miRNAs In Papillary Thyroid Carcinoma Research Articles

Analysis and prognostic significance of miRNAs in papillary thyroid carcinoma

Analysis and prognostic significance of miRNAs in papillary thyroid carcinoma

MicroRNA Deregulation in Papillary Thyroid Cancer and its Relationship With BRAF V600E Mutation.

MicroRNAs (miRNAs) are non-coding regulatory molecules 18-25 nucleotides in length that act as post-transcriptional regulators of gene expression. MiRNAs affect various biological processes including carcinogenesis. Deregulation of miRNAa expression has been described in a variety of tumors including papillary thyroid carcinoma (PTC). The aim of the present study was to investigate the role of selected miRNAs in PTC and find associations between miRNA expression and the BRAF (V600E) mutation. The study group comprised a total of 62 patients with surgically treated PTC. The control group consisted of 30 patients with nodular goitre that were surgically treated in the same time period. The expression status of miR-146b, miR-181a, miR-187, miR-221 and miR-222 was determined using quantitative real-time PCR. BRAF mutation analysis was performed by PCR with reverse hybridization. MiR-146b, miR-181a, miR-187, miR-221 and miR-222 were up-regulated in PTC compared to normal thyroid gland tissue of the same patient. MiR-146b, miR-187, miR-221 and miR-222 were also up-regulated in PTC compared to nodular goitre. The recurrent tumors were statistically significantly associated with up-regulation of miR-221. The mutation V600E of BRAF gene was significantly associated with up-regulation of miR-146b and with down-regulation of miR-187. Over-expression of selected miRNAs in PTC compared to normal thyroid gland tissue and nodular goitre was found. Moreover, miR-221 may serve as a prognostic marker as its over-expression was significantly associated with recurrent tumors.

The Involvement of the hsa_circ_0088494-miR-876-3p-CTNNB1/CCND1 Axis in Carcinogenesis and Progression of Papillary Thyroid Carcinoma.

Recently, growing studies have demonstrated that circular RNAs (circRNAs) function as critical players in multiple human tumors, including papillary thyroid carcinoma (PTC). However, the expression and underlying potential mechanism of circRNAs in PTC are still not fully elucidated. In this study, 14 candidate differentially expressed circRNAs (DECs) between normal thyroid tissues and benign thyroid tissues or PTC were first screened using the GSE93522 dataset by the GEO2R online tool. Then, the structural loop graphs of these 14 circRNAs were obtained through the CSCD database. After performing miRNA co-prediction by combination of CSCD and CRI databases, a potential circRNA-miRNA sub-network, consisting of 9 circRNAs and 21 miRNAs, was successfully constructed. Subsequently, the expression and prognostic values of these miRNAs were further determined by starBase, and two miRNAs, namely, miR-605-5p and miR-876-3p, were identified as key miRNAs in PTC. Then, their downstream target genes were predicted by the miRNet database. CTNNB1 and CCND1 were found to be two most potential targets of miR-876-3p by combination of multiple in silico analyses, including protein–protein interaction (PPI), hub gene screening, correlation analysis, and expression analysis. Conclusively, we established a key hsa_circ_0088494-miR-876-3p-CTNNB1/CCND1 axis linked to carcinogenesis and progression of PTC, which may provide promising therapeutic targets in treating PTC in the future.

Expressions of miRNAs in Papillary Thyroid Carcinoma and Their Associations with the BRAFV600E Mutation and Clinicopathological Features

Objectives The microRNAs (miRNAs) are known to be commonly expressed in papillary thyroid carcinoma. The BRAFV600E mutation is the most common genetic mutation in thyroid cancer. The main aim of this study was to determine the possible association between expression of the three miRNAs and that of BRAFV600E mutation and the clinicopathological features in papillary thyroid carcinoma. Methods This study was conducted on 51 paraffin-embedded tissues (42 thyroid cancer, 9 benign tumor) obtained from patients undergoing thyroidectomy at the Endocrine Center of OOO University Hospital. Results miRNAs expression was significantly high in patients with cervical lymph node metastasis and advanced TNM stage. In addition, miR-146b expression levels were significantly higher in papillary thyroid carcinoma patients with BRAFV600E mutation. The relative quantification (2-ΔΔCt) of miR-146b was also high among the miRNAs. Individually, the AUCs for miRNA-146b was 0.923 (cutoff value −1.97, sensitivity 88.9%, specificity 85.7%). Conclusions Especially, expression of miR-146b increased higher in PTC patients with BRAFV600E mutation. These findings showed a role of miR-146b as potential biomarkers in differentiating PTC from benign tumor and as a prognostic indicator of PTCs. Further investigation will need for the roles of miRNAs in the pathogenesis of papillary thyroid carcinomas.

