Journal: Physiological Reports

Article Title: Tadalafil, a long acting phosphodiesterase inhibitor, promotes bone marrow stem cell survival and their homing into ischemic myocardium for cardiac repair

doi: 10.14814/phy2.13480

Figure Lengend Snippet: Tadalafil increased miR‐21 expression of in vitro MSC s under oxidative stress. Tadalafil induced miR‐21 up‐regulation (micro RNA ‐array analysis, (A). miR‐21 expression was increased in MSC s‐treated by tadalafil versus control (B). RT ‐ PCR : miR‐21 expression bands; and quantitative analysis of miR‐21/U6 expression was assessed in ± MAPK (U0126) and PKG 1 ( KT 5823) inhibitors ( PCR ‐gel bands and densitometry assay, (C). Values are mean ± SE ; n = 6; P‐ value <0.05 is significant.

Article Snippet: A precursor miR‐21 expression clone was constructed in a feline immunodeficiency virus‐based lentiviral vector system (pEZX‐MR04‐miR‐21) with a luciferase reporter construct containing the 3′‐UTR of Fas (TNF receptor super family member) designed to encompass the miR‐21 binding sites (pEZX‐Luc‐Fas 3′‐UTR, GeneCopoeia™, Rockville, MD) (Suzuki et al. ).

Techniques: Expressing, In Vitro, Reverse Transcription Polymerase Chain Reaction

Journal: Physiological Reports

Article Title: Tadalafil, a long acting phosphodiesterase inhibitor, promotes bone marrow stem cell survival and their homing into ischemic myocardium for cardiac repair

doi: 10.14814/phy2.13480

Figure Lengend Snippet: Cytoprotective effects of tadalafil were abrogated by miR‐21 inhibition in in vitro MSC s under oxidative stress. MSC s were transfected with pEZX ‐Luc vector containing Fas 3′‐ UTR together with a plasmid encoding miR‐21; or MSC s were transfected with anti‐miR‐21 using si PORT ™ NeoFx™ transfection agent. Number of TUNEL + cells (A‐B) and cell viability ( CCK ‐8 assay, C), luciferase activity % (D), STAT 3 and Fas expressions (western blots, E) were assessed after treatment with ± miR‐21 inhibitors. Also, p‐ VASP , PKG 1, Fas and BcL‐xl expressions (western blots, F) were assessed under oxidative stress in control and tadalafil in ± FasL inhibitors. Values are mean ± SE ; n = 6; P‐ value < 0.05 is significant.

Article Snippet: A precursor miR‐21 expression clone was constructed in a feline immunodeficiency virus‐based lentiviral vector system (pEZX‐MR04‐miR‐21) with a luciferase reporter construct containing the 3′‐UTR of Fas (TNF receptor super family member) designed to encompass the miR‐21 binding sites (pEZX‐Luc‐Fas 3′‐UTR, GeneCopoeia™, Rockville, MD) (Suzuki et al. ).

Techniques: Inhibition, In Vitro, Transfection, Plasmid Preparation, TUNEL Assay, CCK-8 Assay, Luciferase, Activity Assay, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: Therapy free of cells vs human mesenchymal stem cells from umbilical cord stroma to treat the inflammation in OA

doi: 10.1007/s00018-022-04580-z

Figure Lengend Snippet: Characterisation of MSCs and their derived EV from human umbilical cord estroma. A One representative fluorescence-activated cell sorting (FACS) assay is shown. The antibody is indicated at the top of each plot and its linked fluorochrome at the bottom. Positive MSC markers (CD105, CD90 and CD73) and negative haematopoietic markers (CD34 and CD45). B Representative pictures of immunohistochemical analysis ( OR Oil Red O, SafO safranin O and AR Alizarin Red from human umbilical cord estroma after 14 days with specific differentiation medium (DMEM, AD adipocyte medium, CH chondrocyte medium and OS osteocyte medium). C Histogram represents gene expression of pluripotency markers, Nanog , Sox9 and Oct4 in MSC in front of TC28a2 cell line. Real-time reverse transcriptase PCR (qRT-PCR) analysis normalized by expression of HPRT gene used as housekeeping. * P value less than 0.05 was considered statistically significant using 2ANOVA test. D Representative result from the NTA assay of MSC-derived EV. E Electron micrograph of MSC-derived EV (scale bar 100 nm). F Immunoblot staining for exosome markers CD63 and Calnexin as a negative control. G Histogram represents expression of miR-21 in MSC in front of MSC-miR21 − (left) and MSC-derived EV in front of MSC-miR21 − -derived EV Real-time reverse transcriptase PCR (qRT-PCR) analysis normalized by expression of U6 gene used as housekeeping. * P value less than 0.05 was considered statistically significant using 2ANOVA test

