Papillary Thyroid Carcinoma Tissue Samples Research Articles

Descriptive Analysis of Common Fusion Mutations in Papillary Thyroid Carcinoma in Hungary

Thyroid cancer is the most common type of endocrine malignancy. Papillary thyroid carcinoma (PTC) is its predominant subtype, which is responsible for the vast majority of cases. It is true that PTC is a malignant tumor with a very good prognosis due to effective primary therapeutic approaches such as thyroidectomy and radioiodine (RAI) therapy. However, we are often required to indicate second-line treatments to eradicate the tumor properly. In these scenarios, molecular therapies are promising alternatives, especially if specifically targetable mutations are present. Many of these targetable gene alterations originate from gene fusions, which can be found using molecular diagnostics like next-generation sequencing (NGS). Nonetheless, molecular profiling is far from being a routine procedure in the initial phase of PTC diagnostics. As a result, the mutation status, except for BRAF V600E mutation, is not included in risk classification algorithms either. This study aims to provide a comprehensive analysis of fusion mutations in PTC and their associations with clinicopathological variables in order to underscore certain clinical settings when molecular diagnostics should be considered earlier, and to demonstrate yet unknown molecular–clinicopathological connections. We conducted a retrospective fusion mutation screening in formalin-fixed paraffin-embedded (FFPE) PTC tissue samples of 100 patients. After quality evaluation by an expert pathologist, RNA isolation was performed, and then NGS was applied to detect 23 relevant gene fusions in the tumor samples. Clinicopathological data were collected from medical and histological records. To obtain the most associations from the multivariate dataset, we used the d-correlation method for our principal component analysis (PCA). Further statistical analyses, including Chi-square tests and logistic regressions, were performed to identify additional significant correlations within certain subsets of the data. Fusion mutations were identified in 27% of the PTC samples, involving nine distinct genes: RET, NTRK3, CCDC6, ETV6, MET, ALK, NCOA4, EML4, and SQSTM1. RET and CCDC6 fusions were associated with type of thyroidectomy, RAI therapy, smaller tumor size, and history of Hashimoto’s disease. NCOA4 fusion correlated with sex, multifocality, microcarcinoma character, history of goiter, and obstructive pulmonary disease. EML4 fusion was also linked with surgical procedure type and smaller tumor size, as well as the history of hypothyroidism. SQSTM1 fusion was associated with multifocality and a medical history of thyroid/parathyroid adenoma. NTRK3 and ETV6 fusions showed significant associations with Hashimoto’s disease, and ETV6, also with endometriosis. Moreover, fusion mutations were linked to younger age at the time of diagnosis, particularly the fusion of ETV6. The frequent occurrence of fusion mutations and their associations with certain clinicopathological metrics highlight the importance of integrating molecular profiling into routine PTC management. Early detection of fusion mutations can inform surgical decisions and therapeutic strategies, potentially improving clinical outcomes.

MicroRNA Profiling in Papillary Thyroid Cancer.

Genetic alterations are well known to be related to the pathogenesis and prognosis of papillary thyroid carcinoma (PTC). Some miRNA expression dysregulations have previously been described in the context of cancer development including thyroid carcinoma. In our study, we performed original molecular diagnostics on tissue samples related to our own patients. We aimed to identify all dysregulated miRNAs in potential association with PTC development via sequencing much higher numbers of control-matched PTC tissue samples and analyzing a wider variety of miRNA types than previous studies. We analyzed the expression levels of 2656 different human miRNAs in the context of 236 thyroid tissue samples (118 tumor and control pairs) related to anonymized PTC cases. Also, KEGG pathway enrichment analysis and GO framework analysis were used to establish the links between miRNA dysregulation and certain biological processes, pathways of signaling, molecular functions, and cellular components. A total of 30 significant differential miRNA expressions with at least ±1 log2 fold change were found related to PTC including, e.g., miR-551b, miR-146b, miR-221, miR-222, and miR-375, among others, being highly upregulated, as well as miR-873 and miR-204 being downregulated. In addition, we identified miRNA patterns in vast databases (KEGG and GO) closely similar to that of PTC including, e.g., miRNA patterns of prostate cancer, HTLV infection, HIF-1 signaling, cellular responses to growth factor stimulus and organic substance, and negative regulation of gene expression. We also found 352 potential associations between certain miRNA expressions and states of clinicopathological variables. Our findings-supported by the largest case number of original matched-control PTC-miRNA relation research-suggest a distinct miRNA expression profile in PTC that could contribute to a deeper understanding of the underlying molecular mechanisms promoting the pathogenesis of the disease. Moreover, significant miRNA expression deviations and their signaling pathways in PTC presented in our study may serve as potential biomarkers for PTC diagnosis and prognosis or even therapeutic targets in the future.

