Figures & data
Figure 1. Expression of DNMT3A1/2 in HT and NT2 cell lines. (A) DNMT3A1 isoform expression and (B) DNMT3A2 isoform expression in HT and NT2 cells were assessed by RT-PCR. (C) DNMT3A1/2 protein levels in HT and NT2 cells.
Figure 2. miR-199a-3p directly targets DNMT3A. (A) The putative human DNMT3A 3′-UTR fragment containing miR-199a-3p binding sequence (837bp ~843bp) was inserted into the luciferase report vector pmirGLO downstream. (B) Dual luciferase assay of NT2 cells co-transfected with the luciferase constructs containing the DNMT3A 3′-UTR as well as miR-199a-3p mimics or scrambled oligonucleotides as the negative control.
Figure 3. miR-199a-3p regulates DNMT3A expression. (A) DNMT3A1 mRNA and (B) DNMT3A2 mRNA expression after the transfection with miR-199a-3p mimics or negative control (NC) in NT2 cells. (C) DNMT3A1/2 protein expression of NT2 cells transfected with miR-199a-3p mimics or NC. (D) DNMT3A1 mRNA (E) DNMT3A2 mRNA expression after the transfection with an anti-miRNA inhibitor specific for miR-199a-3p or negative control (NC) in HT cells. (F) DNMT3A1/2 proteins expression of HT cells transfected with an anti-miRNA inhibitor specific for miR-199a-3p or NC.
Figure 4. miR-199a-3p expression and DNMT3A2 mRNA levels inversely correlate in human TGCT tissues. (A) DNM3A1 mRNA expression (B) DNMT3A2 mRNA expression (C) miR-199a-3p expression in TGCT tissues compared with Normal testis tissues. (D) miR-199a-3p and DNMT3A2 mRNA expression levels inversely correlated in 25 clinical samples. Statistical analysis to evaluate correlation was performed using Pearson's correlation analysis (r = −0.591, p = 0.002).
Figure 5. DNMT3A does not negatively regulate miR-199a expression via promoter methylation in NT2 cells. DNM3A1 mRNA expression (A), DNMT3A2 mRNA expression (B) and their protein levels (C) after transfection of siDNMT3A into NT2 cells. (D) MiR-199a-3p mRNA expression and (E) miR-199a-5p mRNA expression after transfection of siDNMT3A into NT2 cells. (F) pre-miR-199a promoter methylation on chromosome 1 and chromosome 19 in NT2 cells after siDNMT3A transfection.
Figure 6. Enforced miR-199a-3p expression decreases methylation of some gene promoters in NT2 cell lines. (A) PRSS21, RASSF1A, MGMT, SCGB3A1, HIC1, HOXA9, APC, and RUNX1 mRNA expression levels in NT2 cells after transfection of miR-199a-3p mimics. (B) APC, HIC1, HOXA9, MGMT and SCGB3A1 mRNA expression levels in HT cells after transfection of miR-199a-3p inhibitor. (C) APC, HIC1, HOXA9, MGMT and SCGB3A1 mRNA expression levels in NT2 cells after transfection of siDNMT3A. (D) The promoter methylation pattern of APC, MGME and SCGB3A1 in NT2 cells after transfection of miR-199a-3p mimics.
Figure 7. miR-199a-3p does not significantly affect the expression of other DNMTs and TETs in NT2 cells. DNMT1, DNMT3B, TET1, TET2 and TET3 mRNA expression levels after transfection of miR-199a-3p mimics into NT2 cells.
