<i>CYR61</i> and <i>CTGF</i> mRNAs expression in human cervical samples, and their correlations with <i>miR-205</i> expression.

<p>Relatively lower expression of <i>CYR61</i> (A) and <i>CTGF</i> (B) was found in a majority of tumor samples as compared to their normal counterparts (n = 28). (C) The expression of <i>CYR61</i> and <i>CTGF</i> was significantly lower in the tumors than the normal samples (<i>P</i> = 0.002 and <i>P</i><0.001, respectively; paired t-test). (D) Inverse correlation between the expression level of <i>miR-205</i> and <i>CYR61</i> (upper) or <i>CTGF</i> (lower). The expression relationship was evaluated by Pearson’s correlation analysis. <i>P</i><0.05 was considered statistically significant.</p

<em>miR-205</em> Expression Promotes Cell Proliferation and Migration of Human Cervical Cancer Cells

<div><p>MicroRNAs (miRNAs) are short non-coding RNA regulators that control gene expression mainly through post-transcriptional silencing. We previously identified <em>miR-205</em> in a signature for human cervical cancer using a deep sequencing approach. In this study, we confirmed that <em>miR-205</em> expression was frequently higher in human cervical cancer than their matched normal tissue samples. Functionally, we demonstrate that <em>miR-205</em> promotes cell proliferation and migration in human cervical cancer cells. To further understand the biological roles of <em>miR-205</em>, we performed <em>in vivo</em> crosslinking and Argonaute 2 immunoprecipitation of miRNA ribonucleoprotein complexes followed by microarray analysis (CLIP-Chip) to identify its potential mRNA targets. Applying CLIP-Chip on gain- and loss-of-function experiments, we identified a set of transcripts as potential targets of <em>miR-205</em>. Several targets are functionally involved in cellular proliferation and migration. Two of them, CYR61 and CTGF, were further validated by Western blot analysis and quantification of mRNA enrichment in the Ago2 immunoprecipitates using qRT-PCR. Furthermore, both <em>CYR61</em> and <em>CTGF</em> were downregulated in cervical cancer tissues. In summary, our findings reveal novel functional roles and targets of <em>miR-205</em> in human cervical cancer, which may provide new insights about its role in cervical carcinogenesis and its potential value for clinical diagnosis.</p> </div

Functional analyses of <i>miR-205</i> regulation in cervical cancer cell lines.

<p>(A) Cell proliferation was assessed in human cervical cancer cell lines transfected with a <i>miR-205</i> mimic (Pre-miR-205), inhibitor (Anti-miR-205) or corresponding negative control (Anti-miR Neg control or Pre-miR Neg control) using WST-1 assay. Relative cell growth was normalized to its respective control-treated cells. (B) Graphs showing relative cell migration in both <i>miR-205</i> inhibition and overexpression experiments as evaluated by Transwell migration assay. (C) Representative images of cell migration evaluated by wound healing assay. Scratch wounds were made on confluent monolayer cultures after 48 h of transfection. Images of wound repair were taken at 0, 18 and 24 h after wound (left panel). The percentage of wound closure was normalized by wound area at 0 h (right panel). Data presented represent mean of three independent experiments. Error bars represent standard deviations from the mean. All comparisons were evaluated using t-test. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; <i>n.s.</i> = not significant.</p

Evaluation of <i>CYR61</i> and <i>CTGF</i> as targets of <i>miR-205</i>.

<p>(A) Representative Western blot showing the protein expression levels of CYR61 and CTGF in cells transfected with a <i>miR-205</i> mimic, <i>miR-205</i> inhibitor, or corresponding scramble and mock transfection controls. (B) CYR61 protein expression was significantly repressed in <i>miR-205</i>-overexpressing (treated with Pre-miR-205) cells and significantly increased in <i>miR-205</i>-depleting (treated with Anti-miR-205) cells as compared to their respective negative controls. CTGF protein expression was slightly repressed in HeLa cells treated with Pre-miR-205, and slightly increased in CaSki cells treated with Anti-miR-205, but the effect was not statistically significant. Data presented represent mean of at least four independent experiments. qRT-PCR analysis of <i>CYR61</i> (C) and <i>CTGF</i> (D) mRNA in the Ago2-immunoprecipitated RNAs of <i>miR-205</i>-overexpressing or -depleted cells as compared to mock-transfection control. Relative expression level of individual mRNAs was normalized to <i>miR-21</i> expression (as endogenous control for Ago2 IP RNA). Fold change was calculated by dividing the normalized expression values of Ago2-immunoprecipiated samples by the normalized expression values of its respective input samples. Data presented represent mean of at least three independent experiments. Error bars represent standard deviations from the mean. All comparisons were evaluated using <i>t</i>-test. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; <i>n.s.</i> = not significant.</p

Real time quantitative RT-PCR of <i>miR-205</i> expression in human cervical tumors, normal cervices and cervical cancer cell lines, normalized to the geometric mean of <i>RNU6B</i> and <i>RNU43</i>.

<p>(A) <i>miR-205</i> expression was significantly higher in the tumors than the normal samples (<i>P</i><0.001; paired t-test). (B) Relatively higher expression of <i>miR-205</i> was found in a majority of tumor samples as compared to their normal counterparts. (C) High expression of <i>miR-205</i> was detected in ME-180, C4I and CaSki cells, and low or undetectable expression level was found in HeLa, SW756, SiHa and C33A cells. Data presented represent mean of three independent experiments with triplicates. Error bars represent standard deviations from the mean.</p

Most common recurrent copy number alterations detected by a-CGH in CDC73-mutated parathyroid tumors (T2–T8) with corresponding region of LOH.

*<p>Genes written in bold are known cancer genes according to Cancer Gene Census, Welcome Trust, Sanger Institute.</p

Sequencing chromatograms of <i>CDC73</i> mutations in parathyroid tumors: T1 (IVS1+1 G>C), T5 constitutional (c128-IVS1+1 delG) and T5a (c.71del 72–90).

<p>Wild-type (WT) sequences are indicated above the mutated sequences.</p

Characteristics of the 9 parathyroid tumors.

<p>M = Male, F = Female, Ex = Exon, Int = Intron, n.a = not available.</p><p>LOH =  Loss of heterozygocity, n.d. = not determined.</p>*<p>CDC73/HRPT2 mutations were previously published for cases T2–T4, and T6–T8 (References <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046325#pone.0046325-Carpten1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046325#pone.0046325-Shattuck1" target="_blank">[5]</a>).</p><p>The constitutional mutation in T5 was revised from a previous publication (Reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046325#pone.0046325-Juhlin4" target="_blank">[30]</a>).</p

Figure 2

<p>(A) Bar graph showing predicted DNA copy number of the <i>CDC73</i> gene using TaqMan DNA copy number analysis. Each bar represents one parathyroid sample and the height of the bar represents the predicted <i>CDC73</i> gene DNA copy number. T2, T3 and T4 had 1 copy of <i>CDC73</i> gene, while T6 and T8 had more than 2 copies. N1, N2 and N3 refer to the normal parathyroid samples used as calibrators. (B) Individual value plot illustrating the methylation density at the three analyzed CpG dinucleotides of the <i>HPRT2</i> promoter. Each red dot represents one CpG site in one parathyroid tumor numbered from T2–T8 along with the three normal references denoted as N1, N2 and N3.</p

LOH karyogram.

<p>Karyogram showing all LOH events detected in (A) the parathyroid carcinomas (T6–T8) and in (B) the parathyroid adenomas (T2–T5) using a 250K SNP array.</p