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Interaction of microRNA Molecules and Hedgehog-GLI Signaling Pathway Genes in High-Grade Serous Ovarian Cancer

Ovarian cancer is the leading cause of death from gynecological malignancies in the Western world. Its high death rate is a result of the fact that >60% patients are diagnosed in advanced stage of the disease. Hedgehog-GLI (HH-GLI) signaling pathway is involved in embryonal ovarian development, but its atypical activation can lead to different types of ovarian tumors. Our previous studies showed aberrant HHGLI activity in some ovarian tumor types and hypermethylation in the promoter of tumor suppressor gene PTCH1 (Ozretić et al. Int J Oncol. 2017 ; Musani et al. Gene. 2013 ; Sabol et al. Int J Oncol. 2012 ; Maurac et al. Int J Gynecol Pathol. 2012 ; Cretnik et al. Int J Mol Med. 2007). We hypothesize that changes in the expression of miRNA molecules related to the HH-GLI signaling pathway genes contribute to the development of high-grade serous ovarian cancer (HGSOC ; the most malignant type and most difficult to detect at an earlier stage. We conducted miRNA and gene expression profiling of 16 HGSOC and 8 healthy Fallopian tube fresh frozen tissue samples as a control (Reade et al. J Obstet Gynaecol Can. 2014) with Agilent SurePrint G3 Human miRNA 8x60K Microarray Kit and Agilent SurePrint G3 Human Gene Expression v3 8x60K Microarray Kit, respectively. Microarray data was analyzed using R/Bioconductor packages AgiMicroRna and limma. Online tools DianaMicroT, miRDB, RNA22- HSA and TargetScan were used to predict target genes of discovered differentially expressed miRNAs. Expression of selected miRNAs and genes was verified using TaqMan and SYBR Green qPCR, respectively, on an extended set of 10 Fallopian tube and 47 HGSOC samples. Data filtration gave a list of 55 miRNAs differentially expressed in HGSOC: 32 up- and 23 downregulated. In addition, 1, 090 genes were significantly over- and 1, 692 were under-expressed in HGSOC. By comparing a list of predicted miRNA target genes and differentially expressed HH-GLI pathway genes we revealed a couple of combinations of miRNAs and their target genes: GRK3 / hsa-miR-513a-5p ; BCL2 / hsa-miR-96- 5p and hsa-miR-21-5p ; IHH / hsa-miR-103a-3p, hsa- miR-107 and hsa-miR-16-5p ; and GLI3 / hsa-miR- 200b-3p. Co-expression of all those target genes / miRNAs has been confirmed in an extended set of samples. Additional in vitro verification will be continued with in vivo approach. Our results highlighted several candidate miRNAs potentially targeting HH-GLI pathway genes. When additionally verified, they could be used as potential diagnostic and prognostic markers or even therapeutic targets for HGSOC. This study was funded by the Terry Fox Foundation, City of Zagreb and Croatian Science Foundation (IP-2016-06-1268).

Hedgehog-GLI signaling ; microRNA ; mRNA ; expression ; ovarian cancer

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