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Annales d'Endocrinologie
Volume 75, n° 4
pages 187-193 (septembre 2014)
Doi : 10.1016/j.ando.2014.01.002
MiR-618 inhibits anaplastic thyroid cancer by repressing XIAP in one ATC cell line
MiR-618, un inhibiteur de prolifération du cancer anaplasique de la thyroïde par répression de la protéine inhibitrice de l’apoptose liée au chromosome X
 

Qianpeng Cheng, Xingguang Zhang, Xiuping Xu, Xiaofeng Lu
 Department of endocrinology, The Military General Hospital of Beijing PLA, 100700 Beijing, PR China 

Corresponding author.
Abstract

X-linked inhibitor of apoptosis protein (XIAP) is a major factor in cancer growth and progression. Reduction of XIAP induces apoptosis of anaplastic thyroid cancer (ATC), which accounts for more than 50% of thyroid cancer mortality. MicroRNAs (miRNAs) are short non-coding RNA molecules, which modulate gene expression via interaction with mRNA by binding to the 3′-untranslated region (3′-UTR), playing a critical role in cell proliferation, migration, and invasion. In this study, we recruited the ATC cell line 8305C and normal human thyroid cell Nthy-ori 3-1, aiming to find the miRNA which could regulate XIAP and therefore inhibit the growth and invasion of ATC. We first used quantitative real-time PCR (qPCR) to reveal that XIAP mRNA expression was 4.6±0.56 folds (P =0.029) up-regulated in 8305C cells, compared with Nthy-ori 3-1 cells. Then miR-618, predicted to target XIAP directly, was detected 0.24±0.06 folds (P =0.019) down-regulated in 8305C cells. Next we used Luciferase assay showing that XIAP was a target gene of miR-618, which could repress the XIAP expression at both mRNA and protein levels. After that, CCK-8 assay was performed to show that over-expression of miR-618 could inhibit the growth of 8305C cells. Finally, we employed transwell method to prove that miR-618 could prevent the invasion and migration of 8305C cells. In conclusion, our collective data showed that over-expression of miR-618 could inhibit ATC cells by targeting XIAP gene.

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Résumé

La protéine inhibitrice de l’apoptose, liée au chromosome X (XIAP), est un facteur important de prolifération et de progression de cancers. La réduction de l’activité XIAP conduit à l’apoptose dans le cancer anaplasique de la thyroïde (ATC), dont la mortalité dépasse 50 % des cas. Les microARN (miARN) sont de courtes séquences non codantes d’ARN qui modulent l’expression de gènes par interaction avec l’ARNm en se liant à la région 3′ non traduite (3′-UTR). Ils jouent un rôle essentiel dans la prolifération cellulaire, la migration et l’invasion. Dans cette étude, nous avons utilisé la lignée cellulaire ATC 850C et une lignée de cellules de la thyroïde humaine normale (Nthy-ori 3-1), dans le but d’identifier si un miRNA pouvait réguler l’expression de XIAP et, par conséquent, inhiber la croissance et l’invasion de la lignée ATC. Nous avons d’abord utilisé la PCR quantitative en temps réel (qPCR) pour montrer que l’expression d’ARNm de XIAP était très augmentée dans les cellules 8305C, par rapport aux cellules Nthy-ori 3-1 (×4,6±0,56 ; p =0,029). La présence de miR-618 a été retrouvée dans les cellules ATC mais en quantité moins abondante que dans les cellules 8305C (0,24±0,06 ; p =0,019). La technique de la luciférase a permis de montrer que le gène XIAP est une cible de miR-618 qui réduit son expression et sa concentration dans les cellules tumorales. Le test CCK-8 a été permis de montrer que la surexpression de miR-618 était capable d’inhiber la croissance des cellules de 8305C. Finalement, par la technique dite du « transwell » nous avons montré que miR-618 pouvait limiter l’invasion et la migration des cellules de 8305C. En conclusion, l’ensemble de ces données suggèrent que la surexpression de miR-618 en ciblant le gène XIAP est une approche thérapeutique potentielle pour réprimer la prolifération des cellules ATC.

