Abstract
The abundance of miR-132 ranges from constitutively high in the brain where it is necessary for neuronal development and function, to inducible expression in haematopoietic and endothelial cells where it controls angiogenesis and immune activation. We show that expression of AGO2, a protein central to miRNA-mediated gene silencing and miRNA biogenesis, is negatively regulated by miR-132. Using HeLa cells, we demonstrate that miR-132 interacts with the AGO2 mRNA 3′UTR and suppresses AGO2 expression and AGO2-dependent small RNA-mediated silencing. Similarly, miR-132 over-expression leads to AGO2 suppression in primary human dermal lymphatic endothelial cells (HDLECs). During phorbol myristate acetate (PMA)-activation of HDLECs, miR-132 is induced in a CREB-dependent manner and inhibition of miR-132 results in increased AGO2 expression. In agreement with the role of AGO2 in maintenance of miRNA expression, AGO2 suppression by miR-132 affects the steady state levels of miR-221 and miR-146a, two miRNAs involved in angiogenesis and inflammation, respectively. Our data demonstrate that the miRNA-silencing machinery is subject to autoregulation during primary cell activation through direct suppression of AGO2 by miR-132.
Highlights
The mature miR-132 is derived from the primary miR-132/miR-212 cluster, found in the intergenic region of chromosome 17p13.3. miR-132 and miR-212 have the same seed sequence and potential targets, miR-132 is the preferentially expressed miRNA from the cluster (Lagos et al, 2010)
The transcription of miR-132/miR-212 cluster is dependent on cAMP response element-binding (CREB) protein phosphorylation (Vo et al, 2005), which is inducible in an ERK-1/2 and MSK-1/2 dependent manner (Remenyi et al, 2010). miR-132 has been implicated in neuronal function and development (Remenyi et al, 2010; Wayman et al, 2008), circadian rhythm control (Alvarez-Saavedra et al, 2011), angiogenesis (Anand et al, 2010), and regulation of innate immune responses (Lagos et al, 2010; Shaked et al, 2009)
Our findings indicate that steady state expression of some miRNAs is highly sensitive to changes in AGO2 expression in human dermal lymphatic endothelial cells (HDLECs)
Summary
The mature miR-132 (miR-132-3p) is derived from the primary miR-132/miR-212 cluster, found in the intergenic region of chromosome 17p13.3. miR-132 and miR-212 have the same seed sequence and potential targets, miR-132 is the preferentially expressed miRNA from the cluster (Lagos et al, 2010). The expression of miR-132 is low under basal conditions in HDLECs (approximately 50–100 copies per cell), endothelial activation leads to an early upregulation of miR-132 required for endothelial cell proliferation and angiogenesis (Anand et al, 2010; Lagos et al, 2010). In the context of miRNA biogenesis, such regulatory loops have been identified in the regulation of DICER by let-7 in mammalian cell lines (Tokumaru et al, 2008), AGO1 regulation by miR-168. We identify an autoregulatory feedback mechanism that involves AGO2 suppression by miR-132 We have examined this mechanism in both transformed cell lines and activated primary human dermal lymphatic endothelial cells (HDLECs). Our findings reveal a novel mechanism regulating AGO2 expression and provide mechanistic insight into the function of miR-132 in human primary endothelial cells. Pri-miR-132 (TaqMan primers from Applied Biosystems) and using the SYBR Green Master Mix (Applied Biosystems) for GAPDH (forward: 5 -GGAGTCAACGGATTTGGTCGTA3 ; reverse: 5 -GGCAACAATATCCACTTTACCAGAGT-3 ) and primiR-126
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