Edition 68 - 2018, June / Subject Review

Subject Review – Ed. 68

Marco A. Rivarola and Alicia Belgorosky. Servicio de Endocrinología, Hospital de Pediatría Garrahan, Buenos Aires, Argentina.

For the Subject Review Section of this issue of Endopedonline, we have selected to comment on publications supporting the impressive contribution of molecular biology to general biology, and to clinical medicine in particular. For this, we have chosen, first, two recent publications, first on the role of the microRNA transcriptome in diagnosing papillary thyroid cancer (Int. J. Mol Sci, 2017), and second, a description of a receptor-associated kinase (IRAK1) with functional roles in the inhibition of various aggressive papillary thyroid cancer (PTC) cell activities (J Clin Endocrinol Metab, 2016). Moreover, a 10-year-old publication, predicting the importance of molecular diagnoses to improve management and to avoid unnecessary surgery, in PTC, will also follow.


First Publication.

Int J Mol Sci. 2017 Mar 15;18(3), E636. doi: 10.3390/ijms18030636.

MicroRNA-146b: A Novel Biomarker and Therapeutic Target for Human Papillary Thyroid Cancer. Chou CK1,2, Liu RT3, Kang HY4,5Division of Endocrinology and Metabolism, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung City 833, Taiwan. 2 Graduate Institute of Clinical Medical Sciences, Chang Gung University, Kaohsiung City 833, Taiwan. 3 Division of Endocrinology and Metabolism, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung City 833, Taiwan. 4 Graduate Institute of Clinical Medical Sciences, Chang Gung University, Kaohsiung City 833, Taiwan. 5 Hormone Research Center and Department of Obstetrics and Gynecology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung City 833, Taiwan.


Papillary thyroid cancer (PTC) is the most common tumor subtype of thyroid cancer. However, not all PTCs are responsive to current surgical and radioiodine treatment. The well-established clinical prognostic factors include tumor size, lymph node/distal metastasis, and extrathyroidal invasion. The RET/PTC-RASBRAF linear molecular signaling cascade is known to mediate PTC pathogenesis. However, whether presence of BRAF mutation, the most common genetic alteration in PTC, can affect PTC behavior and prognosis is controversial. MicroRNAs (miRNAs) have been labeled as promising molecular prognostic markers in several tumor types. Recent studies demonstrated that microRNA-146b (miR-146b) deregulation is associated with PTC aggressiveness and prognosis. Here authors summarize the current knowledge related to the functional roles, regulated target genes, and clinical applications of miR-146b in PTC, and discuss how these studies provide insights into the key role of miR-146b as an oncogenic regulator promoting cellular transformation as well as a prognosis marker for tumor recurrence in PTC. In conjunction with the current perspectives on miRNAs in a wide variety of human cancers, this review will hopefully translate these updated findings on miR-146b into more comprehensive diagnostic or prognostic information regarding treatment in PTC patients before surgical intervention, and follow up strategies.

Extracts and Comments

“Although most of PTCs could be managed successfully with a combination of radioactive iodide and levothyroxine suppression therapy after complete surgical intervention, a certain proportion of PTCs remain irresponsive to treatment and result in comorbidity and mortality. Rearrangements in RET/PTC and NTRK and point mutations in RAS and BRAF constitutively activate the mitogen-activated protein kinase (MAPK) pathway, which are cumulatively found in over 70% PTCs. Numerous clinicopathological and molecular studies provide compelling genetic evidence that constitutively active MAPK signaling is a key component of thyrocyte transformation in PTC. The most common genetic alteration among these mutations is the BRAFV600E point mutation, which is demonstrated to be significantly associated with extrathyroidal invasion, advanced disease stages, lymph node metastasis, and tumor recurrence in PTC.”

