Journal: Journal of Translational Medicine

Article Title: Identification of the regulatory role of lncRNA HCG18 in myasthenia gravis by integrated bioinformatics and experimental analyses

doi: 10.1186/s12967-021-03138-0

Figure Lengend Snippet: Upregulation of HCG18 is a target of miR-145-5p in MG. a The expression of HCG18 was examined in MG patients and control subjects by RT-PCR. b Transfection efficiency of miR-145-5p mimics was determined by RT-PCR. c The relative expression level of HCG18 after transfecting with miRNA NC or let-7c-5p mimics into Jurkat cells was measured using RT-PCR. d The putative miR-145-5p binding sequence of the wild type (WT) and mutation (MUT) sequence of HCG18. e The luciferase reporter plasmid containing HCG18-WT or HCG18-MUT was co-transfected with miR-145-5p mimics or miRNA NC into 293 T cells. The ratio of firefly/renilla activities represented luciferase activitie. The experiment was repeated at least three times, and data are presented as the mean ± SD, * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: The fragments of HCG18-WT or HCG18-MUT were cloned into the Renilla luciferase gene pmirGLO Luciferase Reporter Vectors (Promega, USA) to generate pmirGLO-HCG18-WT and pmirGLO-HCG18-MUT vectors, respectively.

Techniques: Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Transfection, Binding Assay, Sequencing, Mutagenesis, Luciferase, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: miR-200a attenuated oxidative stress, inflammation, and apoptosis in dextran sulfate sodium-induced colitis through activation of Nrf2

doi: 10.3389/fimmu.2023.1196065

Figure Lengend Snippet: Primer sequences used for qPCR.

Article Snippet: First, Future Biotherapeutics constructed a target Keap1 dual luciferase reporter gene vector and a mutant with a mutation in the miR-200a binding site and transfected them into NCM460 cells.

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: miR-200a attenuated oxidative stress, inflammation, and apoptosis in dextran sulfate sodium-induced colitis through activation of Nrf2

doi: 10.3389/fimmu.2023.1196065

Figure Lengend Snippet: miR-200a overexpression reduced oxidative stress and protected against barrier disruption in DSS-induced mice. (A, B) The level of CAT and the activity of SOD in colon samples of mice were detected by commercial kits. (C, D) The level of MDA and the activity of MPO in colon samples of mice were detected by commercial kits. (E) The ratio of GSH/GSSG in colon samples of mice was detected by commercial kits. (F) The mRNA expression of the Nrf2-regulated gene was detected by qRT-PCR in mice. (G) Representative immunoblot bands for the Nrf2, HO-1, Keap1, and NQO1 proteins in the colon of mice. GAPDH was used as a loading control. (H–K) Histogram of relative expression of Nrf2, HO-1, Keap1 and NQO1 proteins in the colon of mice. (L) Representative images of immunofluorescence staining of ZO-1(red) and Occludin (green) in colon samples from different experimental groups. (Original magnifications, ×200, scale bars =50 μm; ×400, scale bars =20 μm). Data are provided as mean ± SEM and reflect six experiments, analyzed using one-way ANOVA with Tukey post-hoc analysis. ∗ P < 0.05; ns, not significant.

Article Snippet: First, Future Biotherapeutics constructed a target Keap1 dual luciferase reporter gene vector and a mutant with a mutation in the miR-200a binding site and transfected them into NCM460 cells.

Techniques: Over Expression, Disruption, Activity Assay, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: miR-200a attenuated oxidative stress, inflammation, and apoptosis in dextran sulfate sodium-induced colitis through activation of Nrf2

doi: 10.3389/fimmu.2023.1196065

Figure Lengend Snippet: miR-200a overexpression attenuated DSS-induced colonic injury in vitro . (A) The expression of miR-200a in cells transfected with the miR-200a mimic or the miR-scramble control for 48 h. (B–E) The levels of inflammatory cytokines (IL-1β, TNF-α, IL-6, IL-10) were analyzed by ELISA kits in the NCM460 cells. (F) The production of MDA in Enterocytes was detected by commercial kits. (G) The activity of ROS in Enterocytes was detected by commercial kits. (H, I) The expression of 4-HNE and GSH/GSSG in Enterocytes was detected by commercial kits. (J–M) Representative immunoblot bands for the Nrf2, Keap1, and NQO1 proteins in Enterocytes. GAPDH was used as a loading control. (N) The mRNA levels of the Nrf2-regulated gene were detected by qRT-PCR in the NCM460 cells. (O) Cell viability detected by MTT in Enterocytes from different experimental groups. (P, R) Representative images of TUNEL staining in the NCM460 cells from different experimental groups. (Original magnifications, ×200, scale bars =50 μm);. The images of TUNEL positive for quantification were analyzed using Image J software. (Q, S) Representative images of the percentage of apoptosis were examined by flow cytometry. Data are provided as mean ± SEM and reflect three experiments, analyzed using one-way ANOVA with Tukey post-hoc analysis. ∗ P < 0.05; ns, not significant.

Article Snippet: First, Future Biotherapeutics constructed a target Keap1 dual luciferase reporter gene vector and a mutant with a mutation in the miR-200a binding site and transfected them into NCM460 cells.

Techniques: Over Expression, In Vitro, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot, Quantitative RT-PCR, TUNEL Assay, Staining, Software, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: miR-200a attenuated oxidative stress, inflammation, and apoptosis in dextran sulfate sodium-induced colitis through activation of Nrf2

doi: 10.3389/fimmu.2023.1196065

Figure Lengend Snippet: miR-200a targeted Keap1 and activated Nrf2. (A) Predicted miR-200a binding sites in the Keap1 3’-UTR. (B) Dual-luciferase reporter gene assay. (C, D) The mRNA levels of Keap1 and Nrf2 detected by qRT-PCR in the Enterocytes. (E) Representative immunoblot bands for the Nrf2 and Keap1 proteins in the NCM460 cells. GAPDH was used as a loading control. (F–G) Histogram of relative expression of Nrf2 and Keap1 proteins in in the NCM460 cells. (H–J) The expression of Nrf2 was tested by qRT-PCR and western blotting in cells. (K) Cell viability detected by MTT in Enterocytes from different experimental groups. (L) The TNF-α mRNA level detected by qRT-PCR in Enterocytes. (M, N) The ROS and MDA production in the NCM460 cells. (O) The caspase 3 activity in the NCM460 cells. Data are provided as mean ± SEM and reflect three experiments, analyzed using one-way ANOVA with Tukey post-hoc analysis. ∗ P < 0.05; ns, not significant. UTR, untranslated region; WT, wild type; MUT, mutant.

Article Snippet: First, Future Biotherapeutics constructed a target Keap1 dual luciferase reporter gene vector and a mutant with a mutation in the miR-200a binding site and transfected them into NCM460 cells.

Techniques: Binding Assay, Luciferase, Reporter Gene Assay, Quantitative RT-PCR, Western Blot, Expressing, Activity Assay, Mutagenesis