Decreased sirtuin 4 levels promote cellular proliferation and invasion in papillary thyroid carcinoma.

This study examined the effect of sirtuin 4 (SIRT4), a NAD+-dependent deacetylase, on the proliferation and progression of papillary thyroid carcinoma (PTC). Data from The Cancer Genome Atlas (TCGA) were analyzed to identify SIRT4 expression in thyroid cancer. Subsequently, the correlation between SIRT4 expression and clinical characteristics was examined in 205 PTC tissue samples. In vitro assays using three human thyroid cancer cell lines (B-CPAP, TPC-1, and SNU-790) were conducted to assess the effects of regulated SIRT4 expression on cell growth, apoptosis, invasion, and migration. Furthermore, in vivo experiments were performed in a xenograft mouse model. Gene Expression Omnibus (GEO) and TCGA data indicated that SIRT4 expression is lower in thyroid cancer and SIRT4 downregulation is associated with poor overall survival. In PTC tissues, positive SIRT4 expression was associated with decreased extracapsular extension. In in vitro experiments using three human thyroid cancer cell lines, overexpression of SIRT4 decreased cell survival, clonogenic potential, and invasion and migratory capabilities, as well as inducing apoptosis and increasing reactive oxygen species levels. SIRT4 overexpression upregulated E-cadherin and downregulated N-cadherin, suggesting its potential involvement in the regulation of epithelial-mesenchymal transition. These findings were confirmed in vivo using a xenograft mouse model. This study provides novel insight into the potential contribution of SIRT4 to the regulation of the pathological progression of PTC. The data suggest that SIRT4 plays a tumor-suppressive role in PTC by inhibiting growth, survival, and invasive potential. Future research should investigate the molecular mechanisms underlying these effects of SIRT4.

GLUT1 and ASCT2 Protein Expression in Papillary Thyroid Carcinoma Patients and Relation to Hepatitis C Virus: A Propensity-Score Matched Analysis.

PurposeRecently, glucose and amino acid transporters have gradually become a hot topic in thyroid gland biology and cancer research. We aimed to investigate the expressions of glucose transporter 1 (GLUT1) and glutamine transporter 2 (ASCT2) in papillary thyroid carcinoma (PTC) and their clinical significance and relation to HCV-related hepatitis.Patients and MethodsScreening 202 TC tissue samples against the selection criteria using a propensity-score matched analysis to adjust for age, sex, side of tumor, histopathological variants, TNM staging system, and the positivity for HCV yielded 51 matched (17 HCV positive and 34 HCV negative) PTC samples. The expressions of GLUT1 and ASCT2 expressions were detected by immunohistochemical staining. Kaplan–Meier survival curves were generated for disease-free and overall survival, and multivariate Cox regression analysis was applied to identify predictors for mortality.ResultsOf 51 thyroid cancer tissues, 85% showed positive GLUT1 cytoplasmic staining, and 26% had a high expression score. All thyroid cancer specimens demonstrated ASCT2 cytoplasmic staining with membranous accentuation. Of these, 78% showed a high expression score, and 22% showed weak staining. On stratifying the study cohort based on the HCV status, HCV negative cohort showed a significantly higher immunoreactivity score for GLUT1 (p = 0.004) but not ASCT2 (p = 0.94) than HCV positive group. The expressions of the studied transporters showed no significant associations with the prognostic features of PTC nor the disease-free/overall survival.ConclusionGLUT1 and ASCT2 immunohistochemical staining showed positive expression with variable intensity in nearly 85% and 100% of PTC tissue samples compared to normal ones, respectively. Furthermore, GLUT1 protein expression, not ASCT2, showed a higher immunoreactivity score in PTC patients who are negative for HCV than cancer patients with positive HCV. Meanwhile, the expression of both protein markers was not associated with the clinicopathological characteristics of the studied PTC patients. Further large-scale multicenter studies are recommended to validate the present findings.

Identification of prognostic biomarkers for papillary thyroid carcinoma by a weighted gene co-expression network analysis.

