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Abstract

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 well-recognized 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.

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).

Table 1. Clinicopathological features of included MTC patients

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

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 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 real-time 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.

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 ≤ 0.05). From all the differentially expressed lncRNAs, 8 downregulated and 7 upregulated lncRNAs had not been published yet in association with any thyroid carcinoma (Table 2).

Table 2. Aberrant LncRNAs in MTC tissues: All significant lncRNAs obtained by qRT-PCR (7900HT Taqman system) through the RT2 lncRNA PCR Arrays. RQ represents the fold-change. From the normalized value of 1.00 of non-tumoral tissues, we represent those lncRNAs downregulated (< 2.00) and those ones upregulated (< 2.00) in our study. The significant p-value was < 0.05. All available information about their implication in other type of cancers is also compiled on the last column of the table, with special mention when they have been linked with thyroid cancer.

Sample	Detector	Avg Ct	Avg Delta Ct	Delta Delta Ct SD	RQ	Described in cancer (is it described into thyroid cancer)?
Non-tumoral	ZFAS1	29.275	3.833	0.000	1.000	Not associated with thyroid cancer but it is related with colorectal, gastric, ovarian, prostate, hepatic, bladder, esophagus and breast cancers.
Tumoral	ZFAS1	23.903	2.997	-0.836	1.785
Non-tumoral	RMST	30.659	5.217	0.000	1.000	Not associated with thyroid cancer but it is related with breast cancer.
Tumoral	RMST	24.888	3.982	-1.235	2.353
Non-tumoral	SNHG16	31.328	5.885	0.000	1.000	Not associated with thyroid cancer but it is related with esophageal squamous cell carcinoma, gastric, lung, glioma, bladder, breast, colorectal and cervical cancers.
Tumoral	SNHG16	25.200	4.294	-1.591	3.014
Non-tumoral	FTX	30.498	5.056	0.000	1.000	Not associated with thyroid cancer but it is related with hepatocellular, colorectal, renal, breast cancers as well as in leukemia and melanoma.
Tumoral	FTX	23.914	3.008	-2.048	4.135
Non-tumoral	GAS5	30.056	4.613	0.000	1.000	Associated with thyroid cancer, among other tumors (Low expression of long non-coding RNA GAS5 is associated with poor prognosis of patients with thyroid cancer.
Tumoral	GAS5	23.297	2.390	-2.223	4.668
Non-tumoral	IPW	30.182	4.739	0.000	1.000	Not associated with thyroid cancer.
Tumoral	IPW	23.908	3.002	-1.738	3.335
Non-tumoral	MALAT1	31.932	6.489	0.000	1.000	Associated with different cancers and other pathologies, and with thyroid cancer (Upregulation of long noncoding RNA MALAT1 in papillary thyroid cancer and its diagnostic value. Liu J et al. Future Oncol. 2018 Jul 10; MicroRNA-21 and long non-coding RNA MALAT1 are overexpressed markers in medullary thyroid  carcinoma.
Tumoral	MALAT1	26.359	5.453	-1.036	2.051
Non-tumoral	MEG3	31.919	6.477	0.000	1.000	Associated with different cancers and other pathologies, and with thyroid cancer (Long noncoding RNAs: emerging players in thyroid cancer pathogenesis.
Tumoral	MEG3	22.835	1.928	-4.548	23.397
Non-tumoral	PTCSC1	28.910	3.467	0.000	1.000	Associated with thyroid cancer Long noncoding RNAs: emerging players in thyroid cancer pathogenesis.
Tumoral	PTCSC1	26.362	5.455	1.988	0.252
Non-tumoral	PTCSC3	29.431	3.989	0.000	1.000	The polymorphism rs944289 predisposes to papillary thyroid carcinoma through a large intergenic noncoding RNA gene of tumor suppressor type.
Tumoral	PTCSC3	26.283	5.377	1.388	0.382
Non-tumoral	TUG1	29.886	4.443	0.000	1.000	LncRNA TUG1 influences papillary thyroid cancer cell proliferation, migration and EMT formation through targeting miR-145.
Tumoral	TUG1	24.500	3.593	-0.849	1.802
Non-tumoral	ADAMTS9-AS2	31.702	6.259	0.000	1.000	Not associated with thyroid cancer but it is related with lung and glioma cancers.
Tumoral	ADAMTS9-AS2	27.908	7.002	0.743	0.598
Non-tumoral	PRNCR1	31.918	6.475	0.000	1.000	Not associated with thyroid cancer but it is related with gastric, colorectal and prostate cancers.
Tumoral	PRNCR1	28.308	7.402	0.927	0.526
Non-tumoral	RMRP	22.623	-2.820	0.000	1.000	Not associated with thyroid cancer but it is related with breast, lung, gastric and colon cancers.
Tumoral	RMRP	18.382	-2.524	0.295	0.815
Non-tumoral	H19	31.640	6.197	0.000	1.000	Associated with thyroid cancer, among other tumors (Epigenetic Modifications in Thyroid Cancer Cells Restore NIS and Radio-Iodine Uptake and Promote Cell Death.
Tumoral	H19	27.891	6.984	0.787	0.580