In-depth characterization of miRNome in papillary thyroid cancer with BRAF V600E mutation

MicroRNAs (miRNAs) are small non-coding RNAs, which play a critical regulatory role in papillary thyroid carcinoma (PTC). BRAF V600E is a hotspot mutation occurring in a majority of PTC cases and is proposed to be associatedwith poor clinical outcomes. The relationship between BRAF V600E status and miRNA expression in PTC has not been comprehensively studied. In this study, we aimed to identify the differentially expressed miRNAs in PTCs with and without BRAF V600E in an unbiased manner. Five fresh frozen thyroid cancer tissues paired with their respective adjacent normal tissues from PTC patients were subjected to BRAF V600E genotyping using Sanger sequencing and small RNA deep sequencing (miRNAseq). MiRNAs differentially expressed between BRAF V600E-positive and BRAF V600E-negative PTC tissues were validated in silico using The Cancer Genome Atlas (TCGA) THCA datasets containing 420 samples. MiRNA target prediction and pathway enrichment analysis were performed to identify biological pathways altered in this cancer. We identified 174 differentially expressed miRNAs; 80 were significantly over-expressed, while 94 were underexpressed (adj. p-value < 0.1; log2 fold change ? -1 or ? 1). Fifteen miRNAs were significantly differentially expressed only in BRAF V600E-positive PTC, and eight of these were validated in TCGA THCA dataset (hsa-miR-212, -132, -135b-3p/5p, -200b, -200a-3p/5p, -27a-3p/5p, -29a and -1296). Subsequent analysis revealed significant enrichment of cancer-relatedpathways including proteoglycans in cancer, ECM-receptor interaction and MAPK pathways in BRAF V600E-positive PTC. Using the miRNAseq and in silico validation using TCGA THCA study, we identified eight miRNAs that were differentially expressed in PTC tissues with BRAF V600E. This study also complemented the existing knowledge about deregulated miRNAs in PTC development.

MiR-326 inhibits the progression of papillary thyroid carcinoma by targeting MAPK1 and ERBB4.

Papillary thyroid carcinoma (PTC) is the prevalent histotype of thyroid cancer, with increasing incidence worldwide. MicroRNAs (miRNAs) could play an important role in the development and progression of human cancers. Interestingly, miR-326 was validated as one of the downregulated miRNAs in PTC. Therefore, it is necessary to research the function of miR-326 involved in the progression of PTC. In the current study, we detected the downregulation of miR-326 in PTC tissues and cell lines. The miR-326 overexpression or knockdown was conducted in TPC-1 or HTh83 PTC cells. miR-326 mimics decreased the proliferation, clone formation ability and caused G1-phase accumulation. In addition, the reduction of migration and invasion abilities was induced by miR-326 mimics. Western blot analysis showed that the cells with miR-326 mimics exhibited the inhibition of vimentin and N-cadherin, as well as enhancement of E-cadherin. Importantly, miR-326 could directly target mitogen activated protein kinase 1 (MAPK1) and epidermal growth factor receptor 4 (ERBB4). MAPK1 or ERBB4 overexpression rescued the effects of miR-326 on proliferation, migration, and invasion in PTC cells. Notably, miR-326 reduced tumorigenesis in vivo, including the decrease of tumor volume and weight, suppression of Ki-67, N-cadherin, MAPK1 and ERBB4. In all, these results might provide a new therapeutic target for the diagnosis of PTC.

Identification of key miRNAs in papillary thyroid carcinoma based on data mining and bioinformatics methods.

