Locked nucleic acid in situ hybridization analysis of miR-21 expression during colorectal cancer development

Nobutake Yamamichi, Ryoichi Shimomura, Ken Ichi Inada, Kouhei Sakurai, Takeshi Haraguchi, Yuka Ozaki, Shuji Fujita, Taketoshi Mizutani, Chihiro Furukawa, Mitsuhiro Fujishiro, Masao Ichinose, Kazuya Shiogama, Yutaka Tsutsumi, Masao Omata, Hideo Iba

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Abstract

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 andmight be emphasized by self-reinforcing regulatory systems integrated with the miR-21 gene, which has been previously shown in cell culture.

Original languageEnglish
Pages (from-to)4009-4016
Number of pages8
JournalClinical Cancer Research
Volume15
Issue number12
DOIs
Publication statusPublished - 15-06-2009

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All Science Journal Classification (ASJC) codes

  • Oncology
  • Cancer Research

Cite this

Yamamichi, N., Shimomura, R., Inada, K. I., Sakurai, K., Haraguchi, T., Ozaki, Y., Fujita, S., Mizutani, T., Furukawa, C., Fujishiro, M., Ichinose, M., Shiogama, K., Tsutsumi, Y., Omata, M., & Iba, H. (2009). Locked nucleic acid in situ hybridization analysis of miR-21 expression during colorectal cancer development. Clinical Cancer Research, 15(12), 4009-4016. https://doi.org/10.1158/1078-0432.CCR-08-3257