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Abstract
Lysophasphatidic acid (LPA) is a mitogenic phospholipid present within the ovarian tumor microenvironment that induces ovarian cancer progression through multiple intracellular signaling cascades, leading to cell growth, motility and proliferation. MicroRNAs (miRNAs) are small, non-protein-coding entities with important roles in post-transcriptional regulation of most of the human genome. Previously, we found that the expression of miR-30c-2-3p is induced by LPA and has an important role in the regulation of cell proliferation in ovarian cancer cells. The goals of this study were to examine the correlation between LPA and miR-30c-2-3p expression as well as mechanisms of miR-30c-2-3p antitumor effects. We observed that Dicer and epigenetic modifications, particularly DNA methylation and histone methylation, were not the major regulators of miR-30c-2-3p overexpression. Applying 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. LPA triggers the expression of both miR-30c-2-3p and ATF3 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. Furthermore, the presence of anti-miR-30c-2-3p enhanced ATF3 mRNA and protein after LPA stimulation. Thus, the data suggest that after the expression of ATF3 and miR-30c-2-3p are elicited by LPA, 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 LPA signaling. Our in vivo pilot study shows evidence that miR-30c-2-3p can be a potential therapy for ovarian cancer. To date, there is limited information on miRNA mechanisms associated with LPA. Thus our findings bring in more understanding about the signaling circuits initiated by LPA, especially at the level of post-transcriptional silencing regulated by miRNAs. Furthermore, we provide experimental data to support the regulation of ATF3, another gene transcript targeted via miR-30c-2-3p, extending the current list, which includes BCL-9, HIF2A, X-box binding protein 1, Cyclin E1 and an adaptor protein of the NF-B signaling pathway.