MiR-15a and Mir-16-1 Affects the Angiogenesis of Multiple Myeloma by Targeting VEGF-A

Abstract 4039

MicroRNAs (miRNAs) are non-coding small RNAs that negatively modulate protein expression at a post-transcriptional level and are deeply involved in the pathogenesis of a variety of cancers. Two miRNAs, miR-15a and miR-16, which act as putative tumor suppressor by targeting the oncogene BCL2, have been implicated in cell cycle, apoptosis and proliferation. Here we investigated the possible role of miR-15a/miR-16 in the angiogenesis of multiple myeloma (MM). Using stem-loop quantitative reverse transcription-PCR analysis, we showed that miR-15a/miR-16 are significantly underexpressed in primary MM cells as well as MM cell lines (RPMI 8226, ARH-77, OPM-2, KM3, U266 and NIH929). The aberrant expression of miR-15a/miR-16 were detected especially in advanced stage multiple myeloma. The expression of miR-15a and miR-16-1 were remarkably lower in stage III MM patients (n=23), whereas it were significantly higher in healthy individuals (n=18) and stage ², II MM patients (n=14) (P<0.01 and P<0.001, respectively). In human MM cell lines and normal plasma cell, expression of miR-15a/miR-16 inversely correlated with the expression of vascular endothelial growth factor (VEGF). Consistently with the proposed role of miRNAs as key regulators of angiogenesis, here we identified VEGF-A as a target for miR-15a and -16. Enforced miR-15a or miR-16 expression reduced VEGF-A protein level and the luciferase reporter assays demonstrated that miR-15a and -16 specifically suppress expression of VEGF-A by directly interacting with its 3′-untranslated region. Moreover, Ectopic overexpression of miR-15a and -16 led to decreased pro-angiogenic activity of MM cells. Conditioned medium of pri-miR-15a- and pri-miR-16- transfected RPMI 8226 cells inhibited human bone marrow microvascular endothelial cell (BMEC-1) proliferation, chemotactic motility and capillary formation in vitro as compared with conditioned medium of scramble probe-transfected RPMI 8226 cells (P<0.05). Finally, miR-15a/miR-16 was shown to greatly inhibit the process of tumor formation in an animal model. Infection of lentivirus-miR-15a or lentivirus-miR-16 can significantly inhibit the xenograft tumor growth in nude mice. The average tumor volume after 4 weeks for GFP-transfected cells was 372.5 mm³. While the average tumor volume for miR-15a-transfected cells was 47 mm³ (P<0.05) and for miR-16-transfected cells was 93.6 mm³ (P<0.05). Of interest, neoangiogenesis analysis by immunohistochemistry staining of anti-CD31 showed that there were significant reductions in microvessel density present in the miR-15a group and miR-16 group as compared to control (P<0.05). Take together, our findings suggested that miR-15a and miR-16 could play a role in the tumorigenesis of MM at least in part by modulation of angiogenesis through targeting VEGF-A. These findings have therapeutic implications and may be exploited for future treatment of MM.