Fig. 6.

EZH2/DNMT3a is assisted by lncRNA ELFN1-AS1 in locating the promoter of MEIS1 to suppress MEIS1 expression. a Venn diagram between EZH2-binding lncRNAs identified by catRAPID and upregulated lncRNAs in the TCGA CRC dataset (p < 0.05 and | logFC | ≥ 1). b qPCR analysis of MEIS1 expression levels in CRC cells transfected with shRNAs targeting lncRNAs. c, d qPCR and western blot analysis of the ELFN1-AS1 mRNA and MEIS1 protein levels, respectively, were determined in cells with overexpression (c) or knockdown of ELFN1-AS1 (d). e After separation of the nuclear and cytosolic fractions, the RNA expression levels were measured by qPCR. GAPDH was used as a cytoplasmic control gene, and U6 was used as the nuclear control gene. Data are presented as percentages of GAPDH, U6, and ELFN1-AS1 levels, and total levels for each were taken as 100%. f RIP experiments for IgG, EZH2, and DNMT3a were performed, followed by qPCR to examine the enrichment of ELFN1-AS1. g, h The interaction of full-length (g) and truncated (h) ELFN1-AS1 with EZH2 and DNMT3a was confirmed by RNA pull-down and immunoblotting. i, j ChIP-qPCR analysis of the enrichment of EZH2, DNMT3a and H3K27 methylation status at the MEIS1 promoter in CRC cells with overexpression (i) or knockdown (j) of ELFN1-AS1. k, l BSP analysis of the DNA methylation status of the MEIS1 locus in CRC cells with overexpression (k) or knockdown (l) of ELFN1-AS1. m Correlation of ELFN1-AS1 expression with the MEIS1 DNA methylation status in CRC and normal tissues from the TCGA 450K array by the Pearson correlation coefficient. n Western blot of EZH2, MEIS1, and FEN1 expression in Caco2 and SW480 cells transfected with EZH2 shRNA, oe-ELFN1-AS1, and their corresponding controls. o Western blot of DNMT3a, MEIS1 and FEN1 expression in Caco2 and SW480 cells transfected with DNMT3a shRNA, oe-ELFN1-AS1, and their corresponding controls. Data are presented as the mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001