Cells grown in monolayers (a, left image) and tumorspheres (a, right image) show different ability to form secondary tumorspheres (b), resistance to doxorubicin (c), expression of MRP1 (d), and similar ability to retain BCECF and exclude PI (e).

<p>Bars represent means ± SE, *<i>p</i> < 0.05.</p

Kinetic and immunophenotyping analysis of MCF-7 TICs and bulk cells: (a) images taken at 10 min intervals illustrate MDR efflux from cells loaded with fluorescein; (b) immunostaining of the same cells for CD44; (c) immunostaining of the same cells for CD24; (d) 2-D plots of the fluorescence intensity of CD44 and CD24 signals in monolayer- ( left) and tumorsphere-derived cells (right); (e) histograms of distribution of Michaelis parameters within CD44^high/CD24^low cells (gray bars) and bulk cells (white bars) demonstrating two ways of MDR activation in TICs; (f) cumulative data summarizing Michaelis parameters in CD44^high/CD24^low cells (gray bars) and bulk cells (white bars) in 5 independent

<p><b>experiments </b>(<b>total 1089 cells, <i>p</i> < 0.01)</b>. </p

Kinetic and morphologic analysis of MCF-7 TICs and bulk cells: (a) typical image of toluidine blue-stained cells; (b) distribution of brightness of stained cells showing discrimination between pale and dark cells; (c, d) histograms of distribution of Michaelis parameters within pale (undifferentiated) cells (gray bars) and dark (differentiated) cells (white bars) demonstrating two ways of MDR activation in TICs; (e) cumulative data summarizing Michaelis parameters in pale (undifferentiated) cells (gray bars) and dark (differentiated) cells (white bars) in 4 independent experiments

<p>(<b> total 821 cells, <i>p</i> < 0.01)</b>. </p

Kinetic and immunophenotyping analysis of 4T1 TICs and bulk cells: (a) histograms of distribution of Michaelis parameters within CD44^high/CD24^low cells (gray bars) and bulk cells (white bars); (b) cumulative data summarizing Michaelis parameters in CD44^high/CD24^low cells (gray bars) and bulk cells (white bars) in 4 independent experiments (total 709 cells, <i>p</i> < 0.02 for <i>V</i>_max).

<p>Kinetic and immunophenotyping analysis of 4T1 TICs and bulk cells: (a) histograms of distribution of Michaelis parameters within CD44^high/CD24^low cells (gray bars) and bulk cells (white bars); (b) cumulative data summarizing Michaelis parameters in CD44^high/CD24^low cells (gray bars) and bulk cells (white bars) in 4 independent experiments (total 709 cells, <i>p</i> < 0.02 for <i>V</i>_max).</p

XB130, a New Adaptor Protein, Regulates Expression of Tumor Suppressive MicroRNAs in Cancer Cells

<div><p>XB130, a novel adaptor protein, promotes cell growth by controlling expression of many related genes. MicroRNAs (miRNAs), which are frequently mis-expressed in cancer cells, regulate expression of targeted genes. In this present study, we aimed to explore the oncogenic mechanism of XB130 through miRNAs regulation. We analyzed miRNA expression in XB130 short hairpin RNA (shRNA) stably transfected WRO thyroid cancer cells by a miRNA array assay, and 16 miRNAs were up-regulated and 22 miRNAs were down-regulated significantly in these cells, in comparison with non-transfected or negative control shRNA transfected cells. We chose three of the up-regulated miRNAs (miR-33a, miR-149 and miR-193a-3p) and validated them by real-time qRT-PCR. Ectopic overexpression of XB130 suppressed these 3 miRNAs in MRO cells, a cell line with very low expression of XB130. Furthermore, we transfected miR mimics of these 3 miRNAs into WRO cells. They negatively regulated expression of oncogenes (miR-33a: MYC, miR-149: FOSL1, miR-193a-3p: SLC7A5), by targeting their 3′ untranslated region, and reduced cell growth. Our results suggest that XB130 could promote growth of cancer cells by regulating expression of tumor suppressive miRNAs and their targeted genes.</p> </div

