Dissecting the 5' Untranslated Region of the PTCH1b Tumor Suppressor Gene (CROSBI ID 613401)
Prilog sa skupa u časopisu | sažetak izlaganja sa skupa | međunarodna recenzija
Podaci o odgovornosti
Ozretić, Petar ; Bisio, Alessandra ; Musani, Vesna ; Trnski, Diana ; Sabol, Maja ; Levanat, Sonja ; Inga, Alberto
engleski
Dissecting the 5' Untranslated Region of the PTCH1b Tumor Suppressor Gene
Background PTCH1 tumor suppressor gene encodes for a 12-pass transmembrane receptor with a negative regulatory role in Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, disorder characterized by developmental abnormalities and tumor susceptibility. Most of the malformations are caused by PTCH1 haploinsufficiency, indicative of fine-tuning needed to properly regulate the activity of pathway which is involved in pathogenesis of various tumors. Since in our previous study we identified 5 to 8 CGG repeats located 4 bases upstream of a translation initiation site, our aim in this study was to examine how 5’ untranslated region (5’UTR) regulates the expression of PTCH1 transcript 1b. Material and methods All potential cis-regulatory elements in PTCH1b 5’UTR were studied by various in silico tools and gene reporter assays. We tested the influence of 5’UTR length and CGG-repeat polymorphism in a way that in pGL3-P plasmid we cloned either 188- or 300bp-long 5’UTR, each harboring 5 to 8 CGG repeats, upstream of firefly luciferase gene. Site-directed mutation (SDM) was used to test the significance of predicted upstream open reading frames (uORF). We also constructed bicistronic pRuF vectors by cloning each PTCH1b 5’UTR between Renilla and firefly luciferase gene, since the last 76bp in both 5’UTR were predicted as an internal ribosome entry site (IRES). Dual luciferase assays and qPCR were performed in MCF-7, HCT 116 and HEK 293T cells. Results Reporter assays showed that shorter 5’UTR significantly increased reporter activity, with a subtle reduction with higher number of repeats. Longer 5’UTR led to much reduced reporter activity, without a difference among repeats. Luciferase mRNA quantification showed that both 5’UTR lengths significantly increased transcription. SDM proved hypothesis that 2 potential uORF, contained only in the first 112bp of 300bp-long 5’UTR, might account for this severe reduction in reporter activity. Both 5’UTR lengths significantly increased firefly luciferase activity of pRuF vectors (proved by PCR this is not due to alternative splicing), with no difference among repeats. Firefly luciferase activity was significantly reduced when predicted IRES motif was removed from pRuF plasmid. Firefly/Renilla luciferase mRNA ratios were the same as for empty vector, indicating that observed higher firefly luciferase activity should be due to a post-transcriptional regulation, i.e., cap-independent translation of firefly luciferase mRNA. Conclusions All our results point to the exceptionally complex and so far unexplored role of 5’UTR in the regulation of PTCH1b expression, whilst the existence of an IRES motif would enable Ptch1 protein to be synthesized under conditions when the general level of protein synthesis is reduced, such as in hypoxia, which is known activator of Hedgehog-Gli pathway.
Hedgehog-Gli; PTCH1; 5’UTR; CGG repeats; uORF; uAUG; IRES
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Podaci o prilogu
S114-S114.
2014.
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objavljeno
Podaci o matičnoj publikaciji
European journal of cancer (1990)
Eggermont, Alexander M.M.
Amsterdam: Elsevier
0959-8049
Podaci o skupu
23rd Biennial Congress of the European Association for Cancer Research
poster
05.07.2014-08.07.2014
München, Njemačka