LNA-i-miR-221 antiproliferative activity and target silencing in retrieved MM xenografted tumors.

<p>A) q-RT-PCR of p27Kip1 mRNA levels in treated tumors retrieved from mice after intravenous LNA-i-miR-221 treatment. Raw Ct values were normalized to GAPDH housekeeping mRNA and expressed as ΔΔCt values calculated using the comparative cross threshold method (miRNA expression in LNA-i-miR-NC treated animals) ±SD. B) Western blot analysis of p27Kip1 protein in retrieved tumors from mice treated with LNA-i-miR-221 inhibitors or LNA-i-miR-NC. GAPDH was the protein loading control. C) H&E (200-fold magnification), p27Kip1 (200-fold magnification) and Ki-67 (10–fold magnification) immunohistochemistry staining of xenografted tumors retrieved from treated animals. Representative images are shown.</p

Molecular effects induced by LNA-i-miR-221 transfection in MM cells.

<p>miR-221(A) q-RT-PCR 24, 48 and 72 hours after transfection with LNA-i-miR-221 and LNA-i-miR-NC in NCI-H929 cells. The results are shown as miRNA expression levels after normalization with RNU44 and ΔΔCt calculations. Data represent the average of 3 independent experiments ±SD. B) q-RT-PCR of p27Kip1 mRNA expression 24 and 48 hours after transfection with LNA-i-miR-221 or scrambled control in NCI-H929 cells. Data represent the average of 3 independent experiments ±SD after normalization with GAPDH mRNA and ΔΔCt calculations. (*) P<0.05, (**) P<0.01. C) Western blot analysis of p27Kip1 protein in NCI-H929 cells 24, 48 and 72 hours after transfection with LNA-i-miR-221 or control. GAPDH was used as protein loading control.</p

LNA-i-miR-221 specifically recognizes the miR-221 complementary sequence.

<p>Luciferase reporter assay of NCI-H929 cells co-transfected with pLightSwitch_3′UTR Reporter Vector containing the miR-221-3p synthetic sequence cloned downstream of the Renilla luminescent reporter gene (RenSP) and miR-221/222 mimics (A) or LNA-i-miR-221 (B) or scrambled sequences as control. The firefly luciferase activity was normalized to Renilla luciferase activity. The data are shown as relative luciferase activity of miR-221/222-transfected cells versus the control (miR-NC) or LNA-i-miR-221-transfected cells versus the scrambled control (LNA-i-miR-NC). C) Dual-luciferase assay of NCI-H929 cells co-transfected with firefly luciferase constructs containing the 3′UTR of p27Kip1 and LNA-i-miR-221 or scrambled oligonucleotides (NC) as indicated. Firefly luciferase activity was normalized to r Renilla luciferase activity. The data are shown as relative luciferase activity of LNA-i-miR-221-transfected cells versus the control (NC).</p

Additional file 1: of Aurora Kinase A expression predicts platinum-resistance and adverse outcome in high-grade serous ovarian carcinoma patients

Figure S1. Correlation of platinum sensitivity status with survival. Kaplan Meier curves of EOC patients grouped according to platinum sensitive (PS) or resistant (PR) disease. Patients with a platinum sensitive ovarian cancer presents a significant longer survival thus confirming the reliability of the sample. (JPG 182 kb

Effects of miR-34a replacement on survival pathways and apoptosis occurrence.

<p>A) SKMM-1 cells were transfected with miR-34a (34a) or scramble miR-NC (NC) and after different times from the transfection were collected for Western blot analysis. Thereafter, the expression and phosphorylation of Erk, the activity and expression of Akt and pro-caspase-6 and -3 expression were evaluated after blotting with specific antibodies, as described in “Material and Methods”. The house-keeping protein α-tubulin was used as loading control. Each point is representative of 3 different evaluations performed in 3 different experiments. B) Scan of the bands associated with pErk-2 expression and Akt activity normalized for total Erk-2 or Akt expression, respectively, and of pro-caspase-3 and pro-caspase-6 expression, normalized with the housekeeping protein α-tubulin in SKMM-1 cells, was performed with ImageJ software. The intensities of the bands were expressed as % of changes based upon determination of arbitrary units (%, mean of three different experiments). Each point is the mean of 3 different evaluations performed in at least 3 different experiments. Bars, s.e.’s. C) SKMM-1 cells after transfection with miR-34a (34a) or scramble miR-NC (NC). The cells were collected after the indicated times from the transfection and apoptosis was evaluated with TUNEL assay by FACScan as described in “Materials and Methods”. Results are shown as percentage of apoptotic cells. Data are the average ‘SD of 3 independent experiments.</p

H&E staining of livers and kidney indicates absence of systemic toxicity.

<p>Hematoxylin and eosin staining (40-fold magnification) of kidney and liver retrieved from SNALP empty (A, B), SNALP miR-NC (C, D) and SNALP miR-34a (E, F) treated mice, respectively. No significant damage was detected in the different groups of treatment. Representative image are shown.</p

SNALP miR-34a reduces Akt activation and induces apoptosis in MM <i>in vivo</i>.

<p>TUNEL assay of SKMM-1 xenograft retrieved from SNALP miR-NC (A, B) and SNALP miR-34a (E, F) treated mice. The TUNEL positive cells are colored in brown. Representative image at 40-fold (A, E) and 60-fold (B, F) magnification are shown. p-Akt immunostaining SKMM-1 xenograft retrieved from SNALP miR-NC (C, D) and SNALP miR-34a (G, H) treated mice. Representative image at 40-fold (C, G) and 60-fold (D, H) magnification are shown.</p