Naslov
Regulation of PTCH1b Tumor Suppressor Gene Expression by 5' Untranslated Region

Autori
Ozretić, Petar ; Bisio, Alessandra ; Musani, Vesna ; Sabol, Maja ; Trnski, Diana ; Levanat, Sonja ; Inga, Alberto

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
1. regionalni kongres Edukacija i znanost u onkologiji : knjiga sažetaka / Šamija, Mirko - Zagreb : Zaklada Onkologija, 2013, 79-80

Skup
1. regionalni kongres Edukacija i znanost u onkologiji / 1st Regional Congress Education and Research in Oncology

Mjesto i datum
Zagreb, Hrvatska, 20.11.2013. - 23.11.2013

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
HH-Gli signaling pathway; PTCH1; 5' UTR; CGG repeats; uORF; IRES

Sažetak
PTCH1 is a tumor suppressor gene that 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 pathway that is involved in pathogenesis of various tumors (e.g., medulloblastoma, meningioma, esophageal carcinoma, squamous cell carcinoma, trichoepithelioma, breast carcinoma, etc.). In this study we wanted to examine the role of 5’ untranslated region (5'-UTR) in the regulation of expression of main PTCH1 transcript 1b. 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 number of repeats. 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 constructed 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. The existence of an IRES motif would enable Ptch1 protein to be synthesized under conditions when general level of protein synthesis is decreased, such as in hypoxia. All these results point to the exceptionally complex and so far unexplored role of 5'-UTR in the regulation of PTCH1b expression.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

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