Article Snippet: PCR products were amplified using specific primers for miRNAs, miR-21-5p (rno481342_mir) and U6 (Rn01526055_g1) small nuclear RNA (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Derivative Assay, Fluorescence, FACS, Immunohistochemical staining, Gene Expression, Reverse Transcription, Quantitative RT-PCR, Expressing, Western Blot, Staining, Negative Control

Journal: Cellular and Molecular Life Sciences

Article Title: Therapy free of cells vs human mesenchymal stem cells from umbilical cord stroma to treat the inflammation in OA

doi: 10.1007/s00018-022-04580-z

Figure Lengend Snippet: miR21 modified the MEK1/2 family. A In silico study using Target Scan V8.0 public repository ( https://www.targetscan.org/vert_80/ ) revealed that MAPK family is a target of miR-21 in mammals. B Western blot analysis of ERK1/2, AKT, phoshor-ERK1/2, phosphor-AKT and GAPDH was used as housekeeping. C Plot showing their densitometry analysis normalized with respect to GAPDH. The gels were run under the same experimental conditions. D Representative Immunofluorescence analysis of SDC1 in liver form OA model. The photos above are at × 10 magnification and the photos below are at × 20. E Plot showing their densitometry analysis normalized with respect to DAPI. F ELISA analysis of shed SDC1 in serum from AO model. EV-miR21 − = MSC-miR21 − -derived EV. * P < 0.05 compared with PBS was considered statistically significant

Article Snippet: PCR products were amplified using specific primers for miRNAs, miR-21-5p (rno481342_mir) and U6 (Rn01526055_g1) small nuclear RNA (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Modification, In Silico, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Experimental neurology

Article Title: Increased precursor microRNA-21 following status epilepticus can compete with mature microRNA-21 to alter translation

doi: 10.1016/j.expneurol.2016.10.003

Figure Lengend Snippet: (A) A schematic diagram of miR-21 biogenesis. MiR-21 is transcribed as a long primary transcript (~3400 bp), pri-miR-21, from its own promoter. Drosha cleaves pri-miR-21 into a precursor transcript (~ 70 bp), pre-miR-21. Dicer further cleaves pre-mi-21 into short strands of ~22 nt, containing guide/mature miR-21 (green) and passenger miR-21 (miR-21*) (magenta). (B–C) qRT-PCR showing changes of pri-, pre-, and mature miR-21 levels at 4, 12, and 48 h after SE. Both pri- and mature miR-21 levels show significant increase only at 12 h and 48 h, respectively (B & D), whereas pre-miR-21 levels are elevated at all time points (C). Relative fold change is normalized to PPIA (cyclophilin) mRNA expression for pri-miR-21 and to 4.5S (ribonuclear small RNA) expression for pre-miR-21 and for mature miR-21. Shown are mean±SEM. Kruskal-Wallis test, *p<0.05, **p<0.01, *** p<0.001. (E & F) Small RNA northern blots detecting pre-miR-21 and mature miR-21 at 12 and 48 h post SE. The blots used LNA probe that detected both pre-miR-21 (red arrowhead) and mature miR-21 (circle). At 12 h time point, the intensity of the pre-miR-21 bands in the SE groups was stronger than that in the control group, but the intensities of the mature miR-21 bands were comparable between the SE and control groups (E). At 48 h time point, the levels of pre-miR-21 and mature miR-21 in the SE group were higher than that in the control group (F). (G & H) Quantification for northern blots. L= ladder for RNA sizes in nucleotides. U6 serves as a loading control. For pri-miR-21: 4h (Control N=10, SE N=12), 12h (Control N=7, SE N=8), 48h (Control N =11, SE N=10). For pre-miR-21: 4h (Control N= 7, SE N=11), 12h (Control N=7, SE N=9), 48h (Control N=5, SE N=6). For mature miR-21: 4h (Control N= 6, SE N=6), 12h (Control N=4, SE N=5), 48h (Control N=6, SE N=6).

