Anaplastic thyroid carcinoma (ATC) represents one the most aggressive neoplasias in humans, and, nowadays, limited advances have been made to extend the survival and reduce the mortality of ATC. Thus, the identification of molecular mechanism underlying its progression is needed. Here, we evaluated the long non-coding RNA (lncRNA) expression profile of nine ATC in comparison with five normal thyroid tissues by a lncRNA microarray. By this analysis, we identified 19 upregulated and 28 downregulated lncRNAs with a fold change >1.1 or <−1.1 and p-value < 0.05, in ATC samples. Some of them were subsequently validated by qRT-PCR. Then, we investigated the role of the lncRNA Prader Willi/Angelman region RNA5 (PAR5), drastically and specifically downregulated in ATC. The restoration of PAR5 reduces proliferation and migration rates of ATC-derived cell lines indicating that its downregulation contributes to thyroid cancer progression. Our results suggest that PAR5 exerts its anti-oncogenic role by impairing Enhancer of Zeste Homolog 2 (EZH2) oncogenic activity since we demonstrated that PAR5 interacts with it in thyroid cancer cell lines, reducing EZH2 protein levels and its binding on the E-cadherin promoter, relieving E-cadherin from the negative regulation by EZH2. Consistently, EZH2 is overexpressed in ATC, but not in differentiated thyroid carcinomas. The results reported here define a tumor suppressor role for PAR5 in undifferentiated thyroid neoplasias, further highlighting the pivotal role of lncRNAs in thyroid carcinogenesis.

Klhl14-AS is a long noncoding RNA expressed since early specification of thyroid bud and is the most enriched gene in the mouse thyroid primordium at E10.5. Here, we studied its involvement in thyroid carcinogenesis by analyzing its expression in cancer tissues and different models of neoplastic transformation. Compared with normal thyroid tissue and cells, Klhl14-AS was significantly downregulated in human thyroid carcinoma tissue specimens, particularly the anaplastic histotype, thyroid cancer cell lines, and rodent models of thyroid cancer. Downregulating the expression of Klhl14-AS in normal thyroid cells decreased the expression of thyroid differentiation markers and cell death and increased cell viability. These effects were mediated by the binding of Klhl14-AS to two miRNAs, Mir182-5p and Mir20a-5p, which silenced Pax8 and Bcl2, both essential players of thyroid differentiation. MIR182-5p and MIR20a-5p were upregulated in human thyroid cancer and thyroid cancer experimental models and their effects on Pax8 and Bcl2 were rescued by Klhl14-AS overexpression, confirming Klhl14-AS as a ceRNA for both Pax8 and Bcl2. This work connects deregulation of differentiation with increased proliferation and survival in thyroid neoplastic cells and highlights a novel ceRNA circuitry involving key regulators of thyroid physiology. Significance: This study describes a new ceRNA with potential tumor suppression activity and helps us better understand the regulatory mechanisms during thyroid differentiation and carcinogenesis.

Background: We have recently reported the downregulation of the Metallophosphoesterase-domain-containing protein 2 (MPPED2) gene and its cognate long non-coding RNA, MPPED2-AS1, in papillary thyroid carcinomas. Functional studies supported a tumor suppressor role of both these genes in thyroid carcinogenesis. We then decided to investigate their role in breast carcinogenesis. Methods: In order to verify MPPED2 expression, 45 human breast carcinoma samples have been investigated by quantitative real-time polymerase chain reaction (qRT-PCR). Then, MPPED2 has been transfected in several human breast carcinoma cell lines, analyzing its role in cell proliferation, migration and invasion. To study the regulation of MPPED2 expression the methylation of its promoter was investigated by targeted bisulfite sequencing. Results: MPPED2 expression was decreased in breast cancer samples, and this was confirmed by the analysis of data available in The Cancer Genome Atlas (TCGA). Interestingly, the hypermethylation of MPPED2 promoter likely accounted for its downregulation in breast cancer. Additionally, MPPED2-AS1 was also found downregulated in breast cancer tissues and, intriguingly, its expression decreased the hypermethylation of the MPPED2 promoter by inhibiting DNA methyltransferase 1 (DNMT1). Furthermore, the restoration of MPPED2 expression reduced cell proliferation, migration and invasion capability of breast carcinoma cell lines. Conclusion: Taken together, these results propose MPPED2 downregulation as a critical event in breast carcinogenesis.

Loss of CBX7 expression has been described in several malignant neoplasias, including human colon and thyroid carcinomas proposing CBX7 as a tumor suppressor gene with a key role in cancer progression. This role is supported from the development of benign and malignant neoplasias in Cbx7 null mice. The aim of our work has been to investigate the mechanisms underlying the CBX7 oncosuppressor activity by analyzing the microRNAs (miRNAs) regulated by CBX7. The miRNA expression profiles of the mouse embryonic fibroblasts (MEFs) null for Cbx7 and the wild-type counterpart were analyzed by the miRNACHIP microarray and then validated by qRT-PCR. To asses KRAS as target of miR-155 we evaluated the protein levels after transfection of the synthetic miR-155. Human colon carcinoma samples have been investigated for the expression of CBX7 and miR-155. Twenty miRNAs were found upregulated and nine, including miR-155, downregulated in cbx7-null MEFS in comparison with the wild-type ones. Then, we focused on miR-155 since several studies have shown its deregulated expression in several human malignancies and, moreover, was the most downregulated miRNA. Subsequently, we searched for miR-155 target genes demonstrating that KRAS protein levels are directly modulated by miR-155. A direct significant correlation (r = 0.6779) between CBX7 and miR-155 expression levels was found in a set of human colon carcinoma tissue samples. miR-155 is positively regulated by CBX7 in MEFs and colon carcinomas, and has KRAS as one of the target genes likely accounting for the anti-apoptotic activity ascribed to miR-155 in some tissue contexts.

Background: Well-differentiated papillary thyroid carcinoma (PTC) represents the thyroid neoplasia with the highest incidence. Long non-coding RNAs (lncRNAs) have been found deregulated in several human carcinomas, and hence, proposed as potential diagnostic and prognostic markers. Therefore, the aim of our study was to investigate their role in thyroid carcinogenesis. Methods: We analysed the lncRNA expression profile of 12 PTC and four normal thyroid tissues through a lncRNA microarray. Results: We identified 669 up- and 2470 down-regulated lncRNAs with a fold change >2. Among them, we focused on the down-regulated RP5-1024C24.1 located in an antisense position with respect to the MPPED2 gene which codes for a metallophosphoesterase with tumour suppressor activity. Both these genes are down-regulated in benign and malignant thyroid neoplasias. The restoration of RP5-1024C24.1 expression in thyroid carcinoma cell lines reduced cell proliferation and migration by modulating the PTEN/Akt pathway. Inhibition of thyroid carcinoma cell growth and cell migration ability was also achieved by the MPPED2 restoration. Interestingly, RP5-1024C24.1 over-expression is able to increase MPPED2 expression. Conclusions: Taken together, these results demonstrate that RP5-1024C24.1 and MPPED2 might be considered as novel tumour suppressor genes whose loss of expression contributes to thyroid carcinogenesis.