The full text of this article is available in PDF format.
Introduction

Thyroid carcinoma is the most common endocrine malignancy, and the incidence of thyroid cancer is increasing worldwide. Histologically, thyroid cancer can be mainly divided into papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), medullary thyroid carcinoma (MTC), and anaplastic thyroid carcinoma (ATC). Though ATC only makes up less than 2% of all the cases of thyroid cancer [1], it accounts for more than 50% of thyroid cancer mortality [2], because it does not respond to radioiodine, radiotherapy or chemotherapy [3]. Therefore, new therapeutic approaches are needed, including gene therapy. A network of abnormal genes acts on the biological behavior of tumors, including the formulation, growth, differentiation, proliferation, even apoptosis. Among these, X-linked inhibitor of apoptosis protein (XIAP), belonging to the human inhibitors of apoptosis (IAP) gene family, plays an important role for cancer progression. And ATC cells themselves express high levels of XIAP [4]. In human malignant glioma cells, XIAP inhibits cell death via direct inhibition of caspases [5]. In human myeloid leukemia HL-60 cells, the over-expression of XIAP could attenuate the apoptosis induced by transfection of Apaf-1, which could enforce the sequential cleavage and activity of caspase-9 and caspase-3 [6]. High level of XIAP also relates to the development of other cancer cells, such as B-cell chronic lymphocytic leukemia (B-CLL) cells [7], breast cancer cells [8], ovarian and prostate cancer cells [9]. Reduction of XIAP levels induces to apoptosis of ATC and other cancer cells [10].

MicroRNAs (miRNAs) are short non-coding RNA molecules, commonly existing in plant and animal cells as important post-transcriptional regulators of gene expression via direct or indirect interaction with mRNA targets by binding to their 3′-UTRs [11]. Function studies manifest that miRNAs play a crucial role in regulating major biological behaviors of tumors, including the formulation, proliferation, differentiation, invasion, metastasis, chemosensitivity, and cell death. In HeLa and SKBr3 cells, over-expression of miR-328 decreases PTPRJ (Protein tyrosine phosphatase, receptor type, J) expression and therefore significantly enhances cells proliferation [12]. MiR-124a could impair proliferation of PTC cells (NPA-87-1) by post-transcriptionally regulating Androgen receptor (AR) [13]. And in human lung adenocarcinoma cells A549, up-regulation of miR-451 expression could inactivate the Akt signaling pathway and thereby enhance cisplatin-induced apoptosis [14].

As miRNAs regulate approximate one third of protein-coding genes [15], we purpose to find out some miRNAs, which could repress the expression of XIAP and therefore inhibit the growth of ATC cells. In this study, we use anaplastic thyroid cancer cells 8305C and normal human thyroid cells Nthy-ori 3-1 to elucidate that miR-618 could inhibit ATC cells by targeting XIAP.

Materials and methods
Cell culture and transfection

The human anaplastic thyroid cancer cell line 8305C (HPACC) and normal human thyroid cell line Nthy-ori 3-1 (HPACC) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Hyclone, America) and antibiotics (100μg/mL penicillin and 100μg/mL streptomycin). HEK 293T cells (ATCC) were cultured in DMEM medium (Hyclone, America) supplemented with the same as 8305C cells. All cells were incubated in a humidified atmosphere containing 5% CO2 at 37°C.

Cell transfection was performed using jetPRIME (PolyPlus, France), according to the manufacturer's protocol. Sixteen hours after being plated, cells in each well were transfected with miR-618 mimics, negative control duplex (NC), or small interfering RNA for XIAP (siXIAP) at a final concentration of 20nM. For western blot assay and quantitative real-time PCR analysis (qPCR), transfection was performed in 6-well plates, while luciferase assay and transwell assay in 24-well plates, and CCK-8 (cell counting kit-8, Dojindo, Kumamoto, Japan) assay in 96-well plates. The mature miR-618 mimics (sense: 5′-AAACUCUACUUGUCCUUCUGAGU-3′, Anti-sense: 5′-UCAGAAGGACAAGUAGAGUUUUU-3′); XIAP siRNA (sense: 5′-GUGCUU UCACUGUGGAGGATT-3′, Anti-sense: 5′-UCCUCCACAGUGAAAGCACTT-3′); and negative control duplex (NC, sense: 5′-UUCUCCGAACGUGUCACGUTT-3′, Anti-sense: 5′-ACGUGACACGUUCGGAGAATT-3′) were designed and synthesized by GenePharma (GenePharma, Shanghai, China).