AimWhole transcriptome analysis was conducted to identify differentially expressed RNAs and regulatory networks associated with papillary thyroid carcinoma (PTC).MethodsA weighted gene co‐expression network analysis based on high‐throughput sequencing data for six pairs of PTC and adjacent tissue samples was conducted to understand the biological functions and regulatory networks involving long non‐coding RNAs (lncRNAs), circular RNAs (circRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs).ResultsWe detected 131, 338, 31, and 556 differentially expressed circRNAs, lncRNAs, miRNAs, and mRNAs, respectively. We identified modules that were significantly positively and negatively related to cancer and lymph node metastasis. Gray and turquoise modules were positively correlated with cancer phenotypes (p < 0.05), whereas yellow, brown, and blue modules were negatively correlated with cancer (p < 0.05). Gray module was positively correlated with lateral lymph node metastasis (p = 0.02). Kaplan–Meier analyses revealed that the levels of transmembrane protein 63C (TMEM63C), lysyl oxidase‐like 1 (LOXL1), collagen type V alpha 1 chain (COL5A1), ADAM metalloproteinase with thrombospondin type I motif 2 (ADAMTS2), and LysM‐domain containing 3 (LYSMD3) were significantly associated with overall survival (p < 0.05). Significant increase in the expression of COL5A1 and LOXL1 in tumor tissues was validated by quantitative real‐time polymerase chain reaction (p < 0.05). COL5A1 and LOXL1 promoted PTC cell growth and invasion in vitro.ConclusionsWe identified COL5A1 and LOXL1 as potential prognostic biomarkers, providing new insights into the occurrence and progression of PTC.

Long Non-Coding RNA NLIPMT as a Tumor Suppressor and Inhibitor of Cell Proliferation and Metastasis in Papillary Thyroid Carcinoma.

IntroductionGene expression association studies of tumor samples have uncovered several long non-coding RNAs (lncRNAs) closely related to various types of cancer. Several lncRNAs have been reported to play essential roles in the progression of papillary thyroid carcinoma (PTC). Novel lncRNA inhibiting proliferation and metastasis (lnc-NLIPMT) is a known regulator of mammary cell proliferation and motility, but its involvement in PTC is unclear.Materials and MethodsWe investigated the role of lnc-NLIPMT in PTC by quantitative real-time polymerase chain reaction (qRT-PCR) on various PTC tissue samples and cell lines. We assessed the effects of overexpression or knockdown of lnc-NLIPMT on the proliferation, migration, and invasion of PTC cells using CCK-8, cell clone formation, and transwell assays. Changes in the expression of N-cadherin and vimentin were detected by immunoblotting.ResultsOur results revealed a downregulation of the expression of lnc-NLIPMT in PTC and a negative correlation between lnc-NLIPMT expression and tumor size (P=0.006). Overexpression of lnc-NLIPMT in TPC-1 and B-CPAP cells significantly suppressed cell proliferation, migration, and invasion, while lnc-NLIPMT knockdown had the opposite effect. In addition, lnc-NLIPMT played an important role in the regulation of the expression of N-cadherin and vimentin.Conclusionlnc-NLIPMT inhibits cell proliferation and metastasis of PTC cells and is a potential diagnostic and prognostic biomarker in PTC.

Potential five-mRNA signature model for the prediction of prognosis in patients with papillary thyroid carcinoma.

Although the mortality rate of papillary thyroid carcinoma (PTC) is relatively low, the recurrence rates of PTC remain high. The high recurrence rates are related to the difficulties in treatment. Gene expression profiles has provided novel insights into potential therapeutic targets and molecular biomarkers of PTC. The aim of the present study was to identify mRNA signatures which may categorize PTCs into high-and low-risk subgroups and aid with the predictions for prognoses. The mRNA expression profiles of PTC and normal thyroid tissue samples were obtained from The Cancer Genome Atlas (TCGA) database. Differentially expressed mRNAs were identified using the ‘EdgeR’ software package. Gene signatures associated with the overall survival of PTC were selected, and enrichment analysis was performed to explore the biological pathways and functions of the prognostic mRNAs using the Database for Visualization, Annotation and Integration Discovery. A signature model was established to investigate a specific and robust risk stratification for PTC. A total of 1,085 differentially expressed mRNAs were identified between the PTC and normal thyroid tissue samples. Among them, 361 mRNAs were associated with overall survival (P<0.05). A 5-mRNA prognostic signature for PTC (ADRA1B, RIPPLY3, PCOLCE, TEKT1 and SALL3) was identified to classify the patients into high-and low-risk subgroups. These prognostic mRNAs were enriched in Gene Ontology terms such as ‘calcium ion binding’, ‘enzyme inhibitor activity’, ‘carbohydrate binding’, ‘transcriptional activator activity’, ‘RNA polymerase II core promoter proximal region sequence-specific binding’ and ‘glutathione transferase activity’, and Kyoto Encyclopedia of Genes and Genomes signaling pathways such as ‘pertussis’, ‘ascorbate and aldarate metabolism’, ‘systemic lupus erythematosus’, ‘drug metabolism-cytochrome P450 and ‘complement and coagulation cascades’. The 5-mRNA signature model may be useful during consultations with patients with PTC to improve the prediction of their prognosis. In addition, the prognostic signature identified in the present study may reveal novel therapeutic targets for patients with PTC.

