engleski

Expression of PTCH1b tumor suppressor gene is controlled by different 5'-untranslated region cis-regulatory elements

Background The PTCH1 tumor suppressor gene encodes for a 12-pass transmembrane receptor with a negative regulatory role in Hedgehog signaling pathway. The 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 patients with different tumors and healthy controls we identified 5 to 8 CGG repeats located 4 bases upstream of a translation initiation site, in this study we wanted to examine how 5’ untranslated region (5’UTR) regulates the expression of PTCH1 transcript 1b. Material and methods Various in silico tools and databases were used to reveal all potential cis-regulatory elements in the PTCH1b 5’UTR. Since PTCH1b transcript has two different-sized 5’UTRs, we built pGL3-P-based plasmids by inserting upstream of firefly luciferase gene either 188- or 300bp-long 5’UTR, each harboring 5 to 8 CGG repeats. As the last 76bp of 5’UTR were predicted as an internal ribosome entry site (IRES), we constructed bicistronic pRuF vectors by cloning each PTCH1b 5’UTR between Renilla and firefly luciferase gene. Reporter gene assays and qPCR were performed in transfected MCF-7, HCT 116 and HEK 293T cells. Results Dual luciferase assays 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 a difference 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. 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 with equal mRNA levels should be due to a post-transcriptional level of regulation, i.e., cap-independent translation of firefly luciferase gene. 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 signaling pathway.

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

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