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MicroRNA-29 Impairs the Early Phase of Reprogramming Process by Targeting Active DNA Demethylation and Wnt Signaling

ABSTRACT: Somatic cell reprogramming by transcription factors and other modifiers such as small molecules or miRNAs has opened broad avenues for the study of developmental processes, cell fate determination, and interplay of molecular mechanisms in signaling pathways. However, many of the mechanisms that drive nuclear reprogramming itself remain yet to be elucidated. Here, we analyzed the role of miR-29 during reprogramming in more detail. Therefore, we evaluated miR-29 expression during reprogramming of fibroblasts transduced with lentiviral OKS and OKSM vectors and we show that addition of c-MYC to the reprogramming factor cocktail decreases miR-29 expression levels. Moreover, we found that transfection of pre-miR-29a strongly decreased OKS-induced formation of GFP+- colonies in MEF-cells from Oct4-eGFP reporter mouse, whereas anti-miR-29a showed the opposite effect. Furthermore, we studied two pathways which are important for reprogramming and which involve miR-29 targets: active DNA-demethylation and Wntsignaling. We show that inhibition of active DNA-demethylation as well as activation of Wnt-signaling leads to decreased reprogramming efficiency. Moreover, transfection of premiR-29 resulted in elevated expression of β-Catenin transcriptional target sFRP2 and increased TCF/LEF-reporter activity. Finally, we report that Gsk3-β is a direct target of miR-29 in MEF-cells. Together, our findings contribute to the understanding of the molecular mechanisms by which miR29 influences reprogramming.

ORGANISM(S): Homo sapiens

PROVIDER: GSE87609 | GEO | 2016/10/05

SECONDARY ACCESSION(S): PRJNA345375

REPOSITORIES: GEO

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Project description:The miR-29 family is an important player in the molecular pathophysiology of distinct types of cancer, with roles that seems to depend on cellular context. Reduced miR-29 levels are associated with more aggressive disease and its overexpression in cancer and leukemic cell lines inhibits proliferation. In contrast, its overexpression in hematopoietic progenitors, promotes leukemogenesis. We explored the potential roles of miR-29a in the molecular pathophysiology of T-cell acute lymphoblastic leukemia (T-ALL). As compared to normal T-cells, miR-29a levels are extremely reduced in T-ALL and in the Jurkat cell line. Microarrays analysis in Jurkat cells, following the introduction of synthetic miR-29a mimics, revealed the down-regulation of several predicted targets, including previously described targets (DNMT3a/b, CDK6, PXDN, MCL1, PIK3R1 and CXXC6), and novel targets with roles in active DNA demethylation, such as members of the ten-eleven-translocation (TET) family and TDG. Reduced miR-29a levels contribute to altered epigenetics, as its introduction in Jurkat cells, promotes the demethylation of the AHR gene (commonly methylated in T-ALL). In T-ALL patients, miR-29a levels are significantly associated with blast counts and disease free survival. Our results highlight the relevance of miR-29 in T-ALL physiopathology, and may help to clarify some contrasting findings reported in the literature. In order to identify potential miR-29a targets in T-cell lymphoblastic leukemia, Jurkat cells were electroporated with synthetic RNA molecules corresponding to miR-29a mimics (pre-miR) or inhibitors (anti-miR); and microarray profiles were compared to the profiles of Jurkat cells eletroporated with the corresponding negative controls. Transcripts with levels reduced following the introduction of the pre-miR-29a (as compared to pre-miR-Control), or transcripts acumulated folowing introduction of the anti-miR-29a inhibitor (as compared to anti-miR-Control), were considered potential targets; and were further compared to microRNA target prediction databases. Two paired samples were used for this analysis.

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MicroRNA-29 Impairs the Early Phase of Reprogramming Process by Targeting Active DNA Demethylation and Wnt Signaling

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