Lysine-Specific Demethylase 1 Affects the Progression of Papillary Thyroid Carcinoma via HIF1α and microRNA-146a.

Lysine-specific demethylase 1 (LSD1) stabilizes hypoxia-inducible factor 1α (HIF1α) to advance tumor progression, while HIF1α functions as a transcription factor to increase the expression of microRNA-146a (miR-146a). We aim to investigate whether LSD1 affects the development of papillary thyroid carcinoma (PTC) via HIF1α and miR-146a. In vitro assays were performed with Nthy-ori 3-1, BHP5-16, BCPAP, K1, and BHP2-7 cell lines. In vivo assays were conducted with established xenograft tumors in nude mice. This study was conducted at our lab. PTC tissues and corresponding adjacent normal tissues were obtained from 45 patients hospitalized in Sun Yat-Sen Memorial Hospital. Assays were conducted using Nthy-ori 3-1, BHP5-16, BCPAP, K1, and BHP2-7 cell lines, as well as 50 male BALB/c nude mice. Cells were transfected with sh-LSD1, sh-GABPA, oe-LSD1, oe-HIF1α, miR-146a mimic, and miR-146a inhibitor. In addition, K1 cells expressing lv-oe-LSD1, lv-miR-146a inhibitor, lv-oe-LSD1 or miR-146a inhibitor were injected into the right side of the mice. LSD1 gene and protein expression patterns were analyzed in 45 clinical PTC tissue samples. Expression of LSD1, HIF1α, miR-146a, and GA-binding protein transcription factor alpha (GABPA), as well as their effects on PTC. LSD1 was highly expressed in clinical PTC tissues. LSD1 stabilized HIF1α and inhibited the degradation of its ubiquitin proteasome. HIF1α was enriched in the promoter region of miR-146a, an upregulated miRNA in PTC. HIF1α increased miR-146a expression to promote PTC progression in vitro, which was achieved by inhibiting GABPA, a target gene of miR-146a. LSD1 upregulated miR-146a to enhance the development and metastasis of PTC in nude mice. Our results show that LSD1 functions as an oncogene in PTC by upregulating HIF1α and miR-146a, elucidating an understanding of undefined mechanisms associated with tumor progression in PTC.

Hypermethylated RASSF1 and SLC5A8 promoters alongside BRAFV600E mutation as biomarkers for papillary thyroid carcinoma.

Circulating cell-free DNA (cfDNA) has been considered as a diagnostic source to track genetic and epigenetic alterations in cancer. We aimed to study mutation in addition to the methylation status in the promoter regions of RASSF1 and SLC5A8 genes in tissues and circulating free DNA samples of patients affected with papillary thyroid carcinoma (PTC) and thyroid nodules as controls. BRAFV600E mutation was studied by ARMS-scorpion real-time polymerase chain reaction method in 57 PTC and 45 thyroid nodule cases. Methylation status of RASSF1 and SLC5A8 promoter regions was analyzed by methylation-specific high-resolution melting curve analysis. BRAFV600E mutation was found in 39 (68.4%) out of 57 PTC tissue samples, while in 33 (49.1%) cases of cfDNA, this mutation was detected. The frequency of BRAFV600E mutation in cfDNA was significantly different between metastatic and nonmetastatic PTC cases (22 of 33 PTC cases vs. 5 of 34 thyroid nodule samples). Methylation levels of three promoter regions of SLC5A8 and proximal promoter region of RASSF1 was significantly different between PTC and thyroid nodule cases in both cfDNA and tissue DNA. In addition, the methylation status of these two genes in tissue DNA was reflected in methylation status observed in cfDNA. This study confirmed that BRAFV600E mutation is better for discrimination between papillary thyroid carcinoma and thyroid nodules. On the other hand, hypermethylation in the more proximal promoter regions to RASSF1 and SLC5A8 genes showed higher sensitivity and more acceptable specificity for this discrimination.