Quantitative real-time PCR analysis

RNA isolation was performed with Trizol reagent (Invitrogen, America) according to the manufacturer's protocol. To detect XIAP mRNA, its cDNA was synthesized from the isolated RNA by using reverse transcription kit (Promega, America) following the manufacturer's instruction. Then qPCR was performed with SYBR Green PCR Mix kit (Takara, Japan), and the following primers: XIAP forward, 5′-GGGGTTCAGTTTCAAGGACA-3′ and reverse, 5′-CGCCTTAGCTGCTCTTCA GT-3′; GAPDH (endogenous control) forward, 5′-TCAGTGGTGGACCTGACCTG-3′ and reverse, 5′-TGCTGTAGCCAAATTCGTTG-3′.

For miR-618 expression detection, we used miRNA first-strand cDNA synthesis kit (TianGen, Beijing, China) to synthesize its cDNA from isolated total RNA following the manufacturer's instruction. Then qPCR was made by using SYBR Green PCR Mix kit (TianGen, Beijing, China) and the following primers: miR-618 forward, 5′-AAACTCTACTTGTCCTTCTGAGT-3′ and reverse, 5′-GCGAGCACAG AATTAATACGAC-3′. U6 small nuclear RNA (endogenous control) forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′.

All the qPCRs were performed in triplicate. The relative expression levels were calculated by the 2−ΔΔCt formula. And the experiment was carried out three times independently.

Construction of recombination plasmids and mutagenesis

The website TargetScan 6.2 was recruited to confine miRNAs, which could bind to the 3′-UTR of human XIAP gene directly. The 3′-UTR of XIAP mRNA containing the predicted binding sites for miR-618 (position 386–393, 2507–2514, and 4673–4679 of 3′-UTR) was cloned by PCR using the following primers: XIAP1 forward, 5′-AACTGCAGGGCAGTGTTTTAGTTGGCAAT-3′ and reverse, 5′-GCCATATGG CCCCTATAAAACCCCTCTG-3′; XIAP2 forward, 5′-AACTGCAGTTCACCTTTG CACTGTCT GC-3′ and reverse 5′-GCCATATGTCTCGATCTCCTGACCTCGT-3′; XIAP3 forward, 5′-AACTGCAGGGTTGCAAGAGCTCAAGGAG-3′ and reverse 5′-GCCATATGGTCACTTCCAGCCCTGTCAT-3′. Each PCR product was cloned in pGL3 luciferase reporter vector at Pst I and Nde I restriction sites, immediately downstream of the luciferase reporter gene, generating three wild-type XIAP UTR luciferase reporter constructs, pGL3-XIAP1-W, pGL3-XIAP2-W, and pGL3-XIAP3-W. Mutant 3′-UTR of XIAP constructs were designed for all nucleotides (UAGAGUU to AUCUCAA, UAGAGUU to AUCUCAA, AGAGUU to UCUCAA) of the seed region in the putative sites binding to miR-618, giving birth to three corresponding mutants (pGL3-XIAP1-M, pGL3-XIAP2-M, and pGL3-XIAP3-M), by using QuickChange Site-Directed Mutagenesis Kit (Stratagene, America). The wild-type and mutant inserts were confirmed by sequencing.

Luciferase assay

HEK 293T cells were co-transfected in 24-well plates (2×104 cells/well), using jetPRIME according to the manufacturer's protocol, with 200ng of the recombination plasmid or mutant vector, 1ng of pRL-CMV plasmid (Promega, America) for normalization, and 20nM (final concentration) of miR-618 mimics or control duplex. Forty-eight hours after transfection, cells were harvested and assayed with luciferase assay (Promega, America) in triplicate. The experiment was repeated three times independently.

Western blot analysis

Forty-eight hours post-transfection, cells seeded in 6-well plates were harvested and lysed with Lysis Reagent (Sigma, America), supplemented with Protease Inhibitor cocktail (Sigma, America) according to the manufacturer's instruction. The same amount of proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto nitrocellulose membranes. The primary antibodies used for immunodetection were anti-XIAP (Santa Cruz, America) and anti-GAPDH antibody (Santa Cruz, America). Blots were visualized by enhanced chemiluminescence reagents Supersignal (Pierce, America), following the manufacturer's instruction.

