DNMT1 promotes the proliferation and migration of gastric cancer cells by inducing microRNA-125a-5p methylation to promote SERPINE1 protein

Figure 1 The expression and methylation levels of microRNA-125a-5p in gastric cancer tissues and gastric cancer cell lines. A: MicroRNA (miR)-125a-5p expression in clinical samples detected by real-time polymerase chain reaction (RT-qPCR); B: MiR-125a-5p promoter methylation level in clinical samples detected by methylation-specific polymerase chain reaction; C: RT-qPCR detection of miR-125a-5p expression in cells; D: Methylation-specific polymerase chain reaction detection of the miR-125a-5p promoter methylation level in cells. ^cP < 0.001. miR: MicroRNA; GC: Gastric cancer; M: Methylation; U: Unmethylated.

Figure 2 Overexpression of microRNA-125a-5p inhibits gastric cancer cell proliferation, migration, invasion and epithelial mesenchymal transition. A: Transfection efficiency of the microRNA (miR)-125a-5p mimic in HGC27 cells, as detected by real-time polymerase chain reaction (RT-qPCR); B: Proliferation of HGC27 cells detected by cell counting kit 8 assay; C: Migration of HGC27 cells detected by scratch experiments; D: HGC27 cell invasion detected by Transwell assays; E: Western blot detection of the expression of the epithelial mesenchymal transition (EMT)-related proteins E-cadherin, N-cadherin and vimentin in HGC27 cells; F: Immunofluorescence detection of the expression of the EMT-related protein vimentin in HGC27 cells (scale bar = 10 μm). ^bP < 0.01. ^cP < 0.001. EMT: Epithelial mesenchymal transition; NC: Normal control; miR: MicroRNA; OD: Optical density; DAPI: 4’,6-diamidino-2-phenylindole; miR: MicroRNA; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 3 DNMT1 represses microRNA-125a-5p expression through methylation. A: DNMT1 expression in clinical samples detected by real-time polymerase chain reaction (RT-qPCR); B: Positive expression of DNMT1 in clinical samples detected by immunohistochemistry (scale bar = 50 μm); C: Western blot analysis of DNMT1 expression in HGC27 cells; D: Toxicity of 5-aza-2’-deoxycytidine in HGC27 cells; E: RT-qPCR detection of microRNA (miR)-125a-5p expression in HGC27 cells after treatment with different concentrations of 5-aza-2’-deoxycytidine; F: Western blot detection of DNMT1 transfection efficiency in HGC27 cells; G: Methylation of miR-125a-5p in HGC27 cells detected by methylation-specific polymerase chain reaction; H: RT-qPCR detection of miR-125a-5p expression in HGC27 cells. ^cP < 0.001. OE-NC: Overexpression negative control; OE-DNMT1: DNMT1 overexpression; si-NC: Small interfering-negative control; si-DNMT1: Small interfering DNMT1; 5-aza-dC: 5-aza-2’-deoxycytidine; miR: MicroRNA; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; GC: Gastric cancer; M: Methylation; U: Unmethylated.

Figure 4 DNMT1 affects gastric cancer cell proliferation, migration, invasion and epithelial mesenchymal transition by mediating microRNA-125a-5p methylation. A: DNMT1 expression in HGC27 cells detected by Western blot; B: Methylation-specific polymerase chain reaction detection of the microRNA (miR)-125a-5p promoter methylation level in HGC27 cells; C: MiR-125a-5p expression in HGC27 cells detected by real-time polymerase chain reaction; D: Detection of HGC27 cell proliferation by cell counting kit 8 assay; E: HGC27 cell migration detected by scratch test; F: HGC27 cell invasion determined by Transwell assay; G: Western blot detection of the expression of the epithelial mesenchymal transition-related proteins E-cadherin, N-cadherin and vimentin in HGC27 cells; H: Immunofluorescence detection of the expression of the epithelial mesenchymal transition-related protein vimentin in HGC27 cells (scale bar = 10 μm). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. OE-NC: Overexpression negative control; OE-DNMT1: DNMT1 overexpression; 5-aza-dC: 5-aza-2’-deoxycytidine; miR: MicroRNA; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; OD: Optical density; M: Methylation; U: Unmethylated; DAPI: 4’,6-diamidino-2-phenylindole; PBS: Phosphate-buffered saline.

Figure 5 DNMT1 regulates SERPINH1 expression through microRNA-125a-5p. A: Positive expression of SERPINH1 in clinical samples detected by immunohistochemistry (scale bar = 50 μm); B: Western blot detection of SERPINH1 expression in cells; C: A bioinformatics platform (http://starbase.sysu.edu.cn/) was used to predict the target binding site of microRNA (miR)-125a-5p; D: Real-time polymerase chain reaction (RT-qPCR) verification of the targeted binding of miR-125a-5p and SERPINE1; E: RT-qPCR was used to detect the efficiency of miR-125a-5p knockdown in HGC27 cells; F: Western blot detection of SERPINH1 expression in HGC27 cells; G: RT-qPCR detection of miR-125a-5p expression in HGC27 cells; H: Western blot detection of SERPINH1 expression in HGC27 cells. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. WT: Wild-type; MUT: Mutant; NC: Negative control; miR: MicroRNA; GC: Gastric cancer; OE-NC: Overexpression negative control; OE-DNMT1: DNMT1 overexpression.

Figure 6 SERPINH1 knockdown attenuates the effects of DNMT1 on gastric cancer cell proliferation, migration, invasion and epithelial mesenchymal transition. A: Western blot detection of SERPINH1 transfection efficiency in HGC27 cells; B: Western blot detection of SERPINH1 expression in HGC27 cells; C: Proliferation activity of HGC27 cells detected by cell counting kit 8 assay; D: HGC27 cell migration detected by scratch experiments; E: HGC27 cell invasion detected by Transwell assay; F: Expression of the epithelial mesenchymal transition-related proteins E-cadherin, N-cadherin and vimentin in HGC27 cells, as detected by Western blot; G: Expression of the epithelial mesenchymal transition-related protein vimentin in HGC27 cells detected by immunofluorescence (scale bar = 10 μm). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. OE-NC: Overexpression negative control; OE-DNMT1: DNMT1 overexpression; si-NC: Small interfering-negative control; si-SERPINH1: Small interfering SERPINH1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; OD: Optical density; DAPI: 4’,6-diamidino-2-phenylindole.