Identification of new long non-coding RNAs associated with medullary thyroid cancer

Medullary thyroid carcinoma (MTC) represents just 5–10% of all thyroid malignancies. In contrast to the familial MEN2, little is known about the etiology of sporadic MTC. New approaches are required to elucidate the mechanisms underlying the pathogenesis of sMTC. Long noncoding RNAs (lncRNAs), are wellrecognized post-transcriptional regulators of genetic expression and recent studies have described multiple aberrantly expressed non-coding RNAs in thyroid cancers. In the current study we have aimed to perform the first screening of multiple lncRNAs in tumoral tissues from MTC patients by qRT-PCR. Our analysis showed the association of 15 lncRNAs from which 6 where new in association with this disease (RMST, SNHG16, FTX, GAS5, IPW, MEG3). The association of these new lncRNAs with overall survival was analyzed by Kaplan-Meier curve. *Correspondence to: Salud Borrego, MD, PhD, Department of Materno Fetal Medicine, Genetics and Reproduction. University Hospital Virgen del Rocío, Maternal Hospital. 2nd floor. Av. Manuel Siurot n/n. 41013, Seville, Spain, E-mail: salud.borrego.sspa@juntadeandalucia.es Received: February 22, 2019; Accepted: February 27, 2019; Published: March 05, 2019 Introduction Medullary thyroid carcinoma (MTC) it is a tumor originated from C-cells and derived from the neural crest which accounts for only 1%– 2% of thyroid cancers, although it is responsible for about 13% of all thyroid cancer–related deaths [1,2]. MTC can occur either sporadically (75%) or as the dominant component of the type 2 multiple endocrine neoplasia syndromes (MEN2, 25%). It is considered a rare disease, with an estimated prevalence in the general population of 1/14,300 [http:// www.orpha.net; ORPHA No: 1332]. The broad term long non-coding RNA (lncRNA) refers to a class of non-coding RNA transcript of minimum 200 nucleotides in length. They have gained widespread attention in recent years as new players in transcriptional, epigenetic, or post-transcriptional regulation of gene expression [3]. To date, only one study has examined the expression of lncRNAs in patients with MTC [4]. Consequently, lncRNAs are attractive and promising targets in cancer prognosis and treatment. The purpose of this study is to bring insight and deeper understanding into the etiology of sMTC, to a deeper understanding of disease mechanisms, pathogenesis, and searching of new therapeutic targets. To afford this aim, we have analyzed the expression of lncRNAs in this type of tumors. Materials and methods Experimental subjects In this study, we have performed lncRNA expression analysis on four sMTC cases (Table 1). All MTC tissues and their corresponding adjacent non-tumor thyroid tissues were obtained from these patients after undergoing surgical resection. The samples were snap frozen in liquid nitrogen and stored at −80°C until use. A written informed consent was obtained from all the participants for clinical and molecular genetic studies. The study was approved by the Ethics Committee for clinical research in the University Hospital Virgen del Rocío (Seville, Spain) and complies with The Code of Ethics of the World Medical Association (Declaration of Helsinki), printed in the British Medical Journal (18 July 1964). Screening by lncRNA PCR Array Total RNA was obtained from tissues of our patients and commercial cells by using RNEasy Purification Kit (Qiagen), according to the manufacturer’s instructions. The RNA was quantified by Nanodrop (Invitrogen, USA) and 1 μg of total RNA was reverse transcribed into Characteristics N Age at diagnosis, years Median (range) 46.5 Gender Male 2 Female Inheritance Sporadic (absence of any mutation MEN2 related) 4 Tumor size, centimeters Median (range) Nodal metastasis at diagnosis 2.75 Distant metastasis Present at initial diagnosis 3 Table 1. Clinicopathological features of included MTC patients Luzón-Toro B (2019) Identification of new long non-coding RNAs associated with medullary thyroid cancer Oral Health Care, 2019 doi: 10.15761/OHC.1000156 Volume 4: 2-3 cDNA using PrimeScript RT Reagent Kit (Perfect Real Time; TaKaRa, Osaka, Japan) to determine lncRNA expression levels, using GAPDH as internal control. For lncRNA expression analysis, laboratory-verified SYBR®Green qPCR assays (RT2 lncRNA PCR Array, Qiagen) were used. Each plate contains 84 lncRNAs already associated with different cancer pathways (Supplementary Table 1). The quantitative realtime PCR (qRT-PCR) was performed at the 7900HT Fast Real-Time PCR System with the 384-Well Block Module (Applied Biosystems). We used the ∆∆Ct method for relative quantitation of lncRNAs level expression, where a fold-change of at least two times and a corrected P-value of < 0.05 were used as a criterion of selection. Statistical analysis Overall survival rates were calculated by the Kaplan-Meier method with the long-rank test applied for comparison. P-value < 0.05 was considered as statistically significant.


