response to miR-34a forced expression. D–F HASMC were infected either with the lentivirus carrying AGTRAP-specific shRNA or with the control virus (sh-scr). AGTRAP ( n = 6 in each group), miR-34a ( n = 6 in each group), and SIRT1 ( n = 6 in each group). miR-34a and mRNA expression were quantified by RT-qPCR. D shAGTRAP decreased AGTRAP mRNA. E Upon AGTRAP knockdown, miR-34a increased, whereas SIRT1 mRNA decreased. F Representative western blot analysis and densitometric ratio demonstrate that AGTRAP and SIRT1 protein expression levels decreased upon AGTRAP knockdown. Statistical analysis was performed using the Wilcoxon t- test (* p < 0.05; ** p < 0.01)

Article Snippet: Experiments with mice were performed in conformity with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and in accordance with the experimental protocol approved by the Animal Welfare Organization (OPBA) of the Italian National Ministry of Health and Cogentech (824/2020-PR). miR-34a −/− (miR-34a KO) mouse line was purchased from the Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Over Expression, Knockdown, Isolation, Infection, Control, Virus, Expressing, Quantitative RT-PCR, Western Blot, shRNA

Journal: GeroScience

Article Title: Angiotensin II, miR-34a, and AGTRAP crosstalk in arterial smooth muscle cells

doi: 10.1007/s11357-025-02018-5

Figure Lengend Snippet: A negative feedback loop exists between AGTRAP and miR-34a in isolated HASMC. HASMC were co-infected with lentiviruses encoding AGTRAP and miR-34a. Single infection either with AGTRAP or miR-34a was performed together with miR-scramble virus or AGTRAP backbone vector, respectively. A AGTRAP overexpression significantly decreased miR-34a expression under baseline conditions but not when miR-34a was overexpressed (miR-scr, n = 11; AGTRAP, n = 8; miR-34a, n = 7; AGTRAP + miR-34a, n = 4). B AGTRAP mRNA diminished in response to miR-34a, and this effect was abolished when both miR-34a and AGTRAP were overexpressed (miR-scr, n = 12; miR-34a, n = 10; AGTRAP, n = 7; AGTRAP + miR-34a, n = 6); C SIRT1 mRNA diminished in response to miR-34a overexpression both under baseline conditions and when both miR-34a and AGTRAP were co-infected (miR-scr, n = 8; miR-34a, n = 8; AGTRAP, n = 6; AGTRAP + miR-34a, n = 7). D Representative Western blot analysis of SIRT1 and AGTRAP protein expression levels in HASMC. E–F Densitometric ratio of AGTRAP and SIRT1, respectively, normalized to tubulin protein levels; E AGTRAP forced expression abolished the effect of miR-34a to decrease AGTRAP protein (miR-scr, n = 10; miR-34a, n = 10; AGTRAP, n = 4; AGTRAP + miR-34a, n = 4). F SIRT1 protein decreased in response to miR-34a forced expression and increased when AGTRAP was overexpressed. The effect of AGTRAP to downmodulate SIRT1 persisted when both AGTRAP and miR-34a were co-infected ( n = 7 in each group). Statistical analysis was performed using the Wilcoxon t- test (* p < 0.05; ** p < 0.01)

Article Snippet: Experiments with mice were performed in conformity with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and in accordance with the experimental protocol approved by the Animal Welfare Organization (OPBA) of the Italian National Ministry of Health and Cogentech (824/2020-PR). miR-34a −/− (miR-34a KO) mouse line was purchased from the Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Isolation, Infection, Virus, Plasmid Preparation, Over Expression, Expressing, Western Blot

Journal: GeroScience

Article Title: Angiotensin II, miR-34a, and AGTRAP crosstalk in arterial smooth muscle cells

doi: 10.1007/s11357-025-02018-5

Figure Lengend Snippet: AGTRAP forced expression decreases inflammation induced by Angiotensin II and miR-34a in isolated HASMC. A HASMC was treated with Ang II (1 µM for 24 h). Total RNA was extracted and assayed for IL-6, COX2, MCP-1, and MFG-E8 by RT-qPCR. Expression levels of IL-6 (Ctrl, n = 6; Ang II, n = 8), COX2 ( n = 6 in each group), MCP-1 ( n = 6 in each group), and MFGE8 ( n = 6 in each group) mRNA increased in response to Ang II, and this effect was either abolished or markedly inhibited in cells overexpressing AGTRAP. B HASMC were single-infected with lentiviruses encoding control scramble (miR-scr), miR-34a, AGTRAP, and double-infected with miR-34a and AGTRAP. Total RNA was extracted and assayed for IL-6, COX2, MCP-1, and MFGE8 by RT-qPCR. miR-34a overexpression increased IL-6 ( n = 6), COX2 ( n = 7), MCP-1 ( n = 6), and MFGE8 ( n = 6) mRNA expression; under these conditions, simultaneous expression of AGTRAP abolished the pro-inflammatory action of miR-34a and restored the baseline expression of these genes. Statistical analysis was performed using the Wilcoxon t- test (* p < 0.05)

