MicroRNA in Papillary Thyroid Carcinoma: A Systematic Review from 2018 to June 2020

Simple Summary The most common form of endocrine cancer - papillary thyroid carcinoma, has an increasing incidence. Although this disease usually has an indolent behavior, there are cases when it can evolve more aggressively. It has been known for some time that it is possible to use microRNAs for the diagnosis, prognosis and even treatment monitoring of papillary thyroid cancer. The purpose of this study is to summarize the latest information provided by publications regarding the involvement of microRNAs in papillary thyroid cancer, underling the new clinical perspectives offered by these publications. Abstract The involvement of micro-ribonucleic acid (microRNAs) in metabolic pathways such as regulation, signal transduction, cell maintenance, and differentiation make them possible biomarkers and therapeutic targets. The purpose of this review is to summarize the information published in the last two and a half years about the involvement of microRNAs in papillary thyroid carcinoma (PTC). Another goal is to understand the perspective offered by the new findings. Main microRNA features such as origin, regulation, targeted genes, and metabolic pathways will be presented in this paper. We interrogated the PubMed database using several keywords: “microRNA” + “thyroid” + “papillary” + “carcinoma”. After applying search filters and inclusion criteria, a selection of 137 articles published between January 2018–June 2020 was made. Data regarding microRNA, metabolic pathways, gene/protein, and study utility were selected and included in the table and later discussed regarding the matter at hand. We found that most microRNAs regularly expressed in the normal thyroid gland are downregulated in PTC, indicating an important tumor-suppressor action by those microRNAs. Moreover, we showed that one gene can be targeted by several microRNAs and have nominally described these interactions. We have revealed which microRNAs can target several genes at once.


Materials and Methods
A literature analysis was performed in MEDLINE using PubMed for studies published from 2018 to June 2020. The following keywords were used: "microRNA"+ "papillary"+ "thyroid" + "carcinoma", which resulted in 466 articles in English. All related abstracts were reviewed and relevant studies that were published in English were selected. We only included papers that had full text available and described the exact method and results regarding microRNA's signatures in PTC epigenetic mechanism. A selection of 137 eligible articles was the result of our search ( Figure 1).

Results
Data on microRNAs, the sample source, the regulatory mode of microRNAs, the target genes/proteins of microRNAs, and their effect on PTC cells from the 137 studies were selected and presented in Table 1  .

Up-and Downregulated microRNAs in Papillary Thyroid Cancer
Out of 139 microRNAs, 106 are downregulated and 33 are upregulated (Table 1). This means that more than a quarter of the described microRNAs have an oncogenic role (oncomiR's) and the rest of them have a tumor-suppressive role. The dysregulation of microRNA is an important event during the development of papillary thyroid carcinoma. Overexpression of certain microRNA can result in the tumor suppressor genes repression. Down-regulation of specific microRNA can lead to increased expression of oncogenes. Overexpression and downregulation induce malignant effects on cell cycle progression, proliferation, migration, and apoptosis, leading to tumor growth and progression in PTC and other types of malignancies [1].

