A Panel of Plasma Exosomal miRNAs as Potential Biomarkers for Differential Diagnosis of Thyroid Nodules.
ABSTRACT: Background: A liquid biopsy using circulating exosomal genetic materials provides new insights for thyroid cancer diagnosis. This study aimed to identify plasma-derived exosomal biomarkers that could be used for early detection of papillary thyroid carcinoma (PTC). Method: Exosomal miRNAs in plasma were isolated from patients with benign thyroid nodules and patients with PTC. Profiling of exosomal miRNA was performed using RNA sequencing (RNA-seq) to identify miRNA candidates and differentiate the benign from malignant. The validation cohort consisted of 30 patients with benign thyroid nodules, 35 PTC patients, and 31 healthy individuals. Real-time PCR was used to quantify the expression of miRNA candidates. The diagnostic potential of the candidates was evaluated by receiver operating characteristic (ROC) curves. Results: After RNA-seq, eight plasma exosomal miRNAs were selected as candidates. Further validation indicated that the levels of exosomal miR-16-2-3p, miR-223-5p, miR-34c-5p, miR-182-5p, miR-223-3p, and miR-146b-5p were significantly lower in nodules compared to healthy controls (p < 0.0001), whereas miR-16-2-3p and miR-223-5p were significantly higher in the PTC cases than in those with benign nodules (p < 0.05). ROC analyses revealed that the above six miRNAs were potent indicators for detection of thyroid nodules. Meanwhile, miR-16-2-3p and miR-223-5p can be utilized for detecting PTC from benign nodules. Additionally, combined miRNA panels showed increased diagnostic sensitivities and specificities compared to single miRNA markers. Conclusion: Six aberrantly expressed plasma exosomal miRNAs may be used as diagnostic biomarkers to differentiate thyroid nodules from healthy individuals. The panel consisting of miR-16-2-3p, miR-223-5p, miR-101-3p, and miR-34c-5p are eligible for discriminating benign from malignant thyroid nodules.
Project description:Thyroid cancers are the most common malignancy of the endocrine system; however, there is no reliable blood biomarkers for thyroid cancer diagnosis and even for aggressive and nonaggressive thyroid cancers as well as benign nodule discrimination. The present study is aimed at evaluating whether circulating microRNA (miRNA) can differentiate aggressive and nonaggressive thyroid cancer from benign thyroid nodules. In this study, we performed a multiphase, case-control study to screen serum miRNA expression profile in 100 patients with papillary thyroid cancer (PTC), 15 patients with aggressive medullary thyroid carcinoma (MTC), 91 patients with benign nodules, and 89 healthy controls using TaqMan low-density array followed by extensive reverse transcription quantitative real-time PCR validation. The results showed that the serum levels of miR-222-3p, miR-17-5p, and miR-451a were markedly increased, while miR-146a-5p, miR-132-3p, and miR-183-3p were significantly decreased in the PTC and benign nodule groups compared with the control group. There was no difference in the miRNA expression profile between the PTC group and the benign nodule group. Nevertheless, the serum levels of miR-222-3p and miR-17-5p were significantly increased in the MTC group than the benign nodule and control group. Moreover, receiver operating characteristic curve analyses demonstrated that the 2 miRNAs and their panel can accurately discriminate MTC from the benign nodule group and healthy controls. These findings indicated that the altered circulating miRNAs may discriminate PTC and benign thyroid nodules from controls, and serum miR-222-3p and miR-17-5p have the potential to serve as auxiliary tools for diagnosing more aggressive thyroid carcinomas, such as MTC.
