The role of microRNAs in the pathogenesis of thyroid cancer

Thyroid cancer is the most frequent type of cancers originating from the endocrine system. Early diagnosis leads to good clinical outcome in differentiated types of thyroid cancer. Yet, there are few treatment options for patients with medullary or anaplastic thyroid cancer. Thus, identification of molecular markers that explain the pathologic process during evolution of this cancer has practical significance. MicroRNAs (miRNAs) have been shown to influence the activity of thyroid cancer-related signaling pathways such as MAPK pathway and RET gene. These small transcripts not only can differentiate malignant tissues from non-malignant tissues, but also have differential expression in different stages of thyroid cancer. Assessment of serum levels of miRNAs is a practical noninvasive method for follow-up of patients after thyroidectomy. Moreover, the therapeutic effects of a number of miRNAs have been verified in xenograft models of thyroid cancer. In the current review, we summarize the data regarding the role of miRNAs in thyroid cancer.


Introduction
Thyroid cancer comprises the majority of tumors that originate from the endocrine system [1]. Based on the histological characteristics, thyroid cancers can be classified to differentiated thyroid cancer (DTC) originating from epithelial cells of the thyroid follicles, medullary thyroid cancer (MTC) and anaplastic thyroid cancer (ATC). Papillary thyroid cancers (PTCs) include most of DTCs. Other histological types of DTCs are follicular thyroid cancer (FTC) and Hürthle cells cancers [1]. Early detection of DTC and the appropriate surgical treatment and administration of radioiodine have improved prognosis of DTC. Yet, resistance to radioactive iodine is a major obstacle in the management of a proportion of patients with DTC. Besides, there are few treatment options for patients with MTC or ATC [1]. Thus, identification of molecular mechanisms for evolution of thyroid cancer is a necessity particularly for the management of histological subclasses that are less sensitive to the routine therapeutic options [2]. MicroRNAs (miRNAs) have recently attracted much attention for putative applications as tumor biomarkers and regulators of the carcinogenic process. Several studies have evaluated expression profiles of these~20 nucleotide transcripts in thyroid cancer cell lines and clinical specimens. Based on their expression pattern in these tissues compared with non-malignant tissues and their effects on cell proliferation and apoptosis, miRNAs have been classified to oncogenic (oncomiRs) and tumor suppressor miRNAs. In the current review, we summarize the role of these transcripts in the pathogenesis of thyroid cancer and their possible application as biomarkers for thyroid malignancy.

