Lysophosphatidic Acid Mediates Activating Transcription Factor 3 Expression Which Is a Target for Post-Transcriptional Silencing by miR-30c-2-3p.

Ha T Nguyen,Aaron M Beedle,Wei Jia,Eileen J Kennedy,Mandi M Murph,Chunhong Yan

doi:10.1371/journal.pone.0139489

Ha T Nguyen, Aaron M Beedle + Show 4 more

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https://doi.org/10.1371/journal.pone.0139489

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Abstract

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.

Highlights

MicroRNAs are small non-protein-coding RNA molecules approximately 20–24 nucleotides in length that post-transcriptionally regulate gene expression
Since ATF3 expression peaked 1 h after lysophosphatidic acid treatment, we assessed a range of concentrations (1 to 40 μM) at the 1 h time point and found 5–20 μM to be effective in stimulating ATF3 mRNA transcription (Fig 1C) and ATF3 protein expression (Fig 1D) in SKOV-3 cells
We selected 5 μM for the remainder of the study because it is the lowest concentration that produces the response. This data suggests that in ovarian cancer cells, lysophosphatidic acid stimulation induces the expression of ATF3

Summary

Introduction

MicroRNAs (miRNAs) are small non-protein-coding RNA molecules approximately 20–24 nucleotides in length that post-transcriptionally regulate gene expression. Via non-canonical base pairing with the 3 ́UTR region of specific mRNAs, a single miRNA molecule is capable of inhibiting the expression of hundreds of different target genes. Massive gene repression by miRNAs only occurs occasionally, numerous mRNA targets are usually modestly repressed by miRNAs [3], further substantiating their role as the genome’s fine-tuners of protein expression. Profiles of human tumor specimens demonstrate that poorly differentiated tumors are far better classified by miRNA profiles, rather than mRNA [4]. Among human specimens of invasive epithelial ovarian cancer, the levels of RNA-interference proteins Dicer and Drosha are decreased in 60% and 51%, respectively, and this correlates with poor clinical outcome [5]

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