Identification and validation of mRNA 3′untranslated regions of DNMT3B and TET3 as novel competing endogenous RNAs of the tumor suppressor PTEN

PTEN inactivation is a frequent event in oncogenesis. Multiple regulatory mechanisms such as promoter hypermethylation, antisense regulation, histone modifications, targeting by microRNAs (miRNAs/miRs) and regulation by transcription factors have all been shown to affect the tumor suppressor functions of PTEN. More recently, the functional involvement of competing endogenous RNAs (ceRNAs) in miRNA-dependent and coding-independent regulation of genes shed light on the highly nuanced control of PTEN expression. The present study has identified and validated DNA methyltransferase 3β (DNMT3B) and TET methylcytosine dioxygenase 3 (TET3) as novel ceRNAs of PTEN, with which they share multiple miRNAs, in HCT116 colorectal cancer cells. miR-4465 was identified and characterized as a miRNA that directly targets and regulates all 3 transcripts via their 3′untranslated regions (3′UTRs) through a combination of luciferase reporter assays, abrogation of miRNA response elements (MREs) via site-directed mutagenesis, target protection of MREs with locked nucleic acids, RT-qPCR assays and western blot analysis. Competitive miRNA sequestration was demonstrated upon reciprocal 3′UTR overexpression and siRNA-mediated knockdown of their respective transcripts. Overexpression of DNMT3B or TET3 3′UTR promoted apop-tosis and decreased migratory capacity, potentially because of shared miRNA sequestration and subsequent activation of PTEN expression. Knockdown of TET3 and DNMT3B decoupled their protein-coding from miRNA-dependent, coding-independent functions. Furthermore, the findings suggested that the phenotypic outcome of ceRNAs is dictated largely by the number of shared miRNAs, and predictably, by the existence of other ceRNA networks in which they participate. Taken together, the findings of the present study identified DNMT3B and TET3 as novel ceRNAs of PTEN that may impact its dose-sensitive tumor suppressive function.