MicroRNA transcriptome analysis identifies miR-365 as a novel negative regulator of cell proliferation in Zmpste24-deficient mouse embryonic fibroblasts

Abstract

Zmpste24 is a metalloproteinase responsible for the posttranslational processing and cleavage of prelamin A into mature laminA. Zmpste24(-/-) mice display a range of progeroid phenotypes overlapping with mice expressing progerin, an altered version of lamin A associated with Hutchinson-Gilford progeria syndrome (HGPS). Increasing evidence has demonstrated that miRNAs contribute to the regulation of normal aging process, but their roles in progeroid disorders remain poorly understood. Here we report the miRNA transcriptomes of mouse embryonic fibroblasts (MEFs) established from wild type (WT) and Zmpste24(-/-) progeroid mice using a massively parallel sequencing technology. With data from 19.5 × 10(6) reads from WT MEFs and 16.5 × 10(6) reads from Zmpste24(-/-) MEFs, we discovered a total of 306 known miRNAs expressed in MEFs with a wide dynamic range of read counts ranging from 10 to over 1 million. A total of 8 miRNAs were found to be significantly down-regulated, with only 2 miRNAs upregulated, in Zmpste24(-/-) MEFs as compared to WT MEFs. Functional studies revealed that miR-365, a significantly down-regulated miRNA in Zmpste24(-/-) MEFs, modulates cellular growth phenotypes in MEFs. Overexpression of miR-365 in Zmpste24(-/-) MEFs increased cellular proliferation and decreased the percentage of SA-β-gal-positive cells, while inhibition of miR-365 function led to an increase of SA-β-gal-positive cells in WT MEFs. Furthermore, we identified Rasd1, a member of the Ras superfamily of small GTPases, as a functional target of miR-365. While expression of miR-365 suppressed Rasd1 3' UTR luciferase-reporter activity, this effect was lost with mutations in the putative 3' UTR target-site. Consistently, expression levels of miR-365 were found to inversely correlate with endogenous Rasd1 levels. These findings suggest that miR-365 is down-regulated in Zmpste24(-/-) MEFs and acts as a novel negative regulator of Rasd1. Our comprehensive miRNA data provide a resource to study gene regulatory networks in MEFs.