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MicroRNA down-regulated in human cholangiocarcinoma control cell cycle through multiple targets involved in the G1/S checkpoint.

ABSTRACT: MicroRNAs (miRs) recently emerged as prominent regulators of cancer processes. In the current study we aimed at elucidating regulatory pathways and mechanisms through which miR-494, one of the miR species found to be down-regulated in cholangiocarcinoma (CCA), participates in cancer homeostasis. miR-494 was identified as down-regulated in CCA based on miR arrays. Its expression was verified with quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). To enforce miR expression, we employed both transfection methods, as well as a retroviral construct to stably overexpress miR-494. Up-regulation of miR-494 in cancer cells decreased growth, consistent with a functional role. mRNA arrays of cells treated with miR-494, followed by pathway analysis, suggested that miR-494 impacts cell cycle regulation. Cell cycle analyses demonstrated that miR-494 induces a significant G1/S checkpoint reinforcement. Further analyses demonstrated that miR-494 down-regulates multiple molecules involved in this transition checkpoint. Luciferase reporter assays demonstrated a direct interaction between miR-494 and the 3'-untranslated region of cyclin-dependent kinase 6 (CDK6). Last, xenograft experiments demonstrated that miR-494 induces a significant cancer growth retardation in vivo.Our findings demonstrate that miR-494 is down-regulated in CCA and that its up-regulation induces cancer cell growth retardation through multiple targets involved in the G1-S transition. These findings support the paradigm that miRs are salient cellular signaling pathway modulators, and thus represent attractive therapeutic targets. miR-494 emerges as an important regulator of CCA growth and its further study may lead to the development of novel therapeutics.

SUBMITTER: Olaru AV

PROVIDER: S-EPMC3212606 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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MicroRNA down-regulated in human cholangiocarcinoma control cell cycle through multiple targets involved in the G1/S checkpoint.

Olaru Alexandru V AV Ghiaur Gabriel G Yamanaka Sumitaka S Luvsanjav Delgermaa D An Fangmei F Popescu Irinel I Alexandrescu Sorin S Allen Sarah S Pawlik Timothy M TM Torbenson Michael M Georgiades Christos C Roberts Lewis R LR Gores Gregory J GJ Ferguson-Smith Anne A Almeida Maria I MI Calin George A GA Calin George A GA Mezey Esteban E Selaru Florin M FM

Hepatology (Baltimore, Md.) 20111201 6

<h4>Unlabelled</h4>MicroRNAs (miRs) recently emerged as prominent regulators of cancer processes. In the current study we aimed at elucidating regulatory pathways and mechanisms through which miR-494, one of the miR species found to be down-regulated in cholangiocarcinoma (CCA), participates in cancer homeostasis. miR-494 was identified as down-regulated in CCA based on miR arrays. Its expression was verified with quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). ...[more]

PMID: 21809359

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Project description:Improper regulation of signaling in vascular smooth muscle cells (VSMCs) by angiotensin II (AngII) can lead to hypertension, vascular hypertrophy and atherosclerosis. The extent to which the homeostatic levels of the components of signaling networks are regulated through microRNAs (miRNA) modulated by AngII type 1 receptor (AT1R) in VSMCs is not fully understood. Whether AT1R blockers used to treat vascular disorders modulate expression of miRNAs is also not known. To report differential miRNA expression following AT1R activation by AngII, we performed microarray analysis in 23 biological and technical replicates derived from humans, rats and mice. Profiling data revealed a robust regulation of miRNA expression by AngII through AT1R, but not the AngII type 2 receptor (AT2R). The AT1R-specific blockers, losartan and candesartan antagonized >90% of AT1R-regulated miRNAs and AngII-activated AT2R did not modulate their expression. We discovered VSMC-specific modulation of 22 miRNAs by AngII, and validated AT1R-mediated regulation of 17 of those miRNAs by real-time polymerase chain reaction analysis. We selected miR-483-3p as a novel representative candidate for further study because mRNAs of multiple components of the renin-angiotensin system (RAS) were predicted to contain the target sequence for this miRNA. MiR-483-3p inhibited the expression of luciferase reporters bearing 3'-UTRs of four different RAS genes and the inhibition was reversed by antagomir-483-3p. The AT1R-regulated expression levels of angiotensinogen and angiotensin converting enzyme 1 (ACE-1) proteins in VSMCs are modulated specifically by miR-483-3p. Our study demonstrates that the AT1R-regulated miRNA expression fingerprint is conserved in VSMCs of humans and rodents. Furthermore, we identify the AT1R-regulated miR-483-3p as a potential negative regulator of steady-state levels of RAS components in VSMCs. Thus, miRNA-regulation by AngII to affect cellular signaling is a novel aspect of RAS biology, which may lead to discovery of potential candidate prognostic markers and therapeutic targets.

Dataset Information

MicroRNA down-regulated in human cholangiocarcinoma control cell cycle through multiple targets involved in the G1/S checkpoint.

Publications

MicroRNA down-regulated in human cholangiocarcinoma control cell cycle through multiple targets involved in the G1/S checkpoint.

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