However, the subject is controversial. “Evidence also supports the concept that the presence of the BRAF mutation may not be the fundamental event in tumorigenesis or an independent predictor of outcome. Therefore, identification of a new marker associated with BRAF-positive tumors and poor prognosis is required and may serve as a new therapeutic target and follow-up marker in PTC.

“MicroRNAs (miRNAs) are small noncoding RNA molecules that function as negative regulators of gene expression by binding to the 30-untranslated region of candidate mRNAs, and blocking the translation or degradation of target mRNAs that regulate various pathophysiological courses. Regulation of classical oncogenes and tumor suppressor genes by miRNAs was effortlessly identified as a hallmark of cancer research, transforming this class of small RNAs into potential targets for cancer therapy, diagnosis, and prognosis. Recent studies on miRNA deregulation have demonstrated increased aberrant miRNA expression (particularly, miR-222, miR-221, and miR-146b) in PTCs compared to that in normal thyroid tissues. These data indicated the distinct miRNA profile associated with PTC carcinogenesis. Furthermore, the deregulation or aberrant expression of several miRNAs in PTC has been implicated in disease development, progression, and prognosis.”

“A microRNA array study demonstrated that deregulation of microRNA-146b (miR-146b) was significantly associated with aggressive tumor behavior in BRAF-positive clinical PTC specimens. In the context of BRAF mutation and miR-146b, authors noticed that patients harboring BRAF mutations had a higher expression level of miR-146b than that in BRAF wild-type patients. Furthermore, Geraldo et al. [MicroRNA miR-146b-5p regulates signal transduction of TGF-TGF-beta by repressing SMAD4 in thyroid cancer. Oncogene 2012, 31, 1910–1922] demonstrated that transformation of BRAF mutation resulted in significant elevation of miR-146b levels. The discovery of BRAF-miRNA regulation opened a new perspective in the understanding of PTC biology.”

“MiR-146a and miR-146b are known as post-transcriptional gene silencers, which play an important role in regulating inflammatory responses. They also share identical sequences except for a difference of two nucleotides in the 3´-end and hence could translationally down-regulate similar candidate genes. Mature forms of miR-146a and miR-146b are encoded by two separate genes—MIR146A and MIR146B—localized to human chromosomes 5 and 10, respectively.”

“It is known that miRNAs can be regulated by multiple genes or factors, sequentially or simultaneously, and previous studies have identified the up-stream regulators of miR-146b. miR-146b has also been demonstrated as one of the downstream targets of nuclear factor-κB (NF-κB), and is up-regulated by toll-like receptor ligand treatment as well as in response to tumor necrosis factor-α or interleukin-1β stimulation. Furthermore, PDGF (platelet-derived growth factor) regulates the transcription of miR-146b and provides evidence for a miR-dependent feedback mechanism balancing growth factor receptor signaling in cancer cells.”

On the basis of histological features, emerging evidence indicates that dysregulated miR-146b is implicated in the different variants of PTC. PTC is a very heterogeneous group of tumors that can be further categorized into several subtypes: such as classical variant of PTC (cPTC), follicular variant of PTC (fvPTC), and tall cell variant (tcPTC). The expression level of miR-146b is higher in cPTC than fvPTC, but no significant difference was noted between cPTC and tcPTC. A similar expression level of miR-146b in infiltrative fvPTC and noninvasive follicular thyroid tumor with papillary-like nuclear features (NIFTP) was also reported recently.

Overexpression of miR-146 has also been observed in follicular thyroid carcinoma and poorly differentiated thyroid carcinoma, suggesting that this miRNA is full of potential to be further investigated for its pathogenesis roles in the malignant thyroid neoplasms.

