mirvana mirna mimic/inhibitor/scrambled control  (Thermo Fisher)

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Small RNA Sequencing of Human Urinary Extracellular Vesicles Reveals Association of High‐Sodium Diet With Renal Proinflammatory Pathways

doi: 10.1161/JAHA.124.040091

Figure Lengend Snippet: We prospectively recruited 14 subjects with high‐risk normotension who underwent 5 to 7 days of a low‐sodium diet (<40 mmol/day or 0.92 g/day) or high‐sodium diet (>200 mmol/day or 4.6 g/day). At the end of each diet, we isolated uEVs from 24‐hour urine collection through serial centrifugation and ultracentrifugation. We then performed small RNA sequencing of the uEVs and bioinformatic analysis to select the miRNA‐mRNA target for in vitro validation. We finally validated miRNA‐target regulation in human tubular cell lines (human kidney 2 cells). EV indicates extracellular vesicle; and uEVs, urinary extracellular vesicles.

Article Snippet: Human kidney 2 cells (ATCC, CRL‐2190–passage 7–10) were transfected with mirVana miRNA inhibitors for hsa‐miR‐320b and hsa‐miR‐10b‐5p (Applied Biosystems, number 4464084) with Lipofectamine 2000 Reagent (Invitrogen, number11668027).

Techniques: Isolation, Centrifugation, RNA Sequencing, In Vitro, Biomarker Discovery

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Small RNA Sequencing of Human Urinary Extracellular Vesicles Reveals Association of High‐Sodium Diet With Renal Proinflammatory Pathways

doi: 10.1161/JAHA.124.040091

Figure Lengend Snippet: A and B , The ring chart shows the proportion of all the expressed ( A ) and differentially expressed small RNAs ( B ) highlighted for type. C and D , Volcano plots reporting expression levels of all small RNAs ( C ) and only miRNAs ( D ) expression levels when the HSD and LSD were compared. The dashed line is set at q value=0.05. Significantly differentially enriched small RNAs or miRNAs are highlighted in yellow (with violet border). The y axis is expressed as −log ( q value); the x axis is expressed as log 2 (fold change). E and F , Dot and box plots of significantly differentially expressed miRNAs upregulated in the LSD ( E ) and HSD ( F ). EV indicates extracellular vesicle; HSD, high‐sodium diet; and LSD, low‐sodium diet.

Article Snippet: Human kidney 2 cells (ATCC, CRL‐2190–passage 7–10) were transfected with mirVana miRNA inhibitors for hsa‐miR‐320b and hsa‐miR‐10b‐5p (Applied Biosystems, number 4464084) with Lipofectamine 2000 Reagent (Invitrogen, number11668027).

Techniques: Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Small RNA Sequencing of Human Urinary Extracellular Vesicles Reveals Association of High‐Sodium Diet With Renal Proinflammatory Pathways

doi: 10.1161/JAHA.124.040091

Figure Lengend Snippet: A and B , Pathway enrichment analysis conducted with EnrichR showing the enriched terms for miRNA target genes expected to be upregulated in the high‐sodium diet ( A ) or in the low‐sodium diet ( B ). Adjustment for multiple testing of the P value was performed with Benjamini‐Hochberg to calculate q values; the −log ( q value) was calculated for pathway bar graph and network analysis. Pathways uniquely enriched in each condition were considered for pathway and network analysis. C and D , Network cluster analysis showing pathways enriched in the high‐ ( C ) and low‐sodium diet ( D ). The node size is proportional to –log ( q ‐value) of each pathway. For clarity of representation, pathways with –log ( q value) <1.5 were excluded in the low‐sodium diet network and –log ( q value) <2.0 in the high‐sodium diet. Connection thickness is proportional to the Jaccard Index. The main functional communities were named on the basis of the principal enriched pathways for each cluster. E and F , miRNA‐mRNA target networks showing miRNA‐target interactions in the high‐ ( E ) and low‐sodium diet ( F ). Yellow dots indicate miRNAs downregulated in the high‐sodium diet ( E ) and red dots in the low‐sodium diet ( F ). Blue dots indicate mRNA targets and are connected to regulating miRNAs. mRNA targets involved in immune‐related clusters (adaptive immune system, innate immune system, interleukin signaling, and interferon signaling) are highlighted in green in the high‐sodium diet ( E ). mRNA targets involved in PPAR‐related clusters are highlighted in green in the low‐sodium diet ( F ). In the high‐sodium diet ( E ), the connection between miR‐320b and ICAM‐1 was added manually since being validated by a previously published study. ²⁶ EGFR indicates epidermal growth factor receptor; ER, endoplasmic reticulum; ESR, estrogen receptor; FLT3, Fms‐like tyrosine kinase 3; GTP, guanosine triphosphate; HOX, homeobox; ICAM‐1, intercellular adhesion molecule 1; MAPK, mitogen‐activated protein kinase; PDGF, Platelet‐Derived Growth Factor; PI3K/akt, phosphoinositide 3‐kinase/protein kinase B; PPAR, peroxisome proliferator‐activated receptor; ROBO, roundabout; RT, regulatory target; SMAD, suppressor of mothers against decapentaplegic; SUMO, small ubiquitin‐like modifier; TGF‐β, transforming growth factor‐beta; TP53, tumor protein P53; and TR, transcriptional regulation.