Article Snippet: Experiments with mice were performed in conformity with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and in accordance with the experimental protocol approved by the Animal Welfare Organization (OPBA) of the Italian National Ministry of Health and Cogentech (824/2020-PR). miR-34a −/− (miR-34a KO) mouse line was purchased from the Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Infection, Control, Over Expression

Journal: GeroScience

Article Title: Angiotensin II, miR-34a, and AGTRAP crosstalk in arterial smooth muscle cells

doi: 10.1007/s11357-025-02018-5

Figure Lengend Snippet: Age-associated increase of miR-34a in monkey and rat central arteries. A miR-34a positively correlated with age in the CCA of NHP 8 to 26 years of age ( n = 10) ( Y = 0.09097 x + 0.1310; Rs = 0.6727; * p < 0.05). B miR-34a expression was higher in the CCA of 30-month (old) vs 8-month (young) old rats ( n = 5 in each group). C A higher miR-34a expression was also found in the aorta of old vs young rats ( n = 13 in each group), and this difference was present both in the adventitia ( n = 4 young, n = 5 old) and in the media ( n = 5 in each group). Statistical analysis was performed using a Mann–Whitney U test (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: miR-34a overexpression in HASMC was achieved by lentiviral infection using pre-miR-34a lentiviral supernatants (pMIRH34a, System Biosciences, SBI) or a control sequence (miR-scramble).

Techniques: Expressing, MANN-WHITNEY

Journal: GeroScience

Article Title: Angiotensin II, miR-34a, and AGTRAP crosstalk in arterial smooth muscle cells

doi: 10.1007/s11357-025-02018-5

Figure Lengend Snippet: Angiotensin II induces miR-34a upregulation and AGTRAP downmodulation in isolated arterial rat and human aortic smooth muscle cells. A VSMC isolated from 8-month (young) and 30-month (old) old rats were treated with Ang II (100 nM for 48 h). Ang II enhanced miR-34a expression in VSMC from old rats but had no effect in VSMC from young rats ( n = 8 in each group). B HASMC ( n = 6 in each group) were treated with Ang II alone (1 µM for 24 h), or with Valsartan (1 µM for 1 h), followed by treatment with Ang II (1 µM for 24 h). Ang II increased miR-34a expression and this effect was abolished by pretreatment with Valsartan. C HASMC was treated with Ang II (1 µM for 24 h). Ang II decreased AGTRAP ( n = 6 in each group) and SIRT1 mRNA ( n = 6–7). D Representative western blot analysis of AGTRAP and SIRT1 and densitometric ratio of AGTRAP and SIRT1 normalized to tubulin protein levels ( n = 7 in each group). Ang II decreased both AGTRAP and SIRT1 protein levels. The statistical analysis was performed using the Wilcoxon t -test ( * p < 0.05; ** p < 0.01)

Article Snippet: miR-34a overexpression in HASMC was achieved by lentiviral infection using pre-miR-34a lentiviral supernatants (pMIRH34a, System Biosciences, SBI) or a control sequence (miR-scramble).

Techniques: Isolation, Expressing, Western Blot

Journal: GeroScience

Article Title: Angiotensin II, miR-34a, and AGTRAP crosstalk in arterial smooth muscle cells

