Abstract 5239: MiR-148a promotes apoptosis in urothelial cell carcinoma of the bladder cells in part by targeting DNMT1

Abstract

Abstract Introduction: MiRNAs are now recognized as major players in cancer. MiR-148a has been shown to be a tumor suppressor in the context of several cancers including prostate, gastric, and lung but a study investigating its role in bladder cancer has never been performed. Here we characterized the expression and role of miR-148a in bladder cancer. Methods: Real time PCR was used to assess the levels of miR-148a across several bladder cancer cell lines. MiR-148a mimics were employed in T24 and UM-UC-3 cells to assess proliferation by CCK-8 proliferation assay, colony formation assay, and flow cytometry. Mimics were also used to assess apoptosis by immunoblot and annexin-V staining coupled to flow cytometry. Immunoblot was used to probe for targets of miR-148a and siRNA was used to knock-down DNMT1 to phenocopy miR-148a treatment results. CCK-8 proliferation assays and crystal violet staining were used to assess the additive effects of miR-148a with either cisplatin or doxorubicin on T24 and UM-UC-3 cells and CCK-8 proliferation assays were also used to assess the role DNMT1 plays in cisplatin sensitivity. Results: All bladder cancer cell lines had significantly reduced expression of miR-148a than that of a control immortalized urothelial cell line. MiR-148a mimic treatment of T24 and UM-UC-3 cells led to large decreases in viability. Flow cytometry demonstrated an increase in S-phase and no major G1 or G2/M arrests in miR-148a mimic treated cells versus control oligo treated cells suggesting that the effects observed on viability were due to increased apoptosis. Immunoblots for cleaved caspase-3 and cleaved PARP along with annexin-V staining coupled to flow cytometry showed as expected a strong induction of apoptosis in miR-148a mimic treated cells. Immunoblots demonstrated that miR-148a targets DNMT1 and DNMT1 knock-down was able to partially phenocopy results from miR-148a mimic treatment suggesting DNMT1 is downstream of miR-148a. Finally, we found that miR-148a can be used with either cisplatin or doxorubicin for an additive effect in inducing apoptosis and that DNMT1 targeting is involved in sensitivity to cisplatin. Conclusions: Our work demonstrates for the first time that miR-148a plays a tumor suppressive role in bladder cancer and suggests it could be used as a novel therapeutic agent either alone or in conjunction with chemotherapeutics. Citation Format: Alan Lombard, Ben Mooso, Steve Libertini, Rebecca Lim, Nicole Costanzo, Paramita Ghosh, Maria Mudryj. MiR-148a promotes apoptosis in urothelial cell carcinoma of the bladder cells in part by targeting DNMT1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5239. doi:10.1158/1538-7445.AM2014-5239