Article Snippet: Human kidney 2 cells (ATCC, CRL‐2190–passage 7–10) were transfected with mirVana miRNA inhibitors for hsa‐miR‐320b and hsa‐miR‐10b‐5p (Applied Biosystems, number 4464084) with Lipofectamine 2000 Reagent (Invitrogen, number11668027).

Techniques: Functional Assay, Derivative Assay, Ubiquitin Proteomics

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Small RNA Sequencing of Human Urinary Extracellular Vesicles Reveals Association of High‐Sodium Diet With Renal Proinflammatory Pathways

doi: 10.1161/JAHA.124.040091

Figure Lengend Snippet: A through C , Relative expression levels of miR‐320b ( A ), mRNA of ICAM‐1 ( B ), and protein levels of ICAM‐1 ( C ) in HK‐2 cells after treatment with miR‐320b inhibitor. D through F , Relative expression levels of miR‐10b‐5p ( D ), mRNA of PPARα ( E ), and protein levels of PPARα ( F ) in HK‐2 cells after treatment with miR‐10b‐5p inhibitor. Expression levels were normalized to miR‐16‐5p expression (a.u.) for miRNAs, to GAPDH expression (a.u.) for mRNA. Protein abundance was normalized for vinculin expression (a.u.). A, B, D , and E , Quantified by quantitative real‐time polymerase chain reaction, median fluorescence of each experiment was normalized by Z score (a.u.). C and F , Protein expression was quantified by flow‐cytometry and Western blot respectively. Representative Western blot analysis of PPARα and vinculin is presented in Figure . In all experiments, the control group was treated with the empty vehicle (Lipofectamine 2000) alone. Data are represented with dot plots. Each experiment was repeated twice with 3 technical replicates (n=6). Statistical differences were assessed by the use of an unpaired t test. * P <0.05. a.u. indicates arbitrary unit; Ctr, control; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; HK‐2, human kidney 2; ICAM‐1, intercellular adhesion molecule 1; and PPARα, peroxisome proliferator‐activated receptor α.

Article Snippet: Human kidney 2 cells (ATCC, CRL‐2190–passage 7–10) were transfected with mirVana miRNA inhibitors for hsa‐miR‐320b and hsa‐miR‐10b‐5p (Applied Biosystems, number 4464084) with Lipofectamine 2000 Reagent (Invitrogen, number11668027).

Techniques: Expressing, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Fluorescence, Flow Cytometry, Western Blot, Control

Journal: BMC Cancer

Article Title: The MAP kinase negative regulator DUSP2 (dual specificity phosphatase 2) is controlled by oncogenic microRNA cluster miR-17-92, miR-106a-363 and miR-106b-25

