Project description:KRAS activation drives DNA methylation and silencing of specific tumor suppressor genes (TSGs). We previously showed that the ERK pathway induces transcriptional repression of TET1, which results in conversion of TSG promoters from a hydroxymethylated, active state to a hypermethylated and silenced state. Here we identified miR-29b as a KRAS-induced molecule that represses TET1 expression. In KRAS-transformed cells, ectopic miR-29b inhibition restores expression of TET1, thereby reactivating TSGs by reducing methylation and restoring hydroxymethylation. Mining gene expression data of lung cancer cell lines identified additional TSGs suppressed by KRAS signaling whose expression was restored by inhibition of miR-29b and re-expression of TET1. Because KRAS changes TSG promoters from hydroxymethylated to hypermethylated with miR-29b-dependent silencing of TET1, we demonstrate a model in which DNMT1 is present on target promoters prior to KRAS transformation. We also propose miR-29b as a potential circulating biomarker and target for rational treatment of specific malignancies.
Project description:Overexpression and inhibition of miR-29 (pre-miR and anti-miR to miR-29b) in murine aortic smooth muscle cells, analysis of their secretome (conditioned media after serum starvation), n=3 for all four groups (pre-miR control, pre-miR-29b, anti-miR control, anti-miR-29b).
Project description:KRAS mutation is one of the major genetic alterations in colorectal cancer (CRC). Treating patients with KRAS mutant CRC remains one of the biggest challenges in oncology. In this study, we aimed to discover an effective therapeutic microRNA (miRNA) that could target KRAS mutant CRC. For this purpose, we investigated the functional relevance of dysregulated miRNAs in KRAS mutant cancers. We transfected exogenous KRASG12V into human embryonic kidney 293 (HEK293) cells and human lung fibroblasts (MRC5) cells, and performed comprehensive microRNA expression profiling by microarray analysis. The results showed that 6 miRNAs are significantly upregulated in KRAS-transfected HEK293 and MRC5 cells. Among 6 miRNAs, we identified mature miRNA-29b-1-5p as potent growth inhibitor in CRC cell proliferation. However, miRNA-29b-1-5p was found to be a passenger strand with a star form (miRNA-29b-1-5p*), and did not function in CRC cells. Proliferation assay revealed that completely opposite complementary strand to miRNA-29b-1-5p possessed a potent anti-tumor effect. We named this novel anti-tumor siRNA sequence “MIRTX”. MIRTX induced apoptosis and significantly inhibited cell proliferation in KRAS mutant CRC in vitro. In addition, MIRTX suppressed NF-κB signaling pathway, which is downstream effector of KRAS in CRC. Furthermore, MIRTX directly targeted 3'-UTR of PIK3R1 and CXCR2 mRNA, and indirectly suppressed KRAS itself. In vivo xenograft mouse models, systemic administration of MIRTX significantly inhibited the tumor growth with no particular toxicity, using carbonate apatite as a vehicle. These findings indicate that inhibition of NF-κB signaling by siRNA-based therapeutic could be a promising strategy against KRAS mutant CRC.
