Fig. 4

Down-regulating lncRNA DLX6-AS1 increases CADM1 expression via blockade of methylation of the CADM1 gene promoter region. a, subcellular localization of lncRNA DLX6-AS1 predicted by lncATLAS website; b, subcellular localization of LncRNA DLX6-AS1 detected by FISH, scale bar = 25 μm; c, Potential binding sites between LncRNA DLX6-AS1 and CADM1 analyzed by Blast; d, prediction of target genes of lncRNA DLX6-AS1 by MEM website; e, regulation of CADM1 by LncRNA DLX6-AS1 determined by dual-luciferase reporter gene assay, ^*, p < 0.05, vs. the NC group; f, fold enrichment of DNMT1, DNMT3a, and DNMT3b in the CADM1 promoter determined by CHIP-RT-qPCR, ^*, p < 0.05, vs. the Input group; g, fold enrichment of DNMT1, DNMT3a, and DNMT3b in the blank, oeLncRNA DXL6-AS1, and shLncRNA DXL6-AS1 groups determined by RIP-RT-qPCR, ^*, p < 0.05, vs. the blank group; h, the methylation of CADM1 promoter by BSP (black circle, methylation site; white circle, unmethylation site); i, distribution of CpG island in CADM1 gene promoter region; j, the methylation of CADM1 promoter detected by MSP (U, Unmethylation; M, methylation); k-l, expression of DLX6-AS1 and CADM1 determined by RT-qPCR, ^*, p < 0.05, vs. the blank group; m-n, protein levels of CADM1 determined by western blot analysis, ^*, p < 0.05, vs. the blank group; the statistical data were expressed as mean value of standard error, the differences between two groups were analyzed by the t test, and others were analyzed by one-way ANOVA; the experiments were conducted 3 times; blast, basic local alignment search tool; CADM1, cell adhesion molecule 1; ANOVA, analysis of variance; RT-qPCR, reverse transcription quantitative polymerase chain reaction; lncRNA, long non-coding RNA; DLX6-AS1, DLX6 antisense RNA 1; FISH, fluorescence in situ hybridization; CHIP, chromatin immunoprecipitation; DNMT1, DNA methyltransferase-1; DNMT3a, DNA methyltransferase-3a; DNMT3b, DNA methyltransferase-3b; BSP, bisulfite sequencing PCR; MSP, methylation specific PCR