252 FUNCTIONAL CHARACTERIZATION OF miR-130 FAMILY DURING BOVINE PRE-IMPLANTATION EMBRYO DEVELOPMENT

Abstract

Small noncoding RNA are known to play a regulatory role in various biological processes including development. Here we aimed to investigate the spatiotemporal expression pattern of the miR-130 microRNA family (miR-130a, miR-130b, and miR-301b) throughout the bovine pre-implantation- stage embryos and to experimentally validate the target of miR-130b. For this, triplicate pools of in vitro-produced immature or mature oocytes (n = 100), zygote (n = 100), 2-cell (n = 50), 4-cell (n = 30), 8-cell (n = 30), morula (n = 20), and blastocyst (n = 20) stage embryos were used to profile the 3 miRNA and one of the predicted target gene MSK1 using SYBR green-based real-time PCR system. Furthermore, based on its expression profile result, miR-130b was selected for localization in all stages of embryos using 3′digoxigenin labeled, LNA-modified oligonucleotide probes. Reporter assay was conducted in cultured bovine cumulus cells to validate the target of miR-130b. The predicted MSK1 gene 3′ untranslated region- containing dual-luciferase miRNA target expression vector (pmirGLO; 600 ng), having renilla luciferase as a control reporter (Promega, Madison, WI, USA), was cotransfected with miR-130b precursor (20 pmol) and/or miR-130b inhibitor (20 pmol) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in Opti-MEM1 Medium with 4 independent transfections. Cells were lysed 48 h post-transfection, and luciferase assay was performed in luminometer. Normalization of firefly luciferase activity was based on renilla luciferase activity, and data were presented as mean ± SD. The expression profiling result shows that miR-130b was abundant (>8 to 12-fold) at morula and blastocyst stages. This was further validated by in situ localization, where high fluorescent intensity was seen in the same stages. Greater abundance of miR-301b was evident immediately after fertilization at zygote stage, whereas miR-130a was abundant in equal amount from oocyte until 8-cell stage, after which expression reduced at morula and blastocyst stages. Verification of MSK1 as target of miR-130b using MSK1-pmirGLO-vector showed a significant reduction (>45%) in expression of luciferase when cells were cotransfected with miR-130b precursor compared with cotransfection with miR-130b inhibitor (95%) or only MSK1 construct transfection (100%). MSK1, which was validated as target of miR-130b in the current study for the first time, is known to regulate the phosphorylation of CREB and ATF1 and is required for inhibits of Wnt-fi-catenin pathway and cell proliferation in colon cancer cells (Morán OP et al. 2008 J. Cell Biol. 183(4), 697-710). The results from this study evidenced the spatiotemporal expression of members of the miR130 family during bovine embryo development and their potential regulatory mechanism in the expression of developmentally important gene. The overexpression or inhibition of these miRNA in bovine oocytes and embryos might help in fully understanding their specific role in bovine embryogenesis.