Data from Locked Nucleic Acid <i>In situ</i> Hybridization Analysis of miR-21 Expression during Colorectal Cancer Development

Abstract

<div>Abstract<p><b>Purpose:</b> To better understand microRNA miR-21 function in carcinogenesis, we analyzed miR-21 expression patterns in different stages of colorectal cancer development using <i>in situ</i> hybridization (ISH).</p><p><b>Experimental Design:</b> Locked nucleic acid (LNA)/DNA probes and a biotin-free tyramide signal amplification system were used in ISH analyses of miRNA expression. Conditions for specific detection of miR-21 were determined using human cell lines and miR-21–expressing lentiviral vectors. Expression was determined in 39 surgically excised colorectal tumors and 34 endoscopically resected colorectal polyps.</p><p><b>Results:</b> In the surgical samples, miR-21 expression was much higher in colorectal cancers than in normal mucosa. Strong miR-21 expression was also observed in cancer-associated stromal fibroblasts, suggesting miR-21 induction by cancer-secreted cytokines. Protein expression of PDCD4, a miR-21 target, was inversely correlated with miR-21 expression, confirming that miR-21 is indeed a negative regulator of PDCD4 <i>in vivo</i>. In the endoscopic samples, miR-21 expression was very high in malignant adenocarcinomas but was not elevated in nontumorigenic polyps. Precancerous adenomas also frequently showed miR-21 up-regulation.</p><p><b>Conclusion:</b> Using the LNA-ISH system for miRNA detection, miR-21 was detectable in precancerous adenomas. The frequency and extent of miR-21 expression increased during the transition from precancerous colorectal adenoma to advanced carcinoma. Expression patterns of miR-21 RNA and its target, tumor suppressor protein PDCD4, were mutually exclusive. This pattern may have clinical application as a biomarker for colorectal cancer development and might be emphasized by self-reinforcing regulatory systems integrated with the <i>miR-21</i> gene, which has been previously shown in cell culture.</p></div>