A Double-Negative Feedback Interaction between MicroRNA-29b and DNMT3A/3B Contributes to Ovarian Cancer Progression

Abstract

Background: Epigenetic abnormalities are increasingly observed in multiple malignancies, including epithelial ovarian cancer (EOC), and their effects can be significantly counteracted by tumor-suppressor microRNAs, namely epi-miRNAs. Here, we investigated the role of miR-29b, a well-established epi-miRNA, in the DNA methylation regulation of EOC cells. Methods: The correlation between miR-29b and DNMT3A/3B expression was evaluated by RT-qPCR, western blotting and immunohistochemical analysis. The functional roles of miR-29b and DNMT3A/3B were tested by anti-miRs and microRNA precursors. A luciferase reporter assay was employed to detect the direct binding of miR-29b to DNMT3A/3B 3′ UTRs. Co-IP was utilized for investigating Id-1 binding activity. Results: miR-29b was negatively correlated with DNMT3A/3B expression at the cellular/histological levels. miR-29b silencing was correlated with increased DNMT3A/3B levels, whereasmiR-29b over-expression caused DNMT3A/3B down-regulation. Luciferase reporter assays confirmed that the miR-29b-mediated downregulation of DNMT3A/3Boccurred through the direct targeting of theirmRNAs'3'-UTRs,whereasBGS assays found that DNMT3A/3B knockdown increased miR-29b expression via CpG island promoter hypomethylation, thus suggesting a crucial crosstalk betweenmiR-29b and DNMT3A/3B via a double-negative feedback loop. Co-IP assay confirmed direct binding between DNMT3A and Id-1. Conclusion: Taken together, our study sheds light on a novel epigenetic circuitry regulating EOC progression and may provide novel options for miR-29b-based epi-therapeutic approaches for future EOC treatment.