<i>In vitro</i> inhibition of miR-221 and miR-222 reduces tumor growth of PC3 derived tumors in SCID mice.

<p>A Northern blot analysis of total RNA extracted from PC3 cells transfected <i>in vitro</i> with anti-miR-221+anti-miR-222 LNA oligonucleotides (anti-221/222). The hybridisation to snRNA U6 was used as a loading control. B Tumor growth curves measured after the injection of PC3 cells transfected with either anti-miR-221 and anti-miR-222 LNA oligonucleotides (anti-221/222) or a control LNA oligo (ctrl). The tumor volumes were calculated as v = L×l²×0.5, where L is the longer diameter, and l the shorter one. C Average volume fold increase of tumors derived from PC3 cells transfected with anti-miR-221+anti-miR-222 LNA oligonucleotides (anti-221/222) or with a negative control LNA oligonucleotide (ctrl). Values represent the ratio between the volumes at the day of sacrifice and the volumes measured 54 days before, when all tumors were clearly detectable and measurable. N = 4 for ctrl tumors and n = 5 for anti-miR treated tumors. Data in B and C are presented as the means±SEM. *, P<0.05.</p

MiR-221 ectopic overexpression enhances the growth of LNCaP-derived tumors.

<p>A Northern blot analysis of LNCaP cells permanently transfected with p-221 or empty vector pCDNA3.1. The expression of miR-221 in the highly aggressive PC3 prostate carcinoma cell line is also shown, as a positive control. Hybridization to snRNA U6 is included as a loading control. Under each lane, a number indicates the relative miR-221 expression as compared to LNCaP cells transfected with the empty vector pCDNA3.1, where miR-221 endogenous expression is set as  = 1. B <i>In vivo</i> tumor growth in SCID mice. Average tumor volumes are represented (n = 6 for both experimental groups) starting from the first time point when tumor volumes were clearly measurable (t0) until the last measurement before sacrifice, performed 6 weeks later. C Average volume fold increase of the same tumors as in B at the moment of sacrifice (i.e. 6 weeks after the first measurement) as compared to values measured at time 0. Data are presented as the mean±SEM *, P<0.05, and are representative of 2 independent experiments. D–E Proliferation markers: mitotic index and Ki-67 expression. D Graph of the mitotic index and Ki-67 expression as percent of positive cells (10 fields, 2 sections for each tumor. *, P<0.01; **, P≪0.001). Grey bars: pCDNA3.1 transfected LNCaP cells; black bars: p-221 transfected LNCaP cells. E, left upper panel: LNCaP cells transfected with empty vector pCDNA3.1, haematoxylin eosin stained section (magnification 600×). Cells have epithelioid phenotype with low mitotic index. Right upper panel: tumor tissue from LNCaP cells transfected with p-221, haematoxylin-eosin stained section (magnification 600×). Cells have epithelioid phenotype with high mitotic index; arrows indicate mitotic pictures. Left lower panel: immunohistochemistry of the proliferation marker Ki-67 in tumor tissue from LNCaP ctrl cells; scattered cells with brown, granular nuclear staining considered to be positive for Ki-67 (magnification 200×). Right lower panel: immunohistochemistry of the proliferation marker Ki-67 in tumor tissue from LNCaP cells transfected with p-221: numerous cells with brown, granular nuclear staining positive for Ki-67 (magnification 200×). F Northern and Western blot analysis of RNA and proteins extracted from p-221 and control vector transduced tumors from two mice sacrificed at 6 weeks from the first measurement (as in B). The upper part of the panel (Northern blot) shows the persistent expression of miR-221 in p-221 transduced tumors, and the lower part (Western blot) shows the downregulation of p27 in miR-221 expressing tumors. U6 and β-actin are shown as loading controls for Northern and Western blot, respectively. The numerical values under each lane indicate the relative expression of miR-221 and of p27, where each p-221 transfected tumor is compared to its controlateral control (pCDNA3.1) tumor, whose miR-221 and p27 expression levels are set as  = 1. G p27 mRNA 3′UTR sites targeted by miR-221 and miR-222. The core annealing regions are located at nucleotides 201–208 and 274–281 of p27 3′UTR. Dotted vertical lines indicate G-U bonds.</p

Clinical aspects of prostate cancer cases used in this study.

a<p>Case: patients treated with radical prostatectomy.</p>b<p>PSA: Prostate Specific Antigen.</p>c<p>Gleason grade: Gleason's score.</p>d<p>TNM: Tumor, Nodes, Metastasis.</p><p>pT: pT category, N: lymphnodes, N0: not involved; M: metastasis, Mx: not reliable.</p

MiR-221 is strongly expressed in prostate carcinoma-derived primary cells and its expression inversely correlates with that of p27.

