MiR-15 a is underexpressed and inhibits the cell cycle by targeting CCNE 1 in breast cancer

MicroRNAs (miRNAs) are a small class of noncoding RNAs that are widely dysregulated in various cancers. They act as either oncogenes or tumor suppressor genes in human cancer. The purpose of this study was to examine the expression levels of miR-15a in human breast cancer and its potential role in disease pathogenesis. The expression levels of miR-15a were measured by quantitative polymerase chain reaction (qPCR) in 40 breast cancer specimens and adjacent normal breast tissues. MTT assays, colony formation assays, transwell chamber migration assays, cell cycle and apoptosis assays were used to explore the potential func tion of miR-15a in MDA-MB-231 human breast cancer cells. Luciferase reporter assays were performed to validate the regulation of a putative target of miR-15a, in corroboration with qPCR and western blot assays. We found that the expression of miR-15a was lower in breast cancer specimens compared with adjacent normal tissues (p<0.05). Overexpression of miR-15a inhibited cellular growth, suppressed migration and arrested cells at the G1 phase, but did not promote cellular apoptosis. Luciferase assays indicated that miR-15a can bind with its putative target site in the 3'-untranslated region (3'-UTR) of CCNE1, suggesting that CCNE1 is a direct target of miR-15a. qPCR and western blot analysis indicated that the overexpression of miR-15a results in the downregulation of CCNE1 at the mRNA and protein levels. Taken together, the upregulation of miR-15a expression causes cellular growth inhibition, suppression of migration and G1 phase arrest by targeting CCNE1. These findings suggest that miR-15a may act as a tumor suppressor gene in breast cancer and that, in the future, it could be used as a therapeutic target for the treatment of breast cancer. Introduction MicroRNAs (miRNAs) are composed of approximately 20-25 nucleotide-non-coding RNAs that may silence post-transcriptional protein expression through two axes: first, by binding to complementary target messenger RNAs to target them for degradation; and second, inhibiting mRNA translation into proteins (1). There has been significant evidence showing that miRNAs regulate as many as 30% of the human protein coding genes (2). Moreover, they can function as oncogenes or tumor suppressor genes by altering regulation of their targets in many cancers (3,4). Indeed, studies have shown that miRNAs are involved in a variety of processes including tumor cell proliferation, differentiation, and apoptosis. Moreover, their differing expressions can lead to different human cancers (5,6). Subsequently, miRNA research has become a hot spot in breast cancer research whereby miRNAs are believed to have broad prospects in terms of diagnosis and treatment of this disease (7-10). Recent studies indicate that miR-15a is downregulated in chronic lymphocytic leukemia (11), prostate cancer (12), osteosarcoma (13), keratocystic odontogenic tumors (14) and breast cancer (15). Furthermore, through overexpression of miR-15a, curcumin can reduce the expression of Bcl-2 and subsequently induce apoptosis in MCF-7 breast cancer cells (15). However, the mechanism by which miR-15a contributes to breast cancer tumorigenesis is still unclear. Cyclin E1 (CCNE1), one member of the cyclin E family, can associate with and activate cyclin-dependent kinase 2 (CDK2). CCNE1 is a positive regulator of G1/S phase transition and is essential for cell cycle re-entry from G0 phase. In many human tumors, CCNE1 is overexpressed and the level of both protein and kinase activity is often deregulated relative to the cell cycle, as demonstrated in human breast epithelial cells (16). One study has indicated that deregulation of CCNE1 is an early event in the development of breast cancer (17). Furthermore, overexpression of CCNE1 in patients with breast cancer is associated with worse prognosis (18,19). CCNE1 can be repressed by some miRNAs, for example, a study has demonstrated the involvement of miR-16 in its regulation in human endothelial progenitor cells (20). In this study, we first demonstrated that miR-15a expression is significantly lower in breast cancer specimens when MiR-15a is underexpressed and inhibits the cell cycle by targeting CCNE1 in breast cancer QIFENG LUO1, XIAOYU LI1, JIA LI2, XIANGJIE KONG1, JUNFENG ZHANG1, LEI CHEN1, YIXIANG HUANG1 and LIN FANG1 1Department of General Surgery, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P.R. China; 2Department of Microbiology and Genetic Institute, Paris-Sud 11 University, Paris, France Received May 24, 2013; Accepted July 5, 2013 DOI: 10.3892/ijo.2013. 2034 Correspondence to: Dr Lin Fang, Department of General Surgery, Shanghai Tenth People's Hospital, Tongji University School of Medicine, 301 Middle Yanchang Road, Zhabei District, Shanghai