Coexistence of BRAFV600E mutation and EGFR overexpression is highly associated with adverse clinicopathological features of papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) generally has a good prognosis, but in a subset of patients it progresses to aggressive forms. Analysis of molecular alterations in relation to clinical phenotype may help in risk stratification of patients by predicting tumor aggressiveness. We analyzed the expression profiles of epidermal growth factor receptor (EGFR) using immunohistochemistry and the presence of BRAF(V600E) mutation by mutant allele-specific PCR in PTC tissue samples (n=92) in relation to clinicopathological parameters. BRAFV600E was detected in 46.7% of patients and correlated with the presence of lymph node metastasis (LNM, p=0.035) and extrathyroid invasion (EI, p&lt;0.0001). EGFR overexpression was detected in 52.2% of the patients and also correlated with LNM (p&lt;0.0001) and EI (p=0.027). Among patients with a single alteration, the presence of BRAFV600E impacted EI, while EGFR overexpression alone had a greater impact on LNM. The strongest association with adverse features was found in PTC patients with coexisting BRAFV600E and EGFR overexpression (28.3%), among whom LNM and EI were evident in 73% and 69%, respectively (p&lt;0.0001, for both). Thus, the coexistence of BRAFV600E mutation and EGFR overexpression identifies high-risk PTC patients, who should be considered for combined molecular therapy offering a better long-term therapeutic outcome.

Clinical prognostic significance of cancer stem cell markers in patients with papillary thyroid carcinoma.

The recent development of the cancer stem cell (CSC) model has been heralded as a new era in thyroid cancer research. The aim of this study was to evaluate the presence of CD44+ and CD24− tumor cells in papillary thyroid carcinoma (PTC) as markers of aggressiveness and poor prognosis. Patients with PTC, who underwent successful surgical resections between January 2003 and December 2012 at a single tertiary hospital, were included in this study. Tissue arrays were prepared from 454 primary tumor tissues. Immunohistochemistry (IHC) was performed to detect the CSC markers CD24 and CD44 on the tissue arrays. IHC was graded using a semi-quantitative histology scoring system based on the extent and intensity of staining. Subsequently, the association between IHC results and clinicopathological characteristics and recurrence-free survival (RFS) was analyzed. In 454 patients, 39 cases recurred during the 70-month median follow-up period, with some patients exhibiting multiple sites of relapse. The results of a Kaplan-Meier survival analysis and univariate log-rank test demonstrated that sex (P=0.008), age (P=0.002), cN1b, defined as metastasis to unilateral, bilateral, or contralateral neck lymph nodes or retropharyngeal lymph nodes (P<0.001), pN1, defined as pathologically proven lymph node metastasis >5 (P<0.001), tumor size >2 cm (P<0.001), extrathyroidal extension (P=0.001) and CD24− (P<0.001) were prognostic factors for RFS. CSC marker combinations (CD44+/CD24−) also exhibited statistical significance in the log-rank test. In conclusion, expression of the CSC markers CD44+ and CD24− in PTC tissue samples was associated with RFS. The combination of CD44+ and CD24− exhibited a statistically significant negative association with RFS and a strong association with gross extra-thyroidal extension.

Identification of potential pathogenic candidates or diagnostic biomarkers in papillary thyroid carcinoma using expression and methylation profiles.

The mechanisms underlying the pathogenesis of papillary thyroid carcinoma (PTC) have not yet been elucidated. The aim of the current study was to identify potential pathogenic biomarkers in PTC by comprehensively analyzing gene expression and methylation profiles, and to increase the understanding of PTC pathogenesis. The gene expression profiles of the GSE97001 and GSE83520 datasets, the miRNA expression profiles of the GSE73182 dataset, and the DNA methylation profiles of the GSE86961 and GSE97466 datasets were downloaded from Gene Expression Omnibus database. The differentially expressed genes (DEGs) and the differentially expressed microRNAs (DEMs) were identified using the limma package in R, and the differentially methylated sites (DMSs) were identified using the β distribution and two-sample t-tests. The Database for Annotation, Visualization and Integrated Discovery, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome were subsequently used to perform functional and pathway enrichment analysis. The miRNA target genes were predicted using the online databases miRWalk. The protein-protein interactions (PPI) were analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins. The regulatory network was constructed, and the gene expression and methylation levels of the key nodes were detected using reverse-transcription quantitative-polymerase chain reaction (PCR) and methylation-specific PCR. A total of 155 overlapping DEGs were identified between the GSE97001 and GSE83520 datasets, and 19 DEMs between PTC tissue and normal tissue samples were identified in the GSE73182 set. In the GSE86961 and GSE97466 datasets, 2,910 overlapping DMSs that were associated with 38 downregulated methylated genes were identified. The overlapping DEGs were enriched in 46 Gene Ontology terms and one KEGG pathway. A total of 60 PPI pairs were identified for the overlapping DEGs and 12 negative miRNA-gene pairs were identified for the DEMs. The expression levels of hsa-miR-199a-5p and decorin (DCN) were decreased in patients with PTC. C-X-C motif chemokine ligand 12 (CXCL12) was hypermethylated and had a decreased expression level in PTC tissues. LDL receptor related protein 4 (LRP4) and carbonic anhydrase 12 (CA12) were hypomethylated and had an increased expression level. The present study revealed that hsa-miR-199a-5p, DCN, CXCL12, LRP4 and CA12 may serve important roles in the pathogenesis of PTC.

