Estudio de la regulación y del papel oncogénico de lisis oxidasa (LOX) en los tumores de Ewing

Tesis doctoral inédita realizada en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 19 Julio de 2010.Bibliografía, p. 111-12

miR-16 suppresses growth of hepatoma cells in vitro and tumorigenicity <i>in vivo.</i>

<p>(<b>A</b>) Hep3B cells were transfected with 50 nM miR-16 or In-miR-16. The effect on cell proliferation was determined by MTT assay at 48 h after transfection. <i>*p< 0.05 </i><i>vs</i>. the control and # <i>p< 0.05 </i><i>vs</i>. the miR-16 transfection condition. (<b>B</b>) Tumor growth curves measured after subcutaneous injection of WRL68 cells transiently transfected with miR-16, miR-NC or miR-16 with a hCOX-2 expression vector lacking 3′ UTR (miR-16+COX-2). Tumor volume (V) was monitored by measuring the length (L) and width (W) with calipers and calculated with the formula (L×W²)×0.5. Tumor growth was measured every 2–3 days. <i>*p< 0.05 </i><i>vs</i>. the miR-NC and # p<0.05 <i>vs.</i> miR-16 (<b>C</b>) Tumor weight and a representative picture of the tumors. At 21 days after injection, mice were killed and tumors were weighed after necropsy. <i>*p< 0.05 </i><i>vs</i>. the miR-NC or # <i>vs.</i> the miR-16+ COX-2 condition. (<b>D–E</b>) miR-16 and human COX-2 expression in tumors using real-time PCR normalized against U6 RNA levels and refers to miR-NC as 1 or 36b4 mRNA, respectively. <i>*p< 0.05 </i><i>vs</i>. the miR-NC Data are reported as means±SD of three independent experiments or five animals per condition.</p

miR-16 and COX-2 correlate inversely in HCC cell lines.

<p>Cells were plated in 100-mm dishes and grown to 60–70% confluence in culture medium supplemented with 10% FBS. (<b>A</b>) Total cellular extracts were prepared from HCC cells and protein (30–50 µg/lane) was analyzed by Western blot. A representative Western blot showing COX-2 protein. The expression of target protein was normalized to that of α-tubulin. Densitometric analysis of COX-2 expression (black bars) is referring to HH as 1 and expressed as relative expression (RE). Total RNA was prepared from HCC cell lines and COX-2 mRNA was analyzed by real-time PCR. COX-2 mRNA amounts (white bars), normalized to the expression of 36b4 mRNA, and miR-16 expression (grey bars), normalized against U6 RNA levels, were calculated. Values represent fold change relative to human hepatocytes (HH) as 1. Data are reported as means±SD of three independent experiments. **<i>p<0.01</i> and <i>*p< 0.05 vs</i>. the HH. (<b>B</b>) The inverse correlation between COX-2 protein/mRNA ratio and miR-16 expression in HCC cells is graphically depicted. The coefficient of determination (R²) was calculated.</p

miR-16 regulates COX-2 expression in HCC cell lines.

<p>WRL68 and Hep3B cells were transfected with: 30 nM siRNA anti-COX2 (siCOX-2) or 50 nM of miR-16, miR-16 inhibitor (In-miR-16), miR negative control (miR-NC) or miR negative control inhibitor (In-miR-NC). (<b>A–B</b>) COX-2 protein was analyzed by Western blot 48 h after transfection and normalized against α-tubulin protein. COX-2 mRNA and miR-16 expression were analyzed by real-time PCR. COX-2 mRNA and miR-16 expression were normalized against 36b4 mRNA and U6 RNA levels, respectively. Relative expression of each sample refers to control as 1 (cells transfected only with lipofectamine). (<b>C–D</b>) PGE₂ concentration was determined by enzyme immunoassay in the supernatant of the cells. Data are reported as means±SD of four independent experiments. <i>*p< 0.05 vs</i>. the control condition and # <i>p< 0.05 vs</i>. the miR-16 transfection condition.</p

Effect of miR-16 on COX-2 mRNA and protein stability.