Project description:<h4>Objective</h4>There is no effective and reliable biomarker to distinguish benign thyroid nodules from papillary thyroid carcinomas (PTC). This study aimed at examining the levels of plasma miRNAs in patients with PTC or benign nodules to explore the potential miRNA biomarkers for PTC.<h4>Patients and methods</h4>Genome-wide plasma miRNA expression profiles were determined by the miRNA Microarray and the significantly higher levels of miRNAs were validated in plasma and tissues by quantitative RT-PCR. The levels of two miRNAs were further tested in seven patients before and after tumor excision and the potential values for the diagnosis of PTC were evaluated by receiver operating characteristic curve (ROC).<h4>Results</h4>In comparison with that in the patients with benign nodules, eight significantly higher and three lower levels of plasma miRNAs were detected in the PTC patients. Further validation indicated that the levels of plasma miR-25-3p, miR-451a, miR-140-3p and let-7i were significantly higher in the PTC cases than in those with benign nodules or the healthy controls. Significantly higher levels of miR-25-3p and miR-451a were detected in the thyroid tissues from the PTC patients. The levels of plasma miR-25-3p and miR-451a in seven patients significantly decreased after tumor excision. ROC analyses revealed that the levels of plasma miR-25-3p at cut-off 1.41 and miR-451a at 1.38 had sensitivity of 92.8% and 88.9%, and specificity of 68.8% and 66.7% for distinguishing PTC from benign nodules, respectively.<h4>Conclusion</h4>Our findings suggest that the levels of plasma miR-25-3p and miR-451a may be valuable for the diagnosis of PTC.
Project description:OBJECTIVES:Papillary thyroid cancer (PTC) is increasing in incidence. Fine needle aspiration is the gold standard for diagnosis, but results can be indeterminate. Identifying tissue and serum biomarkers, like microRNA, is therefore desirable. We sought to identify miRNA that is differentially expressed in the serum of patients with PTC. METHODS:Serum miRNA was quantified in 31 female thyroidectomy patients: 13 with benign disease and 18 with PTC. qPCR results were compared for significant fold-changes in 175 miRNAs, against a pooled control. RESULTS:128 miRNA qualified for analysis. There were identifiable fold-changes in miRNA levels between benign and control, and between PTC and control. There were statistically significant fold changes in the level of four miRNAs between benign and PTC: hsa-miR-146a-5p and hsa-miR-199b-3p were down-regulated, while hsa-let7b-5p and hsa-miR-10a-5p were up-regulated. CONCLUSIONS:MicroRNA is differentially expressed in the serum of patients with PTC. Serum miRNA has the potential to aid in thyroid cancer diagnosis.
Project description:This study screened microRNAs (miRNAs) that are abnormally expressed in papillary thyroid carcinoma (PTC) tissues to identify PTC and nodular goiter and the degree of PTC malignancy. A total of 51 thyroid tumor tissue specimens paired with adjacent normal thyroid tissues were obtained from the Department of Surgical Oncology of Hangzhou First People's Hospital from June-December 2011. miRNA expression profiles were examined by microarrays and validated by quantitative real-time PCR (qRT-PCR). Expression levels of the miRNAs were analyzed to assess if they were associated with selected clinicopathological features. Eleven miRNAs were significantly differentially expressed between nodular goiter and PTC and between highly invasive and low invasive PTC. miR-199b-5p and miR-30a-3p were significantly differentially expressed among the three groups. miR-30a-3p, miR-122-5p, miR-136-5p, miR-146b-5p and miR-199b-5p were selected for further study by qRT-PCR and miR-146b-5p, miR-199b-5p and miR-30a-3p were different between the PTC and nodular goiter groups. miR-199b-5p was over-expressed in PTC patients with extrathyroidal invasion and cervical lymph node metastasis. In conclusion miR-146b-5p, miR-30a-3p, and miR-199b-5p may serve as biomarkers for the diagnosis of PTC and miR-199b-5p is associated with PTC invasiveness.