Introduction
Medullary thyroid carcinoma (MTC) it is a tumor originated from C-cells and derived from the neural crest which accounts for only 1%-2% of thyroid cancers, although it is responsible for about 13% of all thyroid cancer-related deaths [1,2]. MTC can occur either sporadically (75%) or as the dominant component of the type 2 multiple endocrine neoplasia syndromes (MEN2, 25%). It is considered a rare disease, with an estimated prevalence in the general population of 1/14,300 [http:// www.orpha.net; ORPHA Nº: 1332].
The broad term long non-coding RNA (lncRNA) refers to a class of non-coding RNA transcript of minimum 200 nucleotides in length. They have gained widespread attention in recent years as new players in transcriptional, epigenetic, or post-transcriptional regulation of gene expression [3]. To date, only one study has examined the expression of lncRNAs in patients with MTC [4]. Consequently, lncRNAs are attractive and promising targets in cancer prognosis and treatment.
The purpose of this study is to bring insight and deeper understanding into the etiology of sMTC, to a deeper understanding of disease mechanisms, pathogenesis, and searching of new therapeutic targets. To afford this aim, we have analyzed the expression of lncRNAs in this type of tumors.

Experimental subjects
In this study, we have performed lncRNA expression analysis on four sMTC cases (Table 1). All MTC tissues and their corresponding adjacent non-tumor thyroid tissues were obtained from these patients after undergoing surgical resection. The samples were snap frozen in liquid nitrogen and stored at −80°C until use. A written informed consent was obtained from all the participants for clinical and molecular genetic studies. The study was approved by the Ethics Committee for

Screening by lncRNA PCR Array
Total RNA was obtained from tissues of our patients and commercial cells by using RNEasy Purification Kit (Qiagen), according to the manufacturer's instructions. The RNA was quantified by Nanodrop (Invitrogen, USA) and 1 μg of total RNA was reverse transcribed into We used the ∆∆Ct method for relative quantitation of lncRNAs level expression, where a fold-change of at least two times and a corrected P-value of < 0.05 were used as a criterion of selection.

Statistical analysis
Overall survival rates were calculated by the Kaplan-Meier method with the long-rank test applied for comparison. P-value < 0.05 was considered as statistically significant.

Results
The expression profiles of 84 lncRNAs, already associated with different cancer pathways, in 4 tumoral and non-tumoral paired tissues were determined by SYBR ® Green qPCR assays. Fifteen differentially expressed lncRNAs were detected in our samples (all adjusted P ≤  (Table 2).
In addition, analysis of overall survival was performed by using Kaplan-Meier curve although it is not significant (available under request).

Discussion
Many efforts are being made to establish the biological and clinical relationships between lncRNAs and cancer. They are involved in a variety of biological processes through the regulation of gene expression [5,6]. In this manner, lncRNAs regulate transcription and epigenetic events, leading cells adapting to a changing environment.
It is important to highlight that one of the upregulated lncRNAs that we have obtained in this study was MALAT1, which has been already associated with MTC [4]. This fact reinforces the validity of our approach. In this study, we have evaluated 84 different lncRNAs, already associated with cancer pathways, in 4 MTC patients through qRT-PCR, showing the significant association of 3 downregulated and 4 upregulated new lncRNAs that had not been published yet in association with neither MTC nor any thyroid carcinoma.
This study is not devoid of limitations. We have compared by qRT-PCR the expression levels of different lncRNAs in a group of MTC patients and normalizing to the levels detected in normal adjacent thyroid tissues (with mostly follicular cells). Although normal C-Cells would be our perfect control tissue, there is very little number of them in the normal thyroid. Thus, we decided to use thyroid follicular cells because they are very close to the MTCs and they express the thyroid transcription factor 1, as well as C-Cells do. Then, we consider that this comparison approach was a good alternative, as some previous studies also confirmed [4,7,8].