doi: 10.1186/s12885-025-14434-z

Figure Lengend Snippet: Combined approach of in silico target prediction and pan-cancer correlation analysis exhibiting all significant negatively correlated microRNAs– DUSP2 pairs in cancer context. ( A ) Flow chart depicting the combined analysis approach for rigorous preselection of potential microRNA– DUSP2 interactions. ( B ) Box plots showing the DUSP2 mRNA expression levels across 32 TCGA cancer types ordered by median expression of cancer type. ( C ) In the upper part, the left column presents on top, the 17 highly expressed microRNAs (CPM > 1 in > 90% of all TCGA tumour samples) and, below, the 17 microRNAs highly expressed in individual cancer types which were predicted to bind DUSP2 at least by two prediction tools including TargetScan (middle column). The right columns present the negative Spearman correlations calculated between microRNA and DUSP2 mRNA expression in TCGA cancer samples of respective cancer types. The lower part exhibited the Spearman correlations between DUSP2 mRNA expression and abundance of phosphorylated MAPK proteins determined by RPPA in subsets of TCGA samples of respective cancer types. The blue colour indicates significant negative correlations, red indicates significant positive correlations, grey indicates no significant negative correlation and light grey indicated low expression < 1 CPM

Article Snippet: To determine the intracellular effect of microRNAs on DUSP2 mRNA expression 2 × 10 6 WSU-DLCL2 cells were transfected witheither 100nM mirVana miRNA inhibitor negative control #1 (4464076, Thermo Fisher Scientific) or the mirVana miRNA inhibitors for miR-17-5p (MH12412, Thermo Fisher Scientific), miR-20b-5p (MH10975, Thermo Fisher Scientific) or miR-106b-5p (MH10067, Thermo Fisher Scientific) using the Amaxa SE Cell Line 4D-Nucleofector X Kit S (VAXC-1032, Lonza Bioscience, Cologne, Germany) on an Amaxa 4D Nucleofector device (Lonza Bioscience) according to the manufacturer’s instructions with the CL-120 program.

Techniques: In Silico, Expressing

Journal: BMC Cancer

Article Title: The MAP kinase negative regulator DUSP2 (dual specificity phosphatase 2) is controlled by oncogenic microRNA cluster miR-17-92, miR-106a-363 and miR-106b-25

doi: 10.1186/s12885-025-14434-z

Figure Lengend Snippet: Reporter gene assays determined regulatory effects of miR-29b-3p cluster, members of the C19MC cluster as well as miR-122-5p and miR-340-5p on DUSP2 . A ) Reporter gene assay confirmed the interaction of pre-miR-29b-3p (10 nM) with the DUSP2 3’UTR by reducing the relative reporter gene activity by 30%. The effects were abrogated by introducing of mutations (MUT1) in the binding site. B , C ) Transfection with pre-miR-520a-3p or pre-miR-520c-3p caused reductions in relative reporter gene activity by 26% or 23%, respectively, through binding to wild type DUSP2 3’UTR. The introduction of mutations (MUT3) into the predicted binding region of the DUSP2 3’UTR abolished the inhibitory effects of both microRNAs. D ) Transfection with pre-miR-122-5p also led to reduction of relative reporter gene activity by 24%. The effects were abrogated by introducing mutations (MUT2) in the binding site. E ) Pre-miR-340-5p transfection resulted in a significant reduction of the relative reporter gene activity by 21%, which was reversed by introducing mutations (MUT3) into the binding region. All activities ( n = 12) (median ± interquartile range) were determined 48 h after transfection and were shown relative to empty control vector identically transfected and normalized as 3’UTR target sequence vectors. Mann–Whitney U-test; *** p ≤ 0.001

Article Snippet: To determine the intracellular effect of microRNAs on DUSP2 mRNA expression 2 × 10 6 WSU-DLCL2 cells were transfected witheither 100nM mirVana miRNA inhibitor negative control #1 (4464076, Thermo Fisher Scientific) or the mirVana miRNA inhibitors for miR-17-5p (MH12412, Thermo Fisher Scientific), miR-20b-5p (MH10975, Thermo Fisher Scientific) or miR-106b-5p (MH10067, Thermo Fisher Scientific) using the Amaxa SE Cell Line 4D-Nucleofector X Kit S (VAXC-1032, Lonza Bioscience, Cologne, Germany) on an Amaxa 4D Nucleofector device (Lonza Bioscience) according to the manufacturer’s instructions with the CL-120 program.

Techniques: Reporter Gene Assay, Activity Assay, Binding Assay, Transfection, Control, Plasmid Preparation, Sequencing, MANN-WHITNEY