Ion [48]. For the first time, we used a luciferase activity assay and Western blotting to validate that HIF-3 was truly a direct target of let-7a in MM. Therefore, we suggest that let-7a might promote myeloma angiogenesis by inhibiting its target (i.e. HIF-3) and subsequently triggering VEGF expression. Taken together, these data suggest the strong likelihood that the dysregulation of the above-discussed AGO2-induced angiogenic miRNAs leads to AGO2-mediated angiogenesis in MM.Conclusions The present study delineated the mechanistic roles of AGO2 in MM angiogenesis. AGO2 can drive MM neovessel formation in vitro and in vivo by dysregulating the expression of some angiogenic miRNAs. The proangiogenic let-7 family miRNAs, the miR-17/92 cluster and the anti-angiogenic miRNA miR-145 play crucial roles in AGO2-mediated angiogenesis by targeting angiogenesisrelated genes.Wu et al. Journal of Hematology Oncology 2014, 7:40 http://www.jhoonline.org/content/7/1/Page 10 ofFigure 7 AGO2-related let-7a targets HIF-3. The real-time PCR (A) and Western blot (B) detection of the HIF-3 expression levels in LP-1-si-AGO2 cells after transfection with a negative control and let-7a mimics. (C) The effect of let-7a on the luciferase activities of HIF-3 3-UTR or HIF-3 3-UTR with a mutated let-7a-binding site (mUTR). let-7a overexpression resulted in a significant decrease in the luciferase activity of the pmirGLO-reporter vector containing the HIF-3 3-UTR but not of the vector containing the corresponding mutant.MethodsStudy subjectsThis research was approved by the Hospital Review Board of the First Affiliated Hospital of Nanjing Medical University. All participants provided written informed consent in accordance with the Declaration of Helsinki. Bone marrow biopsy samples were obtained from 53 MM patients (33 males, 22 females) with a median age of 61.7 years (range, 38?0 years) who were recruited to this study between July 2010 and January 2013. MM was diagnosed X-396 site According to standard morphological and immunophenotypical criteria. The monoclonal component was IgG in 18 cases, IgA in 12 cases, IgD in 1 case, IgM in 1 case, light chain in 19 cases and no secretion in 2 cases. According to the Durie almon (DS) staging system, 5 patients were stage I, 5 were stage II and the remaining 43 were stage III. According to the International Staging System (ISS), 9 patients were stage I, 15 were stage II and the remaining 29 were stage III. The MM cell lines (LP-1, H929, U266 and OCI-My5) were gifted from Dr Tian (University of Arkansas for Medical Science, USA) and cultured in RPMI-1640 media (Gibco, Grand Island, NY, USA). HUVECs were cultured in ECM media with 10 heat-inactivated foetal bovine serum (FBS, Gibco), 2-mM L-glutamine (Gibco), penicillin (100 U/mL) and streptomycin (100 g/mL) at37 in a humidified chamber with 95 air and 5 carbon dioxide. HUVECs from passages 3? were used in all experiments.Immunohistochemical analysis and MVD assessment of bone marrow biopsiesBone marrow biopsy samples were fixed in 10 formalin and decalcified in 10 nitric acid. Anti-CD138 was used to detect myeloma cells. An anti-AGO2 monoclonal antibody (EAU32; Novocastra Laboratories, Ltd., Newcastleupon-Tyne, UK) was used to detect AGO2 expression in the myeloma cells from these PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27864321 samples. AGO2 staining was evaluated by 2 independent observers. The immunoreactive scores were determined according to the sum of the stained area and the intensity. Specifically, a score of 0 was as.