Article Snippet: The reaction mix in each well contained 10 ul of the TaqMan® Universal Master Mix II (Thermo Fisher Scientific, MA), 1 ul of the FAM-MGB probe/primer mix (20x) for gene of interest (purchased from Thermo Fisher Scientific, MA; pri-miR-21: Rn03464993_pri; TGFBR2: Rn00579682_m1; NT-3: Rn00579280; pre-miR-21: CS51000; mature miR-21: 000397), 9 ul of cDNA/ddH2O.

Techniques: Quantitative RT-PCR, Expressing, RNA Expression, Northern Blot

Journal: Experimental neurology

Article Title: Increased precursor microRNA-21 following status epilepticus can compete with mature microRNA-21 to alter translation

doi: 10.1016/j.expneurol.2016.10.003

Figure Lengend Snippet: (A & B) A schematic shows the energetically favorable binding sties (in green for mature miR-21 and magenta for miR-21*) of pre-miR-21 to TGFBR2 mRNA for human (A) and for mouse (B). The minimum free energy change (ΔG) of human and mouse pre-miR-21 binding to TGFBR2 3′UTR are −54.0 kcal/mol and −52.4 kcal/mol, respectively. (C & D) A schematic shows the energetically favorable binding sties (in green for mature miR-21 and magenta for miR-21*) of pre-miR-21 to NT-3 mRNA for human (C) and in mouse (D). The ΔG of human and mouse pre-miR-21 binding to NT-3 3′UTR are −37.4 kcal/mol and −41.1 kcal/mol, respectively.

Article Snippet: The reaction mix in each well contained 10 ul of the TaqMan® Universal Master Mix II (Thermo Fisher Scientific, MA), 1 ul of the FAM-MGB probe/primer mix (20x) for gene of interest (purchased from Thermo Fisher Scientific, MA; pri-miR-21: Rn03464993_pri; TGFBR2: Rn00579682_m1; NT-3: Rn00579280; pre-miR-21: CS51000; mature miR-21: 000397), 9 ul of cDNA/ddH2O.

Techniques: Binding Assay

Journal: Experimental neurology

Article Title: Increased precursor microRNA-21 following status epilepticus can compete with mature microRNA-21 to alter translation

doi: 10.1016/j.expneurol.2016.10.003

Figure Lengend Snippet: Quantitative RT-PCR of TGFBR2 mRNA and NT-3 mRNA from the rat hippocampal lysate at 12 and 48 h after SE. (A) The TGFBR2 mRNA level in SE group was elevated by 1.7-fold at 12 h and 2.14-fold at 48 h compared to the control group. The 48 h SE group showed statistically significant increase. (B) The NT-3 mRNA level was significantly reduced at both 12 h (0.4±0.089) and 48 h (0.46±0.076) post SE compared to the control group. Relative fold change was normalized to housekeeping PPIA (cyclophilin) mRNA expression. Shown are mean±SEM. Control N= 7 samples and SE N= 9 samples. One-way ANOVA, Kruskal-Wallis test. *p<0.05, ** p<0.01, N.S.= not significant.

Article Snippet: The reaction mix in each well contained 10 ul of the TaqMan® Universal Master Mix II (Thermo Fisher Scientific, MA), 1 ul of the FAM-MGB probe/primer mix (20x) for gene of interest (purchased from Thermo Fisher Scientific, MA; pri-miR-21: Rn03464993_pri; TGFBR2: Rn00579682_m1; NT-3: Rn00579280; pre-miR-21: CS51000; mature miR-21: 000397), 9 ul of cDNA/ddH2O.

Techniques: Quantitative RT-PCR, Expressing

Journal: Experimental neurology

Article Title: Increased precursor microRNA-21 following status epilepticus can compete with mature microRNA-21 to alter translation

