Locked Nucleic Acid In situ Hybridization Analysis of miR-21 Expression during Colorectal Cancer Development

• Published in: Clinical cancer research Vol. 15; no. 12; pp. 4009 - 4016
• Main Authors: Yamamichi, Nobutake, Shimomura, Ryoichi, Inada, Ken-ichi, Sakurai, Kouhei, Haraguchi, Takeshi, Ozaki, Yuka, Fujita, Shuji, Mizutani, Taketoshi, Furukawa, Chihiro, Fujishiro, Mitsuhiro, Ichinose, Masao, Shiogama, Kazuya, Tsutsumi, Yutaka, Omata, Masao, Iba, Hideo
• Format: Journal Article
• Language: English
• Published: United States American Association for Cancer Research 15.06.2009
• Subjects: Adenocarcinoma - diagnosis; Adenocarcinoma - genetics; Adenocarcinoma - pathology; Apoptosis Regulatory Proteins - metabolism; cancer-associated stromal fibroblasts; colorectal cancer; Colorectal Neoplasms - diagnosis; Colorectal Neoplasms - genetics; Colorectal Neoplasms - pathology; DNA Probes - genetics; Genetic Vectors - metabolism; HeLa Cells; Humans; hybridization; in situ; In Situ Hybridization; LNA; MicroRNAs - analysis; MicroRNAs - genetics; miR-21; Oligonucleotides - genetics; PDCD4; Precancerous Conditions - diagnosis; Precancerous Conditions - genetics; Precancerous Conditions - pathology; RNA-Binding Proteins - metabolism
• DOI: 10.1158/1078-0432.CCR-08-3257
• ISSN: 1078-0432

Purpose: To better understand microRNA miR-21 function in carcinogenesis, we analyzed miR-21 expression patterns in different stages of colorectal cancer development using in situ hybridization (ISH). Experimental Design: 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. Results: 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 in vivo . 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. Conclusion: 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 miR-21 gene, which has been previously shown in cell culture.