doi: 10.1007/s11357-025-02018-5

Figure Lengend Snippet: Aging and miR-34a modulate AGTRAP expression. Experiments in ( A–B ) were performed with VSMC isolated from 8-month-old (young) and 30-month-old (old) rats. A AGTRAP mRNA expression was not modulated by aging in VSMC of young ( n = 9) and old ( n = 6) rats. B AGTRAP protein was lower in VSMC of old rats. Representative western blot analysis of AGTRAP in young and old rat VSMC and densitometric ratio of AGTRAP normalized to vinculin protein levels ( n = 5, young; n = 6, old). C–D AGTRAP expression was modulated by miR-34a and aging in vivo. Thoracic aortas of WT and miR-34a KO male mice, 2-month-old (young) and 18-month-old (old), were tested for AGTRAP expression by immunohistochemistry. C Aging was associated with markedly lower AGTRAP protein expression in both WT and miR-34a KO mice. AGTRAP was higher in young miR-34a KO mice compared to the WT control. In old mice, AGTRAP exhibited a trend toward higher expression in miR-34a KO vs WT, but the difference was not statistically significant. The percentage of positive area per AGTRAP was calculated by determining the ratio of positive AGTRAP area to total aortic cross-sectional area in one section per animal and multiplying by 100. Each circle or square in the graph represents data from a single mouse (WT young, n = 8; WT old, n = 4; miR-34a KO, young, n = 9; miR-34a KO, old, n = 5). D Representative images (one mouse per group) of the quantification shown in ( C ) of an aortic section field stained for AGTRAP (brown signal) with a specific antibody and counterstained with hematoxylin (purple signal). Calibration bar applied to all panels = 20 µm. E AGTRAP is a direct target of miR-34a. HEK293 were transfected with firefly luciferase constructs containing the Human 3′UTR-AGTRAP wild-type (wt) or mutated in miR-34a seed sequence (mut). The panel shows a schematic representation of miR-34a binding sites. The wt and mut seed sequences are indicated in bold, whereas mut nucleotides are indicated in small letters in red. The seed sequence nucleotides indicated are specific for the 3′UTR of Human AGTRAP ENST00000376627.2. F These constructs were co-transfected with a plasmid encoding either miR-34a or miR-scramble (miR-scr) sequences as control. Values were normalized according to Renilla luciferase activity ( n = 6 in each group). The overexpression of miR-34a decreased luciferase activity of 3′UTR-AGTRAP wt but had no effect on 3′UTR-AGTRAP mut. In ( A ), ( B ), and ( C ) the statistical analysis was performed using Mann–Whitney U test. In ( F ), statistical analysis was performed using Wilcoxon t -test ( *p<0.05 ; ** p < 0.01; *** p < 0.001)

Article Snippet: miR-34a overexpression in HASMC was achieved by lentiviral infection using pre-miR-34a lentiviral supernatants (pMIRH34a, System Biosciences, SBI) or a control sequence (miR-scramble).

Techniques: Expressing, Isolation, Western Blot, In Vivo, Immunohistochemistry, Control, Staining, Transfection, Luciferase, Construct, Sequencing, Binding Assay, Plasmid Preparation, Activity Assay, Over Expression, MANN-WHITNEY

Journal: GeroScience

Article Title: Angiotensin II, miR-34a, and AGTRAP crosstalk in arterial smooth muscle cells

doi: 10.1007/s11357-025-02018-5

Figure Lengend Snippet: Effect of miR-34a overexpression and AGTRAP knockdown on miR-34a targets in isolated HASMC. A–C HASMC were infected for 48 h either with a lentivirus encoding miR-34a or with a control virus (miR-scr); miR-34a ( n = 8 in each group), AGTRAP ( n = 9 in each group), and SIRT1 ( n = 8 in each group). miR-34a and mRNA expression were quantified by RT-qPCR. A miR-34a increased, and B AGTRAP and SIRT1 mRNA levels were lower in miR-34a overexpressing cells compared to scramble control. C Representative western blot analysis and densitometric ratio of AGTRAP and SIRT1, normalized to β-actin protein levels ( n = 6 in each group), demonstrated a significant decrease of AGTRAP and SIRT1 protein in response to miR-34a forced expression. D–F HASMC were infected either with the lentivirus carrying AGTRAP-specific shRNA or with the control virus (sh-scr). AGTRAP ( n = 6 in each group), miR-34a ( n = 6 in each group), and SIRT1 ( n = 6 in each group). miR-34a and mRNA expression were quantified by RT-qPCR. D shAGTRAP decreased AGTRAP mRNA. E Upon AGTRAP knockdown, miR-34a increased, whereas SIRT1 mRNA decreased. F Representative western blot analysis and densitometric ratio demonstrate that AGTRAP and SIRT1 protein expression levels decreased upon AGTRAP knockdown. Statistical analysis was performed using the Wilcoxon t- test (* p < 0.05; ** p < 0.01)

Article Snippet: miR-34a overexpression in HASMC was achieved by lentiviral infection using pre-miR-34a lentiviral supernatants (pMIRH34a, System Biosciences, SBI) or a control sequence (miR-scramble).

