RESUMO
Wnt/ß-catenin signaling pathway plays essential roles in mammalian development and tissue homeostasis. MicroRNAs (miRNAs) are a class of regulators involved in modulating this pathway. In this study, we screened miRNAs regulating Wnt/ß-catenin signaling by using a TopFlash based luciferase reporter. Surprisingly, we found that miR-142 inhibited Wnt/ß-catenin signaling, which was inconsistent with a recent study showing that miR-142-3p targeted Adenomatous Polyposis Coli (APC) to upregulate Wnt/ß-catenin signaling. Due to the discordance, we elaborated experiments by using extensive mutagenesis, which demonstrated that the stem-loop structure was important for miR-142 to efficiently suppress Wnt/ß-catenin signaling. Moreover, the inhibitory effect of miR-142 relies on miR-142-3p rather than miR-142-5p. Further, we found that miR-142-3p directly modulated translation of Ctnnb1 mRNA (encoding ß-catenin) through binding to its 3' untranslated region (3' UTR). Finally, miR-142 was able to repress cell cycle progression by inhibiting active Wnt/ß-catenin signaling. Thus, our findings highlight the inhibitory role of miR-142-3p in Wnt/ß-catenin signaling, which help to understand the complex regulation of Wnt/ß-catenin signaling.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/genética , Via de Sinalização Wnt , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Proliferação de Células , Humanos , Camundongos , MicroRNAs/química , Conformação de Ácido Nucleico , Biossíntese de Proteínas/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
MicroRNA-155 (miR-155) plays significant role in various physiological processes involving both innate and adaptive immunity. miR-155 expression level changes dynamically during various immune responses. However, current approaches for miR-155 detection at the RNA level do not precisely reflect the real-time activity. Herein, we generated a transgenic mouse line (R26-DTR-155T) for determination of miR-155-5p activity in vivo by inserting miR-155-5p target sequence downstream of a reporter transgene comprising Diphtheria Toxin Receptor and TagBlue fluorescence protein. Using this approach, R26-DTR-155T mice were able to measure variation in levels of miR-155-5p activity in specific cell types of interest. The DTR expression levels were inversely correlated with the endogenous miR-155 expression pattern as detected by quantitative RT-PCR. Our data demonstrate a novel transgenic mouse line which could be useful for tracing miR-155-5p activity in specific cell types through measurement of miR-155-5p activity at single cell level.
