MicroRNA Expression and Maturation

The first step in the expression of a miRNA is the transcription of the miRNA gene into the primary miRNA transcript (Pri-miRNA) by an RNA polymerase (Fig. 1). Following transcription, still within the nucleus, the Drosha-DGCR8 (DiGeorge critical region 8) microprocessor complex cleaves both strands of the stem on the Pri-miRNA [12]. The product of the cleaved Pri-miRNA is the shortened precursor miRNA (Pre-miRNA) [13].

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Figure 1.

Schematic diagram depicting miRNA production. The Pri-miRNA is transcribed from a miRNA gene within the nucleus. The Pri-miRNA is then cleaved to become the Pre-miRNA, which is exported into the cytoplasm. Dicer processes the Pre-miRNA into the mature miRNA duplex. One of the strands is the guide strand, which is incorporated into the miRNA effector machine, known as the RNA-induced silencing complex. Cleavage or translational repression of the target mRNA occurs depending on the complementarity of the miRNA to the mRNA. Figure copyright Beth Croce, Bioperspective. Reproduced with permission.

Abbreviations: AGO, Argonaute protein; miRNA, microRNA; Pre-miRNA, precusor microRNA; Pri-miRNA, primary microRNA; RISC, RNA-induced silencing complex.

The Pre-miRNA produced by the nuclear processing is then exported into the cytoplasm by exportin-5 [12]. In the cytoplasm, the Pre-miRNA is cleaved into a double-stranded miRNA duplex by Dicer, an RNase enzyme [14]. One of the strands of the duplex is the guide strand, whereas the complementary strand is the passenger strand. The guide strand is incorporated into the miRNA effector machine, known as the RNA-induced silencing complex (RISC), and the passenger strand is degraded.

The RISC is the machinery responsible for gene silencing and consists of a miRNA strand, a member of the Argonaute (AGO) protein family, and other structural and facilitating proteins [12]. The guide miRNA strand recognizes the target mRNA transcript, whereas the AGO protein (with other facilitating proteins) mediates the corresponding action [12, 15]. The nature of the mediated action depends on the degree of complementarity between the miRNA and mRNA. If there is significant yet incomplete pairing of the miRNA and mRNA sequences, mRNA translation is repressed. It is this incomplete nature of miRNA-mRNA matching that allows a single miRNA to target multiple mRNAs and multiple miRNAs to target the same mRNA sequence [16]. In the less commonly occurring event of complete base pairing, the mRNA transcript is degraded [15].

MicroRNA Deregulation

MicroRNAs contribute to oncogenesis either as tumor suppressors or oncogenes. Their deregulation is a result of genomic abnormalities similar to those for protein-coding genes: chromosomal rearrangements, genomic amplifications or deletions, and mutations [17]. In any given cancer, a combination of abnormalities in protein-coding and noncoding genes can be identified [18]. Among the possible mechanisms, the contemporary view is that aberrant gene expression is the main mechanism underlying the functional changes of miRNAs in cancer cells. This is characterized by abnormal levels of expression for mature and/or precursor miRNA sequences compared with the normal tissue of that organ. The abnormal expression level is otherwise known as deregulation [17]. In addition, defects in miRNA processing and maturation also contribute to their deregulation and subsequently cause diseased states [12].

MicroRNA in PTC Diagnosis

Although the clinical diagnosis of PTC is not usually a problem, it is important to appreciate the development of miRNA research in PTC, as a foundation for other clinical applications.

He et al., in their seminal paper published in 2005, were the first to apply a miRNA profiling capability to PTC [23]. One of the significant findings was that in PTC, the key differentially expressed miRNAs appeared to be overexpressed, although global under-expression of miRNA was normally associated with cancers in general. This finding has since been confirmed in other reports [24, 25].

He et al. compared the miRNA expression of 30 PTC tumors and the normal tissue samples of their corresponding thyroid glands, using the Affymetrix HG-U133 plus two array chips [23]. The pairwise comparisons indicated that 23 miRNAs showed differential expression, with 17 of them overexpressed and 6 underexpressed. Of the overexpressed miRNAs, 6 demonstrated a fold change of >1.5-fold. The most overexpressed miRNAs were miR-146b, miR-221, and miR-222, showing increases of 11- to 19-fold. These findings were further confirmed on semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and Northern blotting. All underexpressed miRNAs had a fold-change of less than twofold [23]. Incidentally, in a 2006 publication, Calin and Croce pointed out that a less-than-twofold change in miRNA expression in some instances might also be biologically significant [17]. Using as few as 5 miRNAs, He et al. were able to define cancer status in 12 blind tumor samples with 100% accuracy [23].

He et al. also found that miR-221 expression was increased in nonmalignant adjacent thyroid tissue, albeit to a lesser degree [23]. This suggested that miR-221 overexpression was either an early genetic event in PTC carcinogenesis or that some carcinogenic miRNA changes were global phenomena within the thyroid gland [23].

