<i>ZNF367</i> Inhibits Cancer Progression and Is Targeted by miR-195

<div><p>Background</p><p>Several members of the zinc finger protein family have been recently shown to have a role in cancer initiation and progression. Zinc finger protein 367 (<i>ZNF367</i>) is a member of the zinc finger protein family and is expressed in embryonic or fetal erythroid tissue but is absent in normal adult tissue.</p><p>Methodology/Principal Findings</p><p>We show that <i>ZNF367</i> is overexpressed in adrenocortical carcinoma, malignant pheochromocytoma/paraganglioma and thyroid cancer as compared to normal tissue and benign tumors. Using both functional knockdown and ectopic overexpression in multiple cell lines, we show that <i>ZNF367</i> inhibits cellular proliferation, invasion, migration, and adhesion to extracellular proteins <i>in vitro</i> and <i>in vivo</i>. Integrated gene and microRNA expression analyses showed an inverse correlation between <i>ZNF367</i> and miR-195 expression. Luciferase assays demonstrated that miR-195 directly regulates <i>ZNF367</i> expression and that miR-195 regulates cellular invasion. Moreover, integrin alpha 3 (<i>ITGA3</i>) expression was regulated by <i>ZNF367</i>.</p><p>Conclusions/Significance</p><p>Our findings taken together suggest that <i>ZNF367</i> regulates cancer progression.</p></div

<i>ZNF367</i> regulates <i>ITGA3</i> expression.

<p>(<b>A</b>) <i>ZNF367</i> knockdown upregulates <i>ITGA3</i> expression in SW13 cells. Error bars represent ± SEM. (<b>B</b>) The correlation between <i>ITGA3</i> and <i>ZNF367</i> mRNA expression in adrenocortical tumor samples. X and Y axes represent log 2–transformed values. (<b>C</b>) Western blot quantification of ITGA3 protein expression with <i>ZNF367</i> knockdown. (<b>D</b>–<b>E</b>) ITGA3 expression with ZNF367 overexpression. Error bars represent ± SEM.</p

<i>ZNF367</i> mRNA and protein levels in adrenocortical carcinoma, papillary thyroid cancer, and pheochromocytoma/paraganglioma compared to benign and normal tissue samples for each cancer type.

<p>(A and B) Expression level in the normal adrenal cortex, benign adrenocortical adenomas, and adrenocortical carcinomas; (C and D) normal adrenal medulla, benign and malignant pheochromocytoma/paraganglioma tissue samples; and (E and F) normal thyroid and papillary thyroid cancer tissue samples. The Y axis on each graph represents the percentage of mRNA expression using the 2^∧-ΔCt*100% method ± SEM. *p<0.05, **p<0.001, ***p<0.001 (Kruskal-Wallis test). Representative immunohistochemistry images are from normal, benign, and malignant tumor samples at 20X magnification.</p

Effect of <i>ZNF367</i> knockdown and overexpression.

<p><i>ZNF367</i> overexpression decreases cellular invasion and migration. (<b>A</b>) SW13, (<b>B</b>) BD104A, (<b>C</b>) TPC-1, and (<b>D</b>) HEK293 cell lines. After transfection, cells were plated inside a Boyden chamber for 48 hours. Cells were stained and counted in 4 fields. The left panel shows the representative image (12.5X) from the siRNA knockdown and negative control groups. The right panel indicates the quantitative measurement of invaded and migrated cells in knockdown and the negative control. *p<0.05 and error bars indicate ± SD. <i>ZNF367</i> overexpression decreases colony number, cellular invasion, and migration in HEK293 cells. (<b>E</b>) Western blot of <i>ZNF367</i> overexpression in HEK293 and SW13 cells (Empty vector, XL4). GAPDH was used as a loading control. (<b>F</b>) Representative clonogenic image for cells with ectopic <i>ZNF367</i> expression and its corresponding control (Empty vector, XL4). (<b>G</b>) Cellular invasion and migration decreased with <i>ZNF367</i> overexpression in HEK293 cells. The quantitative measurement of invaded and migrated cells per group (HEK293-HEK293-Empty vector or <i>ZNF367</i>) is represented in the bar graph on the right panel. (<b>H</b>) The extracellular protein attachment of SW13 cells with <i>ZNF367</i> knockdown. Cells were transfected with <i>ZNF367</i> siRNA and negative control siRNA, and were plated into adhesion plates and incubated for 90 minutes. The Y axis represents absorbance at 540 nM of cells adherent to each protein. Error bars represent ± SEM. *p<0.05, ***p<0.001.</p

The effect of <i>ZNF367</i> knockdown on growth in SW13 cells in vitro and in vivo.

<p>(A) <i>ZNF367</i> knockdown increases cellular proliferation. The Y axis represents relative fluorescent units (RFU), and the X axis indicates days post-transfection. *p<0.05 relative to the negative control. Error bars represent ± SD. (B) <i>ZNF367</i> knockdown enhances tumor growth in vivo. SW13 cells were transfected with the negative control (n = 4) and siRNA (n = 4) into the right and left flank of each mouse. After 48 hours of transfection, 3×10⁶ cells were injected in athymic nude mice, and tumor growth was measured weekly. The Y axis represents the tumor volume and X axis the weeks of tumor measurement after flank injection. *p<0.05 and error bars represent ± SD.</p

MiR-195 targets ZNF367.

<p>(<b>A</b>) The correlation between miR-195 and <i>ZNF367</i> expression in an adrenocortical tumor. The Pearson correlation coefficient is indicated by r with its p value. (<b>B</b>) Ectopic overexpression of pre-miR-195 results in downregulation of <i>ZNF367</i>. <i>ZNF367</i> mRNA expression in SW13 cells transfected with pre-miR-195 and the pre-negative control at 5 nM for 24 hours (presented as a fold-change relative to the pre-negative control). Error bars represent ± SEM. The right panel shows the Western blot of the ZNF367 protein from SW13 cells transfected with pre-miR-195 and the negative control. (<b>C</b>) Pre-miR-195 overexpression increases invasion in SW13 cells compared to the negative control. The right panel shows the mean number of invaded cells on the Y axis. (<b>D</b>) <i>ITGA3</i> mRNA expression is increased after transfection of miR-195 and the negative control in SW13 cells. Error bars represent ± SEM. (<b>E</b>) The binding site of miR-195 in the <i>ZNF367</i> 3′UTR, along with the mutant construct in the predicted seed region. In the right panel, the luciferase assay demonstrates decreased luminescence in SW13 cells co-transfected with miR-195 and the negative control at 5 nM, with the empty vector, wild-type <i>ZNF367</i> 3′UTR, or MUT-<i>ZNF367</i> 3′UTR vector (mutated in the first three nucleotides of the seed sequence). The luminescence was read after 24 hours of transfection. The Y axis represents the ratio of <i>ZNF367</i> 3′UTR to the empty vector. *p value < 0.05 compared to the negative control. Error bars represent ± SEM.</p