<p>A MiR-221 and miR-222 expression measured by quantitative real-time PCR in primary cell lines from prostate carcinomas (T samples) or normal prostate (N samples). The graph shows the log fold change of miR-221 and miR-222 expression as compared to the value obtained for non-tumoral control sample N1. B Representative Western blot analysis showing p27 expression in 4 tumor- and 2 non-tumor-derived primary cell lines. β-actin immunoreactivity is shown as a loading control. Under each lane, the numerical values represent p27 relative amount, that was set  = 1 in the normal control sample N1. C Spearman correlation analysis performed between miR-221, miR-222, and p27 levels in 18 primary cell lines derived from prostate carcinoma tissues. P<0.05.</p

The intratumoral injection of anti-miR-221 and anti-miR-222 antagomirs into PC3-derived tumors reduces tumor growth and has long lasting effects on miR-221 and miR-222 endogenous expression.

<p>A Tumor growth curves depicting the average±SEM values of PC3 derived tumors injected either with a negative control antagomir (ctrl) or with a mixture of anti-miR-221 and anti-miR-222 antagomirs (anti-miR221/222). Each tumor was treated with three injections of 1 µg of each antagomir at days 0, 5, and 9 (arrows). Day 0 is the day of the first antagomir injection. Each mouse (n = 8) was bearing a negative control injected tumor on one flank, and an antagomir treated one on the controlateral flank. Data are presented as the means±SEM of two independent experiments. *, P = 0.009. B Average volume fold increase of the same tumors as in A. The data presented are the means±SEM, and represent the ratio between the volumes at the sacrifice (33 days from the first antagomir injection) and the volumes measured at the day of the first antagomir injection. *, P = 0.009. C Quantitative real-time PCR of miR-221 (upper panel) or miR-222 (lower panel) in tumors excised from four representative mice (A10, D3, D10, E3) at the day of sacrifice, 24 days after the last antagomir injection. The data are presented as the means±SD of three independent experiments, each performed in triplicate. *, P<0.05. The values presented are the miRNA expression fold changes, as compared to the expression detected in negative control tumors grown in each mouse, set as  = 1. D Representative p27 Western blot analysis on total proteins extracted from the same tumors as in c. For each mouse, the proteins from the negative control tumor and the antagomir-treated tumor are shown. β-actin immunoreactivity is shown as a loading control. Under each lane, the numerical values represent p27 relative amount, that was set  = 1 in each control tumor (ctrl), and the p27 expression in the respective controlateral anti-miR221/222 tumor was calculated.</p

Multifunctional Core–Shell Nanoparticles: Discovery of Previously Invisible Biomarkers

Many low-abundance biomarkers for early detection of cancer and other diseases are invisible to mass spectrometry because they exist in body fluids in very low concentrations, are masked by high-abundance proteins such as albumin and immunoglobulins, and are very labile. To overcome these barriers, we created porous, buoyant, core–shell hydrogel nanoparticles containing novel high affinity reactive chemical baits for protein and peptide harvesting, concentration, and preservation in body fluids. Poly(<i>N</i>-isopropylacrylamide-co-acrylic acid) nanoparticles were functionalized with amino-containing dyes via zero-length cross-linking amidation reactions. Nanoparticles functionalized in the core with 17 different (12 chemically novel) molecular baits showed preferential high affinities (<i>K</i>_D < 10^–11 M) for specific low-abundance protein analytes. A poly(<i>N</i>-isopropylacrylamide-co-vinylsulfonic acid) shell was added to the core particles. This shell chemistry selectively prevented unwanted entry of all size peptides derived from albumin without hindering the penetration of non-albumin small proteins and peptides. Proteins and peptides entered the core to be captured with high affinity by baits immobilized in the core. Nanoparticles effectively protected interleukin-6 from enzymatic degradation in sweat and increased the effective detection sensitivity of human growth hormone in human urine using multiple reaction monitoring analysis. Used in whole blood as a one-step, in-solution preprocessing step, the nanoparticles greatly enriched the concentration of low-molecular weight proteins and peptides while excluding albumin and other proteins above 30 kDa; this achieved a 10,000-fold effective amplification of the analyte concentration, enabling mass spectrometry (MS) discovery of candidate biomarkers that were previously undetectable