Abstract 2081: Frequent silencing of tumor suppressive MicroRNA-34b/c by aberrant methylation in malignant mesothelioma
Authors: Kubo Takafumi , Toyooka Shinichi , Tsukuda Kazunori , Asano Hiroaki , Sakaguchi Masakiyo , Soh Junichi , Ueno Tsuyoshi , Yamamoto Hiromasa , Yamane Masaomi , Oto Takahiro , Nasu Yasutomo , Matsui Hideki , Huh Nam ho , Miyoshi Shinichro 
Published: February 2011

Background: Altered expression of microRNA (miRNA) is strongly implicated in human malignancies. Some miRNAs are classified as oncogenic or tumor suppressive miRNAs according to their function in cellular transformation. Similar to encoding genes, tumor suppressive miRNAs are silenced with DNA methylation. The miR-34a and b/c are direct transcriptional targets of p53 and sometimes down-regulated in malignancies. Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with a dismal prognosis. Unlike other malignant tumors, mutation of P53 gene is rare and p53 expression is generally intact in MPM. In this study, we examined methylation and expression status of miR-34a and b/c in MPM. We also investigated the effect of miR-34b/c induction into MPM cells. Methods: We examined 6 MPM cell lines (H28, H290, 211H, H2052, H2452, and HP1), 20 primary MPMs and 7 non-malignant mesotheial tissues. Methylation and expression status of miR-34a and b/c were determined with methylation-specific PCR and quantitative PCR, respectively. Colony formation assay was applied to examine the anti-proliferative effect by induction of miR-34b/c and scramble sequence control with plasmid vector. Protein expression and cell cycle were analyzed with western blotting and flow cytometry, respectively.

Results: Aberrant methylation was found in 2 (33.3%) of 6 MPM cell lines and 6 (33.3%) of 20 primary MPMs in miR-34a and in all 6 MPM cell lines (100%) and 18 (90%) of 20 primary MPMs in miR-34b/c. No methylation was found in 7 non-malignant mesothelial tissues. Expression of miR-34a and b/c in all methylated cell lines was silenced and restored with 5-Aza-CdR treatment, indicating that methylation caused gene silencing. Because the frequency of methylation was particularly high in miR-34b/c, we focused on miR-34b/c in following study. Ectopic induction of miR-34b/c gene in examined 3 MPM cell lines (H28, H290, and 211H) showed significant inhibition of colony formation compared with induction of control vector, demonstrating anti-proliferative effect of miR-34b/c on MPM. Western blotting analysis for all 3 MPM cell lines after induction of miR-34b/c showed suppression of total and phospho-c-met, cdk4, cdk6, and cyclin E2 collated genes of cell proliferation, suggesting that these genes were interfering targets of miR-34b/c. Cell cycle analysis showed that G2-M fractions were reduced with flow cytometry in all 3 MPM cell lines with miR-34b/c induction.

Conclusion: Our results demonstrated that miR-34b/c methylation is frequent in MPM and resulting miR-34b/c silencing plays an important role in pathogenesis of MPM. The miR-34b/c can be a promising candidate as a therapeutic miRNA for MPM.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2081.