Project description:<h4>Background</h4>Non-coding circular RNAs (circRNAs) have displayed dysregulated expression in several human cancers. Here, we profiled the circRNA expression of papillary thyroid carcinoma (PTC) tumors to improve our understanding of PTC pathogenesis.<h4>Methods</h4>Microarray profiling was performed on 18 thyroid samples, consisting of six PTC tumors, six matching contralateral normal samples, and six benign thyroid lesions. After low-intensity filtering, hierarchical clustering revealed the circRNA expression patterns. Statistical analysis followed by qRT-PCR validation identified the differential circRNAs. MicroRNA (miRNA) target prediction software identified putative miRNA response elements (MREs), which were used to construct a network map of circRNA-miRNA interactions for the differential circRNAs. Bioinformatics platforms predicted cancer-related circRNA-miRNA associations and putative downstream target genes, respectively.<h4>Results</h4>A total of 88 circRNAs and 10 circRNAs were significantly upregulated and downregulated, respectively, in PTC tumors relative to normal thyroid tissue, while 129 circRNAs and 226 circRNAs were significantly upregulated and downregulated, respectively, in PTC tumors relative to benign thyroid lesions. A total of 12 upregulated and four downregulated circRNAs were overlapping between the foregoing comparisons. One downregulated circRNA (hsa_circRNA_100395) showed interactive potential with two cancer-related miRNAs (miR-141-3p and miR-200a-3p). From this analysis, we identified several promising cancer-related genes that may be targets of the dysregulated hsa_circRNA_100395/miR-141-3p/miR-200a-3p axis in PTC tumors.<h4>Conclusions</h4>circRNA dysregulation may play a role in PTC pathogenesis, and several key circRNAs show promise as candidate biomarkers for PTC. The hsa_circRNA_100395/miR-141-3p/ miR-200a-3p axis may be involved in the pathogenesis of PTC.
Project description:Papillary thyroid cancer (PTC) can be divided into classical variant of PTC (cPTC), follicular variant of PTC (fvPTC), and tall cell variant (tcPTC). These variants differ in their histopathology and cytology; however, their molecular background is not clearly understood. Our results shed some new light on papillary thyroid cancer biology as new direct miRNA-gene regulations are discovered. The Cancer Genome Atlas (TCGA) 466 thyroid cancer samples were studied in parallel datasets to discover potential miRNA-mRNA regulations. Additionally, miRNAs and genes differentiating PTC variants (cPTC, fvPTC, and tcPTC) were indicated. Putative miRNA regulatory pairs were discovered: hsa-miR-146b-5p with PHKB and IRAK1, hsa-miR-874-3p with ITGB4 characteristic for classic PTC samples, and hsa-miR-152-3p with TGFA characteristic for follicular variant PTC samples. MiRNA-mRNA regulations discovery opens a new perspective in understanding of PTC biology. Furthermore, our successful pipeline of miRNA-mRNA regulatory pathways discovery could serve as a universal tool to find new miRNA-mRNA regulations, also in different datasets.
Project description:Exosomes and exosomal miRNAs from the plasma of volunteers were isolated from thyroid nodules and papillary tyriod cancer patients . Profiling of exosomal miRNA was performed using next-generation sequencing(NGS) to identify miRNA candidates for diagnosis. Overall design: Sequencing small RNA libraries prepared from 8 benign thyroid nodules and 16 PTC patients with or without metastasis.
Project description:Potential use of sonographic TI-RADS classification combined with circulating miRNA expression profiling in the diagnosis of thyroid nodules was explored. Retrospective analysis was performed on clinical data of 121 patients with thyroid nodules. The biopsy specimens of patients obtained through ultrasound-guided aspiration and blood specimens were evaluated in Zhengzhou Central Hospital Affiliated to Zhengzhou University from June 2018 to June 2019. In addition, the blood specimen test results of 121 healthy volunteers (control group) who underwent physical examination were retrospectively analyzed. Results of sonographic TI-RADS classification and circulating miRNA expression profiling were compared with the pathological results. Of the 212 nodules, 2 fell into TI-RADS category 2 and were diagnosed as benign. Malignant nodules accounted for 4.35, 37.14, 84.78, 93.33 and 96.77% of those nodules that fell into TI-RADS categories 3, 4a, 4b, 4c and 5, respectively. Of the 121 patients, 92.55% had with nodular goiter, 3.31% had inflammatory nodules, 2.48% toxic nodular goiter, 0.83% thyroid cysts and 0.83% thyroid tumors. A nodule that fell into a higher TI-RADS classification category had a higher risk of malignancy. The expression levels of miRNA146b, miRNA187, miRNA375, miRNA-222-3p and miRNA-151a-5p were higher, while the level of miRNA138 was lower, in patients with either benign or malignant thyroid nodules compaed to those in the control group. The expression levels of miRNA146b, miRNA187, miRNA375, miRNA-222-3p and miRNA-151a-5p were higher, while the level of miRNA138 was lower, in patients with malignant thyroid nodules than those in patients with benign thyroid nodule (P<0.05). The AUC of the combined diagnostic method was 0.973, which was significantly different from the AUCs of the individual diagnostic method (P<0.05). In conclusion, sonographic TI-RADS classification combined with circulating miRNA expression profiling can improve the diagnosis of thyroid nodules.
