Fig 2: piR-823 activated Wnt signaling through regulating DNA methylation of APC promoter in MCF-7 breast cancer. (A) Upregulation of DNMT1, DNMT3A, and DNMT3B by piR-823 at the mRNA levels in MCF-7 cells. (B) Upregulation of DNMT1, DNMT3A, and DNMT3B by piR-823 at the protein levels in MCF-7 cells. (C) Increased global DNA methylation in MCF-7 cells by overexpression of piR-823. DNMT inhibitor 5-Aza-2’-deoxycytidine (5-AzaDC) was used as a positive control. (D) TOP/FOP assay demonstrated activated Wnt signaling by overexpression of piR-823. (E) Immunofluorescence staining assay indicating relocation of β-catenin from the cytoplasm to the nucleus in MCF-7 cells after overexpression of piR-823. (F) piR-823 promoted DNA methylation of gene APC promoter in MCF-7 cells. DNA methylation-specific primers were applied for PCR analysis to distinguish methylated and unmethylated APC. (G,H) Suppressed expression of APC at both mRNA (G) and protein (H) levels by piR-823. (I) Western blot analyses indicating the upregulation of APC and downregulation of DNMT 1, DNMT 3A, and DNMT 3B in the tumor samples of mice treated with anti-piR-823. (J) Schematic representation of the mechanism through which piR-823 promotes tumorigenesis in the luminal subtype of breast cancer by methylating the APC promoter and activating Wnt signaling, thereby regulating breast cancer stem cells. Data are presented as mean ± SEM (N = 3), *p < 0.05, **p < 0.01.

Fig 3: miR-29b overexpression inhibits cholangiocarcinoma tumorigenesis in vivo. (A) Representative images of subcutaneous tumors excised from nude mice (day 63 post-inoculation) and tumor volume curve. Measurements began on day 15 post-implantation and were repeated every 7 days until sacrifice. (B) Tumor weight at sacrifice. (C) Representative images of immunohistochemical analysis of DNMT3B and CDKN2B expression in excised tumors. (D–G) Corresponding semiquantitative expression analyses of DNMT3B and CDKN2B. DNMT3B mRNA (D) and protein (E) levels. CDKN2B mRNA (F) and protein (G) levels. Scale bars = 100 μm. The values presented are means ± SD. *P<0.05 compared to the negative control group, as determined by analysis of one-way variance (ANOVA), followed by the repeated measures.

Fig 4: CircASPH regulated the level of the DNMT3b/5mC Axis by sponging miR-370-3p in HCC cells. (A, B) Molecular function and biological process of miR-370-3p target genes in gene ontology analysis. (C) KEGG analysis of miR-370-3p target genes. (D) Heatmap of the expression of the DNMT gene family level in GSE124535. (E) Predicted binding sites between DNMT3b and miR-370-3p. (F) Dual-luciferase reporter assay showed that miR-370-3p could interact with the 3’UTR of DNMT3b mRNA. (G, H) The expression of the DNMT3b level with miR-370-3p overexpression and knockdown was detected by RT-qPCR and Western blot. (I) Images of 5hmC-, 5mC-, and DAPI-stained HCC cells with different levels of miR-370-3p; scale bar = 20 μm. (J, K) The expression of DNMT3b level with circASPH overexpression and knockdown was detected by RT-qPCR and Western blot. (L) Images of 5hmC-, 5mC-, and DAPI-stained HCC cells with different levels of circASPH; scale bar = 20 μm. **p < 0.01, ***p < 0.001.

Fig 5: CircASPH promoted the HCC process via the miR-370-3p/DNMT3b/5mC/HAO2 axis. (A) The level of HAO2 expression was evaluated by RT-qPCR in 20 paired HCC tumor tissues and normal tissues. (B) Pearson analysis indicated the relationship between circASPH and HAO2. (C) EDU staining assays indicated the proliferation of cells expressing a combination of circASPH, sh-miR-370-3p, and HAO2. (D, E) Wound-healing assays and Transwell assays indicated the ability of migration and invasion of cells by expressing a combination of circASPH, sh-miR-370-3p, and HAO2. **p < 0.01.