One Gene Can Be Targeted by Several microRNAs
Analyzing, individually, in each study, the interaction between microRNAs and the genes targeted by them, we noticed that the same gene can be targeted by different microRNAs. For example, HMGB1 has been reported to play an important role in promoting both cell survival and death by regulating multiple signaling pathways, including proliferation, autophagy, inflammation, invasion, and metastasis. The study by Ding. C et al. [127] indicates that microRNA-let-7e downregulates HMGB1 expression by directly targeting the HMGB1 3 -UTR, downregulated HMGB1 inhibits PTC cell proliferation and metastasis [127]. MicroRNA-1179 interacted with the 3 UTR of HMGB1 and suppressed HMGB1 expression at the post-transcriptional level and indicates that the microRNA-1179/MHGB1 pathway plays a tumor suppressor role in PTC [97]. The same gene-HMGB1 is involved in ANRIL/HMGB1/ microRNA-320a pathway. Propofol-mediated ANRIL downregulation competed with HMGB1 to bind microRNA-320a, thus inhibiting PTC cell malignant behaviors [42].
A study by Chen et al. [18] has shown that enforced expression of microRNA-202-3p inhibited WNT signaling by downregulating β-catenin expression in PTC. Again, the same gene is regulated by microRNA-3619-3p to promote cell migration and invasion in PTC [102]. WNT1 has been shown to promote cancer progression because it triggers cell proliferation and metastasis, microRNA-329 inhibits papillary thyroid cancer progression via direct targeting WNT1 [90]. WNT5a, an important signaling molecule in the non-canonical Wnt family, has been involved in nearly all parts of the non-canonical Wnt pathway. The invasion and metastasis of PTC cells were inhibited by microRNA-26a-5p via Wnt5a [66].
One more targeted gene by multiple microRNAs in PTC is AKT, the human homolog of the viral oncogene v-Akt is related to protein kinases A (PKA) and C (PKC) in humans. The pathway that involves AKT inactivates several proapoptotic factors, AKT also activates transcription factors which promote anti-apoptotic genes. Overexpression of microRNA-15a inhibited the activation of the AKT pathway, which inhibited cell proliferation and promoted the process of apoptosis [39]. Upregulated microRNA-203 suppresses epithelial-mesenchymal transition (EMT), invasion, proliferation, and migration as well as induces apoptosis of PTC cells via downregulated AKT3 [101]. lncRNA n384546 could regulate the expression of AKT3 by sponging microRNA-145-5p [119]. lncRNA HOTTIP modulated Akt1 expression by regulating microRNA-637 in PTC cell lines [103].
Another example is the Sphingosine kinase (SPHK), an enzyme, catalyzing the formation of the prosurvival second messenger sphingosine-1-phosphate (S1P) from the pro-apoptotic lipid sphingosine. High SPHK expression is correlated with a significant decrease in survival rate in patients with several forms of cancer, including PTC. LncRNA LINC00460 promoted PTC progression via modulating SphK2 through sponging microRNA-613 in PTC [26]. lncRNA LINC00520 accelerates the progression of papillary thyroid carcinoma by serving as a competing endogenous RNA of microRNA-577 to increase SphK2 expression [72]. MicroRNA-128 targets SPHK1 to induce apoptosis and reduce cell proliferation, migration in thyroid cancer cell lines, and inhibits tumor growth [14].