Proliferation assay

8305C cells were seeded into 96-well plates with the density of 1500 cells/well. Six, 24, 48, 72 or 96hours after transfection, the viability of cells was detected using CCK-8 according to the manufacturer's instruction. The experiment contained four groups: mock, transfected with nothing; negative control (NC), transfected with control miRNA mimics; miR-618, transfected with mature miR-618 mimics; XIAP siRNA, transfected with XIAP siRNA. Each group was plated in decuplicate. The experiment was performed three times independently.

Cell invasion and migration assay

The migration and invasion activities of tumor cells were assessed by transwell method. For invasion assay, 8305C cells (1.0×105) suspended in serum-free medium were added to the upper chamber (Costar, Corning, America) which owned a 6.5mm diameter polyvivyl/pyrrolidone (PVP)-free polycarbonate filter of 8μm pore size, and which was coated with 30mg/cm2 Matrigel (Sigma). Medium containing 10% FBS was placed in the lower compartment of the chamber. Cells were further incubated for 24hours to observe the number of tumor cells transferring to the filter under microscope after the membrane surface was totally wiped off, fixed with 4% paraformaldehyde for 1hour and then stained with 0.25% crystal violet solution for 30minutes. Migration assay was under the same way as invasion assay except that the upper chamber was coated with no matrigel and the permeating time for cells was 12hours.

Statistical analysis

All data was reported as the mean±standard deviation. Differences were assessed by Student's t -test. P <0.05 was considered statistically significant.

Results
Over-expression of XIAP mRNA and protein in ATC cells

Due to XIAP gene linking with cancer progression, we choose it as our candidate to do further research. First, we used qPCR and western blot assay to verify the higher level of XIAP in anaplastic thyroid cancer cells than that in normal human thyroid cells. As shown in Fig. 1A, the mRNA level of XIAP was 4.6±0.56 folds (P =0.029) up-regulated in 8305C cells compared with Nthy-ori 3-1 cells, and the protein level in 8305C cells was significantly higher (Fig. 1B). These data confirms that XIAP gene plays a role in anaplastic thyroid cancer.



Fig. 1


Fig. 1. 

XIAP mRNA and protein expression levels in Nthy-ori 3-1 and 8305C cells. Cells cultured in 6-well plates were harvested to perform qPCR and western blot analysis. Results were shown as the mean level of relative XIAP gene expression in Nthy-ori 3-1 and 8305C cells for three independent experiments. GAPDH was used for normalization. The XIAP mRNA in 8305C cells was 4.6±0.56 times that of Nthy-ori 3-1 cells (A, *P <0.05). And 8305C cells made significantly more XIAP protein than Nthy-ori 3-1 cells (B).

Zoom

MiR-618 complementarily binding to XIAP gene

In order to find the miRNAs which may repress XIAP expression, we recruited the target prediction website (TargetScan 6.2), which indicated that miR-618 could complementarily bind to the 3′-UTR of human XIAP gene in three regions (Fig. 2).



Fig. 2


Fig. 2. 

The seed matches of miR-618 predicted by TargetScan. We used TargetScan to predict the target gene of miR-618 and the target miRNAs of XIAP gene, finding miR-618 could directly bind to the 3′-UTR of XIAP in three positions (386–393, 2507–2514, and 4673–4679).

Zoom

Low level of miR-618 in ATC cells

As miR-618 may target XIAP, its expression should suffer from a parallel change of XIAP. So we used qPCR to compare miR-618 expression in 8305C cells and Nthy-ori 3-1 cells. As shown in Fig. 3, miR-618 was 0.24±0.06 folds (P =0.019) down-regulated in 8305C cells compared with Nthy-ori 3-1 cells, implying that miR-618 is probably related to XIAP and that it could act a role in ATC progression.



Fig. 3


Fig. 3. 

Relative expression levels of miR-618 in Nthy-ori 3-1 cells and 8305C cells. Results were shown as the mean level of relative miR-618 expression in Nthy-ori 3-1 and 8305C cells for three independent experiments, U6 small nuclear RNA was used for normalization. MiR-618 in 8305C cells was only 24±6% of that in Nthy-ori 3-1 cells (*P <0.05).

