Nguyen Ha T, Jia Wei, Beedle Aaron M, Kennedy Eileen J, Murph Mandi M
Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, College of Pharmacy, 240 W. Green Street, Athens, Georgia 30602, United States of America.
PLoS One. 2015 Sep 29;10(9):e0139489. doi: 10.1371/journal.pone.0139489. eCollection 2015.
Although microRNAs (miRNAs) are small, non-protein-coding entities, they have important roles in post-transcriptional regulation of most of the human genome. These small entities generate fine-tuning adjustments in the expression of mRNA, which can mildly or massively affect the abundance of proteins. Previously, we found that the expression of miR-30c-2-3p is induced by lysophosphatidic acid and has an important role in the regulation of cell proliferation in ovarian cancer cells. The goal here is to confirm that ATF3 mRNA is a target of miR-30c-2-3p silencing, thereby further establishing the functional role of miR-30c-2-3p. Using a combination of bioinformatics, qRT-PCR, immunoblotting and luciferase assays, we uncovered a regulatory pathway between miR-30c-2-3p and the expression of the transcription factor, ATF3. Lysophosphatidic acids triggers the expression of both miR-30c-2-3p and ATF3, which peak at 1 h and are absent 8 h post stimulation in SKOV-3 and OVCAR-3 serous ovarian cancer cells. The 3´-untranslated region (3´-UTR) of ATF3 was a predicted, putative target for miR-30c-2-3p, which we confirmed as a bona-fide interaction using a luciferase reporter assay. Specific mutations introduced into the predicted site of interaction between miR-30c-2-3p and the 3´-UTR of ATF3 alleviated the suppression of the luciferase signal. Furthermore, the presence of anti-miR-30c-2-3p enhanced ATF3 mRNA and protein after lysophosphatidic acid stimulation. Thus, the data suggest that after the expression of ATF3 and miR-30c-2-3p are elicited by lysophosphatidic acid, subsequently miR-30c-2-3p negatively regulates the expression of ATF3 through post-transcriptional silencing, which prevents further ATF3-related outcomes as a consequence of lysophosphatidic acid signaling.
尽管微小RNA（miRNA）是小的非蛋白质编码实体，但它们在大多数人类基因组的转录后调控中发挥着重要作用。这些小实体在mRNA表达中产生微调调整，这可以轻微或大量影响蛋白质的丰度。此前，我们发现溶血磷脂酸可诱导miR-30c-2-3p的表达，并且其在卵巢癌细胞的细胞增殖调控中具有重要作用。这里的目标是确认ATF3 mRNA是miR-30c-2-3p沉默的靶点，从而进一步确立miR-30c-2-3p的功能作用。通过结合生物信息学、qRT-PCR、免疫印迹和荧光素酶测定，我们揭示了miR-30c-2-3p与转录因子ATF3表达之间的调控途径。溶血磷脂酸触发miR-30c-2-3p和ATF3的表达，它们在SKOV-3和OVCAR-3浆液性卵巢癌细胞中于刺激后1小时达到峰值，并在8小时后消失。ATF3的3´非翻译区（3´-UTR）是miR-30c-2-3p的预测推定靶点，我们使用荧光素酶报告基因测定法确认其为真正的相互作用。引入到miR-30c-2-3p与ATF3的3´-UTR之间预测相互作用位点的特定突变减轻了荧光素酶信号的抑制。此外，抗miR-30c-2-3p的存在在溶血磷脂酸刺激后增强了ATF3 mRNA和蛋白质。因此，数据表明，在溶血磷脂酸引发ATF3和miR-30c-2-3p的表达后，随后miR-30c-2-3p通过转录后沉默负调控ATF3的表达，这防止了溶血磷脂酸信号传导导致的进一步ATF3相关结果。