Discussion
Each microRNA can regulate hundreds of messenger RNAs (mRNAs), while various microRNA can control the same mRNA. Additionally, many microRNAs regulate and are regulated by other species of non-coding RNAs, such as circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs). For this reason, it is extremely difficult to predict, study, and analyze the precise role of a single microRNA involved in human cancer, considering the complexity of its connections. Focusing on a single microRNA molecule represents a limited approach. Additional information could come from network analysis, which has become a common tool in the biological field to better understand molecular interactions [1].
Most studies assess the level of expression of the microRNA in question, show the actions of its overexpression/silencing on cell lines, find the gene targeted by the microRNA, and how the metabolic pathway microRNA / target gene works. Although complex information is presented, at the end of the discussion chapter we find the same dry phrase "microRNA-X could be a potential therapeutic/diagnostic/prognostic target for PTC treatment". Despite this, there are several articles with a different study design that offer something more than "could be". One of them is the study of Zhao. L et al. [134] which finds Matrine, a traditional Chinese medicine, as an alternative drug for PTC treatment. Treatment with matrine at the concentrations of 1, 2, 5, 10, and 20 mg/ml inhibited TPC 1 cell proliferation by up to 95.8% (for 20 mg/ml matrine). Matrine induced apoptosis and G1 cell cycle arrest through downregulating microRNA-21 to affect the PTEN/Akt signaling in TPC 1 human thyroid cancer cells. Liu. F et al. [121] discovered that microRNA-206 contributed to euthyrox resistance in PTC cells through blockage p38 and JNK signaling pathway by targeting MAP4K3. Another study by Liu et al. [49], found the promoting gene and the signaling pathway regulating RAI-resistance in PTC. The results attested that NEAT1 was upregulated in RAI-resistant PTC accompanied microRNA-101-3p inhibition, FN1 overexpression, and PI3K/AKT signaling pathway abnormal activation. Fang. T et al. [23] discovered that Shenmai injection (SMI), a traditional Chinese formula mainly made up of Red Ginseng and Radix Ophiopogonis. SMI inhibits the differentiation of CD4 + T cells into Treg cells via the microRNA-103/GPER1 axis, which improves the immunological function of PTC patients with postoperative 131-Iodine ablation. Although few, these studies differ in the classical approach to the use of microRNAs in papillary thyroid carcinomas and should be recognized as at least promising.
Even if in the world of publications microRNA is a hot topic, when we talk about PTC, most international guidelines regarding thyroid cancer management, do not even mention microRNA.
Here we refer to the NCCN 2018 [149], ETA 2019 [7], and ESMO 2019 [11] guidelines. The exception is the ATA 2015 guideline, which, although published several years before the above-listed guidelines, mentions microRNA as an additional diagnostic molecular marker strategy under development. microRNA markers have shown initial diagnostic utility in FNA samples with indeterminate cytological diagnoses, but they have not been thoroughly validated. It is also mentioned about microRNA, also in this guide, in the chapter "Directions for future research", as possible progress in identifying markers of thyroid cancer. To result in a significantly improved accuracy of cancer detection in thyroid nodules as compared to the currently available clinical tests [8].
Hence, which are the most challenging parts in PTC management where we could use microRNA? After the clinical and ultrasound evaluation of a thyroid nodule, if malignancy criteria are present, a fine needle biopsy is performed for cytological examination. Some results of the cytological examination can be inconclusive. In such cases, there is a need to assess molecular markers to make a presurgical differentiation of benign and malign lesions. MicroRNAs are one of the novel classes of molecular markers that are being used to improve the diagnosis of thyroid cancer. Several studies have shown that a microRNA-based signature in FNABs can be used to discriminate benign from malignant thyroid nodules. MicroRNA profiling of thyroid cancers can also provide prognostic information useful for defining optimal management strategies. Expression levels of certain microRNA in thyroid tumor tissues are associated with clinicopathological characteristics, such as tumor size, multifocality, capsular invasion, extrathyroidal extension, and both lymph node and distant metastases [150]. Treatment options have been proposed and implemented based on the results obtained from research conducted on epigenetic alterations. Therefore, the development of new therapeutic strategies based on targeting epigenetic changes to restore the expression of tumor suppressor microRNAs or to blunt overexpressed oncogenic microRNAs may provide a new landscape for the treatment of aggressive PTC [151].
Although PTMC generally has an excellent prognosis, the long-term rate of recurrence of PTMC has been reported to be as high as 10% [9]. Currently, there are no reliable clinical features including molecular markers, that can differentiate PTMC in patients who develop progressive disease from indolent PTMC. Even so, searching the PubMed database, regarding microRNA signatures in PTMC, there is only one study by Zhang et al. which combines serum microRNA with ultrasound profile as predictive biomarkers of diagnosis and prognosis for PTMC. In this study, microRNAs were found to be significantly associated with a poor prognosis of patients with PTMC and could be used as prognostic molecular markers or patients with PTMC before and after surgery. These results suggest that circulating microRNAs may be useful as non-invasive molecular biomarkers of diagnosis and prognosis for PTMC [9], selecting those cases that need aggressive therapies, despite the histology of PTMC. Given the need for more studies in this field, this topic could be a research idea for the future, in the era of personalized medicine.

Conclusions
Research regarding microRNAs in PTC is undergoing a tremendous shift, suggesting rapid maturation of this field. In this review, we tried to represent as briefly as possible the interactions of microRNAs with target proteins. We also showed microRNAs regulation mode and its effect on PTC cells. Our results showed that a gene can target multiple microRNAs simultaneously, and vice versa. All this information can be used to find the most effective therapeutic targets/biomarkers in PTC. For future research, we indicated a possible niche, namely microRNA signatures in PTMC.