MicroRNAs (miRNAs) are a class of short (approximately 22 nucleotides), non-coding and endogenous RNA molecules that play pivotal roles in the occurrence and development of cancer. The present study aimed to investigate key miRNAs involved in papillary thyroid carcinoma (PTC). Two independent datasets (GSE73182 and GSE113629) were obtained from the GEO database. The differentially expressed miRNAs (DEmiRNAs) between PTC tissues and normal thyroid tissues were analyzed by GEO2R with the Limma R package. Key miRNAs in PTC were identified by the VennDiagram R package. The targets of the key miRNAs were predicted by miRWalk and were functionally enriched by clusterProfiler R package. Five miRNAs including hsa-miR-146b-5p, hsa-miR-15a-5p, hsa-miR-21-5p, hsa-miR-221-3p and hsa-miR-222-3p were identified as key miRNAs in PTC. The expression levels of these key miRNAs were upregulated in PTC. This finding was also confirmed in the other dataset. Target prediction of miRNAs indicated that hsa-miR-146b-5p, hsa-miR-15a-5p, hsa-miR-21-5p, hsa-miR-221-3p and hsa-miR-222-3p exhibited 2, 41, 3, 14 and 8 target genes, respectively. Enrichment analysis indicated that these key miRNAs were mainly involved in nine biological processes, such as regulation of MAP kinase activity, JNK cascade signaling and regulation of protein serine/threonine kinase activity) and in 28 pathways, including the mitogen associated protein kinase, the sphingolipid, ErbB, Ras and the C-type lectin receptor signaling pathways. In conclusion, the present study identified several key miRNAs in PTC, which serve as potential targets for PTC diagnosis and treatment.

MiR-222 expression is correlated with the ATA risk stratifications in papillary thyroid carcinomas.

Background:miR-222 is one of the most consistently overexpressed miRNAs in papillary thyroid carcinoma (PTC). Previous studies demonstrated that miR-222 overexpression conferred high-risk features in PTC patients, suggesting its value in risk-stratification. However, studies in term of miR-222's utility on stratifying PTCs are lacking.Methods:One hundred patients including 10 with multinodular goiter and 90 with PTC were enrolled. Formalin-fixed paraffin-embedded samples were exploited for miR-222 quantitative reverse transcriptase- polymerase chain reaction (RT-PCR) analysis. Correlations between miR-222 expression and different clinicopathological features, Tumor-node-metastasis (TNM) staging and ATA risk level were analyzed.Results:miR-222 expression of the PTC group was significantly higher than that of the goiter group (P < .001). Furthermore, miR-222 expression was significantly higher in PTCs with advanced features like larger tumor, capsular invasion, vascular invasion and lymph nodes metastasis. The majority of patients (61%) were in stage I group (similar to ATA low-risk) by TNM staging system. As to the ATA system, the majority (73%) were in intermediate-risk group (similar to TNM stage II and III roughly). Contrary to previous report, here we found that miR-222 expression was correlated with the ATA risk level (P < .001), but not with the TNM staging (P = .122).Conclusion:In the present study, we demonstrated that miR-222 overexpression was correlated with advanced features like capsular invasion, vascular invasion, larger tumor size and lymph node metastasis in PTCs. Most importantly, miR-222 expression was correlated with ATA risk levels, suggesting its potential value in PTC risk-stratification.

DNA methylation-mediated silencing of miR-204 is a potential prognostic marker for papillary thyroid carcinoma.

BackgroundPapillary thyroid carcinoma (PTC) is the most common endocrine malignancy and its incidence has increased over the last few decades. The molecular mechanisms underlying PTC tumorigenesis and progression are still unclear.Patients and methodsThe microRNA (miRNA) expression patterns of PTC were revealed by miRNA microarray analysis and validated with The Cancer Genome Atlas (TCGA) data. Promoter DNA methylation rates of miR-204 were analyzed by Agena Methylation MassAR-RAY analysis and validated with TCGA data. The underlying molecular mechanisms of miR-204 involved in PTC were studied by bioinformatics analyses.ResultsA total of 181 differentially expressed miRNAs (89 downregulated and 92 upregulated miRNAs) between PTC and normal tissues were detected in this study. We identified miR-204 as one of the most significantly downregulated miRNAs in PTC. Downregulation of miR-204 was related to PTC extrathyroidal extension, high T-stage, lymph metastasis, BRAF V600E mutation, and aggressive tall cell variant. The Agena MassARRAY results indicated that 12 CpG sites located at the promoter of miR-204 were hypermethylated in PTC tissues compared to normal tissues. The promoter methylation rates of miR-204 in PTC were negatively correlated with the expression levels of miR-204 and its host gene TRPM3. Downregulated miR-204 expression was related to several important pathways and mechanisms involved in tumorigenesis and progression.ConclusionPromoter DNA methylation-silenced miR-204 could serve as a potential diagnostic biomarker of PTC. Downregulation of miR-204 may play an important role in PTC via its involvement in many tumor-related pathways. Novel target genes and putative mechanisms of miR-204 in PTC need to be further validated.