Validation of Reference Genes for Normalization of Relative qRT-PCR Studies in Papillary Thyroid Carcinoma

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) in thyroid tumors require accurate data normalization, however, there are no sufficient studies addressing the suitable reference genes for gene expression analysis in malignant and normal thyroid tissue specimens. The purpose of this study was to identify valid internal control genes for normalization of relative qRT-PCR studies in human papillary thyroid carcinoma tissue samples. The expression characteristics of 12 candidate reference genes (GAPDH, ACTB, HPRT1, TBP, B2M, PPIA, 18SrRNA, HMBS, GUSB, PGK1, RPLP0, and PGM1) were assessed by qRT-PCR in 45 thyroid tissue samples (15 papillary thyroid carcinoma, 15 paired normal tissues and 15 multinodular goiters). These twelve candidate reference genes were selected by a systematic literature search. GeNorm, NormFinder, and BestKeeper statistical algorithms were applied to determine the most stable reference genes. The three algorithms were in agreement in identifying GUSB and HPRT1 as the most stably expressed genes in all thyroid tumors investigated. According to the NormFinder software, the pair of genes including ‘GUSB and HPRT1’ or ‘GUSB and HMBS’ or ‘GUSB and PGM1’ were the best combinations for selection of pair reference genes. The optimal number of genes required for reliable normalization of qPCR data in thyroid tissues would be three according to calculations made by GeNorm algorithm. These results suggest that GUSB and HPRT1 are promising reference genes for normalization of relative qRT-PCR studies in papillary thyroid carcinoma.

MicroRNA-26a suppresses the malignant biological behaviors of papillary thyroid carcinoma by targeting ROCK1 and regulating PI3K/AKT signaling pathway.

Papillary thyroid carcinoma (PTC) is an endocrine malignancy, the morbidity of which has kept rising in recent years. MicroRNA (miRNA/miR) is emerging as a key regulator in carcinogenesis, including PTC. The current study concentrates on the biological roles and mechanisms of miR-26a in the PTC progression. 51 pairs of PTC tissue samples and matched adjacent thyroid tissues were collected from PTC patients who received surgical excisions at The People's Hospital of Linqing between July 2015 and June 2018 with informed consent. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to detect expressions of miR-26a and rho-associated coiled-coil-containing protein kinase 1 (ROCK1) mRNA in PTC tissues and cells. Functional assays, including (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) MTT assays and transwell assays were performed to determine the roles of miR-26a in the PTC progression. Western blot was used to detect expression levels of the related proteins. Findings demonstrated prominently down-regulated miR-26a in PTC tissues and cells. Down-regulated miR-26a indicated malignant clinicopathologic characteristics and shorter overall survival rate of PTC patients. MTT assay and transwell assay indicated that miR-26a up-regulation significantly repressed PTC cell viability, invasion, and metastasis. Western blot analysis revealed that miR-26a exerted its anti-PTC effects via phosphatidylinositol 3-kinase/protein kinase B pathway (PI3K/AKT) and epithelial-to-mesenchymal transition (EMT). ROCK1 was a target of miR-26a in PTC cells and ROCK1 was mediated by miR-26a, as a regulatory mechanism in PTC. Taken together, these findings demonstrated the anti-tumor functions of miR-26a in PTC, providing novel strategies for PTC diagnosis and therapy.

