INTERPLAY BETWEEN PRC2/EZH2 COMPLEX AND LONG NON-CODING RNAS IN THYROID CANCER

INTRODUCTION AND OBJECTIVES: Thyroid cancer is the most common endocrine malignancy, comprising different types of tumors, such as anaplastic thyroid carcinoma (ATC) the rarest type but most lethal. Different genetic and epigenetic alterations are associated with the etiology of ATC, especially those in the MAPK pathway. Long non-coding RNAs are a broad class of non-coding RNAs (>200nt in length) that regulate genes' expression and interact with epigenetic modifiers, such as the EZH2/PRC2 complex. EZH2 catalyzes the trimethylation of histone H3 lysine’s 27 residue, resulting in chromatin compaction and silencing of specific genes. In thyroid cancer, an increase in EZH2 is observed in ATC but the impact of the EZH2 and lncRNAs interaction on thyroid cancer's progression remains unclear. Therefore, we investigated the role of epigenetic regulation in ATC biology and analyze the relationship between lncRNAs and the PRC2/EZH2 complex. MATERIAL AND METHODS: To compare the expression of different lncRNAs in the two types of thyroid tumors, we used five different cell lines of thyroid carcinoma (TPC1, BCPAP, KTC2, SW1736, and 8305C). We profiled the expression of 90 lncRNAs using the Human and Mouse LncProfiles qPCR Array Kit (System Biosciences). For that, we used 1ug of total RNA from each cell line to generate the cDNA, according to the manufacturer's instructions. The gene expression was analyzed by qPCR using SYBR Green and the expression was normalized using 5 endogenous controls. We used TPC1 gene expression as a control to normalize the expression among cell lines. To identify lncRNAs interacting with EZH2 in ATC, we performed an RNA-IP using the SW1736 cell line. The cell lysate was incubated with anti-EZH2 antibodies. Then, we incubated the sample with the magnetic beads and after several washes on a magnetic rack, the total RNA was extracted from the beads with the trizol reagent. The cDNA was transcribed with random primers and the validation of the RNA-IP (lncRNAs interacting with EZH2) was performed by qPCR with specific primers for the lncRNAs (H19, HOTAIR, MALAT1, MEG3, and XIST). RESULTS AND CONCLUSION: Our gene expression analysis identified 36 lncRNAs with differential expression among cell lines. Of all the 90 lncRNAs tested, in this study, we identified differential expression in four of these lncRNAs known to interact with the EZH2/PRC2 complex in other tissues (H19, HOTAIR, TUG1, and XIST). These findings suggest heterogeneity in the expression of lncRNAs within the same tumor histotype. Our EZH2-associated RNA immunoprecipitation data show that HOTAIR, MALAT1, and XIST lncRNAs interact with EZH2 in anaplastic thyroid carcinoma. Thus, additional experiments focusing on these lncRNAs may improve the understanding of the role and functions of such lncRNAs in association with EZH2 and how this regulation affects tumor aggressiveness.