MicroRNA‑509 targets PAX6 to inhibit cell proliferation and invasion in papillary thyroid carcinoma.

MicroRNAs (miRNAs/miRs) negatively regulate the expression of numerous genes and therefore contribute to the occurrence and development of papillary thyroid carcinoma (PTC). Hence, further investigation into the specific roles of miRNAs in PTC is valuable for developing effective therapeutic methods for patients with this disease. MiRNA‑509 is dysregulated and serves pivotal roles in several types of human cancer; however, the expression and roles of miR‑509 in PTC and its underlying mechanism require further investigation. In the present study, the expression of miR‑509 in PTC tissues and cell lines was detected and the specific functions of miR‑509 in the progression of PTC were investigated. Additionally, the molecular mechanisms underlying the action of miR‑509 in PTC were determined. The present study demonstrated that miR‑509 was significantly downregulated in PTC tissues and cell lines. MiR‑509 upregulation inhibited the PTC cell proliferation and invasion. Mechanistically, paired box 6 (PAX6) was identified as a novel target of miR‑509 in PTC cells. In clinical PTC samples, miR‑509 was significantly overexpressed and inversely correlated with PAX6 expression. PAX6 restoration effectively reversed the inhibitory effects of miR‑509 overexpression on PTC cell proliferation and invasion. These results demonstrated that miR‑509 may act as a tumor suppressor in PTC by directly targeting PAX6. Thus, miR‑509 may be a potential therapeutic target for the treatment of patients with PTC.

Expressions of miRNAs in papillary thyroid carcinoma and their associations with the clinical characteristics of PTC.

The sensitivity and specificity of biomarkers which have been used in clinical practice for diagnosis of papillary thyroid carcinoma (PTC) are low, it is essential to develop novel diagnostic and prognostic biomarkers for PTC. To explore the expressions of miR-940, miR-15a, miR-16 and IL-23 in PTC tissues and plasma and their associations with the clinical characteristics of PTC. We investigated the expressions of miR-940, miR-15a, miR-16 and IL-23 in plasma and thyroid tissues of PTC, nodular goiter and healthy people with qRT-PCR, and further analyzed the associations between their levels and the clinical characteristics of PTC. Level of IL-23 expression was higher while levels of miR-940, miR-15a and miR-16 expression in the PTC tissues were lower compared with the nodular goiter tissues and perineoplastic thyroid tissues. And the levels of miR-940, miR-15a, miR-16 and IL-23 expression in the PTC tissues were associated with some clinical characteristics of PTC, including bilateral tumor, multicentricity, extrallyroidal invasion, cervical lymph node metastasis, distant metastasis and clinical advanced stages (III/IV). Expressions of miR-940, miR-15a, miR-16 and IL-23 in PTC tissues might be useful biomarkers and promising targets in the diagnosis of papillary thyroid carcinoma.

Circulating microRNA124-3p, microRNA9-3p and microRNA196b-5p may be potential signatures for differential diagnosis of thyroid nodules.

It is important to develop an effective auxiliary approach to distinguish papillary thyroid carcinoma (PTC) from benign nodules because a considerable proportion cannot be identified by fine-needle aspiration cytology at present, resulting in unnecessary thyroidectomy. Circulating miRNAs are potential biomarkers for differential diagnosis of tumors. We aimed to investigate the dysregulation of circulating miRNAs in PTC and evaluate the diagnostic value for differentiation of PTC from benign nodules. We first assessed the expression of miRNAs in patients with PTC, patients with benign nodules and healthy controls using a miRCURY LNA Array (n = 3 for each group). Expression of circulating miR-124-3p, miR-9-3p and miR-5691 was significantly up-regulated, while miR-4701 and miR-196b-5p were down-regulated in PTC patients. The dysregulation of miR-124-3p, miR-9-3p, miR-4701 and miR-196b-5p was further validated by qRT-PCR in fifty participants from each group. The expression of circulating miR-124-3p and miR-9-3p was significantly up-regulated in PTC patients. Both miR-124-3p and miR-9-3p could distinguish PTC from benign nodules with high sensitivity and specificity. There were no significant differences in the expression of circulating miR-4701 and miR-196b-5p between PTC patients and healthy controls. Nevertheless, patients with benign nodules showed a higher level of miR-196b-5p compared with that of PTC patients and healthy controls. ROC analysis indicated that miR-196b-5p had a good diagnostic value for differentiation of benign nodules from PTC. Our study suggested that miR-124-3p, miR-9-3p and miR-196b-5p may be potential signatures for differential diagnosis of thyroid nodules in eastern coastal areas of China.