Techniques: Over Expression, Knockdown, Isolation, Infection, Control, Virus, Expressing, Quantitative RT-PCR, Western Blot, shRNA

Journal: GeroScience

Article Title: Angiotensin II, miR-34a, and AGTRAP crosstalk in arterial smooth muscle cells

doi: 10.1007/s11357-025-02018-5

Figure Lengend Snippet: A negative feedback loop exists between AGTRAP and miR-34a in isolated HASMC. HASMC were co-infected with lentiviruses encoding AGTRAP and miR-34a. Single infection either with AGTRAP or miR-34a was performed together with miR-scramble virus or AGTRAP backbone vector, respectively. A AGTRAP overexpression significantly decreased miR-34a expression under baseline conditions but not when miR-34a was overexpressed (miR-scr, n = 11; AGTRAP, n = 8; miR-34a, n = 7; AGTRAP + miR-34a, n = 4). B AGTRAP mRNA diminished in response to miR-34a, and this effect was abolished when both miR-34a and AGTRAP were overexpressed (miR-scr, n = 12; miR-34a, n = 10; AGTRAP, n = 7; AGTRAP + miR-34a, n = 6); C SIRT1 mRNA diminished in response to miR-34a overexpression both under baseline conditions and when both miR-34a and AGTRAP were co-infected (miR-scr, n = 8; miR-34a, n = 8; AGTRAP, n = 6; AGTRAP + miR-34a, n = 7). D Representative Western blot analysis of SIRT1 and AGTRAP protein expression levels in HASMC. E–F Densitometric ratio of AGTRAP and SIRT1, respectively, normalized to tubulin protein levels; E AGTRAP forced expression abolished the effect of miR-34a to decrease AGTRAP protein (miR-scr, n = 10; miR-34a, n = 10; AGTRAP, n = 4; AGTRAP + miR-34a, n = 4). F SIRT1 protein decreased in response to miR-34a forced expression and increased when AGTRAP was overexpressed. The effect of AGTRAP to downmodulate SIRT1 persisted when both AGTRAP and miR-34a were co-infected ( n = 7 in each group). Statistical analysis was performed using the Wilcoxon t- test (* p < 0.05; ** p < 0.01)

Article Snippet: miR-34a overexpression in HASMC was achieved by lentiviral infection using pre-miR-34a lentiviral supernatants (pMIRH34a, System Biosciences, SBI) or a control sequence (miR-scramble).

Techniques: Isolation, Infection, Virus, Plasmid Preparation, Over Expression, Expressing, Western Blot

Journal: GeroScience

Article Title: Angiotensin II, miR-34a, and AGTRAP crosstalk in arterial smooth muscle cells

doi: 10.1007/s11357-025-02018-5

Figure Lengend Snippet: AGTRAP forced expression decreases inflammation induced by Angiotensin II and miR-34a in isolated HASMC. A HASMC was treated with Ang II (1 µM for 24 h). Total RNA was extracted and assayed for IL-6, COX2, MCP-1, and MFG-E8 by RT-qPCR. Expression levels of IL-6 (Ctrl, n = 6; Ang II, n = 8), COX2 ( n = 6 in each group), MCP-1 ( n = 6 in each group), and MFGE8 ( n = 6 in each group) mRNA increased in response to Ang II, and this effect was either abolished or markedly inhibited in cells overexpressing AGTRAP. B HASMC were single-infected with lentiviruses encoding control scramble (miR-scr), miR-34a, AGTRAP, and double-infected with miR-34a and AGTRAP. Total RNA was extracted and assayed for IL-6, COX2, MCP-1, and MFGE8 by RT-qPCR. miR-34a overexpression increased IL-6 ( n = 6), COX2 ( n = 7), MCP-1 ( n = 6), and MFGE8 ( n = 6) mRNA expression; under these conditions, simultaneous expression of AGTRAP abolished the pro-inflammatory action of miR-34a and restored the baseline expression of these genes. Statistical analysis was performed using the Wilcoxon t- test (* p < 0.05)

Article Snippet: miR-34a overexpression in HASMC was achieved by lentiviral infection using pre-miR-34a lentiviral supernatants (pMIRH34a, System Biosciences, SBI) or a control sequence (miR-scramble).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Infection, Control, Over Expression