title: Lysophosphatidic Acid Mediates Activating Transcription Factor 3 Expression Which Is a Target for Post-Transcriptional Silencing by miR-30c-2-3p.
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.
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, College of Pharmacy, 240 W. Green Street, Athens, Georgia 30602, United States of America.
authors.forename: Ha T
authors.initials: HT
authors.lastname: Nguyen
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, College of Pharmacy, 240 W. Green Street, Athens, Georgia 30602, United States of America.
authors.forename: Wei
authors.initials: W
authors.lastname: Jia
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, College of Pharmacy, 240 W. Green Street, Athens, Georgia 30602, United States of America.
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Beedle
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, College of Pharmacy, 240 W. Green Street, Athens, Georgia 30602, United States of America.
authors.forename: Eileen J
authors.initials: EJ
authors.lastname: Kennedy
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, College of Pharmacy, 240 W. Green Street, Athens, Georgia 30602, United States of America.
authors.forename: Mandi M
authors.initials: MM
authors.lastname: Murph
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D051700:Activating Transcription Factor 3 / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D015151:Immunoblotting; D008246:Lysophospholipids / Q000494:pharmacology*; D035683:MicroRNAs / Q000235:genetics*; D008856:Microscopy, Fluorescence; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D020413:3' Untranslated Regions; D051700:Activating Transcription Factor 3; D008246:Lysophospholipids; C546086:MIRN30b microRNA, human; D035683:MicroRNAs; C032881:lysophosphatidic acid