Journal: British Journal of Cancer

Article Title: miR-4711-5p regulates cancer stemness and cell cycle progression via KLF5, MDM2 and TFDP1 in colon cancer cells

doi: 10.1038/s41416-020-0758-1

Figure Lengend Snippet: a Comparison of the tumour growth inhibitory effects of miR-4711-5p and four miRNAs (miR-21, miR-152, miR-153, and miR-448) that have been reported to suppress KLF5 expression. We also included a putative anti-oncomiR, miR-34a, as a positive control. MiR-4711-5p was the most effective in suppressing DLD-1 and HCT116 tumour cell growth among the miRNAs tested ( P < 0.05). b , c Comparison of the cell growth inhibitory effects of miR-4711-5p and miR-34a on CRC cells cultured from five different CRC patients (3 stage II and 2 stage III patients). The images of tumour cell cultures derived from two CRC patients (stage II) at 72 h after transfection of miR-NC, miR-34a, and miR-4711-5p ( b ). Cell viability of tumour cells cultured from five different CRC patients at 72 h after transfection. The tumour-suppressive effects of miR-4711-5p were much stronger than those of miR-34a in all patient-derived CRC cell cultures ( P < 0.01) ( c ). All data represent the mean ± SD.

Article Snippet: Mimic-miR-152-5p, mimic-miR-153-3p and mimic-miR-448-3p were purchased from Bioneer Corporation (Daejeon, Korea).

Techniques: Expressing, Positive Control, Cell Culture, Derivative Assay, Transfection

Journal: Cardiovascular Research

Article Title: Pivotal role of miR-448 in the development of ROS-induced cardiomyopathy

doi: 10.1093/cvr/cvv238

Figure Lengend Snippet: Changes in miRs expression in dystrophic heart. (A) Real-time PCR analysis of cardio-specific miRs and miRs predicted to target Cybb and Ncf1 genes. Graphs show changes in miR levels in mdx hearts relative to their levels in WT samples (indicated by dashed line). N = 5–6, *P < 0.05, t-test. (B) Genomic localization of mmu-miR-448 on intron 4 of the Htr2C gene located on X-chromosome of mouse genome. It is further processed to pre-miR-448 and later to the mature miR-448, where miR-448-3p is conserved among species. (C) Left, schematic representation of miR-448-3p binding region (‘seed region’) in mouse Ncf1 gene. On the bottom is the map of quadruple mutant (mutated residuals is in light grey). Right, luciferase activity assay shows the dose-dependent decrease of luciferase activity in cells transfected with miR-448 mimic and NCF1-3′UTR plasmid, but not with construct with the mutated seed region. For each transfection, a total of six wells for each condition were collected and measured individually, and then averaged (n = 6). A total of three separate transfections were assayed and averaged (N = 3). *P < 0.05, t-test.

Article Snippet: To determine the role of miR-448-3p in vivo , we injected WT mice with miRCURY LNA™ miR-448-3p inhibitor (Exiqon, Woburn, MA, 10 mg/kg and 25 mg/kg) or with scrambled LNA-NC (10 mg/kg) through a tail vein according to manufacturer's protocol ( Figure ).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Construct

Journal: Cardiovascular Research

Article Title: Pivotal role of miR-448 in the development of ROS-induced cardiomyopathy

doi: 10.1093/cvr/cvv238

Figure Lengend Snippet: Chronic inhibition of miR-448 in WT mice induces cardiac remodelling. (A) Morphological changes in hearts following chronic LNA treatment. (B) Morphological changes in cardiac myocytes. Left, representative images of LV myocardium of LNA-treated mice stained with haematoxylin-eosin. Right, averaged cross-sectional area of the myocytes in hearts of animals treated with either LNA-NC (N = 3) or LNA-antimiR-448 (N = 3). *P < 0.05, t-test. (C and D) Left, patterns of perivascular and cardiac fibrosis in LV remote myocardium stained with Masson trichrome (C) or picric acid sirius red (D). Right, relative amount of fibrotic patches in hearts of animals treated with either LNA-NC (N = 3) or LNA-antimiR-448 (N = 3). **P < 0.001, t-test. (E) Real-time PCR analysis shows overexpression of pro-fibrotic genes mRNA in cardiac tissues after acute (grey bars) and chronic (black bars) inhibition of miR-448. Dashed line indicates gene expression in LNA-NC samples. N = 4–6. *P < 0.05, **P < 0.001, t-test.

Article Snippet: To determine the role of miR-448-3p in vivo , we injected WT mice with miRCURY LNA™ miR-448-3p inhibitor (Exiqon, Woburn, MA, 10 mg/kg and 25 mg/kg) or with scrambled LNA-NC (10 mg/kg) through a tail vein according to manufacturer's protocol ( Figure ).

