Fig 2: RGS2 regulates CDH1 promoter methylation. A The putative CpG sites within CDH1 promoter are shown. B Methylation status of 11 CpG sites on the promoter region of CDH1 gene. Methylation analysis was performed in ten clones for each group. Each row of circles represents a single clone, and each circle represents a single CpG site. Open circle represents unmethylated cytosine; filled circle represents methylated cytosine. C Western blot analysis for ALKBH1 expression levels in Flag-RGS2-transfected or control vector-transfected (Flag-RGS2, -) HTR-8/SVneo cells. D Western blot analysis for ALKBH1 expression levels in RGS2 siRNA-transfected (siRGS2) or control siRNA-transfected (siRGS2, -) HTR-8/SVneo cells. E Luciferase assay of HTR-8/SVneo cells co-transfected with firefly luciferase constructs containing the promoter construct of CDH1 and ALKBH1 or empty vector (Con) or treatment with decitabine for 24 h. F qRT-PCR analysis for E-Cadherin in HTR-8/SVneo cells transfected with ALKBH1 or treated with decitabine for 24 h. G Luciferase assay of HTR-8/SVneo cells co-transfected with firefly luciferase constructs containing the promoter construct of CDH1 and RGS2 and/or ALKBH1. H Luciferase assay of HTR-8/SVneo cells co-transfected with firefly luciferase constructs containing the promoter construct of CDH1 and miR-3935 mimics and/or TRAF6. I Luciferase assay of HTR-8/SVneo cells co-transfected with firefly luciferase constructs containing the promoter construct of CDH1 and miR-3935 inhibitor or negative control (NC). **p < 0.01 versus Con, vehicle, (RGS2 -, ALKBH1 -), mimics negative control (NC) without TRAF6 or inhibitor negative control (NC); ##p < 0.01 versus RGS2 without ALKBH1 or miR-3935 mimics with TRAF6; data represent the mean ± SD of three independent experiments

Fig 3: Transcriptome analysis of miR-3935-regulated genes. A A heat-map of normalized expression levels of the altered genes measured by RNA-seq comparing negative miRNA control-transfected (NC) and miR-3935 mimics-transfected HTR-8/SVneo cells. Blue indicates low expression levels; red indicates high expression levels. B A volcano plot showing the altered genes in the presence of miR-3935 mimics. The number of genes is summarized in the table. C GO and pathway analysis of miR-3935-regulated genes measured by RNA-seq comparing negative miRNA control-transfected and miR-3935 mimics-transfected HTR-8/SVneo cells. D A table showing the three representative candidate genes, whose expression levels were significantly altered by miR-3935 mimics. E qRT-PCR analysis for RGS2, TRAF6 and CTR9 levels in miR-3935 mimics-transfected or negative miRNA control-transfected (NC) HTR-8/SVneo cells. F qRT-PCR analysis for RGS2, TRAF6 and CTR9 levels in miR-3935 inhibitors-transfected or negative miRNA control-transfected (NC) HTR-8/SVneo cells. **p < 0.01 versus mimics negative control (NC) or inhibitor negative control (NC); data represent the mean ± SD of three independent experiments

Fig 4: TRAF6 is a direct target of miR-3935. A The putative binding site of miR-3935 and RGS2 is shown. B Luciferase assay of HEK293T cells co-transfected with firefly luciferase constructs containing the TRAF6 wild-type 3’-UTRs and miR-3935 mimics, mimics NC, miR-3935 inhibitor or inhibitor NC. C Luciferase assay of HEK293T cells co-transfected with firefly luciferase constructs containing the TRAF6 mutated 3’-UTRs (mut) and miR-3935 mimics, mimics NC, miR-3935 inhibitor or inhibitor NC. D Western blot analysis for TRAF6 in miR-3935 mimics or mimics NC-transfected HTR-8/SVneo cells. E qRT-PCR analysis for RGS2 in HTR-8/SVneo cells co-transfected with TRAF6, CTR9 and miR-3935 mimics or mimics NC. **p < 0.01 versus mimics control (NC), inhibitor control (NC), or mimics control without TRAF6 or CTR9 (NC, TRAF6 -, CTR9 -); ##p < 0.01 versus mimics control with TRAF6 and without CTR9 (NC, TRAF6 + , CTR9 -); data represent the mean ± SD of three independent experiments