Abstract A115: Annexin A1: A novel gene target of miRNA-21
Authors: Bhardwaj Anjana , Ganesan Nivetha , Albarracin Constance T. , Bedrosian Isabelle 
Published: May 2015

Abstract
Introduction: Numerous studies have reported miRNA-21 (miR-21) to be deregulated in a wide variety of cancers, including breast. miR-21 regulates a number of processes and pathways relevant to tumorigenesis including PDCD4, a regulator of apoptosis; PTEN, a suppressor of PI3K/AKT signaling; Smad7 and MSH2, regulators of TGFβ signaling; and Sprouty 1, an inhibitor of ERK-MAPK signaling. Here we describe an additional gene target of miR-21, annexin A1 that may be particularly relevant in the biology of triple negative breast cancer. Methods: To identify post-transcriptionally regulated genes that may play a role in breast cancer progression, we screened proteome and transcriptome of MCF10A breast cancer progression panel, reported by Choong et al (2010) and Rhee et al (2008) respectively. This resulted in the identification of Annexin A1. To identify miRNAs that have the potential to regulate annexin A1, we performed an in silico screen for miRNAs targeting annexin A1 using the miRNA algorithm TargetScan, and found miR-21 as a potential regulator. To validate if miR-21 indeed regulates annexin A1 we performed transient transfections to modulate miRNA levels in cells and subsequently performed western blot analysis and luciferase assay to measure the annexin A1 protein and reporter levels respectively. To measure endogenous and modulated levels of miRNAs and their effect on mRNA targets we performed QPCR on RNA samples obtained cell lines. Results: Our in silico analysis, suggested that annexin A1 was a potential downstream target of miR-21 with probability of conserved site targeting (PCT ) &lt; 0.1. To confirm that miR-21 directly targets annexin A1, we inserted their putative target sequence along with surrounding 3'UTR sequences, into a luciferase reporter vector. Co-transfection of the sequence-specific miR-21 inhibitor increased luciferase activity from the Annexin A1–3' UTR reporter vector by 1.8 fold to 2.5 fold relative to lipid only, in MCF7 cells that expresses relatively high levels of miR-21, whereas ~1.3 fold increase was caused by scramble sequence. In a complementary experiment, we transfected a sequence-specific miR-21 mimic (a double-stranded 21-nt form of miR-21 that is processed into a functional single-stranded miRNA in cells) along with Annexin A1-3'UTR reporter vector, in A293T cells, which expresses moderate levels of miR-21. The miR-21 mimic decreased Annexin A1-3'UTR driven luciferase activity by ~40% relative to lipid only, whereas ~15% increase was caused by scramble sequence. To validate that miR-21 regulates the endogenous expression of annexin A1 protein, we transfected sequence specific miR-21 inhibitors into MCF7 and 293T cells. The miR-21 inhibitor increased annexin A1 protein levels by ~50% in both cell lines compared to lipid only, whereas scramble sequences produced only a marginal increase (&lt;10%) in annexin A1 protein. Our efforts to address if this regulation is posttranscriptional or transcriptional revealed that annexin A1 is posttranscriptionally regulated by miR-21 since we did not observe any significant increase in Annexin A1 (mRNA) levels by miR-21 inhibitors in MCF7 cells. Lastly, we examined the annexin A1 protein and miR-21 levels in a panel of breast cancer cell lines. These analyses revealed although there was no correlation in ER + cell lines, an inverse pattern between miR-21 and annexin A1 expression was suggested in triple negative breast cancer (TNBC) cell lines. Conclusions: Annexin A1 represents a novel target of miR-21. This interaction appears to be predominant in TNBC. These observations require validation in breast tissue samples to assess if this TNBC specific regulation of annexin A1 by miR-21 also holds true in patient samples.

Citation Format: Anjana Bhardwaj, Nivetha Ganesan, Constance T. Albarracin, Isabelle Bedrosian. Annexin A1: A novel gene target of miRNA-21. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A115.