ECE2014 Poster Presentations Thyroid Cancer (70 abstracts)
Functional analysis of newly discovered microRNA deregulated in papillary thyroid carcinoma
Monika Maciag 1 , Anna Kubiak 1 , Michal Swierniak 1, , Kinga Dymecka 1 , Monika Kolanowska 1 , Lukasz Koperski 4 , Barbara Gornicka 4 , Elzbieta Stachlewska 5 & Krystian Jazdzewski 1,
1Genomic Medicine, Department of General, Transplant and Liver Surgery, Medical University of Warsaw, Warsaw, Poland; 2Centre of New Technologies, CENT, University of Warsaw, Warsaw, Poland; 3Department of Nuclear Medicine and Endocrine Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland; 4Department of Pathology, Medical University of Warsaw, Warsaw, Poland; 5Department of Endocrine Oncology and Nuclear Medicine, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland.
Introduction: Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy where alterations in the RET/PTC–RAS–BRAF signaling pathway and deregulation of microRNA (miRNA) expression are suggested to be major mechanism of pathogenesis. MicroRNAs are small non-coding RNA molecules that recognize specific sequences in 3′UTR of target gene and prevent from its translation. Our recent deep sequencing studies of thyroid tissue transcriptome revealed a putative novel small non-coding RNA, encoded within PAX8 gene. In silico analysis confirmed that the short non-coding sequence could be miRNA. We aimed to investigate the role of newly identified microRNA (miR-PAX8) in thyroid carcinogenesis.
Methods and results: To confirm functionality of newly discovered RNA, pcDNA3 vector expressing pri-miRNA was transfected into U2-OS cells. TaqMan assay confirmed the proper processing towards mature miRNA in transfected cells. Analysis of PAX8 mRNA and miR-PAX8 expression in 42 pairs of PTC and matched control tissue showed 28% (P=0.02) decrease of miR-PAX8 and 40% (P=0.001) decrease of PAX8 mRNA in tumor compared with noncancerous tissue (correlation r=0.57, P=0.00008). Using in silico analysis we chose CTBP1 gene as a target gene of miR-PAX8 for further studies. qPCR analysis revealed 1.6-fold (P=0.001) increase of CTBP1 mRNA in PTC compared to control tissue that could potentially results from miR-PAX8 decrease (correlation r=−0.45, P=0.002). Direct interaction of miR-PAX8 with CTBP1 transcript was confirmed by luciferase assay using vector with CTBP1 3′UTR cloned downstream to luciferase. Cells transfected with pGL3-3′UTR construct and vector expressing pre-miRNA revealed 17% (P=0.002) decrease in luciferase expression.
Conclusions: We demonstrated that the newly discovered non-coding RNA is functional microRNA that is downregulated in PTC tissue. As CTBP1 mediates repression of several tumor suppressors, interaction of miR-PAX8 with CTBP1 transcript suggests that novel miRNA could be involved in PTC development. To strengthen this hypothesis further studies including Western blot and flow cytometry analysis will be performed.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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