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].

Conclusions

We describe here six new lncRNAs (RMST, SNHG16, FTX, GAS5, IPW, MEG3) which could play an interesting role in this rare tumor, that to date has any effective therapy or prognosis. Further studies with larger sample sizes would be needed to confirm the role of these new lncRNAs in MTC that maybe can serve as predictive cancer biomarkers or targets for preventive drugs.

Data availability

The expression data from the qPCR assays used to support the findings of this study are available from the corresponding author upon request.

Conflicts of interest

The authors declare that there is no conflict of interest regarding the publication of this article.

Funding statement

This study was supported by Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness, Spain and co-funded by European Union (ERDF/ESF, “Investing in your future”) [PI16/0142]. In adittion, it has been funded by the Regional Ministry of Innovation, Science and Enterprise of the Regional Government of Andalusia [CTS-7447]. CIBERER is an initiative of the ISCIII, Spanish Ministry of Economy and Competitiveness.

Acknowledgments

The authors thank the patients that have participated in this study, as well as the donors and the Hospital Universitario Virgen del Rocío-Instituto de Biomedicina de Sevilla Biobank (Andalusian Public Health System Biobank and ISCIII-Red de Biobancos PT13/0010/0056) for the human specimens used in this study.

Supplementary material

Supplementary table 1: The 84 lncRNAs from the RT² lncRNA PCR Arrays.

Supplementary Table 1. The 84 lncRNAs from the RT2 lncRNA PCR Arrays.

