Research Paper Volume 11, Issue 6 pp 1695—1715

Long non-coding RNA CDKN2B-AS1 reduces inflammatory response and promotes cholesterol efflux in atherosclerosis by inhibiting ADAM10 expression

Figure 5. CDKN2B-AS1 promotes ADAM10 methylation by recruiting DNMT1. (A) the expression of CDKN2B-AS1 and transcription level of ADAM10 in cells infected with lentiviral vector expressing oe-CDKN2B-AS1 or sh-CDKN2B-AS1 determined by RT-qPCR; (B) Western blot analysis was used to detect the protein band and level of ADAM10 in cells infected with lentiviral vector expressing oe-CDKN2B-AS1 or sh-CDKN2B-AS1; (C) FISH was used to detect CDKN2B-AS1 localization in cells (× 200); (D) MS-PCR was used to detect the electrophoresis band of ADAM10 methylation level in atherosclerotic plaque, IMA tissues and ox-LDL-exposed THP-1 macrophages or ox-LDL-exposed THP-1 macrophages treated with 5-aza-dc or M.SssI; (E) CHIP assay to detect the output percentage of ADAM10 in cells infected with lentiviral vector expressing oe-CDKN2B-AS1 or sh-CDKN2B-AS1; (F) RIP assay to detect the output percentage of CDKN2B-AS1 in cells infected with lentiviral vector expressing oe-CDKN2B-AS1 or sh-CDKN2B-AS1; (G) RNA pull down to detect the DNMT1 protein pulled down by lncRNA CDKN2B-AS1 in cells infected with lentiviral vector expressing oe-CDKN2B-AS1 or sh-CDKN2B-AS1; In panel D, U represents the un-methylated lane, and M is representative of the methylation lane; * p < 0.05 vs. the oe-NC group; # p < 0.05 vs. the sh-NC group; the measurement data were expressed in the form of mean ± standard deviation and analyzed by one-way ANOVA, the experiment was repeated 3 times; THP-1, the human monocytic leukemia cell line; RT-qPCR, reverse transcription quantitative polymerase chain reaction; CDKN, cell-dependent kinase inhibitor; ANOVA, analysis of variance; ELISA, enzyme linked immunosorbent assay; RIP, RNA-binding protein immunoprecipitation; FISH, fluorescence in situ hybridization; CHIP, chromatin immunoprecipitation; MS-PCR, methylation-specific PCR.