doi: 10.1016/j.expneurol.2016.10.003

Figure Lengend Snippet: (A & B) Dual-luciferase assay in HEK 293 cells in the presence of mature miR-21 (Sh-21) or both pre-miR-21 and mature miR-21 (CMV-21) to measure binding competition between pre-miR-21 and mature miR-21 to target mRNAs. (A) For binding to TGFBR2 3′UTR, mature miR-21 showed 40% suppression of TGFBR2 3′UTR-luciferase expression, and this suppression was alleviated to 20% in the presence of pre-miR-21 (p-value=0.0009). (B) For binding to NT3 3′UTR, mature miR-21 showed 60% suppression of NT3 3′UTR-luciferase expression, and this suppression was comparable in the presence of pre-miR-21 (p-value=0.19, not significant). Normalization was done with respect to a scramble control (Sh-S) for Sh-21 and to a CMV-driven GFP control (CMV-ctl) for CMV-21. Two-tailed Student’s t-test, and values are mean±SEM. N = 8; data points from two independent experiments measured in quadruplicate. (C & E) Schematics of the predicted loss of competition between pre-miR-21 and mature miR-21 for binding to TGFBR2 3′ UTR due to the deletion of the miR-21* binding site (TGFBR2-del) or the replacement of the miR-21* binding site by a scramble sequence (TGFB2R-3p-scr). (D & F) Dual-luciferase assay of the modified psiCHECK TGFBR2 constructs (TGFBR2-del and TGFB2R-3p-scr) in presence of Sh-21 and CMV-21. m= mature miR-21 and p= pre-miR-21.

Article Snippet: The reaction mix in each well contained 10 ul of the TaqMan® Universal Master Mix II (Thermo Fisher Scientific, MA), 1 ul of the FAM-MGB probe/primer mix (20x) for gene of interest (purchased from Thermo Fisher Scientific, MA; pri-miR-21: Rn03464993_pri; TGFBR2: Rn00579682_m1; NT-3: Rn00579280; pre-miR-21: CS51000; mature miR-21: 000397), 9 ul of cDNA/ddH2O.

Techniques: Luciferase, Binding Assay, Expressing, Two Tailed Test, Sequencing, Modification, Construct

Journal: Experimental neurology

Article Title: Increased precursor microRNA-21 following status epilepticus can compete with mature microRNA-21 to alter translation

doi: 10.1016/j.expneurol.2016.10.003

Figure Lengend Snippet: (A & B) Representative Northern blots of ribosome profiles generated from lysate of U-87 cells that were treated with either cycloheximide (A) or puromycin (B) as described in Methods. (A) Top: Northern analysis to detect pre-miR-21 (red arrowhead) and mature miR-21 (red circle) in each of the fractions across the profile. Bottom: Tracing of the profile at 254 nm shows distinct peaks for ribosome subunits, monosome and polysome as indicated (arrows). (B) Top: Northern analysis to detect pre-miR-21 and mature miR-21 (red arrowhead) and mature miR-21 (red circle) in each of the fractions across the profile. Bottom: Tracing of the profile at 254 nm shows loss of polysomes and an accumulation of the 80S monosomes (arrowhead). Quantification of the levels of pre-miR-21 (C) and mature miR-21 (D) in three independent polysome profiles. Unbound= fractions 1–5, 80S= fraction 6, polysome= fractions 7–14. Percentage calculated is relative to the sum of fractions 1–14. Black, magenta, and green data points represent three independent polysome profiling experiments.

Article Snippet: The reaction mix in each well contained 10 ul of the TaqMan® Universal Master Mix II (Thermo Fisher Scientific, MA), 1 ul of the FAM-MGB probe/primer mix (20x) for gene of interest (purchased from Thermo Fisher Scientific, MA; pri-miR-21: Rn03464993_pri; TGFBR2: Rn00579682_m1; NT-3: Rn00579280; pre-miR-21: CS51000; mature miR-21: 000397), 9 ul of cDNA/ddH2O.

Techniques: Northern Blot, Generated

Journal: Experimental neurology

Article Title: Increased precursor microRNA-21 following status epilepticus can compete with mature microRNA-21 to alter translation

doi: 10.1016/j.expneurol.2016.10.003

Figure Lengend Snippet: A schematic model depicts the novel regulation of increased pre-miR-21 through completing with mature miR-21 to bind to 3′UTR of TGFBR2 mRNA and to regulate its expression following SE.

Article Snippet: The reaction mix in each well contained 10 ul of the TaqMan® Universal Master Mix II (Thermo Fisher Scientific, MA), 1 ul of the FAM-MGB probe/primer mix (20x) for gene of interest (purchased from Thermo Fisher Scientific, MA; pri-miR-21: Rn03464993_pri; TGFBR2: Rn00579682_m1; NT-3: Rn00579280; pre-miR-21: CS51000; mature miR-21: 000397), 9 ul of cDNA/ddH2O.

Techniques: Expressing