Fig. 1: MeCP2 regulates miR-22 expression via binding to miR-22 upstream methylated enhancer.

a MiR-22 levels in MeCP2 knockdown or overexpression GC cells. b ChIP-PCR assay was used to capture the miR-22 enhancer region with MeCP2 antibodies in GC cells. c Schematic of the relative position of the enhancer and MeCP2-binding site to the miR-22 location. d Correlation between miR-22 expression and the methylation level of two CpG sites located in the MeCP2 occupied enhancer from the TCGA database (Pearson analysis). e MiR-22 levels in GC cells treated with different concentrations of 5-azacytidine (5-Aza). f The methylation levels of CpG sites. Genomic DNA was extracted from DMSO- and 5-Aza-treated AGS cells. n = 15, Fisher’s exact test. g Schematic of luciferase reporter vectors (top), and luciferase activity of GC cells transfected with the reporter vectors (bottom). h ChIP-PCR analysis of the enhancer with anti- H3K27ac, P300, and IgG in AGS and MKN45 cells. i Diagram of full-length MeCP2 and different MeCP2 domain deletion vectors (top). After the vectors were transfected into AGS and MKN45 cells, different expression types were detected by western blotting using anti-GFP (bottom). j Immunofluorescence of the expression of plasmids MeCP2-WT, MeCP2ΔMBD, MeCP2ΔTRD, and MeCP2ΔTRD+NLS in AGS cells. A GFP antibody was used to label the exogenous proteins, and DAPI was used to label the nuclei. k AGS and MKN45 cells were transfected with GFP-tagged MeCP2 full length or domain deletion constructs, and the interaction between MeCP2 and the miR-22 enhancer was determined by ChIP-PCR. l Bioinformatics analysis of Hi-C data. All the results are shown as the mean±SD. n = 3, *p < 0.05, **p < 0.01, Student’s t test.