Significant overexpression of miR-146b, miR-221, and miR-222, reported by He et al. [23], was confirmed in many subsequent studies by other groups [25–27]. Using microarray and Northern blotting, for example, Pallante et al. also showed that miR-221 and miR-222 were significantly overexpressed in PTC tissue compared with normal thyroid tissue of the contralateral, unaffected lobe [26]. In their study, the third overexpressed miRNA reported was miR-181b [26]. Further confirmation of these findings was achieved by quantitative RT-PCR comparing the miRNA precursors in an external cohort of 39 PTC and 8 follicular adenoma tissue samples. Means of approximately 13-fold overexpression were seen in all 3 miRNA precursors in the PTC samples; they then extended this technique to fine needle aspiration (FNA) samples [26] (as discussed in MicroRNA in Thyroid FNA).

Between 2005 and 2013, eight publications reported on the differential miRNA expression profiles of human PTC samples compared with normal or benign thyroid tissue (Table 1); however, these publications were heterogeneous in terms of the included sample populations, tissue studied, and methodology. The heterogeneity arose from different study designs and questions, but the common ground was the use of microarray on at least 10 samples including PTC and control tissue types. Most studies included both classical and follicular variants of PTC, whereas some did not define the subtype of PTC samples included. Some studies also included other thyroid malignancies [28, 29]. The control or comparison groups varied from normal thyroid tissue [27, 30] to multinodular goiter (MNG) [25] or benign follicular lesions [28, 29]. Despite these inconsistencies, some similar trends of miRNA deregulation can be observed in PTC samples. The most consistently overexpressed miRNAs in PTC were miR-221, miR-222, and miR-146b. Other commonly reported overexpressed miRNAs were miR-155 and miR-181b. There was less consistency in the downregulated miRNA set.

Table 1.

Overview of studies using miRNA microarray to compare PTC and control

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MicroRNA in Thyroid FNA

Although more histological subtypes of PTC were being profiled, work was also being done on thyroid FNA samples. Pallante et al. were the first to report three highly overexpressed miRNAs (miR-221, miR-222, and miR-181b) in thyroid tumors in the context of FNA cytology samples [26]. All three miRNAs showed higher expression levels in seven of eight PTC FNA samples compared with normal thyroid cells from FNA of non-neoplastic nodules. With this finding, new doors were opened for the utility of miRNA profiling in the diagnosis of thyroid malignancies. The potential role of miRNA profiling in thyroid pathology was transformed from postoperative confirmation or subtyping in tissue samples to preoperative diagnosis or subtyping in FNA samples. This development has major implications for the way PTC patients may be managed, if it enters clinical application.

Currently, approximately 3%–6% of FNA samples are considered indeterminate [32]. It is the management of this small subgroup that can be significantly improved with molecular diagnosis preoperatively. Keutgen et al. reported that by using a panel of 4 miRNAs (miR-222, miR-328, miR-197, miR-21), thyroid malignancies could be diagnosed in FNA samples of indeterminate lesions, with 100% sensitivity and 86% specificity and overall accuracy of 90% [33]. Other studies using a similar study design of indeterminate FNA samples, with single or a panel of miRNAs, failed to achieve such high accuracy [29, 34, 35]. Agretti et al. [35] and Shen et al. [34] were able to distinguish benign thyroid tissue from PTC using miRNA expression of FNA samples; however, the discriminatory power of the technique was poorer in the subgroup with indeterminate cytology, limiting the clinical application [35]. Using 1 miRNA on 125 indeterminate FNA samples, Vriens et al. achieved accuracy of 75% and negative predictive value of 81% [29]. These rates are not yet suitable for clinical use. The consensus is that further work is required before miRNA expression in FNA samples can be of clinical utility [36].

MicroRNA in Staging and Prognostication

One of the challenges in managing PTC patients is identifying the small subgroup with aggressive disease and providing those patients with more extensive treatment and intensive long-term surveillance. As the number patients with indolent PTC increases, there is an even greater need for accurate and efficient identification of those with aggressive disease. Accurate stratification at initial diagnosis and treatment is essential to avoid overtreatment and unnecessary surveillance of the majority and to provide adequately intensive management for the minority with aggressive disease.

Various molecular markers have been investigated in the hope of more accurate risk stratification [37–40]. The most studied marker is BRAF (the B-isoform of the Raf gene). The BRAF^V600E mutation has been shown to be associated with aggressive clinical behaviors of PTC by some investigators [37]; however, the evidence is still inconclusive, with contradictory reports [38]. Other molecular markers being investigated include cell-cycle regulators p27, p21, and cyclin D1 and immunohistochemical markers such as CEACAM-1 (carcinoembryonic antigen-related cell adhesion molecule 1), OPN (osteopontin), and E-Cadh (E-cadherin) [39, 40]. None of these markers have reached wide acceptance in clinical practice so far, including BRAF.

