MiR-20a Is Upregulated in Anaplastic Thyroid Cancer and Targets <i>LIMK1</i>

<div><p>Background</p><p>There have been conflicting reports regarding the function of miR-20a in a variety of cancer types and we previously found it to be dysregulated in sporadic versus familial papillary thyroid cancer. In this study, we studied the expression of miR-20a in normal, benign and malignant thyroid samples, and its effect on thyroid cancer cells <i>in vitro</i> and <i>in vivo</i>.</p><p>Methodology/Principal Findings</p><p>The expression of miR-20a in normal, benign and malignant thyroid tissue was determined by quantitative RT-PCR. Thyroid cancer cells were transfected with miR-20a and the effect on cellular proliferation, tumor spheroid formation, and invasion was evaluated. Target genes of miR-20 were determined by genome-wide mRNA expression analysis with miR-20a overexpression in thyroid cancer cells and target prediction database. Target genes were validated by quantitative PCR and immunoblotting, and luciferase assays. MiR-20a expression was significantly higher in anaplastic thyroid cancer than in differentiated thyroid cancer, and benign and normal thyroid tissues. MiR-20a significantly inhibited thyroid cancer cell proliferation <i>in vitro</i> (p<0.01) and <i>in vivo</i> (p<0.01), tumor spheroid formation (p<0.05) and invasion (p<0.05) in multiple thyroid cancer cell lines. We found that <i>LIMK1</i> was a target of miR-20a in thyroid cancer cell lines and direct knockdown of LIMK1 recapitulated the effect of miR-20a in thyroid cancer cells.</p><p>Conclusions/Significance</p><p>To our knowledge, this is the first study to demonstrate that miR-20a plays a role as a tumor suppressor in thyroid cancer cells and targets <i>LIMK1</i>. Our findings suggest the upregulated expression of miR-20a in anaplastic thyroid cancer counteracts thyroid cancer progression and may have therapeutic potential.</p></div

MiR-20a targets LIMK1 and LIMK1 regulates cellular invasion.

<p>(<b>A</b>) Luciferase activity of pEZX-LIMK1-UTR in FTC-133 cells when co-transfected with miR-20a or miR-NC. All luciferase measurements were made in triplicates and readings were performed at 24 hours post-transfection. Error bars represent standard error of mean (* indicates p<0.05). (<b>B</b>) <i>LIMK1</i> siRNA knockdown in FTC-133 thyroid cancer cell line. LIMK1 mRNA expression by quantitative RT-PCR (top panel). LIMK1 protein expression by Western blot (bottom panel). Data shown is for 72 hours after siRNA transfection. (<b>C</b>) Cellular invasion with LIMK1 knockdown. Transfection of <i>LIMK1</i> siRNAs inhibited FTC-133 thyroid cancer cells invasion. The Y axis represents the invasion index of the thyroid cancer cells. Data shown is for 72 hours after siRNA transfection. Error bars represent standard error of mean (* indicates p<0.05; ** indicates p<0.01). (<b>D</b>) Transfection of <i>LIMK1</i> siRNAs has no effect on the migration of FTC-133 thyroid cancer cells. The Y axis represents the wound distance. Data shown is for 72 hours after siRNA transfection. Error bars represent standard error of mean.</p

Genes identified to be regulated by miR-20a using both microarray analysis and target scan analysis.^*

<p>*Genes listed were common to genome-wide gene expression analysis and target scan database, and based on change in gene expression of 2-fold or greater with adjusted p value of 0.05. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096103#pone.0096103.s001" target="_blank">Table S1</a> is the entire gene list with 1.5-fold or greater change in expression, with adjusted p value of 0.05, and genes which are predicted targets by target scan analysis.</p

MiR-20a overexpression decreases LIMK1 protein expression in thyroid cancer cell lines.

<p>(<b>A</b>) Immunoblots for LIMK1 protein expression in C643, XTC-1, FTC-133, and TPC-1 cell lines, which were transfected with either miR-20a or miR-NC for 72 hours. (<b>B</b>) Immunoblots for endogenous LIMK1 and GAPDH in FTC-133 cells transfected with either miR-20a or miR-NC for 7 days, 14 days and 21days.</p

MiR-20a overexpression inhibits thyroid cancer cell invasion.

<p>(<b>A</b>) TPC-1 thyroid cancer cell line, (<b>B</b>) XTC-1 thyroid cancer cell line, (<b>C</b>) FTC-133 thyroid cancer cell line, and (<b>D</b>) C643 thyroid cancer cell line. The Y axis represents the invasion index of the thyroid cancer cells. Error bars represent standard error of mean (* indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001).</p

MiR-20a overexpression inhibits cellular proliferation.