Expressions of miRNAs in papillary thyroid carcinoma and their associations with the BRAFV600E mutation

Alterations in microRNA (miRNA) expression have been described in thyroid tumors, suggesting a role for miRNAs in thyroid carcinogenesis. BRAF(V600E) is the most frequently identified genetic alteration in papillary thyroid carcinoma (PTC). We investigated the link between BRAF(V600E) status and the expression of miRNAs in PTC and analyzed the associations of these factors with clinicopathological characteristics. Prospective study of patients who underwent thyroid surgery between October 8, 2008 and November 1, 2010. BRAF(V600E) status was determined by mutant allele-specific amplification PCR and direct sequencing of exon 15 of the BRAF gene in 69 PTC tissues and 69 respective paracancerous normal thyroid tissues. Initially, miRNA expression was analyzed in 12 PTC tissues and three associated paracancerous tissues using a miRNA microarray. miRNAs differentially expressed between BRAF(V600E)-positive and -negative PTC tissues were then validated by real-time quantitative PCR on 69 PTC tissues and 69 paracancerous tissues. We also explored the associations between BRAF(V600E) status or differential miRNA expression and clinicopathological characteristics. The mutation rate of BRAF(V600E) in PTC was 47.8%. Twelve miRNAs were upregulated and six were downregulated in PTC tissues, among which miR-15a, 15a*, 34a*, 34b*, 551b, 873, 876-3p, and 1274a were first identified. miR-21* and 203 were significantly dysregulated (P<0.05) in PTC tissues with BRAF(V600E). Additionally, there were significant associations (P<0.05) between BRAF(V600E) and a higher tumor-node-metastasis staging (III/IV), and between miR-21* over-expression and lymph node metastasis. We identified two miRNAs that are differentially expressed in PTC tissues with BRAF(V600E) and revealed their associations with clinicopathological features. These findings may lead to the development of a potential diagnostic biomarker or prognostic indicator of PTC.

MicroRNA miR-146b-5p regulates signal transduction of TGF-β by repressing SMAD4 in thyroid cancer

MicroRNAs (miRNA) are small non-coding RNAs involved in post-transcriptional gene regulation that have crucial roles in several types of tumors, including papillary thyroid carcinoma (PTC). miR-146b-5p is overexpressed in PTCs and is regarded as a relevant diagnostic marker for this type of cancer. A computational search revealed that miR-146b-5p putatively binds to the 3' untranslated region (UTR) of SMAD4, an important member of the transforming growth factor β (TGF-β) signaling pathway. The TGF-β pathway is a negative regulator of thyroid follicular cell growth, and the mechanism by which thyroid cancer cells evade its inhibitory signal remains unclear. We questioned whether the modulation of the TGF-β pathway by miR-146b-5p can contribute to thyroid tumorigenesis. Luciferase reporter assay confirmed the direct binding of miR-146b-5p on the SMAD4 3'UTR. Specific inhibition of miR-146b-5p with a locked nucleic acid-modified anti-miR-146b oligonucleotide significantly increased SMAD4 levels in the human papillary carcinoma cell lines, TPC-1 and BCPAP. Moreover, suppression of miR-146b-5p increased the cellular response to the TGF-β anti-proliferative signal, significantly decreasing the proliferation rate. The overexpression of miR-146b-5p in normal rat follicular PCCL3 cells decreased SMAD4 levels and disrupted TGF-β signal transduction. MiR-146b-5p overexpression in PCCL3 cells also significantly increased cell proliferation in the absence of thyroid-stimulating hormone and conferred resistance to TGF-β-mediated cell-cycle arrest. Additionally, the activation of thyroid most common oncogenes RET/PTC3 and BRAF in PCCL3 cells upregulated miR-146b-5p expression. Our results confirm the oncogenic role of miR-146b-5p in thyroid follicular cells and contribute to knowledge regarding the modulation of TGF-β signal transduction by miRNAs in PTCs.