Improvements to Direct Quantitative Analysis of Multiple MicroRNAs Facilitating Faster Analysis

Studies suggest that patterns of deregulation in sets of microRNA (miRNA) can be used as cancer diagnostic and prognostic biomarkers. Establishing a “miRNA fingerprint”-based diagnostic technique requires a suitable miRNA quantitation method. The appropriate method must be direct, sensitive, capable of simultaneous analysis of multiple miRNAs, rapid, and robust. Direct quantitative analysis of multiple microRNAs (DQAMmiR) is a recently introduced capillary electrophoresis-based hybridization assay that satisfies most of these criteria. Previous implementations of the method suffered, however, from slow analysis time and required lengthy and stringent purification of hybridization probes. Here, we introduce a set of critical improvements to DQAMmiR that address these technical limitations. First, we have devised an efficient purification procedure that achieves the required purity of the hybridization probe in a fast and simple fashion. Second, we have optimized the concentrations of the DNA probe to decrease the hybridization time to 10 min. Lastly, we have demonstrated that the increased probe concentrations and decreased incubation time removed the need for masking DNA, further simplifying the method and increasing its robustness. The presented improvements bring DQAMmiR closer to use in a clinical setting

XB130 shRNA transfected WRO cells showed increased levels of miR-33a, miR-149 and miR-193a-3p by real-time quantitative RT-PCR.

<p>The mature forms (<b>A</b>) and primary transcripts (<b>B</b>) of miR-33a, miR-149 and miR-193a-3p are significantly higher in XB130 shRNA transfected WRO cells (C3-1, C3-4, C4-3 and C4-11) than in WRO (*P<0.05).</p

MiR-33a, miR-149 and miR-193a-3p directly target a potential binding site in the 3′UTR of MYC, FOSL1 and SLC7A5, respectively.

<p>(<b>A</b>) Seed sequences of miR-33a, miR-149 and miR-193a-3p and pairing 3′UTRs sequence of MYC, FOSL1 and SLC7A5 as predicted by TargetScan are shown. The pmirGLO construct with the cloned 3′UTR sequences (luc-MYC, luc-FOSL1 and luc-SLC7A5) and the miR mimic (33a-m, 149-m and 193a-m) or control miR mimic (cont-m) were co-transfected into WRO cells. (<b>B</b>) Overexpression of miR-33a significantly reduced luciferase activity of the pmirGLO construct with cloned 3′UTR sequences of MYC (luc-MYC). Similarly, overexpression of miR-149 and miR-193-3p significantly reduced luciferase activity of the luc-FOSL1 (<b>C</b>) and the luc-SCL7A5 (<b>D</b>), respectively.</p

Suppressive effects of miR-33a, miR-149 and miR-193a-3p on proliferation in WRO cells.

<p>The numbers of viable cells were assessed by MTS assay (A) and cell counting (B) at 24, 48, and 72 h post-transfection of control mimic (cont-m) and specific miR mimic (33a-m, 149-m and 193a-m). The overexpression of miR-33a, miR-149 and miR-193a-3p led to reduction in cell proliferation. (<b>C</b>) Cell proliferation markers, Ki-67 and PCNA levels, were also reduced in miR mimic treated cells (33a-m, 149-m and 193a-3p). (<b>D</b>) Apoptosis was determined by flow cytometry using PI/annexin V double staining. Overexpression of miR-193a-3a significantly induced both early apoptosis (annexin V positive/PI negative) and late apoptosis (annexin V/PI double positive) in WRO cells. *: P<0.05.</p

Significantly upregulated miRNAs in XB130 shRNA transfected Cells.

<p>Note: Two strands come from one stem loop precursor miRNA (pre-microRNA) by dicer processing. One of them is indicated with an asterisk. For example, has-miR-191* and has-miR-191 are two sequences from the same precursor.</p