Journal: Oxidative Medicine and Cellular Longevity

Article Title: Depleted Long Noncoding RNA GAS5 Relieves Intervertebral Disc Degeneration via microRNA-17-3p/Ang-2

doi: 10.1155/2022/1792412

Figure Lengend Snippet: Silencing of GAS5 ameliorates IVDD in vivo by miR-17-3p-mediated inhibition of Ang-2. (a) Observation of the MRI signal intensity and IVDD degree by MRI scanning. (b) Western blot analysis of Ang-2 and ECM metabolism-related proteins in NP tissues extracted from IVDD mice, which was normalized to GAPDH. (c) Statistical results of TUNEL-positive cells in the NP tissues extracted from IVDD mice. (d) The expression of glucosaminoglycan (GAG) in mouse NP tissues detected by Safranin O-Fast Green staining. n = 8. (e) HE histological scores of mouse NP tissues by HE staining. n = 8. ∗ p < 0.05 vs. mice without any injection; # p < 0.05 vs. IVDD mice injected with harboring shRNA against shRNA NC; $ p < 0.05 vs. IVDD mice injected with lentivirus harboring shRNA against GAS5; $ p < 0.05 vs. IVDD mice injected with lentivirus harboring overexpression vector NC; @ p < 0.05 vs. IVDD mice injected with lentivirus harboring oe-GAS5. Data (mean ± standard deviation) were assessed by one-way ANOVA, followed by Tukey's post hoc test. n = 8.

Article Snippet: Afterwards, plasmids of sh-NC, sh-GAS5, oe-NC, oe-GAS5, miR-17-3p mimic, and miR-17-3p inhibitor were packaged into lentivirus vectors (pSIH1-H1-copGFP and pLV-EGFP-N) by GenePharma Co., Ltd. (Shanghai, China) to generate lentiviruses including pSIH1-H1-copGFP-sh-NC, pSIH1-H1-copGFP-sh-GAS5, pLV-EGFP-NC, pLV-EGFP-GAS5, pSIH1-H1-copGFP-miR-17-3p inhibitor, and pLV-EGFP-miR-17-3p, respectively.

Techniques: In Vivo, Inhibition, Western Blot, TUNEL Assay, Expressing, Staining, Injection, shRNA, Over Expression, Plasmid Preparation, Standard Deviation

Journal: EBioMedicine

Article Title: Long non-coding RNA CDKN2B-AS1 contributes to atherosclerotic plaque formation by forming RNA-DNA triplex in the CDKN2B promoter

doi: 10.1016/j.ebiom.2020.102694

Figure Lengend Snippet: CDKN2B-AS1 affects atherosclerosis by forming RNA-DNA triplex with CDKN2B promoter. a, RT-qPCR assay for the transcriptional expression of the CDKN2B gene following treatment with GSK343, an E2H2 inhibitor. b, RNA immunoprecipitation assay for the binding of EZH2 to CDKN2B-AS1 relative to IgG. c, RT-qPCR assay for the expression of CDKN2B mRNA. d, ChIP assay for the enrichment of CTCF in the CDKN2B promoter region relative to IgG. e, ChIP assay for the enrichment of EZH2 and H3K27me3 in the CDKN2B promoter region relative to IgG. f, ChIP assay for the enrichment of CTCF and H3K27me3 in CDKN2B promoter region relative to IgG, following treatment with GSK343, an E2H2 inhibitor. g The CDKN2B-AS1 TFO sequence predicted by LongTarget. h, The binding of CDKN2B-AS1 and CDKN2B promoter detected by in vitro triplex pull-down assay. i, RNA-DNA triplex formed by CDKN2B-AS1 and CDKN2B detected by EMSA. j, The binding of CDKN2B-AS1 and CDKN2B promoter detected by in vivo triplex capture assay. * p < 0.05 vs. the sh-NC, DMSO, oe-control oligo or dGTP group; # p < 0.05 vs. the oe-NC or GSK343 group; ## p < 0.05 vs. the oe-CTCF group. The experiment was repeated 3 times independently. The t -test was used for comparison of data between two groups, and one-way ANOVA was used for comparison of data between multiple groups. sh-NC, cells transduced with pSIH1-H1-copGFP-sh-NC; sh-CDKN2B-AS1, cells transduced with pSIH1-H1-copGFP-sh-CDKN2B-AS1; oe-NC, cells transduced with LV5-GFP empty vector; oe-CTCF, cells transduced with LV5-GFP-CCCTC-binding factor; sh-CTCF, cells transduced with pSIH1-H1-copGFP-sh-CCCTC-binding factor.

Article Snippet: Lentivirus vectors were constructed by Shanghai GenePharma Company (Shanghai, China), including LV5-Green fluorescent protein (GFP) (gene overexpression vector), pSIH1-H1-copGFP (gene silencing vector), CDKN2B-AS1 shRNA, CCCTC-binding factor (CTCF) shRNA, and CDKN2B shRNA.

Techniques: Quantitative RT-PCR, Expressing, Immunoprecipitation, Binding Assay, Sequencing, In Vitro, Pull Down Assay, In Vivo, Transduction, Plasmid Preparation