SAT-LB096 Relationship between MicroRNA Expression Levels and Clinicopathological Parameters and Recurrence of Papillary Thyroid Carcinoma

Introduction: Papillary thyroid carcinoma (PTC) is the most common type of endocrine cancer. Although PTC is a malignancy that has a good prognosis, disease progression occurs in up to 20 % of patients. Currently, recurrence risk stratification is accomplished by using clinicopathologic factors, despite their limited prognostic value. Number of biomarkers for PTC recurrence prediction is still very limited. Identification of possible associations of traditional clinicopathological parameters of disease recurrence with molecular biomarkers of PTC may help better understanding the carcinogenesis and improving the clinical management of patients with PTC. Objectives: The aim of this study was to identify specific miRNAs as biomarkers for predicting the recurrence of PTC and to analyze the association between miRNAs expression levels and clinicopathologic characteristics of PTC. Methods: We selected 5 miRNAs (miRNA- 146b, -222, -21, -221 and -181b) and measured the expression levels of these miRNAs in 400 FFPE PTC tissue specimens and compared the levels of selected miRNAs expression to healthy thyroid tissue. We also analyzed the correlation between miRNAs (- 146b, -222, -21, -221 and -181b) expression and clinicopathologic characteristics of PTC. Results: Levels of miR-146b, miR-222, miR-21, miR-221 and miR-181b expression in PTC tissue samples (n = 400) were compared with those in healthy thyroid tissue (n = 4). All miRNAs were over-expressed in PTC tissue compared to healthy thyroid tissue (p ˂ 0.01, p ˂ 0.01, p ˂ 0.01, p ˂ 0.01 and p = 0.19 respectively). Higher expression levels of miRNAs (miRNA-146b p<0.01; miRNA-222 p<0.01; miRNA-21 p<0.01; miRNA-221 p=0.01; miRNA-181b p<0.01) were observed in recurrence PTC patients (n = 87) compared to non-recurrence PTC patients (n = 313). Expression levels of all miRNAs were significantly higher in lymph node metastases-positive group (n=91) compared to lymph node metastases-negative group (n=309) (p < 0.01). Younger patients had similar expression levels of miR-146b, miR-222, miR-21 and miR-221 as compared to older patients (p=0.11, p=0.944, p=0.485, p=0.314, respectively). Only miR-181b was over-expressed in patients younger than 45 years compared to older patients (p=0.007). Conclusion. The levels of miRNA-146b, - 222, -21, -221 and -181b expression in PTC were strongly associated with PTC recurrence and lymph node metastases. Selected miRNAs did not show age-related differences in expression, suggesting that mechanisms other than age may influence the expression of these miRNAs. However, the prognostic value of these miRNAs is rather limited in individual cases as the distribution of miRNA expression overlaps between patients with high and low risk of PTC recurrence. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

MicroRNA-23a inhibits the growth of papillary thyroid carcinoma via regulating cyclin G1.

The aim of this study was to investigate the regulatory mechanism of miR-23a on biological behaviors of papillary thyroid carcinoma (PTC) cells, such as cell proliferation, cell cycle and apoptosis. The expression of miR-23a in 28 paired of PTC tissue samples and matched adjacent tissues was detected by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Meanwhile, miR-23a expression in PTC cell lines was also detected by qRT-PCR. Subsequently, miR-23a mimics and inhibitor were transfected into PTC cells. The effects of gain or loss of miR-23a on cell proliferation, cell cycle and apoptosis were analyzed. Bioinformatics analysis, Dual-Luciferase activity assay and Western blot were recruited to validate the potential target gene of miR-23a. The expression of miR-23a was significantly decreased in PTC tissue samples and cell lines. Upregulation of miR-23a in PTC cells markedly decreased cell proliferation, induced cell cycle arrest at G0/G1 phase and promoted cell apoptosis. However, decreased miR-23a exerted the opposite effects. Dual-Luciferase, qRT-PCR and Western blot showed that CCNG1 was a target gene of miR-23a. Furthermore, the silence of CCNG1 intensified the suppressive effect of miR-23a on cell growth. MiR-23a was involved in the development of PTC via targeting CCNG1, which might provide a new prospect for PTC diagnosis and therapy.

Microarray-based differential expression profiling of long noncoding RNAs and messenger RNAs in formalin-fixed paraffin-embedded human papillary thyroid carcinoma samples.