Zoom

XIAP is a target of miR-618

First, we cloned three fragments of XIAP 3′-UTR, each containing one of the binding regions for miR-618, named XIAP1 (position 386–393 of XIAP 3′-UTR), XIAP2 (position 2507–2514), and XIAP3 (position 4673–4679). Next we cloned these fragments and their mutant versions into pGL3 vector, generating pGL3-XIAP-W and pGL3-XIAP-M. Then we performed luciferase assay and the point mutant experiment in HEK 293T cells. Forty-eight hours post-transfection with miR-618 mimics or its control duplex in 24-well plates, cells were collected to detect luciferase activity. As shown in Fig. 4, compared with negative control group, miR-618 mimics decreased the relative luciferase activity by 5.3% (P >0.05) in pGL3-XIAP1-W, 46.9% (P <0.05) in pGL3-XIAP2-W, 3.8% (P >0.05) in pGL3-XIAP3-W. While after the binding sites mutation, the relative luciferase activity returned to 97.3% (P >0.05) in pGL3-XIAP1-W, 95.8% (P >0.05) in pGL3-XIAP2-W, and 95.5% (P >0.05) in pGL3-XIAP3-W, indicating that miR-618 could bind to 3′-UTR of XIAP at the locus 2507–2514, and miR-618 could directly target XIAP gene.



Fig. 4


Fig. 4. 

Luciferase activity assay and mutation experiment performed in HEK 293T cells. Wild represented the cells transfected with miR-618 and plasmids containing binding sites for miR-618, while mutant represented those transfected with miR-618 and mutant versions of plasmids. NC represented the cells transfected with control mimics and non-mutant versions. In pGL3-XIAP2 group, miR-618 decreased the luciferase activity of wild by 46.9%, but not the mutant, compared with NC. In other two groups, no apparent decrease existed in wild or mutant. Experiments were performed independently three times. *P <0.05, **P >0.05.

Zoom

MiR-618 regulates XIAP expression in ATC cells

Next, we examined the effects of miR-618 on XIAP expression in ATC cells. Forty-eight hours after transfection 8305C cells in 6-well plates were collected to perform qPCR and western blot analysis. As shown in Fig. 5A, the XIAP mRNA in 8305C cells transfected with miR-618 mimics was 43±8% (P <0.05) down-regulated, compared with its control duplex transfected cells. Western blot analysis manifested that the XIAP protein level in 8305C cells decreased sharply after transfection with miR-618 (Fig. 5B). These findings showed that miR-618 could decrease XIAP mRNA and protein level in A549/CDDP cells.



Fig. 5


Fig. 5. 

The expression level of XIAP mRNA and protein in 8305C cells after transfection. Post-transfection of 48h in 6-well plates, 8305C cells were harvested to do qPCR and western blot analysis. Compared with the negative control mimics (NC), miR-618 reduced XIAP mRNA level by 43±8% (A), and repressed the protein level significantly (B). Experiments were performed independently three times. *P <0.05.

Zoom

MiR-618 inhibits growth and progression of ATC cells

To manifest the role of miR-618 in the growth of ATC cells, we performed proliferation assay. The viability of 8305C cells was detected with CCK-8 assay at different time after transfection (6, 24, 48, 72, and 96h). A curve of cell numbers, represented by the viability, was generated to show the speed of cells’ growth in each group. As shown in Fig. 6A, the growth of 8305C cells transfected with nothing or negative control duplex was almost at the same speed. But the growth was apparently inhibited in cells transfected with miR-618 mimics or XIAP siRNA, compared with those transfected with negative control duplex, indicating that over-expression of miR-618 could inhibit ATC cells growth as well as down-regulating XIAP. Further more, we revealed the effects of miR-618 on invasive and migratory characteristics of ATC cells. Transwell assay demonstrated that over-expression of miR-618 or knocking down of XIAP could decrease the numbers of invasive (Fig. 6B) and migratory (Fig. 6C) cells. All those results showed that XIAP could regulate the growth and progression of ATC cells, and that over-expression of miR-618 mediated the inhibition of ATC cells.



Fig. 6


Fig. 6. 

