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Abstract(s)
Colorectal cancer (CRC) has a high incidence and mortality rates worldwide [1]. CRC carcinogenesis is a continuous accumulation of genetic alterations with concomitant variations in gene expression profiles [2]. To study the variations of gene expression profiles involved in cancer progression, genome-wide analyses have so far focused on the abundance of mRNA as measured either by microarray or RNA sequencing [3,4]. However, neither approach provides information on protein synthesis, which is the true end-point of gene expression [3-5]. Ribosome profiling (Ribo-Seq) emerges to monitor in vivo translation, providing global and quantitative measurements of translation by deep sequencing of ribosome-protected mRNA fragments (RPFs) [5,6]. This technique has revealed unexpected complexity in translation, including the presence of ribosomes outside of classical protein-coding regions of the transcriptome [3].
The main goal of this project is to determine the changes between the translatome of CRC and normal colorectal cells and their role in CRC tumorigenesis. For that, we aim to analyze ribosome profiling data already available for the CRC cell line HCT116, and eventually data from non-neoplasic colorectal cells (if available). Gene ontology and network interaction analysis of the differentially translated mRNAs will elucidate the main molecular pathways through which the corresponding proteins are involved in CRC progression. Furthermore, we aim to analyze the function of translatable small open reading frames (sORFs), such as the upstream ORFs (uORFs), and/or the corresponding encoded peptides in the regulation of CRC progression.
We have performed a computational analysis of HCT116 Ribo-Seq data to detect potential translatable uORFs. For that we are currently determining the number of RPFs in the 5’UTR of transcripts. Meanwhile, and based on previously published data about the prediction/detection of translatable alternative ORFs (AltORFs) in CRC cells [7], ABCE1, ABCF1, ABCF2 and ABCF3 mRNAs were chosen for further studies. To analyze their mRNA expression levels, we performed semi-quantitative RT-PCR analysis using RNA from HCT116, Caco-2 and SW480 CRC cells, as well as from non-neoplasic colorectal NCM460 cells. Our results show that these mRNAs are down-regulated in HCT116 cells comparing to their expression in the other three cell lines. In addition, we have been involved in mapping, by circular rapid amplification of cDNA ends (cRACE), cloning and sequencing, the exact 5’ end of the ABCE1 5’UTR. After getting this information, we will clone this 5’UTRs in a reporter construct that will allow us to test the ABCE1 uORF potential function in CRC progression.
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Genómica funcional e estrutural Expressão génica