OncomiRs in thyroid cancer
In vitro and in vivo experiments have revealed the role of several miRNAs in the pathogenesis of thyroid cancer (Fig. 1). These oncomiRs have been shown to decrease expression of a number of tumor suppressors, thus enhancing cell proliferation and cell cycle progression. The role of these miRNA is exerted through modulation of cancer-related signaling pathways such as PI3K/Akt/mTOR, the adipocytokine signaling pathway, Hippo, Wnt and Jak-STAT signaling pathways.
Among the oncomiRs whose role in thyroid cancer have been assessed is miR-19a. This member of the miR-17-92 cluster is over-expressed in ATC tissues, promoting the de-differentiation and aggressiveness of the corresponding cells. Forced over-expression of this miRNA in the well-differentiated FTC cell line has enhanced cell proliferation and modified the signature of genes associated with thyroid cell differentiation and aggressiveness such as thyroid stimulating hormone receptor and thyroglobulin [3]. The oncogenic effects of the miR-223 in thyroid cancer cells are probably mediated through downregulation of APQ-1 protein. Notably, siRNA-mediated silencing of this miRNA has inhibited cell proliferation and induced apoptosis in these https://doi.org/10.1016/j.ncrna.2020.06.001 Received 13 June 2020; Received in revised form 15 June 2020; Accepted 15 June 2020 cells [4]. Besides, miR-221 has been shown to directly bind with the 3′ untranslated region (3′UTR) of TIMP3, thus inhibiting its expression and promoting proliferation and invasion of PTC cells. The oncogenic effects of this miRNA has been also verified in xenograft model of PTC [5]. miR-222 has been identified as another oncomiR in PTC based on its over-expression on PTC patients compared with goiter group. Besides, its expression levels were higher in patients with larger tumor sizes and invasive properties. Expression of miR-222 was also correlated with the risk levels provided by the American Thyroid Association, but not with the TNM staging [6]. Expression of miR-181a has also been increased in thyroid cancer tissues compared with the paired noncancerous tissues. Functional studies showed that miR-181a silencing decreases cell growth, while its up-regulation inhibits apoptosis and enhances cell cycle progression. This miRNA inhibits expression of RB1 [7]. Another study has demonstrated up-regulation of miR-146b, miR-222, miR-21, miR-221 and miR-181b in PTC tissue samples compared with normal thyroid tissues. Over-expression of these miRNAs were also detected in recurrent PTC tumors compared with non-recurrent samples and in lymph node metastases (LNM)-positive samples compared LNMnegative ones. Yet, distribution expression levels of these miRNAs were not different between PTC patients that have high and low risk of recurrence [8]. Expression of miR-146b-5p, miR-146b-3p, miR-221-3p, miR-222-5p, miR-222-3p has been increased in PTC tissues compared with normal thyroid samples. These were significant associations between up-regulation of miR-146b-5p and miR-222-3p and higher risk of recurrence. Over-expression of miR-146b-5p and miR-146b-3p distinguishes classical type and tall-cell variant but not follicular variant of PTC. Besides, miR-21-5p was remarkably increased only in tall-cell variant. Therefore, expression profile of miRNAs might be used in the molecular classification of PTC [9]. Table 1 summarizes the function and molecular interactions of oncomiRs in thyroid cancer.

Tumor suppressor miRNAs in thyroid cancer
Several miRNAs have been shown to negatively regulate expression of oncogenes, thus inhibiting cell proliferation and migration. MAPK, PI3K, NF-κB, GSK-3β/β-catenin, AKT and PI3K pathways are among cancer-related pathways which are modulated by these miRNAs. An extensive number of these miRNAs have been shown to be downregulated in thyroid cancer cell lines or clinical samples, thus facilitating malignnat behavior of these cells. For instance, miRNome sequencing has shown constant down-regulation of hsa-miR-139-5p in patients with recurrent or metastatic thyroid cancer compared to disease-free patients. Functional studies have shown the roel of this miRNA in attenuation of cell migration and proliferation in ATC cells. RICTOR, SMAD2/3 and HNRNPF have been identified as pssible targets for this miRNA. Moreover, expression of hsa-miR-139-5p has been inversely correlated with the expression of HNRNPF transcript, which codes for an alternative splicing factor participating in cryptic exon inclusion/skipping [34]. Besides, miR-128 has been shown to target sphingosine kinase-1 (SPHK1) thrugh direct interaction with its 3′UTR. Over-expressiion of this miRNA has led to attenuation of tumor growth rate and tumor weight in tumor-bering animals [35]. Up-regulation of miR-let-7e has been shown to suppress cell migration and invasion of thyroid cancer cells. This miRNA inhibits HMGB1 expression through binding with its 3′ UTR. miR-let-7e has been regarded as a tumor suppressor miRNA in PTC and a putative therapeutic candidate for this kind of cancer [36]. miR-129 is another tumor suppressor miRNA in PTC which exerts its function through inhibition of expression. Overexpression of miR-129 inhibits growth and invasion of PTC cells. Thus, miR-129-MAL2 axis is regarded as a therapeutic target in PTC [37]. Expression of miR-26b-5p has been decreased in thyroid cancer tissues compared with adjacent normal tissues in association with lymph node metastasis. In vitro studies showed the role of this miRNA in suppression of cell proliferation, invasion and migration of thyroid cancer cells. The tumor suppressor role of this miRNA might be exertedv through the Gsk-3β/β-catenin pathway [38]. miR-203 has been down-regulated in PTC tissues and cell lines compared with control tissues and cells. Down-regulation of this miRNA was associated with up-regulation of survivin, through which miR-203 modulates Bcl-2 expression [39]. Table 2 summarizes the functions and molecular interaction of the tumor suppressor miRNAs in thyroid cancer.

