Endocrine Abstracts

Introduction: Papillary Thyroid Carcinoma (PTC) exhibits activation of MAPK (Mitogen-activated protein kinases) pathway, leading to uncontrolled cellular division and growth. Concomitantly, thyroid tumors exhibit aberrant expression of numerous microRNAs, which are non-coding RNAs that inhibit expression of protein-coding genes. Employing next-generation sequencing (NGS), we revealed comprehensive miRNA profiles of normal thyroid and PTC, and identified putative novel microRNA genes.

Aim: The aim of this study was to analyze the role of the novel microRNA in physiology and neoplastic transformation of the thyroid gland.

Methods and results: NGS analysis performed in 14 PTC and control samples resulted in identification of a novel microRNA encoded within the thyroglobulin gene (TG), important regulator of thyroid homeostasis. Cloning into the expression vector, cell line transfection and Taqman quantification revealed that the microRNA gene was processed towards mature microRNA. Q20-PCR analysis in 33 PTC and matched non-tumorous tissue samples showed that the levels of miR-TG and thyroglobulin were decreased in tumor by 30% (P=0.001) and 16% (P=0.04), respectively. Levels of both transcripts were positively correlated (r=0.48, P=0.005). In silico analysis revealed that genes putatively regulated by novel microRNA included MAPK and Pi3K/Akt pathway proteins: DUSP6 and MAP4K4. Q20-PCR showed a 12-fold upregulation of DUSP6 (P=0.00001) and a 2.5-fold upregulation of MAP4K4 (P=0.002) in PTC compared to control tissue. Moreover, expression of both genes negatively correlated with expression of novel microRNA: r=−0,44 for DUSP6 (P=0.01) and r=−0.48 for MAP4K4 (P=0.005). Direct binding of studied microRNA to 3’UTRs of MAP4K4 and DUSP6 was confirmed in luciferase assay, leading to reduced luciferase activity by 10% (P=0.0006) and 17% (P=0.0008), respectively.

Conclusion: We propose that the novel microRNA encoded within TG fine-tunes the MAPK pathway, inhibiting expression of DUSP6 and MAP4K4. Severe downregulation of the microRNA leads to activation of MAPK kinases, underlying initiation and progression of thyroid carcinogenesis.