BackgroundLong noncoding RNAs (lncRNAs) can regulate the expression of genes at almost every level. The altered expression of lncRNAs was observed in many kinds of cancers. Until recently, few studies have focused on the function of lncRNAs in the context of papillary thyroid carcinoma (PTC).MethodsIn the current study, we collected seven PTC and nodular goiter tissue samples and explored mRNA and lncRNA expression patterns in these samples by microarray.ResultsWe observed aberrant expression of 94 lncRNAs and 99 mRNAs in the seven PTC samples as compared to the nodular goiter tissue [fold change (FC) ≥2.0; P<0.01]. To confirm these microarray results, quantitative polymerase chain reaction (q-PCR) was performed to assess the expression of three randomly selected differentially expressed mRNAs and lncRNAs, confirming our microarray findings significantly. We then performed gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses to systematically characterize the twelve significantly differential genes. A co-expression analysis revealed that the lncRNAs n382996, n342483, and n409114 were closely related to the regulation of MT1G, MT1H, and MT1F.ConclusionsIn the present study a string of novel lncRNAs associated with PTC were identified. Further study of these lncRNAs should be performed to identify novel target molecules which may improve diagnosis and treatment of PTC.

Microarray-based differential expression profiling of long noncoding RNAs and messenger RNAs in formalin-fixed paraffin-embedded human papillary thyroid carcinoma samples

Background: Long noncoding RNAs (lncRNAs) can regulate the expression of genes at almost every level. The altered expression of lncRNAs was observed in many kinds of cancers. Until recently, few studies have focused on the function of lncRNAs in the context of papillary thyroid carcinoma (PTC). Methods: In the current study, we collected seven PTC and nodular goiter tissue samples and explored mRNA and lncRNA expression patterns in these samples by microarray. Results: We observed aberrant expression of 94 lncRNAs and 99 mRNAs in the seven PTC samples as compared to the nodular goiter tissue [fold change (FC) ≥2.0; P Conclusions: In the present study a string of novel lncRNAs associated with PTC were identified. Further study of these lncRNAs should be performed to identify novel target molecules which may improve diagnosis and treatment of PTC.

Long noncoding RNA H19 is a critical oncogenic driver and contributes to epithelial-mesenchymal transition in papillary thyroid carcinoma.

BackgroundGrowing evidence has indicated that the long noncoding RNA H19 (lncRNA H19), frequently deregulated in almost all tumor types tested, acted as a pivotal contributor to both cancer initiation and progression. However, the role of lncRNA H19 in human papillary thyroid carcinoma (PTC) remains controversial. The aim of the study was to investigate the expression and potential function of lncRNA H19 in human PTC.Patients and methodsThe lncRNA H19 level was determined by quantitative real-time (RT)-PCR analyses in 58 PTC tissue samples and their paired paracancerous tissue samples. RNA interference, RT-PCR analysis, and Western blot assay were used to determine the impact of lncRNA H19 on epithelial-mesenchymal transition (EMT) markers in human PTC cells. The migratory and invasive capacities of PTC cells were determined by wound-healing and transwell migration and invasion assays.ResultslncRNA H19 expression was 2.417-fold higher in PTC tissues than their paired paracancerous tissue (95% CI: 1.898–2.935, P<0.0001). Higher level of lncRNA H19 was correlated to elevated expression of Vimentin, ZEB2, Twist, and Snail2. Inhibition of lncRNA H19 resulted in upregulation of E-cadherin and downregulation of Vimentin both at mRNA and protein levels. Conversely, enforced expression of the exogenous lncRNA H19 led to E-cadherin mRNA and protein downregulation and relative upregulation of Vimentin. Moreover, wound-healing and transwell migration and invasion assays showed that lncRNA H19 could promote the migratory and invasive abilities of PTC cells.ConclusionThe level of lncRNA H19 was significantly higher in PTC tissues than paired paracancerous tissue or normal tissues. Overexpression of lncRNA H19 was correlated with higher tumor burden of PTC. It also contributes to EMT process, as well as promotes migration and invasion of PTC cells.

The LINC01186 suppresses cell proliferation and invasion ability in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy. Long non coding RNAs (lncRNAs) have been involved in regulating tumor progression including PTC. In the present study, we demonstrated that long non coding RNA LINC01186 was significantly downregulated in PTC tissue samples compared with adjacent normal tissue samples in patients. LINC01186 expression was also found to be higher in PTC cells. Lower LINC01186 expression was associated with lymph node metastasis of PTC patients. Functionally, LINC01186 overexpression significantly suppressed cell proliferation, cell colony formation and cell invasion ability in TPC-1 and IHH-4 cells. In addition, we demonstrated that LINC01186 overexpression inhibited large tumor suppressor kinase 1 (LATS1)/YY1 associated protein 1 (YAP) signaling by reducing YAP1 expression, while increasing LATS1 expression in TPC-1 and IHH-4 cells. Collectively, our data suggested that LINC01186 may serve as a potential target for therapy in thyroid carcinoma.