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The role of 5' untranslated region in the regulation of PTCH1b gene expression

Ozretić, Petar
The role of 5' untranslated region in the regulation of PTCH1b gene expression // Godišnja skupština Hrvatskog društtva za istraživanje raka
Zagreb, Hrvatska, 2012. (pozvano predavanje, nije recenziran, neobjavljeni rad, znanstveni)

The role of 5' untranslated region in the regulation of PTCH1b gene expression

Ozretić, Petar

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, neobjavljeni rad, znanstveni

Godišnja skupština Hrvatskog društtva za istraživanje raka

Mjesto i datum
Zagreb, Hrvatska, 21.12.2012

Vrsta sudjelovanja
Pozvano predavanje

Vrsta recenzije
Nije recenziran

Ključne riječi
PTCH1; 5' UTR; CGG repeats; uORF; IRES

PTCH1 is a tumor suppressor gene that encodes for a receptor with a negative regulatory role and one of the key players in Hedgehog signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, disorder characterized by developmental abnormalities and tumor susceptibility. Most of malformations are caused by PTCH1 haploinsufficiency, indicative of fine-tuning needed to properly regulate the pathway that is involved in pathogenesis of various tumors. We wanted to examine the role of 5’ untranslated region (5’UTR) in PTCH1 expression regulation. In patients with different tumors and healthy controls we identified 5 to 8 CGG repeats located 4 bases upstream of translation initiation site. Since PTCH1b transcript has 2 different 5’UTRs, we built pGL3-P-based plasmids by inserting upstream of firefly luciferase gene 188- or 300-bases-long 5’ UTR, each harboring 5 to 8 CGG repeats. Luciferase assays performed in MCF7, HCT116 and HEK293 cells showed that shorter 5’UTR significantly increased reporter activity, with a subtle reduction with increased repeats number. Longer 5’UTR led to much reduced reporter activity without differences among repeats. Luciferase mRNA quantification showed that both 5’UTR lengths significantly increased transcription. Site-directed mutagenesis proved hypothesis that 2 potential upstream open reading frames, contained in first 112bp of longer 5’UTR, might account for this severe reduction in reporter activity. Since last 76bp of 5’UTR were predicted as internal ribosome entry site (IRES) we built bicistronic pRuF vectors (PTCH1b 5’UTR cloned between Renilla and firefly luciferase gene). Both 5’UTR lengths significantly increased luciferase activity (proved by PCR this is not due to alternative splicing) with no differences among repeats. Firefly luciferase activity was significantly reduced when IRES was removed from plasmids. Firefly/Renilla luciferase mRNA ratios were same as for empty vector. This higher luciferase activity with equal mRNA levels appears to be a post-transcriptional effect.

Izvorni jezik

Znanstvena područja
Temeljne medicinske znanosti


Projekt / tema
098-0982464-2461 - Prijenos signala u tumorima: Hh-Gli put, interakcije i potencijalne terapije (Sonja Levanat, )

Institut "Ruđer Bošković", Zagreb

Autor s matičnim brojem:
Petar Ozretić, (295840)