Techniques: Inhibition, Staining, Real-time Polymerase Chain Reaction, Over Expression, Expressing

Journal: Cardiovascular Research

Article Title: Pivotal role of miR-448 in the development of ROS-induced cardiomyopathy

doi: 10.1093/cvr/cvv238

Figure Lengend Snippet: Targeting of miR-448 by LNA-antimiR-448 inhibitor in WT mice results in an increase in Ncf1 gene and p47phox protein expression. (A) Protocol for LNA-antimiR-448 transfer. (B) Real-time PCR analysis of Ncf1 mRNA expression in cardiac tissue form WT mice treated with LNA-antimiR-448 inhibitor at two different concentrations. N = 5, *P < 0.05, t-test. (C) Expression of p47phox subunit of NOX2 in hearts of WT mice treated with LNA-NC and LNA-antimiR 448 inhibitors. N = 5, *P < 0.05, t-test.

Article Snippet: To determine the role of miR-448-3p in vivo , we injected WT mice with miRCURY LNA™ miR-448-3p inhibitor (Exiqon, Woburn, MA, 10 mg/kg and 25 mg/kg) or with scrambled LNA-NC (10 mg/kg) through a tail vein according to manufacturer's protocol ( Figure ).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Cardiovascular Research

Article Title: Pivotal role of miR-448 in the development of ROS-induced cardiomyopathy

doi: 10.1093/cvr/cvv238

Figure Lengend Snippet: Systemic inhibition of miR-448 in WT mice results in an increase in NOX2-dependent ROS production and exacerbated Ca2+ responses in ventricular cardiomyocytes. (A) Representative images of DCF fluorescence in myocytes isolated from mice treated with LNA-NC and LNA-antimiR-448 inhibitor at two different concentrations. Graph in the middle illustrates changes in average DCF signals. Bar graph on the right illustrates the rate of DCF oxidation in six different groups of experiments: LNA-NC (N = 5, n = 17; N = 4, n = 22), LNA-antimiR-448, 10 mg/kg (N = 3, n = 25; N = 3, n = 13), and LNA-antimiR-448, 25 mg/kg (N = 3, n = 22; N = 3, n = 15) without and with incubation with DPI, respectively. (B) Intracellular Ca2+ responses to mild hypo-osmotic shock in ventricular cardiomyocytes from WT mice treated with LNA-NC and LNA-antimiR-448. Left panels are line-scan representations of series of images acquired from cells and converted to a two-dimensional X,t image. Middle panel represents time course of normalized fluo-4 fluorescence in WT cells treated with LNA-NC and LNA-antimiR-448. Right panel shows pooled data of mean values of normalized fluorescence during 60 s after the osmotic shock. Number of cells studied was LNA-NC (N = 4, n = 26), LNA-antimiR-448, 10 mg/kg (N = 4 n = 27; N = 3 n = 10), and LNA-antimiR-448, 25 mg/kg (N = 4 n = 27; N = 3 n = 12) without and with incubation with DPI, respectively. *P < 0.05, **P < 0.001, t-test (for ROS and Ca2+ measurements) and #P < 0.05, ##P < 0.001, t-test (for ROS and Ca2+ measurements after DPI treatment).

Article Snippet: To determine the role of miR-448-3p in vivo , we injected WT mice with miRCURY LNA™ miR-448-3p inhibitor (Exiqon, Woburn, MA, 10 mg/kg and 25 mg/kg) or with scrambled LNA-NC (10 mg/kg) through a tail vein according to manufacturer's protocol ( Figure ).

Techniques: Inhibition, Fluorescence, Isolation, Incubation

Journal: Cardiovascular Research

Article Title: Pivotal role of miR-448 in the development of ROS-induced cardiomyopathy

doi: 10.1093/cvr/cvv238

Figure Lengend Snippet: Chronic inhibition of miR-448 in WT mice impairs cardiac functions. (A) Protocol for LNA-antimiR-448 injection. (B) Left, real-time PCR analysis of Ncf1 mRNA expression in heart of WT mice injected with LNA-antimiR inhibitor of miR-448 (10 mg/kg) compared with the corresponding NC (N = 5). Middle, immunoblot and right, summary of p47phox subunit expression under the same experimental conditions. N = 5, *P < 0.05, t-test. (C) Left, M-mode echocardiographic images of left ventricles (LVs) of LNA-treated adult WT mice. Right, bar graphs illustrate changes in FS and ventricle ejection fraction in animals treated with LNA-NC (N = 7) and LNA-antimiR-448 (N = 4), respectively. *P < 0.05, t-test.

Article Snippet: To determine the role of miR-448-3p in vivo , we injected WT mice with miRCURY LNA™ miR-448-3p inhibitor (Exiqon, Woburn, MA, 10 mg/kg and 25 mg/kg) or with scrambled LNA-NC (10 mg/kg) through a tail vein according to manufacturer's protocol ( Figure ).

Techniques: Inhibition, Injection, Real-time Polymerase Chain Reaction, Expressing, Western Blot