Double-negative feedback interaction between DNA methyltransferase 3A and microRNA-145 in the Warburg effect of ovarian cancer cells

Abstract

Ovarian cancer is the most lethal gynecological malignancy because of its poor prognosis. The Warburg effect is one of the key mechanisms mediating cancer progression. Molecules targeting the Warburg effect are therefore of significant therapeutic value for the treatment of cancers. Many microRNAs (miR) are dysregulated in cancers, and aberrant miR expression patterns have been suggested to correlate with the Warburg effect in cancer cells. In our study, we found that miR-145 negatively correlated with DNA methyltransferase (DNMT)3A expression at cellular/histological levels. miR-145 inhibited the Warburg effect by targeting HK2. Luciferase reporter assays confirmed that miR-145-mediated downregulation of DNMT3A occurred through direct targeting of its mRNA 3'-UTRs, whereas methylation-specific PCR (MSP) assays found that knockdown of DNMT3A increased mRNA level of miR-145 and decreased methylation levels of promoter regions in the miR-145 precursor gene, thus suggesting a crucial crosstalk between miR-145 and DNMT3A by a double-negative feedback loop. DNMT3A promoted the Warburg effect through miR-145. Coimmunoprecipitation assays confirmed no direct binding between DNMT3A and HK2. In conclusion, a feedback loop between miR-145 and DNMT3A is a potent signature for the Warburg effect in ovarian cancer, promising a potential target for improved anticancer treatment.

Keywords: DNMT3A; methylation; miR-145; ovarian cancer; the Warburg effect.

Figure 1

Micro RNA (miR)‐145 was inversely correlated with DNA methyltransferase (DNMT)3A in epithelial ovarian cancer (EOC). A, mRNA levels of miR‐145 in ovarian cancer tissue samples (n = 31) were lower than in normal ovarian tissue samples (n = 15). B, Relative expression of miR‐145 in ovarian cancer tissue samples (I/II‐III/IV) (n = 12/n = 19) and normal ovarian tissue samples (n = 15). C, mRNA level of DNMT3A in ovarian cancer tissue samples (n = 31) was higher than in normal ovarian tissue samples (n = 15). D, Scatter diagram showing DNMT3A expression and miR‐145 expression by qPCR and their correlations (r ² = .156, P = .028) in 31 EOC tissue samples. E, Immunohistochemistry results indicate a negative correlation between miR‐145 and DNMT3A in EOC epithelia. F, Quantitative real‐time PCR results show a negative correlation between DNMT3A and miR‐145 in both SKOV3 and 3AO cells. G, Western blot assays show the expression of DNMT3A in SKOV3 and 3AO cells. Scale bar, 100 μm, *P < .05, t test

Figure 2

Micro RNA (miR)‐145 inhibited aerobic glycolysis by directly targeting hexokinase‐2 (HK2). A, Quantitative real‐time PCR shows that transfection of miR‐145 mimic rescued miR‐145 level in SKOV3 and 3AO cells. B, SKOV3 and 3AO cells expressing either control or mimic145 were cultured for 48 h. Acidification of the culture medium was evaluated by visually inspecting the color of the medium. C, Levels of lactate in the culture medium and glucose consumption were then measured and normalized to cell number. D, Quantitative real‐time PCR analysis shows that expression of metabolic enzyme genes was downregulated in SKOV3 and 3AO cells treated with mimic145 for 48 h. E, Western blot assays show that expression of HK2 was downregulated in SKOV3 and 3AO cells treated with mimic145 for 72 h. F, Luciferase reporter assays show that miR‐145 targeted HK2 directly. G, Western blot assays show that overexpression of HK2 counteracted the decline of HK2 caused by miR‐145 overexpression. H, Levels of lactate in the culture medium and glucose consumption were reduced by miR‐145 overexpression, and the effect was attenuated by overexpression of miR‐145 and HK2. *P < .05, **P < .01, t test. GLUT1, glucose transporter 1; LDH, lactate dehydrogenase; PDK1, pyruvate dehydrogenase kinase; PFK2, phosphofructokinase 2; PKM2, pyruvate kinase M2