Diagnostic/prognostic role of miRNAs in thyroid cancer
Several studies have assessed diagnostic accuracy of miRNAs in thyroid cancer. Among them is the study conducted by Rosignolo et al. which identified serum profile of 754 miRNAs in PTC patients prior to and after thyroidectomy [25]. Notably, expression of eight miRNAs was significantly higher in patients before treatment compared with their levels both in healthy subjects and afer-treatmnet samples. The most promising results were reported for miR-146a-5p and miR-221-3p. Thus, expression of these miRNAs can be used as biomarkers for followup of patients. Prognostic significance of miRNAs in thyroid cancer has been verified through application of Kaplan-Meier analysis and cox regression methods. For instance, Wen et al. have reported consistent down-regulation of miR-486-5p in a number of PTC samples from TCGA, GEO and ArrayExpress datasets. They also reported associations between expression levels of this miRNA and clincal parameter such as cancer stage, lymph node involvement, distant metastsis and most notably overall survival [51]. Mazel et al. have assessed miRNA profiles in thyroid samples using next generation sequencing and multiplexing technologies. They recognized significant differences in miRNA signature between normal and malignant tissues. Notably, expression of 19 miRNAs were significantly different between benign and malignant tissues. In the bvalidation step, these miRNAs could classify 35 other nodules with indeterminate cytology. This panel has sensitivity, specificity and diagnostic power of 91%, 100% and 94%, respectively, which are superior to the existing molecular assays [65]. Table 3 summarizes the results of studies which appraised diagnostic/prognostic significance of miRNAs in thyroid cancer. OncomiRs play important roles in the regulation of different processes in the thyroid cancer and can be used as diagnostic, prognostic, and therapeutic biomarkers in this cancer. These miRNAs induce cell proliferation and growth, invasion and metastasis, whereas, inhibit apoptosis. In addition, high expression of oncomiRs was related to a reduced survival rate.

Role of miRNAs in chemoresistnce in thyroid cancer
The significance of miRNAs in determination of response to anticancer agents has been addressed in thyroid cancer patients. For instance, the tumor suppressor miRNA, miR-199b-5p has been shown to enhance sensitivity of thyroid cancer cells to the chemotherapeutic agent paclitaxel [43]. Moreover, miR-125b has significantly sensitized thyroid cancer cells to the effcts of cisplatin by activating autophagy through an Atg7 dependent route [70]. Most notably, miR-375 expression levels has been associated with reduced cell proliferation and improved sensitivity to vandetanib, a multi-kinase inhibitor which is used as a therapeutic option for metastatic MTC [30]. Table 4 summarizes the results of studies which reported association between expression levels of miRNAs and response to anti-cancer drugs.

