engleski

Functional Analyses of PTCH1 Gene 5’-UTR: The Impact of CGG Triplet Repeat Sequence Variants

PTCH1 tumor suppressor gene encodes for a transmembrane receptor with a negative regulatory role in the Hedgehog-Gli (Hh) signaling pathway. PTCH1 germline mutations cause nevoid basal cell carcinoma syndrome, an autosomal dominant disorder characterized by developmental abnormalities and susceptibility to various tumor types. Most of the malformations are caused by PTCH1 haploinsufficiency, indicative of the fine-tuning needed to properly regulate Hh pathway. Genetic analysis of PTCH1 5’-UTR (transcript variant 1b) in patients with different types of tumor and healthy controls has identified a different number of CGG repeats, located four nucleotides upstream of the translation start site. While 7 repeat alleles were prevalent, 5, 6 and 8 repeats have also been found. The aim of our study is to examine the potential impact of these CGG repeats on PTCH1 expression at transcriptional or post-transcriptional level. Since PTCH1b transcript has two different annotated 5’-UTRs, 188- and 300-nucleotide-long, we constructed pGL3-promoter based plasmids by inserting both 5’-UTR sizes, each harboring different number of CGG repeats, upstream of firefly gene. Luciferase assays, in MCF-7 and HCT116 cells, revealed that the 188nt 5’-UTR significantly increased reporter activity compared to empty vector, with a subtle, if any, reduction with increased CGG repeat number. Interestingly, plasmids with 300nt 5’-UTR showed a much reduced reporter activity, and no difference among repeat number. Relative quantification of luciferase mRNA showed that both types of 5’-UTR increased the reporter gene transcription to a similar level. The presence of a uORF in the 300nt 5’-UTR might account for the severe reduction in reporter activity. Bicistronic dual-luciferase vectors are being used to continue the functional analysis of the CGG repeats, while qPCR and 5' RACE at the endogenous gene level are used to map the actual PTCH1b 5’-UTR and the relative abundance of different transcripts.

Hedgehog-Gli signaling; PTCH1; 5' UTR; CGG repeats

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