Position	UniGene	GenBank	Symbol	Description
A01	N/A	ENST00000437930	ACTA2-AS1	ACTA2 antisense RNA1
A02	N/A	ENST00000460833	ADAMTS9-AS2	ADAMTS9 antisense RNA 2
A03	N/A	ENST00000608442	AFAP1-AS1	AFAP1 antisense RNA 1
A04	N/A	ENST00000601203	AIRN	Antisense of IGF2R non-protein coding RNA
A05	N/A	NR_047671	BANCR	BRAF-activated non-protein coding RNA
A06	Hs.24611	NR_024049	BCAR4	Breast cancer anti-estrogen resistance 4
A07	N/A	NR_103783	BLACAT1	Bladder cancer associated transcript 1 (non-protein coding)
A08	N/A	ENST00000604200	CAHM	Colon adenocarcinoma hypermethylated (non-protein coding
A09	N/A	ENST00000413862	CBR3-AS1	CBR3 antisense RNA 1
A10	N/A	ENST00000500112	CCAT1	Colon cancer associated transcript 1 (non-protein coding)
A11	N/A	NR_109834	CCAT2	Colon cancer associated transcript 2 (non-protein coding)
A12	N/A	ENST00000421632	CDKN2B-AS1	CDKN2B antisense RNA 1
B01	N/A	ENST00000501177	CRNDE	Colorectal neoplasia differentially expressed (non-protein coding)
B02	N/A	NR_002733	DGCR5	DiGeorge syndrome critical region gene 5 (non-protein coding)
B03	Hs.547964	NR_002612	DLEU2	Deleted in lymphocytic leukemia 2 (non-protein coding)
B04	Hs.34969	NR_015448	DLX6-AS1	DLX6 antisense RNA 1
B05	Hs.312592	NR_002791	EMX2OS	EMX2 opposite strand (non-protein coding
B06	N/A	ENST00000418855	FTX	FTX transcript, XIST regulator (non-protein coding)
B07	N/A	ENST00000419650	GACAT1	Gastric cancer associated transcript 1 (non-protein coding)
B08	Hs.736055	NR_002578	GAS5	Growth arrest-specific 5 (non-protein coding)
B09	N/A	NR_044995	GAS6-AS1	GAS6 antisense RNA 1
B10	Hs.122718	NR_002785	GNAS-AS1	GNAS antisense RNA 1
B11	Hs.533566	NR_002196	H19	H19, imprinted maternally expressed transcript (non-protein coding)
B12	Hs.61435	NR_003679	HAND2-AS1	Nbla00301
C01	N/A	NR_045680	HEIH	Hepatocellular carcinoma up-regulated EZH2-associated long non-coding RNA
C02	N/A	ENST00000557544	HIF1A-AS1	HIF1A antisense RNA 1 [Source:HGNC Symbol;Acc:43014]
C03	N/A	NR_045406	HIF1A-AS2	HIF1A antisense RNA 2
C04	Hs.612351	ENST0000043333	HNF1A-AS1	HNF1A antisense RNA 1 (non-protein coding)
C05	Hs.197076	NR_003716	HOTAIR	Hox transcript antisense RNA (non-protein coding)
C06	N/A	ENST00000425358	HOTAIRM1	HOXA transcript antisense RNA, myeloid-specific 1
C07	N/A	ENST00000421733	HOTTIP	HOXA distal transcript antisense RNA
C08	Hs.587427	NR_002795	HOXA11-AS	HOXA11 antisense RNA 1 (non-protein coding)
C09	N/A	ENST00000517550	HOXA-AS2	HOXA cluster antisense RNA 2
C10	N/A	ENST00000503668	HULC	Hepatocellular carcinoma up-regulated long non-coding RNA
C11	N/A	NR_023915	IPW	Imprinted in Prader-Willi syndrome (non-protein coding)
C12	N/A	KC469579	JADRR	JADE1 adjacent regulatory RNA
D01	Hs.741312	NR_002728	KCNQ1OT1	KCNQ1 overlapping transcript 1 (non-protein coding)
D02	N/A	ENST00000407852	KRASP1	Kirsten rat sarcoma viral oncogene homolog pseudogene 1
D03	Hs.652166	NR_024204	LINC00152	Non-protein coding RNA 152
D04	N/A	NR_001558	LINC00261	Long intergenic non-protein coding RNA 261
D05	Hs.433151	NR_024065	LINC00312	Non-protein coding RNA 312
D06	N/A	NR_046189	LINC00538	Long intergenic non-protein coding RNA 538
D07	Hs.606465	NR_024480	LINC00887	Hypothetical LOC100131551
D08	N/A	ENST00000412141	LINC00963	Long intergenic non-protein coding RNA 963
D09	N/A	ENST00000594200	LINC01233	Long intergenic non-protein coding RNA 1233
D10	N/A	ENST00000510694	LINC01234	Long intergenic non-protein coding RNA 1234
D11	N/A	GU228577	LSINCT5	Long stress-induced non-coding transcript 5