Discussion
Recent studies have revealed aberrant expression of miRNAs in tissues or peripheral blood of patients with thyroid cancer. These miRNAs have been involved in the regulation of signaling pathways such as MAPK, PI3K, AKT, GSK-3β/β-catenin, Wnt, mTOR and NF-κB. Recent studies have revealed association between DTC and mutations in the RAS/RAF/MAPK pathway or RET/PTC rearrangements [1]. Moreover, MTC tumors have been linked with activating mutations in the RET gene [1]. The observed dysregulation of MAPK-associated miRNAs in thyroid cancer further shows the complex interactive network between miRNAs and signaling pathways in the context of thyroid cancer. Few studies have shown association between RET and miRNAs in this kind of cancer. For instance, miR-153-3p has been shown to be a RETregulated tumor suppressor miRNA in MTC [62]. Besides, expression of the oncogenic miR-182 has been increased in RET mutated cells. Notably, suppression of RET oncogenic signaling has decreased expression of miR-182. RET induced NF-κB translocation also affects expression of this miRNA. Notably, a known suppressor of the Notch pathway is targeted by miR-182 in mutant RET cell lines [76]. Therefore, miRNAs may serve as functional links between several cancer-related pathways in thyroid cancer.
The possibility of application of miRNA-targeted therapies in thyroid cancer has been assessed in some animal studies. For example, targeted intravenous transport of miR-153-3p has suppressed tumor growth in a xenograft model of MTC. This therapeutic option has been shown to have synergic effects with the tyrosine kinase inhibitor cabozantinib as well [62]. Thus, miRNA-targeted therapies might also reverse resistance to other anti-cancer therapies.
Diagnostic power of miRNAs in thyroid cancer has been evaluated by several groups. miRNAs not only can differentiate malignant tissues from non-malignant tissues, but also have differential expression in different stages of thyroid cancer. Assessment of serum levels of miRNAs is a practical noninvasive method for follow-up of patients after thyroidectomy. Notably, a transcript signature consisting of 19 miRNAs could discriminate benign lesions from malignant thyroid nodules with unknown cytology at better accuracy and lower expense compared with existing molecular assays [65]. However, diagnostic power of these panels of miRNAs should be appraised in different populations to obtain the best panel for each ethnic group. It is worth mentioning that the presence of single nucleotide polymorphisms in both miRNAs and the mRNA targets might alter their bindings. Thus, the significance of each oncomiR or tumor suppressor miRNA in the pathogenesis of thyroid cancer might vary in different populations based on the frequencies of these variants in each population.
Taken together, miRNAs have critical roles in regulation of thyroid cancer-related signaling pathways. Their availability in body fluids provides the possibility of application of non-invasive sampling in diagnosis of thyroid cancer. A number of miRNAs panels have been shown to be applicable in determination of cancer course and patients prognosis in thyroid cancer. Verification of these results in larger induced cell cycle arrest at G0/G1 phase and stimulated cell apoptosis. [71] (PTC: papillary thyroid carcinoma, ANT: adjacent normal tissue, MTC: medullary thyroid cancer, ATC: anaplastic thyroid cancer, FTC: follicular thyroid cancer). - [29] 491 PTC tissues and 59 corresponding normal tissues 0.886 --Poor OS was found in the patients in the high-risk group than in those in the low-risk group for all the patients and subclasses.

-
The miRNA signature was an independent prognostic factor associated with OS. [73] 40 PTC tissues and eight ANTs ---Patients with a lower miR-199b-5p level exhibited a shorter survival, and patients with higher miR-199b-5p expression had a longer survival time.
-- [43] 28FFPE MTC samples along with ANTs (continued on next page) Poor prognosis was associated only with male sex, tumor burden and high plasmatic levels of miR-375.
Downregulation of miR-215 expression was negatively associated with tumor size, differentiation, and lymph node metastasis status.
- [69] 71 paired tissue specimens of human PTC and ANTs ---Patients with primary tumors expressing higher miR-146b levels had a lower DFS rate than those with lower miR-146b expressions.
-miR-146b expression was a prognostic factor for DFS rate in patients with PTC. Advanced tumor stages and cervical LN metastasis were poor prognostic factors of DFS in patients with PTC at followup. [33] (ANT: adjacent normal tissue, OS: overall survival, RFS: relapse-free survival, DFS: disease-free survival, PTC: papillary thyroid carcinoma, HC: healthy control, DTC: differentiated thyroid cancer, MTC: medullary thyroid cancer, LN: lymph node).
samples sizes of patients from various ethnicities would pave the way for their applications in clinical settings.

Declaration of competing interest
The authors declare they have no conflict of interest.

Table 4
Role of miRNAs in response to anti-cancer drugs in thyroid cancer.
Response to anti-cancer drug miRNA Function Reference Paclitaxel sensitivity miR-199b-5p Up-regulation of miR-199b-5p suppresses cell proliferation, enhances apoptosis, and improves the sensitivity of thyroid carcinoma cells to paclitaxel. This miRNA inhibits tumor growth in nude mice. [43] Cisplatin resistance miR-182 miR-182 enhances cell growth through suppressing TRIM8 expression. Up-regulation of miR-182 enhances resistance of ATC cells to cisplatin by the suppression of TRIM8. [31] Cisplatin sensitivity miR-125b Up-regulation of miR-125b enhances sensitivity of thyroid cancer cells to cisplatin through regulation of autophagy.