2012;3:475C480. chemoresistant Hydroxyprogesterone caproate NB cell lines and induced apoptosis in MYCN-amplified cell lines. Moreover, the conditional expression of miR-497 in NB xenografts reduced tumor growth and inhibited vascular permeabilization. MiR-497 targets multiple genes related to the DDR, cell cycle, survival and angiogenesis, which renders this molecule a promising candidate for NB therapy. and analysis carried out to select miRNAs candidates. Hydroxyprogesterone caproate (B) Twenty-eight different mimic miRNAs oligonucleotides were reverse-transfected in the chemoresistant NB cell lines CHLA-90 (MYCN non-amplified, gray bars) and SK-N-BE(2) (MYCN amplified, black bars). Cells were fixed and stained with crystal violet 96 h post-transfection. Absorbance was measured at 590 nm after dissolving the crystals with 15% acetic acid. Proliferation values were normalized versus MOCK-transfected cells. Data represent mean SEM of three independent experiments (six replicates each experiment). Statistical significance was determined by two-tailed unpaired Student’s = 328), the low expression of miR-15a, miR-195, miR-497 and miR-424 correlated with worse progression-free survival (Figure ?(Figure2A).2A). Tumors from patients with MYCN amplification (poor outcome) also showed reduced levels of miR-195, miR-497 and miR-424 (Supplementary Figure 1). These data suggest that replacement of miR-15 family members could be exploited therapeutically. In order to clarify whether the diverse family members could have different therapeutic potential, we compared the effects of transfecting all 6 individual miRNAs on the proliferation of NB cells. MiR-497 was the miR-15 family member which reduced the number of viable NB cells the most (Figure ?(Figure2B2B). Open in a separate window Figure 2 MiR-15 family members expression correlates with NB prognosis and regulates cell proliferation(A) Kaplan-Meier progression-free survival analysis of miR-15 family members in human NB tissues (= 328). (B) The miR-15 family members (miR-15a, miR-15b, miR-16-1/2, miR-195, miR-497 and miR-424) were reverse-transfected in SK-N-BE(2) and LA1-5s cells. 96 h later, cells were fixed and stained with crystal violet. Proliferation values were normalized versus MOCK-transfected cells. Data represent mean SEM of three independent experiments (six replicates per experiment). **< 0.01; ***< 0.001. MiR-497 reduces proliferation of chemoresistant NB cells and induces apoptosis in MYCN-amplified cell lines The effects of miR-497 were then analyzed in a panel of NB cell lines representative of the major subclasses of NB (MYCN-amplified and non-amplified) over a time-course period. A reduction in cell proliferation started to be visible in all cell lines at 72 h post-transfection (Figure ?(Figure3A3A). Open in a separate window Figure 3 MiR-497 overexpression reduces proliferation of chemoresistant NB cells and induces apoptosis in MYCN-amplified NB cells(A) Proliferation time course comparing miR-497 versus miR-Control (25 nM) reverse transfected Hydroxyprogesterone caproate in CHLA-90 and SK-N-AS cells (both non-MYCN amplified) or SK-N-BE(2) and LA1-5s cells (both MYCN amplified). (B) Representative images of nuclear morphology assessment at 96 h post-transfection with Hoechst staining in miR-Control and miR-497 (25 nM) reverse transfected NB cell lines. Arrowheads point at condensed or fragmented nuclei. (C) Quantification of apoptosis was performed from 4 representative images of 3 replicates per condition. (D) Caspase-3/7 activity Hydroxyprogesterone caproate assays and (E) representative Western blot of PARP protein at 72 h post-transfection. SK-N-BE(2) and LA1-5s cells were reverse transfected with 25 nM of miR-Control or miR-497. Data represent mean SEM of three independent experiments *, ** or *** indicated significant differences comparing miR-497 miR-Control at < 0.05, IL22RA1 < 0.01 or < 0.001, respectively. To further ascertain whether the effects of miR-497 were due to a reduction in cell proliferation and/or increased cell death, the induction of apoptosis was analyzed in miR-497-transfected cells. The number of cells with condensed or fragmented chromatin (one of the hallmarks of apoptotic cell death) was found to be increased upon miR-497 transfection in MYCN-amplified (SK-N-BE(2) and LA1-5s) but not.