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|Title: ||MLH1 and DCC, two genes involved in colorectal cancer, have different mechanisms of translation initiation|
|Authors: ||Lacerda, Rafaela|
Luísa Romão, Luísa
|Keywords: ||Genómica Funcional e Estrutural|
|Issue Date: ||22-Nov-2012|
|Publisher: ||Instituto Nacional de Saúde Doutor Ricardo Jorge, IP|
|Abstract: ||Introduction: Colorectal cancer (CRC) is one of the leading causes of death worldwide and its development and progression can be dependent upon regulation of gene expression. MLH1 and DCC are two genes whose expression is altered in CRC. MLH1 is a mismatch repair gene and its expression is repressed in CRC. DCC is a tumor suppressor gene that has one allele deleted in CRC. Regulation of gene expression can occur at translation level, namely at the initial step. Translation initiation can be cap-dependent or internal ribosome entry site (IRES)-dependent. IRESs are structures within the 5’ untranslated region (UTR) of the mRNAs that directly recruit the ribosome without scanning the 5’UTR. This work aims to investigate whether MLH1 and DCC transcripts are translated via IRES or if their translation is exclusively cap-dependent.
Methods: The MLH1 and DCC 5’UTRs were cloned in the dicistronic p_Renilla_Firefly vector. NCM460 cells (derived from normal intestinal mucosa) and SW480 cells (derived from stage IV CRC adenocarcinoma) were transfected with such plasmids. Luciferase activity was measured by luminometry assays and compared to the negative control (dicistronic vector without insert).
Results and Discussion: The levels of Firefly luciferase observed when DCC 5’UTR is present are similar to those of negative control in both cell lines, which means translation of this transcript is exclusively cap-dependent and not mediated by IRES.
Regarding MLH1 5’UTR-containing plasmid, the levels of Firefly luciferase observed are 70-fold greater than that of negative control in NCM460 cells and 18-fold greater in SW480 cells, which might indicate IRES activity. However, when cells were transfected with a promoter-less plasmid containing this 5’UTR, we observed a 482-fold increase in Firefly luciferase levels in NCM460 cells and a 42-fold increase in SW480 cells, which suggests this region works as a cryptic promoter that is much more active in normal cells than in adenocarcinoma cells.|
|Peer Reviewed: ||yes|
|Appears in Collections:||DGH - Posters/abstracts em congressos nacionais|
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