Fig. 2: Methylation and expression profiles of the MEG3-miR-493-5p locus after epigenetic unmasking. | Cell Death & Disease

Fig. 2: Methylation and expression profiles of the MEG3-miR-493-5p locus after epigenetic unmasking.

From: MEG3-derived miR-493-5p overcomes the oncogenic feature of IGF2-miR-483 loss of imprinting in hepatic cancer cells

Fig. 2

a Schematic representation of the imprinted DLK1-MEG3 locus on human chromosome 14. This genomic region consists of protein-coding genes (including DLK1 and retrotransposon Gag like 1 (RTL1)), lncRNAs (including MEG3), small nucleolar RNAs (snoRNAs), and miRNAs. While DLK1 and RTL1 are paternally expressed, MEG3 shows maternal expression. MEG3-DMR covers a genomic segment upstream of the MEG3 TSS and extending to its first exon. The black arrow indicates the MEG3 TSS. The position of the six miRNAs highlighted after epigenetic unmasking is depicted. b Comparison of the MEG3-DMR methylation level between hepatic tumor cells and human hepatocytes. COBRA was performed to evaluate methylation of the CpG sites located upstream of the MEG3 TSS. Twelve CpG sites were analyzed in two distinct CpG-rich regions of MEG3-DMR, ranging from −1864 to −1593 bp for Region 1 (CpG #1 to #7) and −353 to −58 bp for Region 2 (CpG #8 to #12). The circles represent in black the methylation ratio calculated for each CpG analyzed in HCC cells (n = 3 per CpG). The average values of CpG methylation levels measured in human hepatocytes from four different donors were used as a reference. c Quantification of MEG3-DMR methylation status evaluated by COBRA in human hepatocytes. The methylation ratios (%) represent the average values calculated for CpG #1 to #7 and CpG #8 to #12, which delimited Region 1 and Region 2, respectively. d Quantification of MEG3-DMR methylation status after 5-AZA treatment, evaluated by COBRA in Hep3B and HepG2 cells. Genomic DNA was extracted after demethylating treatment with 2.5 µM 5-AZA for 10 days. e Expression levels of MEG3, miR-493-3p, and miR-493-5p after epigenetic unmasking. The relative mRNA and mature miRNA expression levels were determined by RT-qPCR. Non-treated HCC cells were used as controls. The histograms shown in the figure represent the mean ± SD. Significant differences in gene expression and methylation levels were achieved at *p < 0.05, **p < 0.01, and ***p < 0.001 using a t-test and Mann–Whitney U test, respectively

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