Deng et al. [Deng, X et al. MiR-146b-5p promotes metastasis and induces epithelial-mesenchymal transition in thyroid cancer by targeting ZNRF3 (Cell Physiol. Biochem. 2015, 35, 71–82)] demonstrated the involvement of miR-146b-ZNRF3 signal transductions in epithelial-mesenchymal transition (EMT) through modulation of Wnt/beta-catenin signaling. In order to delineate the role of miR-146b in EMT by augmenting PTC cancer cell migration/invasion, they identified a regulatory mechanism linking miR-146b and its target gene IRAK1 in PTC cell lines. The function of the miR-146b-IRAK1 axis may be potentially associated with EMT by regulation of E-cadherin in PTC cancer cell lines. Authors also confirmed that miR-146b promotes aggressive tumor characteristics in PTC by suppressing IRAK1 expression and that restoration of IRAK1 expression reversed this outcome.

Second Publication

Another recent publication supports the role of IRAK1 on thyroid cancer development:

Clin Endocrinol Metab. 2016 Nov;101(11):4357-4366.

IRAK1, a Target of miR-146b, Reduces Cell Aggressiveness of Human Papillary Thyroid Carcinoma. Chou CK1Chi SY1Huang CH1Chou FF1Huang CC1Liu RT1Kang HY1. 1Division of Endocrinology and Metabolism, Department of Internal Medicine (C.-K.C., C.-H.H., R.-T.L.), Graduate Institute of Clinical Medical Sciences (C.-K.C., H.-Y.K.), Chang Gung University, Taiwan, Departments of Surgery (S.-Y.C., F.-F.C.), Pathology (C.-C.H.), and Obstetrics and Gynecology (H.-Y.K.), Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaoshiung City 833.


CONTEXT: MicroRNA (miR)-146b is overexpressed in papillary thyroid carcinoma (PTC) and is associated with extrathyroidal invasion, advanced tumor stage, and poor prognosis. However, the underlying mechanism of miR-146b in relation to its oncogenic behavior in PTC and its putative targets remain unknown. OBJECTIVE: The purpose was to investigate IL-1 receptor-associated kinase 1 (IRAK1) as the potential miR-146b target gene and its involvement in PTC. DESIGN: We used genome-wide microarray, computational analysis, and 3′ UTR reporter gene assays to identify IRAK1 as a miR-146b target gene. In vitro gain/loss-of-function experiments were further performed to determine the effects of IRAK1 on proliferation, colony formation, and wound-healing in PTC cancer cell lines. Expression levels of miR-146b and IRAK1 of 50 cases of PTC and its adjacent normal thyroid specimens were assessed via qRT-PCR. RESULTS: Microarray expression profile revealed that the mRNA level of IRAK1 gene was down-regulated by miR-146b. The 3′ UTR of IRAK1 mRNA was found to be a molecular target of miR-146b posttranscriptional repression in BCPAP cells by reporter gene assays. MiR-146b promoted the migration and proliferation of PTC cells by down-regulating IRAK1 expression, whereas restoration of IRAK1 expression reversed this effect. In addition, the expression of IRAK1 mRNA was significantly lower in PTC clinical tissue samples than normal adjacent thyroid specimens and showed a strong inverse correlation with the expression of miR-146b in PTC specimens.


Our results demonstrated that IRAK1 is a direct target of miR-146b and has functional roles to inhibit various aggressive PTC cell activities. In conjunction with current therapeutic regimens, targeting the miR-146b-IRAK1 axis may provide a potential approach for PTC management.


The fact that a single miRNA can regulate hundreds of genes may play a key role in explaining the complex mechanisms underlying tumorigenesis and the versatile tumor behaviors observed in malignancies. The dysregulation or aberrant expression of several miRNAs in PTC has been implicated in the development, progression, and prognosis of the disease. Several recent studies, have provided evidence that over- expression of miR-146b plays a critical role in PTC progression and patient prognosis. Overexpression of miR-146 has also been observed in other thyroid neoplasms such as follicular carcinoma and poorly differential thyroid carcinoma, suggesting that this miRNA and its putative target genes have a critical role in malignant thyroid neoplasms. Subsequently, It was reported that miR-146b promotes cell proliferation and invasion, and inhibits cell apoptosis in PTC cell lines Therefore, its overexpression could also be used as a predictive marker of poor prognosis in the follow up of PTC patients. Although up-regulation of miR-146b has been described in lung cancer, esophageal cancer, gastric adenocarcinoma, and bladder cancer, miR-146b is frequently down-regulated in other tumors, including breast cancer,  glioma, gallbladder cancer, and large B-cell lymphoma. Thus, these small noncoding RNAs seem to act as either oncogenes or tumor-suppressor genes depending on the tissue origin of the cancer.

