Abstract 5247: Comprehensive and systematic identification of miR-215 targets in colon cancer via a novel translation capture immunoprecipitation (TrIP) approach

Abstract

Abstract The important roles that non-coding miRNAs play in cancer development, makes the investigation of the various genes they target particularly crucial. In addition to targeting mRNA transcripts for degradation, miRNA can also alter gene expression at the translational level to achieve rapid regulation. It has been a technical challenge to study translational control using a small number of cells. We have overcome this hurdle by developing a translational capture immunoprecipitation approach (TrIP) to investigate RNA binding proteins or non-coding RNA regulation at the translational level from a small numbers of cells. The new approach is based on the fact that nascent translated polypeptides are associated with hsp70 chaperones along with actively translating mRNA transcript complexes, called polyribosomes. We are able to isolate actively translating mRNA from free cytoplasmic mRNA by immunoprecipitation of hsp70 associated polyribosome complexes. In this study, we utilize this novel approach to identify both degraded and non-degraded mRNA transcripts targeted by miR-215 in colon cancer. Following miR-215 overexpression in colon cancer cells, actively translating mRNA transcripts were isolated using the TrIP technique and purified following standard RNA isolation techniques. We were able to identify additional non-degraded mRNA targets regulated by miR-215 using a TrIP with transcriptome-wide microarray based approach, when compared to a generic approach based on steady state total RNA that can only identify degraded targets. Specific targets were independently validated using qRT-PCR analysis. We further validated miR-215 targets at the protein level with a comprehensive and high-throughput quantitative SILAC proteomics analysis. Protein targets were further validated using Western blotting with select positive control targets including TYMS, DTL and DHFR. This approach offers the unique opportunity to understand not only the mRNA targets a miRNA may target for degradation, but also those it regulates at the translational level, providing unique insight into the actions of miRNAs. Interest in this type of thorough analysis of different miRNAs or RNA binding proteins will likely continue to grow as the important roles miRNA play in cancer are further investigated. Citation Format: Andrew Fesler, Xiao Xu, Emily Chen, Jingfang Ju. Comprehensive and systematic identification of miR-215 targets in colon cancer via a novel translation capture immunoprecipitation (TrIP) approach. [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 5247. doi:10.1158/1538-7445.AM2014-5247