Following reports on the role that miRNAs play in PTC tumorigenesis, and thus their potential diagnostic utility, investigation also progressed to testing the prognostic ability of miRNA expression profiling. In one of the earliest studies on this subject, Gao et al. demonstrated miRNA differential expression in three subpopulations of a PTC cell line (IHH-4) with increased lymph node metastatic potency compared with the control subpopulations [41]. They found that 11 miRNAs from the array of 509 miRNAs were significantly differentially expressed among 3 pairs of subpopulations with high and low metastatic potential. Five of these miRNAs were upregulated in the metastatic subpopulations, and six were downregulated.

Three miRNAs (miR-146b, miR-221, and miR-222) consistently found to be overexpressed in PTC tissue, compared with normal thyroid tissue, appeared to also confer high-risk features such as extrathyroidal extension, lymph node metastasis, distant metastasis, recurrence, and BRAF^V600E mutation. In 100 PTC samples, Chou et al. showed that tumors with the BRAF^V600E mutation had higher miR-146b expression than those without the mutation [42]. In a follow-up study, Chou et al. reported that patients whose PTC expressed high levels of miR-146b had poorer overall survival. They further demonstrated that the BCPAP human papillary thyroid cancer cell line showed increased ability with regard to cell migration and invasion when transfected with miR-146b mimics [43]. Yip et al. demonstrated that miR-146b and miR-222 were overexpressed in aggressive PTC, defined as PTC associated with distant metastasis or recurrence. They also demonstrated downregulation of miR-34b and miR-130b in the PTC samples associated with aggressive biology [30]. Lee et al. reported that miR-146b and miR-222 were overexpressed in PTC with recurrence and were more strongly associated with PTC recurrence than BRAF expression. They postulated that the quantifiable nature of miRNA expression is more suited to the role of prognostication than the dichotomous nature of BRAF expression [44]. Zhou et al. found that overexpression of miR-221 was associated with extrathyroidal extension, lymph node metastasis, advanced disease stages III and IV, and the BRAF mutation [45].

Three miRNAs (miR-146b, miR-221, and miR-222) consistently found to be overexpressed in PTC tissue, compared with normal thyroid tissue, appeared to also confer high-risk features such as extrathyroidal extension, lymph node metastasis, distant metastasis, recurrence, and BRAF^V600E mutation.

The findings in these studies are exciting, although further refinement through larger, externally validated studies is necessary to uncover the true potential of prognosticating with miRNA expression profiling [46]. Although few studies have reported miRNA profiling as a prognostic tool in PTC risk stratification, miRNA classifiers with promising prognostic potentials have been reported consistently across a variety of other cancers. In a 2012 systematic review of 43 studies, Nair et al. reported that several miRNA classifiers provided more accurate prognostic information, for diverse cancers, than the traditional clinicopathologically based tools [46]. They also reported consistencies in the deregulation of specific miRNAs across classifiers for different cancers, leading them to postulate that some miRNA-coordinated regulatory pathways are common to many cancers. They cautioned that errors and biases can occur at every step of the appraisal of miRNAs for prognostic purposes and concluded that the clinical application of miRNA expression measurement as a biomarker for prognosis has been limited so far.

MicroRNA in Treatment

The majority of nonaggressive PTC is adequately treated by surgery, radioactive iodine ablation, and thyroid-stimulating hormone-suppressive therapy; however, a subgroup of patients present with aggressive disease that can be locally invasive, widely metastatic, or repeatedly recurrent. Disappointingly, external-beam radiotherapy and chemotherapy have shown limited success. Recent trials of tyrosine kinase inhibitors have shown promising results and indicate the need for alternative treatment options for advanced PTC [47, 48].

The therapeutic potential of miRNAs as agents of targeted therapies is currently being explored. The ability of a single miRNA to target multiple genes is of great therapeutic advantage. Simultaneous targeting of multiple components of the same pathway, leading to synergistic effects, would confer therapeutic advantage. However, achieving target organ specificity while limiting off-target effects is a major challenge in the translation from bench to bedside [49]. Although the prospect of manipulating miRNAs to complement current therapeutic strategies in cancer is appealing, the practicality is complex.

Despite the challenges, in April 2013, for the first time, a miRNA mimic reached phase I clinical study. MRX34 aimed to restore the lost tumor suppressor function of endogenous miR-34 in patients with primary liver cancer or metastatic tumors to the liver. By restoring the tumor suppressor pathway via miR-34 replacement therapy, apoptosis was induced in tumor cells in vitro and in mouse models; however, off-target effects, dosing, and pharmacokinetics were all areas that required further study [50]. Currently, there is limited research on the miRNA-targeted treatment of PTC. In a recent early stage in vitro study, Lin et al. showed that restoration of miR-101 expression in the K1 PTC cell line significantly reduced proliferation [51].