D12	N/A	ENST00000511918	LUCAT1	Lung cancer associated transcript 1 (non-protein coding)
E01	Hs.642877	NR_002819	MALAT1	Metastasis associated lung adenocarcinoma transcript 1 (non-protein coding
E02	Hs.654863	NR_002766	MEG3	Maternally expressed 3 (non-protein coding)
E03	Hs.697120	NR_001458	MIR155HG	MIR155 host gene (non-protein coding)
E04	Hs.652877	NR_027349	MIR17HG	MiR-17-92 cluster host gene (non-protein coding)
E05	N/A	ENST00000304425	MIR31HG	MIR31 host gene (non-protein coding)
E06	Hs.326728	NR_027148	MIR7-3HG	Non-protein coding RNA 306
E07	N/A	ENST0000041980	MRPL23-AS1	MRPL23 antisense RNA 1
E08	N/A	NR_102270	NAMA	Non-protein coding RNA, associated with MAP kinase pathway and growth arrest
E09	Hs.559259	NR_003108	NBR2	Neighbor of BRCA1 gene 2 (non-protein coding)
E10	N/A	NR_028272	NEAT1	Nuclear paraspeckle assembly transcript 1 (non-protein coding)
E11	N/A	ENST00000517270	NRON	Non-protein coding RNA, repressor of NFAT
E12	N/A	NR_109836	PANDAR	Promoter of CDKN1A antisense DNA damage activated RNA
F01	Hs.663766	NR_015342	PCA3 Prostate cancer antigen 3 (non-protein coding)
F02	N/A	ENST00000519319	PCAT1	Prostate cancer associated transcript 1 (non-protein coding)
F03	Hs.546994	NR_002769	PCGEM1	Prostate-specific transcript 1 (non-protein coding)
F04	N/A	ENST0000044559	POU5F1P5	POU class 5 homeobox 1 pseudogene 5
F05	N/A	ENST00000519282	PRNCR1	Prostate cancer associated non-coding RNA 1
F06	N/A	AK023948	PTCSC1	Papillary thyroid carcinoma susceptibility candidate 1 (non-protein coding)
F07	N/A	ENST0000055613	PTCSC3	Papillary thyroid carcinoma susceptibility candidate 3 (non-protein coding)
F08	Hs.493716	NR_023917	PTENP1	Phosphatase and tensin homolog pseudogene 1
F09	Hs.675281	NR_003367	PVT1	Pvt1 oncogene (non-protein coding)
F10	N/A	ENST0000036346	RMRP	RNA component of mitochondrial RNA processing endoribonuclease
F11	N/A	NR_024037	RMST	Rhabdomyosarcoma 2 associated transcript (non-protein coding)
F12	N/A	ENST00000455390	RPS6KA2-AS1	RPS6KA2 antisense RNA 1
G01	N/A	NR_038108	SNHG16	Small nucleolar RNA host gene 16 (non-protein coding)
G02	N/A	AK024556	SPRY4-IT1	SPRY4 intronic transcript 1 (non-protein coding)
G03	N/A	NR_002190	SUMO1P3	SUMO1 pseudogene 3
G04	N/A	ENST00000363312	TERC	Telomerase RNA component
G05	N/A	ENST00000431460	TRERNA1	Translation regulatory long non-coding RNA 1
G06	Hs.529901	NR_003255	TSIX	TSIX transcript, XIST antisense RNA (non-protein coding)
G07	Hs.554829	NR_002323	TUG1	Taurine upregulated 1 (non-protein coding)
G08	N/A	ENST00000466156	TUSC7	Tumor suppressor candidate 7 (non-protein coding)
G09	Hs.644234	NR_015379	UCA1	Urothelial cancer associated 1 (non-protein coding)
G10	Hs.567499	NR_023920	WT1-AS	WT1 antisense RNA (non-protein coding)
G11	Hs.529901	NR_001564	XIST	X (inactive)-specific transcript (non-protein coding)
G12	Hs.356766	NR_003604	ZFAS1	ZNFX1 antisense RNA 1
H01	Hs.520640	NM_001101	ACTB	Actin, beta
H02	Hs.534255	NM_004048	B2M	Beta-2-microglobulin
H03	Hs.546285	NM_001002	RPLP0	Ribosomal protein, large, P0
H04	N/A	NR_001445	RN7SK	RNA, 7SK small nuclear
H05	N/A	NR_002907	SNORA73A	Small nucleolar RNA, H/ACA box 73A
H06	N/A	SA_00105	HGDC	Human Genomic DNA Contamination
H07	N/A	SA_00104	RTC	Reverse Transcription Control
H08	N/A	SA_00104	RTC	Reverse Transcription Control
H09	N/A	SA_00104	RTC	Reverse Transcription Control
H10	N/A	SA_00103	PPC	Positive PCR Control
H11	N/A	SA_00103	PPC	Positive PCR Control
H12	N/A	SA_00103	PPC	Positive PCR Control

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