Abstract 2058: Translational regulation of SPARC by the microRNAs mir29a, b, and c

Abstract

Abstract Background: SPARC (Secreted Protein Acidic and Rich in Cysteine) is overexpressed in most cancers, including breast, prostate, lung, brain, head-and-neck, and kidney. SPARC overexpression enhances tumor progression, angiogenesis, and metastasis and is generally associated with poor prognosis. In a previous study, SPARC mRNA was detected among the majority of both normal and cancer tissues using RT-PCR, in contrast to immunohistochemistry (IHC) that showed SPARC protein only in tissue remodeling and in cancers. These results suggested a post-transcriptional regulation of SPARC mRNA. We report here microRNA (miRNA) mediated regulation of SPARC mRNA. Methods: A chimera expression vector with CMV driven Luciferase ORF upstream of SPARC's 3′UTR was constructed and used in an assay to detect miRNA mediated suppression of luciferase translation. The construct was co-transfected into a permissive cell line- HEK293 (embryonic kidney cells) and individual miRNA mimics or inhibitors obtained from Dharmacon (Lafayette, Co, USA). Inhibitors of luciferase ORF were detected by a suppression of the luciferase signal. Deletion analysis on SPARC's 3′UTR was also performed to localize sequence required for translational suppression. Findings using CHO and 293 cells were confirmed using other cell lines including ES-2, SKOV-3, CAOV-3, PA-1, U87-MG, and OVCAR-3. Results: It was noted during forced expression of SPARC that CHO cells did not support SPARC overexpression, whereas HEK293 could. Screening using the luciferase ORF linked to SPARC's 3′UTR revealed that mir29 a, b and c were able to cause translational inhibition of SPARC mRNA. Deletion mutants of 3′UTR from SPARC restored the luciferase activity, whereas inhibition was retained when region 1-236 nt of SPARC's 3′UTR was maintained suggesting that the mir29 binding sequence is located within this region. The finding was not specific to CHO, and mir29 inhibition of SPARC expression was confirmed among all other cell lines tested (ES-2, SKOV-3, CAOV-3, PA-1, U87-MG and OVCAR-3). Conclusions: Human mir29a, b and c down-regulated the SPARC translation across a number of cell lines tested. These data suggest that that miRNAs mir29 a, b and c could play an important role in tumor metastasis and progression by control of SPARC expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2058.