Abstract 4365: miR-106b modulates C1orf24 expression in thyroid tumors
Authors: Nozima Bruno H. , Carvalheira Gianna M. , Cerutti Janete M. 
Published: May 2015

Abstract
BACKGROUND: Thyroid nodules are commonly found in clinical practice. Fine-needle aspiration biopsy (FNA) cytology is currently the most used method for the initial evaluation of a thyroid nodule. Although FNA remains the gold standard method, its accuracy is related to the histological subtype, being much lower for both benign follicular adenoma (FTA) and malignant follicular carcinoma (FTC). Hence, to improve preoperative diagnosis, we previously performed gene expression profiling of FTC and FTA. We have identified 4 genes (ARG2, DDIT3, ITM1 and C1orf24) over-expressed in carcinomas that, in combination, can distinguish FTA from FTC with high sensitivity and specificity. Despite their relevance as molecular marker, little is known about the mechanism that controls their expression and their role on thyroid cancer development. Since microRNA (miR) is an emerging class of small RNAs that regulates gene expression, we hypothesized if over-expression of C1orf24 in thyroid cancer was due to miR deregulation. We here sought to identify miRs that potentially modulate C1orf24 expression, analyze their levels in tumor samples and verify their effect on C1orf24 regulation.
METHODS: C1orf24 expression was evaluated in 64 benign and malignant thyroid samples by quantitative PCR (qPCR). In parallel, candidate miRs were identified in Silico and their endogenous levels were evaluated by qPCR in all thyroid samples. Additionally miRs and C1orf24 expression were measured in 5 thyroid cell lines by qPCR and Western Blot. miR precursor and negative control were transfected by siPORTNeoFX. C1orf24 3′-UTR was cloned in pmiR-Glo Dual Luciferase Vector and luciferase activity was measured by Dual-Luciferase Reporter Assay System. One-way ANOVA or t-test was used for statistical analysis (p&lt; 0.05).
RESULTS: Analysis of miRBase Sequence database suggested two miRs - miR-17-5p and miR-106b - that potentially regulate C1orf24 expression. miR-106b expression was significant lower in thyroid carcinomas (TC) than FTA (p-value 0.0060). On the other hand, C1orf24 expression was higher in TC (p-value 0.0018), suggesting that miR-106b potentially regulates C1orf24 expression. Next, we investigated if miR-106b could directly modulate C1orf24 expression. As thyroid carcinoma cell line (WRO) presented high levels of C1orf24 expression and low levels of miR-106b, we transiently transfected miR-106b precursor into WRO cells and measured C1orf24 expression. Both mRNA (p-value 0.0433) and protein levels (p-value 0.0877) were diminished when compared to control. Luciferase activity was lower in WRO cells transfected with both C1orf24 3′-UTR and miR-106b precursor than negative control (p-value 0.0026), suggesting a direct interaction between them.
CONCLUSION: Our findings suggest that miR-106b, but not miR-17-5p, directly modulates C1orf24 gene expression acting as a tumor suppressor miR, suggesting a novel mechanism of C1orf24 expression in thyroid cancer.
Citation Format: Bruno H. Nozima, Gianna M. Carvalheira, Janete M. Cerutti. miR-106b modulates C1orf24 expression in thyroid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4365. doi:10.1158/1538-7445.AM2014-4365