Signaling and Regulation MicroRNA-30c-2 Expressed in Ovarian Cancer Cells Suppresses Growth Factor–Induced Cellular Proliferation and Downregulates the Oncogene BCL9

MicroRNAs (miRNAs) are small noncoding RNAs that function as master regulators of posttranscriptional gene expression with each miRNA negatively regulating hundreds of genes. Lysophosphatidic acid (LPA) is a mitogenic lipid present within the ovarian tumor microenvironment and induces LPA receptor activation and intracellular signaling cascades like ERK/MAPK, leading to enhanced cellular proliferation. Here, we show that in SKOV-3 and OVCAR-3 cells, LPA stimulation at concentrations ranging from 1 nmol/L to 20 mmol/L for 30 to 60 minutes increases miR-30c-2 , and this effect is mediated through a combination of receptors because knock down of multiple LPA receptors is required for inhibition. The epidermal growth factor and platelet-derived growth factor also increase miR-30c-2 transcript expression, suggesting a broader responsive role for miR-30c-2 . Thus, we investigated the functional role ofmiR-30c-2 through ectopic expression of syntheticmiRNAprecursors ofmature miRNA or antagomir transfection and observed that microRNA-30c-2 reduces, and the antagomir enhances, cell proliferation and viability in OVCAR-3, cisplatin-insensitive SKOV-3 and chemoresistant HeyA8-MDR cells. Ectopic expression of miR-30c-2 reduces BCL9mRNA transcript abundance and BCL9 protein. Consistent with this observation, miR-30c-2 ectopic expression also reduced BCL9 luciferase reporter gene expression. In comparison with IOSE cells, all cancer cells examined showed increased BCL9 expression, which is consistent with its role in tumor progression. Taken together, this suggest that growth factor induced proliferation mediates a neutralizing response by significantly increasing miR-30c-2 which reduces BCL9 expression and cell proliferation in SKOV-3 andOVCAR-3 cells, likely as amechanism to regulate signal transduction downstream.MolCancer Res; 1–14. 2011 AACR. Introduction MicroRNAs (miRNAs) are small noncoding RNAs, 18 to 25 nucleotides in length, that negatively regulate gene expression through perfect or imperfect base pairing with the 30-UTR (untranslated region) of specific target mRNAs, facilitating mRNA degradation, or blocking protein translation, respectively. It is estimated that one miRNA is capable of regulating the expression of several hundred genes through this posttranscriptional process, which ultimately results in protein reduction (1). The mere existence of miRNA in biological systems reveals complex layers of epigenetic regulation govern the outcome of cellular signaling pathway cascades, after the initiation of the primary activating signal and subsequent downregulation of the initiating protein. It also reveals the possibility for an alternative therapeutic strategy for exploitation among disease states, particularly cancer, where multiple pathways are aberrant. However, elucidating the molecular function and systematic influence of individual miRNAs is challenging but has highlighted critical processes regulated through transcript-miRNA interactions (2). In oncogenesis the deregulation of miRNA results in altered tumor suppressor expression through translational inhibition and/or mRNA target degradation; thus an aberrant class of oncomirs that misregulate gene expression occurs through errors in sequence complementation (3) or a dual role in both processes—inhibition and degradation (4). Among expression profiles of human cancers, miRNA performed significantly better than mRNA gene expression transcriptional profiles to classify types (5), further underscoring their importance in malignancy and their tissue specificity. In particular, miRNAs are hypothesized to play an important regulatory role in ovarian cancer disease where observations highlighted the deregulation of miRNAs (6). Comparing epithelial ovarian carcinoma with immortalized ovarian surface epithelial cells, the vast majority of miRNA Authors' Affiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia, Athens, GA Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Corresponding Author: Mandi Murph, University of Georgia, 250 W. Green Street, Athens, GA 30602. Phone: 706-583-0216; Fax: 706-5425358; E-mail: [email protected] doi: 10.1158/1541-7786.MCR-11-0245 2011 American Association for Cancer Research. Molecular Cancer Research www.aacrjournals.org OF1 on June 19, 2017. © 2011 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Published OnlineFirst October 24, 2011; DOI: 10.1158/1541-7786.MCR-11-0245