Figure 3

DNA methyltransferase (DNMT)3A methylated pre‐micro RNA (miR)‐145 and downregulated miR‐145. A, Real‐time PCR shows that expression of miR‐145 was increased in SKOV3 and 3AO cell lines after treatment with 5‐aza‐2′‐deoxycytidine (5‐Aza‐CdR) for 5 days compared with mock‐treated cells. B, Quantitative real‐time PCR (qRT‐PCR) results show that DNMT3A mRNA level was significantly reduced by siDNMT3A transfection. C, qRT‐PCR shows that knockdown of DNMT3A increased miR‐145 in both SKOV3 and 3AO cells. D, Western blot assays show that knockdown of DNMT3A decreased DNMT3A expression. E, Quantitative analysis of methylation‐specific PCR (MSP) results show that the methylated proportion of miR‐145 precursor gene in DNMT3A siRNA‐transfected cells was lower than in control cells. F, qRT‐PCR results show that DNMT3A mRNA level was significantly increased by pcDNA3/Myc‐DNMT3A transfection. G, qRT‐PCR shows that overexpression of DNMT3A decreased miR‐145 in both SKOV3 and 3AO cells. H, Western blot assays show that overexpression of DNMT3A increased DNMT3A expression. I, Quantitative analysis of MSP results show that the methylated proportion of miR‐145 precursor gene in pcDNA3/Myc‐DNMT3A‐transfected cells was higher than in control cells. P < .05, **P < .01, t test

Figure 4

Micro RNA (miR)‐145 directly inhibited DNA methyltransferase (DNMT)3A. Mimic145‐mediated overexpression of miR‐145 markedly repressed DNMT3A at both the mRNA and protein level in SKOV3 and 3AO cells. A, mRNA level. B, protein level. C, Luciferase reporter assays show that miR‐145 targeted DNMT3A directly. *P < .05, t test

Figure 5

DNA methyltransferase (DNMT)3A promoted the Warburg effect in SKOV3 and 3AO ovarian cancer cells. A, DNMT3A expression was significantly decreased after 4 days of ‐aza‐2′‐deoxycytidine (5‐Aza‐CdR) treatment. B, Downregulation of DNMT3A by 5‐Aza‐CdR treatment inhibited glucose uptake and lactate secretion. C, Quantitative real‐time PCR (qRT‐PCR) shows that reduction of DNMT3A expression mainly inhibited the expression of HK2 among other molecules implicated in the Warburg effect. D, Western blot assays show that reduction of DNMT3A expression inhibited the expression of HK2. E, Levels of lactate in the culture medium and glucose consumption were measured and normalized to cell number. F, qRT‐PCR analysis shows that expression of metabolic enzyme genes was increased in SKOV3 and 3AO cells transfected with DNMT3A expression vector for 48 h. G, Western blot assays show that expression of hexokinase‐2 (HK2) was upregulated in SKOV3 and 3AO cells transfected with DNMT3A expression vector for 72 h. H, Coimmunoprecipitation assays show that there was no direct interaction between DNMT3A and HK2. *P < .05, **P < .01, t test

Figure 6

Micro RNA (miR)‐145 overexpression reversed DNA methyltransferase (DNMT)3A‐induced Warburg effect in ovarian cancer cells. A, SKOV3 and 3AO cells expressing negative control, overexpression of DNMT3A, and overexpression of DNMT3A plus miR‐145 were cultured for 48 h. Levels of lactate in the culture medium and glucose consumption were then measured and normalized to cell number. B, Quantitative analysis and (C) western blot results indicate that HK2 increase caused by transfection with DNMT3A expression vector pcDNA3/Myc‐DNMT3A was reversed by miR‐145 overexpression. *P < .05, **P < .01, t test

Figure 7

Expression of hexokinase‐2 (HK2) and DNA methyltransferase (DNMT)3A in ovarian cancer tissue subcutaneous tumors of nude mice. A, Immunohistochemical staining of HK2 and DNMT3A expression in subcutaneous tumor samples (original magnification, × 100; insets, × 400). B, Western blot shows HK2 and DNMT3A expression in subcutaneous tumor samples. Numbers 1‐7 represent 7 transplanted tumors in nude mice subcutaneous tumor models, respectively. C, Schematic representation of the anti‐Warburg effect mechanism of DNMT3A. miR‐145, micro RNA‐145. Scale bar, 100 μm, *P < .05, t test