MiR-146b is Highly Expressed in Adult Papillary Thyroid Carcinomas with High Risk Features Including Extrathyroidal Invasion and the BRAFV600E Mutation

Papillary thyroid carcinoma (PTC) are clinicopathogenetically heterogeneous. Micro-RNAs (miRNAs) are involved in the pathogenesis of diverse human cancers, including PTC. Information regarding associations between clinicopathological features of PTC with the expression of specific miRNAs, however, is sparse. In this study, we compared expression of deregulated miRNAs in PTCs to assess this was associated with selected clinicopathogenetic features. We analyzed the expression levels of three reported deregulated miRNAs (miR-221, miR-222, and miR-146b) using quantitative real-time polymerase chain reaction in 100 cases of PTCs with distinct clinicopathogenetic characteristics and 16 paired normal controls. The tumor samples were categorized into low- and high-risk groups on the basis of the tumor-node-metastasis staging system. The expression levels of miR-221, miR-222, and miR-146b were significantly associated with extrathyroidal invasion (p = 0.013, 0.05, and 0.003, respectively). The expression levels of miR-221 and miR-146b were significantly higher in the high-risk PTC group (p = 0.01 and 0.042, respectively). The miR-146b expression levels in PTCs with BRAF mutation were significantly higher than those without this mutation (p < 0.0001). There were no other associations between the expression of these miRNAs and other clinicopathological parameters. Our results show the potential importance of miR-221, miR-222, and miR-146b in determining the aggressive properties of PTCs and highlight the need to identify the gene targets of these miRNAs.

Lack of Correlation betweenBRAFV600E Mutational Status and the Expression Profile of a Distinct Set of miRNAs in Papillary Thyroid Carcinoma

Recent studies demonstrated a significant upregulation of distinct microRNAs (miRNAs), small endogenous RNAs that regulate gene expression, in papillary thyroid carcinoma (PTC). In the pathogenesis of PTC the T1799A (V600E) BRAF mutation is the most common genetic alteration leading to a constitutive activation of the MAPK pathway. The aim of the present study was to elucidate a possible correlation between BRAF mutational status and a distinct miRNA expression profile. In a series of 221 PTC we determined the BRAF V600E mutational status using DNA-sequencing and correlated the occurrence of the mutation with a variety of clinicopathologcial data. The miRNA expression profile of five selected subtypes (miRNA-146b, -181b, -21, -221, -222) in two matched cohorts of BRAF positive (n=28) and wildtype cases (n=26) was examined by RT-PCR TaqMan miRNA assay. The BRAF V600E mutation was significantly found in PTCs with extrathyroidal extension (p <0.001). Among them, V600E was even significantly associated with smaller tumour size of 1 cm or less (microcarcinomas; p<0.003) and the follicular (p=0.017) and tall cell variant (p=0.015). By calculating relative changes in miRNA gene expression no differences in fold changes could be detected between BRAF positive and wildtype PTC suggesting that BRAF has no regulatory influence on the expression of the five examined miRNAs. However, our study confirmed the diagnostic utility of this distinct set of miRNAs to detect PTC by significant fold changes in at least 3 miRNAs (miRNA-146b, -221, -222) irrespective of its histological variant.

Differential Expression of miRNAs in Papillary Thyroid Carcinoma Compared to Multinodular Goiter Using Formalin Fixed Paraffin Embedded Tissues

microRNAs (miRNAs) are approximately 22 nt RNAs that negatively regulate target gene expression. Their dysregulation has been implicated in the pathogenesis of a number of human cancers, including papillary thyroid carcinoma (PTC). Whereas previous studies using microarray technologies have largely relied on the ability to procure fresh tissue at the time of surgery to characterize miRNA signatures in PTC, we exploited the ability to procure sufficient miRNA from formalin-fixed paraffin-embedded (FFPE) tissue to describe a series of miRNAs whose expression is dysregulated in PTC compared to benign proliferative multinodular goiter (MNG). We identified 13 miRNAs upregulated and 26 miRNAs downregulated in PTC versus MNG. These include miRNA-21, miRNA-31, miRNA-221, and miRNA-222. Their dysregulation was further validated by real time RT-PCR analysis in an independent set of FFPE tissues. Many of these have previously been described in fresh tissue studies as altered in PTC, confirming the utility of this approach. These results further highlight the applicability of miRNA expression patterns as potential markers of human cancer, and our results suggest that FFPE tissues are suitable resources for such miRNA expression analyses. The ability to utilize FFPE tissue in the molecular characterization of human malignancy will unlock a rich resource for future cancer studies.