Project description:<b><i>Background:</i></b> MiR-146b has been reported to be overexpressed in papillary thyroid cancer (PTC) tissues and associated with aggressive PTC. MiR-146b is regarded as a relevant diagnostic marker for this type of cancer. MiR-146b-5p has been confirmed to increase cell proliferation by repressing SMAD4. However, detailed functional analysis of another mature form of miR-146b, miR-146b-3p, has not been carried out. This study aimed to identify the differential expression of miR-146b-5p and miR-146b-3p in more aggressive PTC associated with lymph node metastasis, and further elucidate the contribution and mechanism of miR-146b-3p in the process of PTC metastasis. <b><i>Methods:</i></b> Expression of miR-146b-5p and miR-146b-3p was assessed in formalin-fixed paraffin-embedded tissue samples from PTC patients, and the relationship with lymph node metastasis was analyzed. A variety of PTC cells, including BHP10-3, BHP10-3SC<sub>mice</sub>, and K1 cells, were cultured and treated with miR-146b-5p or miR-146b-3p mimics/inhibitors. The cell migration and invasion abilities were characterized by the real-time cell analyzer assay and Transwell™ assay. PTC xenograft models were used to examine the effect of miR-146b-3p on PTC metastatic ability <i>in vivo</i>. Direct downstream targets of miR-146b-3p were analyzed by luciferase reporter assay and Western blotting. The mechanism by which miR-146b-3p affects cell metastasis was further characterized by co-transfection with merlin, the protein product of the <i>NF2</i> gene. <b><i>Results:</i></b> MiR-146b-5p and miR-146b-3p expression was significantly higher in thyroid cancer tissues and cell lines than in normal thyroid tissue and cells. Moreover, expression of miR-146b-5p and miR-146b-3p was further increased in thyroid metastatic nodes than in thyroid cancer. After overexpression of miR-146b-5p or miR-146b-3p in BHP10-3 or K1 cells, PTC migration and invasion were increased. Notably, miR-146b-3p increased cell migration and invasion more obviously than did miR-146b-5p. Overexpression of miR-146b-3p also significantly promoted PTC tumor metastasis <i>in vivo</i>. Luciferase reporter assay results revealed that <i>NF2</i> is a downstream target of miR-146b-3p in PTC cells, as miR-146b-3p bound directly to the 3' untranslated region of <i>NF2</i>, thus reducing protein levels of NF2. Overexpression of merlin reversed the enhanced aggressive effects of miR-146b-3p. <b><i>Conclusions:</i></b> Overexpression of miR-146b-5p and miR-146b-3p is associated with PTC metastasis. MiR-146b-3p enhances cell invasion and metastasis more obviously than miR-146b-5p through the suppression of the <i>NF2</i> gene. These findings suggest a potential diagnostic and therapeutic value of these miRNAs in PTC metastasis.
Project description:The follicular thyroid carcinoma (FTC) and follicular thyroid adenoma (FTA) are malignant and benign thyroid neoplasms, respectively. MicroRNA (miRNA) expressions have been touted as an indicator for prognostic outcome in thyroid cancer. The study objective was to explore genes suppressed by miRNA-21-3p and miRNA-21-5p for potential therapeutic insights. Differentially expressed genes and their functional enrichment were obtained from 25 FTA and 27 FTC gene microarray dataset GSE82208 using R and Bioconductor tools. The miRNA target sites were obtained from miR-TarBase database. A unique gene list of differentially expressed FTC and FTA were entered into miR-TarBase database to obtain target genes for both miRNA-21-3p and miRNA-21-5p. The result showed that miRNA-21-3p and miRNA-21-5p downregulated TIMP3, MAT2A, TGFBR2, and PLAT gene in FTC and FTA leading to significant expression of acute phase-response to metallothionein, metal ions, and unfolded protein response (UPR). The computational analysis suggests that the suppression of miRNA-21-3p and miRNA-21-5p could be an intervention strategy for therapeutically targeting FTC and FTA treatments.