<p>Hep3B cells were transfected with 50 nM miR-16 or miR-NC, or 30 nM siCOX-2. 5 µg/ml actinomycin-D (Act D) or 10 µg/ml cycloheximide (CHX) were added after transfection. (<b>A–B</b>) COX-2 protein was analyzed by Western blot at different time after actinomycin-D treatment. Corresponding densitometry analysis is shown and the relative expression of each sample is related to sample at 0 h as 1. (<b>C</b>) mRNA COX-2 levels were analyzed by real time PCR. COX-2 mRNA amounts were calculated as relative expression and normalized to the expression of 36b4 mRNA. Values represent fold change relative to sample at 0 h. (<b>D–E</b>) COX-2 protein levels were analyzed by Western blot in the presence or absence of cycloheximide. Corresponding densitometric analysis is shown and the relative expression of each sample is related to the value at 0 h as 1. <b>F)</b> Hep3B cells were transfected with 50 nM miR-16, miR-16 inhibitor (In-miR-16) or lipofectamine and permeabilized with digitonine to obtain soluble and pellet fractions enriched in PB as described in Methods. RNA was isolated from each fraction with Trizol reagent, reverse transcriptased, and PCR amplified with COX-2, Xrn1 and actin primers. Input, RNA extracted from cells prior to fractionation. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. <b>G)</b> The presence of COX-2 mRNA in soluble and PB fractions was assessed and fold differences were plotted. Data are reported as means±SD of three independent experiments. **p<0.01 and *p<0.05 vs. the value of sample at 0 h.</p

miR-16 binds COX-2 mRNA and inhibits its translation.

<p>(<b>A</b>) WRL68 cell extracts (500 µg per lane) were immunoprecipitated with Ago-2 or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 primers. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The presence of COX-2 mRNA in WRL68 cell transfected with miR-16 or Lipofectamine after Ago2 immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; and control, bound mRNA after immunoprecipitation with IgG antiboby. (<b>B</b>) Scheme of pGL3-empty, pGL3-seed and pGL3-mut reporter vectors. In pGL3-seed, the putative binding site of miR-16 on COX-2 mRNA 3′-UTR region (as detected by RNAhybrid software) was introduced downstream luciferase gene. In pGL3-mut this region was mutated in order to avoid the binding between miR-16 and Luc mRNA. (<b>C–D</b>) A luciferase assay was carried out on HuH-7 and HepG2 cell lines using pGL3-seed and pGL3-mut reporter vectors. Firefly luciferase activity was evaluated 48 h after co-transfection with pGL3-empty/seed/mut (750 ng), miR-16 (50 nM), In-miR-16 (50 nM) and miR-NC (50 nM) as indicated. Data were normalized against renilla luciferase activity (all samples were co-transfected with 50 ng pRL vector and refer to the positive control, pGL3 empty vector). Data are reported as means±SD of three independent experiments. <i>*p< 0.05 vs</i>. the pGL3-empty condition and # <i>p< 0.05 vs</i>. the miR-16 transfection condition.</p

Expression of selected miRNAs is evaluated in HCC cell lines.

<p>The miRNAs (miR-16, miR-26b, miR-101, miR-199a, miR-122 and miR-21) were selected by using miRWalk computational analysis as described in Methods. The expression profile was analyzed in HCC cell lines using real-time PCR, normalized against U6 RNA levels and refers to human hepatocytes (HH) as 1. COX-2 protein/mRNA ratio was compared to miRNAs expression in HCC cells and the coefficient of determination (R²) was calculated.</p

Downregulation of COX-2 by miR-16 increases apoptosis in HCC cells.

<p>Hep3B cells were transfected with 50 nM miR-16 or In-miR-16 in the presence or absence of 5 µM PGE₂ (<b>A</b>) Apoptosis was measured with Annexin V-FITC Apoptosis Detection Kit (<b>B</b>) Western blot analysis of caspase-3. Results are the means ± SD of three different experiments. <i>*p< 0.05 vs</i>. the corresponding control cells <i>^# p< 0.05 vs.</i> miR-16 condition.</p

HuR antagonizes the downregulation of COX-2 expression caused by miR-16 in hepatoma cell lines.

<p>WRL68 cell extracts (500 µg per lane) were immunoprecipitated with HuR or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 (<b>A</b>) or miR-16 primers (<b>B</b>). PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The abundance of the transcripts present in WRL68 cells after HuR immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; unbound, unbound mRNA after immunoprecipitation with HuR antibody; bound, bound mRNA after immunoprecipitation with HuR antibody; and control, bound mRNA after immunoprecipitation with IgG antiboby. (<b>C–D</b>) WRL68 and Hep3B cell lines were transfected with miR-16 or In-miR-16 (50 nM). COX-2 and HuR protein levels were analyzed by Western Blot. (<b>E–F</b>) WRL68 and Hep3B cell lines were cotransfected with miR-16 (50 nM) and pcDNA3-HuR-GFP expression vector (4 µg). COX-2 and HuR protein levels were analyzed by Western Blot. Data are reported as means±SD of three independent experiments. <i>*p< 0.05 vs</i>. the control condition and # <i>p< 0.05 vs</i>. the miR-16 transfection condition.</p