<p><b>(A–D)</b>. Thyroid cancer cell line proliferation with miR-20a overexpression. The Y axis represents the cell number. (<b>E–F</b>) Thyroid cancer <i>in vivo</i> growth, <i>ex vivo</i> tumor harvests and weight. Error bars represent standard error of mean (* indicates p<0.05; ** indicates p<0.01).</p

Summary of sequencing changes in KIAA0101.

<p>*Percentage of mutation is for the samples which were analyzed by category and available for sequencing.</p><p>**ND (not detected).</p

KIAA0101 mRNA and protein expressions were decreased with siRNA knockdown in the NCI-H295R adrenocortical carcinoma cell line.

<p><b>A</b>) H295R cells were transfected with KIAA0101 siRNA and negative control and analyzed for KIAA0101 mRNA expression after 48 hours of treatment. Columns represent KIAA0101 mRNA expression percentage relative to negative control ± standard deviation of four experiments. <b>B</b>) Representative Western blot demonstrating protein expression of KIAA0101 after 6 days of siRNA and negative control. <b>C</b>) Relative intensity of western blot using Image J Software. Columns indicate ratio of KIAA0101 and GAPDH intensity measurements. NC40 = Negative control at 40 nM; SI40  =  siRNA at 40 nM; NC80  =  Negative control at 80 nM; SI80  =  siRNA at 80 nM.</p

KIAA0101 siRNA knockdown reduced soft agar anchorage independent growth and invasion in NCI-H295R cells.

<p><b>A</b>) Cells cultured for 15 days in the presence of indicated concentrations of siRNA and negative control. Representative images are shown at 12X and 40X magnifications. <b>B</b>) The distribution and number of colonies in group treated with KIAA0101 siRNA and negative control group at 80 nM (p = 0.001; relative to negative control; NC80 =  Negative control at 80 nM; SI80  = siRNA at 80 nM). Error bars represent ± standard error of mean and is representative of four experiments. <b>C</b>) Invading cells were stained with 0.2% crystal violet and visualized by microscopy. Representative images at 20X following invasion assay. <b>D</b>) The distribution of number of cells in KIAA0101 siRNA and negative control group. Columns represent the mean ± standard deviation (SD) of three independent experiments performed in triplicate. *p<0.05 KIAA0101 siRNA vs. negative control. H295R cells were transfected with negative control or KIAA0101 siRNA at 80 nM for 120 h, followed by an invasion assay for 48 h.</p

Expression of KIAA0101 mRNA in ACC.

<p><b>A</b>) KIAA0101 mRNA expression in normal adrenal cortex (n  =  21), benign adrenocortical tumors (N  =  80), ACC (N  =  10), metastatic and recurrent ACC (n = 30) and the NCI-H295R cell line. KIAA0101 mRNA expression level was determined by quantitative RT-PCR and normalized to GAPDH mRNA expression. Columns represent mean ± standard deviation of replicate determinations. Statistical significant difference is indicated by an asterisk (*) (p<0.05, Kruskal Wallis test). Primary ACC vs. normal (p<0.0001), primary ACC vs. benign adrenocortical tumors (p<0.0001) and primary ACC vs. metastases (p<0.0001). <b>B</b>) ROC curve analysis for KIAA0101 mRNA expression. The receiver operating characteristic curve (ROC) is depicted on graph using RT PCR expression data of KIAA0101 normalized to GAPDH in normal adrenal cortex (n  =  21), benign adrenocortical tumors (n  =  80), ACC (n  =  10) . The area under the curve (AUC) was 0.78. A perfect diagnostic marker without any false-negatives or false-positives would have an AUC of 1. <b>C</b>) KIAA0101 protein expression in ACC. KIAA0101 protein expression levels in normal adrenal cortex (n = 5), benign (n = 13), and malignant adrenocortical tumors (n = 3) as shown in representative Western blot with KIAA0101 specific and β-Actin control antibodies. β-Actin signal was used as a control for protein loading. C  =  ACC, B  =  benign adrenocortical tumor, and N  =  normal adrenal cortex. <b>D</b>) Scatter plot of KIAA0101 mRNA and protein expression level in normal, benign and malignant adrenocortical tissue samples. Y-axis shows normalized mRNA expression by quantitative RT-PCR and X-axis shows protein expression level in the same sample based on band densitometry measurement normalized to β actin expression level. Spearmen correlation coefficient <b>(r)  = 0.61, p<0.001. </b><b>E</b>) Immunohistochemistry for KIAA0101 protein expression in normal adrenocortical tissue, benign tumors and ACC. Representative images are shown for each category at 20X magnification. Arrow indicates the positive nuclear staining for KIAA0101. In normal tissue samples, only the subcupsular region was positive. <b>F</b>) KIAA0101 cellular localization was analyzed by immunoflouroscence. Nuclei were stained by DAPI (blue), red color indicates KIAA0101 expression. Representative images from malignant ACC and normal samples are shown at 20X magnification.</p