MiR-146a and -146b are located on chromosomes 5 and 10, respectively. Although these two miRNAs are encoded by two different genes, they differ by only two bases and seem to function redundantly in many systems. Authors used microarray analysis and loss/gain-of-function studies in vitro in PTC cell lines, They also evaluated the correlation of a candidate target gene and miR-146b expression, in specimens from patients with PTC.

Studies were also carried out in the human thyroid cancer cell line, BCPAP, which harbors a homozygous BRAF mutation, and TPC-1, which harbors RET/ PTC1 rearrangement (provided by Professor Dumont from Belgium). A total of 131 genes were identified to be significantly down-regulated in miR-146b-overexpressing BCPAP cells, 20 genes with the most-significant expression changes were selected as potential target genes of miR-146b.

This work identified a regulatory mechanism linking miR-146b and its target gene IRAK1 in PTC. These results demonstrated that miR-146b promotes aggressive tumor characteristics in PTC by suppressing the expression of IRAK1, and that restoration of IRAK1 expression reversed this effect. Further more, IRAK1 was negatively correlated with miR-146b expression in vivo from specimens collected from PTC patients as part of a clinical study. These results provide insights into the role of miR146b-regulated target genes in PTC and suggest that anti miR-146b drugs or restoration of expression of its target gene such as IRAK1 may orchestrate antitumor immunity and act as novel intervention for developing effective immune-therapeutic strategies against PTC.

Third (old) Publication

Publication 3

The following abstract is an example of a previous seminal publication (10 years earlier) on the role of molecular diagnosis of PTC using the microRNA transcriptome:

Perspectives for improved and more accurate classification of thyroid epithelial tumors. Eszlinger M1Krohn KHauptmann SDralle HGiordano TJPaschke R.

Third Medical Department, University of Leipzig, D-04103 Leipzig, Germany. J Clin Endocrinol Metab. 2008 Sep;93(9):3286-94. doi: 10.1210/jc.2008-0201. Epub 2008 Jul 1.


CONTEXT: Histologic examination of thyroid nodules is the current standard to distinguish benign from malignant thyroid epithelial tumors and to classify histologic subtypes. This review analyzes the problems in histological differential diagnosis as well as contradictions between histology and molecular data and describes possibilities to combine histology with molecular data in an effort to more accurately classify thyroid epithelial tumors. EVIDENCE ACQUISITION: Published literature, addressing the current recommendations for thyroid tumor classification, as well as literature on the application of histology and molecular studies on the etiology of thyroid tumors is analyzed.

EVIDENCE SYNTHESIS: The current histologic criteria to classify thyroid tumors, especially follicular-patterned tumors, are hampered by considerable inter observer variability. The detection of somatic mutations via genotyping and the definition of potentially informative gene expression signatures by microarray analyses, which can distinguish cancer subtypes as well as low- and high-risk cohorts, have recently demonstrated significant diagnostic potential. Moreover, in a routine diagnostic setting, micro-RNA profiling appears most promising due to their relative stability and the high accuracy of their expression profiles. CONCLUSIONS: It is very likely that molecular definitions of thyroid tumors mentioned in the current World Health Organization classification will be further developed, leading to future progress in defining thyroid tumor types by an integrated histologic and molecular approach. These integrated classifications need to be evaluated for their specific impact on thyroid tumor diagnosis and prognosis.

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