Abstract 1184: NADPH oxidase-dependent reactive oxygen species generation regulate micro RNA-21 in prostate cancer
Authors: Jajoo Sarvesh , Mukherjea Debashree , Sheehan Kelly , Kaur Tejbeer , Sheth Sandeep , Rybak Leonard P. , Ramkumar Vickram 
Published: June 2012

Abstract
Introduction: MicroRNAs (miRs) are small non-coding RNAs that regulate the coding RNAs at post-transcriptional level. Some miRs have been shown to act as oncomiRs by promoting growth and invasiveness of tumor cells. One of these oncomiRs, miR-21, has been shown to contribute to tumorigenesis of prostate cancer. Although several effector pathways have been suggested for its oncogenic role, little is known about its upstream regulators. In the present study, we hypothesize that high levels of reactive oxygen species (ROS) produced by the NADPH oxidase regulate the expression of miR-21 and its target proteins, maspin and programmed cell death 4 (PDCD4) in prostate cancer cells and prostate cancer samples.
Methods: The levels of miR-21 and NADPH oxidase subunit p47phox were detected in RNA samples from human prostate cancer tissues (from OriGene Technologies) by TaqMan and real time RT-PCR assays, respectively. The levels of maspin and PDCD4 were determined in the formalin-fixed paraffin-embedded (FFPE) prostate tumor sections by immunostaining. In vivo studies were done in severe combined immunodeficient (SCID) mice to study the role of miR-21 and NADPH oxidase in prostate cancer metastasis by injecting transiently-transfected PC3-MM cells with control antisense oligodeoxynucleotides (ODNs), miR-21 antisense ODNs (anti-miR-21), scramble siRNA or p47phox siRNA via the tail vein and counting the metastatic lesions in the lungs. For in vitro studies androgen-independent human prostate cancer cell line PC3-MM was used after transient-transfections with ODNs or siRNAs (as mentioned above). Lysates were used to determine the levels of miR-21 (by TaqMan assay) and maspin, PDCD4 and p47phox (by Western blotting), while invasion and migration was studied by matrigel and wound-healing assays, respectively.
Results: RNA samples from patients with different stages of prostate cancer showed high levels of miR-21 and p47phox. This was associated with low expression of maspin and PDCD4, targets proteins which are negatively regulated by miR-21. Studies in SCID mice showed reduced number of metastatic lung lesions in mice injected with anti-miR-21 and p47phox siRNA- transfected PC3-MM cells in comparison to control ODNs and scramble siRNA-transfected cells. Further studies in PC3-MM cells showed reduced expression of miR-21, maspin and PDCD4, and decreased invasiveness and migration of PC3-MM cells in presence of anti-miR-21 and p47phox siRNA.
Conclusions: Based on the data from the present study we conclude that NADPH oxidase derived ROS serve as essential regulators of miR-21, its target proteins (maspin and PDCD4), and the oncogenic and pro-invasive functions of miR-21 in prostate cancer cells. The increased expression of p47phox could account for the high levels of ROS generation characteristic of prostate cancer cells and aid in promoting high levels of miR-21 expression and oncogenesis.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1184. doi:10.1158/1538-7445.AM2011-1184