Abstract 3952: MicroRNA miR-34b/c is significantly down-regulated by aberrant promoter methylation and forced expression of miR-34b/c enhances the radiosensitivity in malignant pleural mesothelioma cells
Authors: Maki Yuho , Toyooka Shinichi , Asano Hiroaki , Sho Junichi , Tukuda Kazunori , Muraoka Takayuki , Aokage Keiju , Tanaka Norimitsu , Ueno Tsuyoshi , Yamane Masaomi , Oto Takahiro , Miyoshi Shinichiro 
Published: August 2014

&lt;Background&gt; MicroRNA miR-34b/c, one of the direct transcriptional targets of p53, plays an important role in tumor-suppressor network. It is known that the miR-34b/c can induce cell cycle arrest by blocking CDK4, CDK6, CCND1, CCNE2, E2F, bcl-2 and promote apoptosis. On the contrary, loss of miR-34b/c attenuates p53-mediated cell death. We previously described the miR-34b/c was significantly down-regulated by heavy methylation in the promoter region in malignant pleural mesotheliomas(MPMs). It has been reported miR-34a enhanced the radiosensitivity in pancreatic cancer cells. But there is no report that demonstrates the relationship between miR-34b/c and radiosensitivity. We hypothesized that miR-34b/c might enhance the radiosensitivity by blocking cell cycle. Because miR-34b/c seems to play an important role in pathogenesis in MPMs, we examined the miR-34b/c-mediated radiosensitization in MPM cells.

&lt;Materials and Methods&gt; To test this hypothesis, two MPM cell lines(H28, H2052) stably transfected with miR-34b/c were used in this study. A Real-time PCR for miR-34b and miR-34c was performed to confirm the expression of miR-34b/c in stable transfectants. A Western blotting for CDK4, CDK6, CCND1, CCNE2, E2F, bcl-2 was done in two MPM cell lines(H28, H2052). A clonogenic survival assay and a cell cycle analysis were performed. As a measure of radiation-induced DNA damage, gamma H2AX foci assay was performed at 1hour, 6hour and 24hour after 4Gy irradiation.

&lt;Results&gt; The miR-34b expression was increased by 72.8-fold and 37.8-fold in H28 and H2052 stable miR-34b/c transfectants, respectively, compared to scramble microRNA stable transfectants. The miR-34c expression was increased by 6.7-fold and 15.3-fold in H28 and H2052 stable miR-34b/c transfectants, respectively. Western blotting showed CDK4, CDK6, CCND1, CCNE2 and E2F were down-regulated in stable miR-34b/c transfectants. Cell cycle analysis showed an increase in the G0-G1 fraction and a decrease in the G2-M and S fraction, indicating that miR-34b/c induced G1 cell cycle arrest. Clonogenic survival assay showed the miR-34b/c transfection significantly enhanced the radiosensitivity. Gamma H2AX foci assay demonstrated the average number of gamma H2AX foci at 24 hours after irradiation was 4.94/cell in stable miR-34b/c transfectants and 3.24/cell in stable scramble microRNA transfectants. &lt;Conclusion&gt; These results indicate miR-34b/c can enhance tumor cell radiosensitivity in MPM cells by inducing G1 cell cycle arrest and suggest that this effect may involve in an inhibition of DNA double strand break repair.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3952. doi:10.1158/1538-7445.AM2011-3952