A. The growth curve of 8305C cells after transfection. After transfection with miR-618 or siXIAP, the proliferation of cells was inhibited significantly, compared with the control. And almost no changes of speed were found between the cells transfected with nothing and the control. B and C. The invasion and migration assays of 8305C cells after transfection. After transfection with miR-618 or siXIAP, the invasive (B) and migratory (C) cells decreased significantly, compared with the control. No apparent differences were found between the cells transfected with nothing and the control in both B and C.

Zoom

Discussion

XIAP locating in the region of chromosome Xq24-25, spans approximately 8kb, with 7 exons in the coding region. XIAP gene, encoding 497 amino acids, is associated with tumor genesis, growth, progression, metastasis, and apoptosis. It has been verified high expression in malignancies, e.g. lung cancer, pancreatic cancer, and prostatic cancer [16, 17]. In thyroid carcinoma specimens, positive rates of XIAP expression in follicular variant papillary thyroid carcinoma (PTC), follicular, medullary, poorly differentiated, and anaplastic thyroid carcinoma specimens were 20%, 25%, 38%, 67%, and 38%, respectively[18]. And XIAP expression was associated with lymph node metastasis in PTCs [19].

XIAP gene is regulated by a network of factors. In SK-Hep1 hepatoma cells, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) decreased XIAP level [20]. Tillman explored that rottlerin could induce the release of cytochrome c, with concomitant caspase activiation and down-regulation of XIAP, inducing the apoptosis of human colon carcinoma cell PKO [21]. In ovarian cancer and melanoma cells, phenoxodiol caused XIAP to degrade and sensitized cells to carboplatin [22]. GW8510 is a commercially available synthetic CDK (cyclin-dependent kinase) inhibitor, and it inhibited the growth of the lung cancer cell lines, A549 and H1299, correlating with the down-regulation of the mRNA and protein level of XIAP [23]. Another study revealed that, in leukemic cell lines, hematopoietic cytokines, such as GM-CSF, G-CSF, or SCF, could regulate the expression of XIAP through PI3K and MAPK signal transduction pathways, and therefore induced cell death [24].

To date, some miRNAs have been proved to regulate XIAP level. In human ovarian granulosa cells, miR-23a may play important roles in regulating apoptosis via decreasing XIAP expression [25]. In human umbilical vein endothelial cells (HUVECs), miR-513a-5p mediated TNF-α and LPS induced apoptosis via down-regulation of XIAP [26]. Over-expression of miR-497 reduced XIAP protein level and sensitized cisplatin resistance human lung adenocarcinoma cell A549/CDDP to cisplatin-induced apoptosis [27]. Recently, miR-181a/b was manifested to significantly sensitize chronic lymphocytic leukemia (CLL) cells to fludarabine induced apoptosis by direct inhibiting XIAP protein [28]. Our data reveals that over-expression of miR-618 inhibits the growth, migration, and invasion of ATC cells by targeting XIAP. Abdalla argues that miR-618 is up-regulated in hepatocellular carcinoma (HCC) patients. The sensitivity and specificity of miR-618 for detecting HCC among hepatitis C positive individual was 64% and 68%, respectively [29].

The relationships between miRNA and thyroid carcinoma have been explored in numbers of papers. The down-regulation of miR-25 and miR-30d could contribute to the process of thyroid cancer progression, leading to the development of anaplastic carcinomas targeting EZH2 mRNA [30]. Decapentaplegic homolog 3 (Smad3), a member of the TGF-β pathway that has an inhibitor role in thyroid follicular cell proliferation. MiR-23b or miR-29b, targeting Smad3, could promote thyroid cell growth [31]. Paduano reported that miR-328 was an important player in the regulation of tyrosine phosphatase PTPRJ expression, and could increased thyroid carcinoma cell proliferation [12]. And many other miRNAs, such as miR-124a, miR-7, and miR- miR-886-3p [13, 32, 33] are crucial to the progression of thyroid carcinoma.

In conclusion, our data showed that XIAP is a target gene of miR-618, and that after over-expression of miR-618, the growth of anaplastic thyroid cancer cell slowed down, the migration and invasion capacity also decreased sharply.

Disclosure of interest

The authors declare that they have no conflicts of interest concerning this article.

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