Association of MicroRNA Expression and BRAFV600E Mutation with Recurrence of Thyroid Cancer.
ABSTRACT: Many miRNAs and cancer-related mutations have been proposed as promising molecular markers of papillary thyroid carcinoma (PTC). However, there are limited data on the correlation between miRNA expression, BRAFV600E mutation, and PTC recurrence. Therefore, to evaluate the potential of BRAFV600E mutation and five selected miRNAs (-146b, -222, -21, -221, -181b) in predicting PTC recurrence, these molecular markers were analyzed in 400 formalin-fixed, paraffin-embedded PTC tissue specimens. The expression levels of miRNAs were measured using qRT-PCR. It was demonstrated that expression levels of all analyzed miRNAs are significantly higher in recurrent PTC than in non-recurrent PTC (p < 0.05). Moreover, higher expression levels of miR-146b, miR-222, miR-21, and miR-221 were associated with other clinicopathologic features of PTC, such as tumor size and lymph node metastases at initial surgery (p < 0.05). No significant differences in the frequency of BRAFV600E mutation in recurrent PTC and non-recurrent PTC were determined. Our results suggest that miRNA expression profile differs in PTC that is prone to recurrence when compared to PTC that does not reoccur after the initial surgery while BRAFV600E mutation frequency does not reflect the PTC recurrence status. However, the prognostic value of the analyzed miRNAs is rather limited in individual cases as the pattern of miRNA expression is highly overlapping between recurrent and non-recurrent PTC.
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:We show that numerous miRNAs are transcriptionally up-regulated in papillary thyroid carcinoma (PTC) tumors compared with unaffected thyroid tissue. Among the predicted target genes of the three most upregulated miRNAs (miRs 221, 222 and 146b), only less than 15% showed significant downexpression in transcript level between tumor and unaffected tissue. The KIT gene which is known to be downregulated by miRNAs 221 and 222 displayed dramatic loss of transcript and protein in those tumors that had abundant mir-221, mir-222, and mir-146b transcript. Experiment Overall Design: Total RNA was extracted from paired tumor and normal thyroid tissues from 9 PTC patients. The same set samples were applied to Custom miRNA microarray chips (OSU_CCC version 2.0) and Affymetrix HG-U133 plus 2 chips.
Project description:BACKGROUND:DNA methylation in miRNA genes has been reported as a mechanism that may cause dysregulation of mature miRNAs and consequently impact the gene expression. This mechanism is largely unstudied in papillary thyroid carcinomas (PTC). METHODS:To identify differentially methylated miRNA-encoding genes, we performed global methylation analysis (Illumina 450 K), integrative analysis (TCGA database), data confirmation (pyrosequencing and RT-qPCR), and functional assays. RESULTS:Methylation analysis revealed 27 differentially methylated miRNA genes. The integrative analyses pointed out miR-21 and miR-146b as potentially regulated by methylation (hypomethylation and increased expression). DNA methylation and expression patterns of miR-21 and miR-146b were confirmed as altered, as well as seven of 452 mRNAs targets were down-expressed. The combined methylation and expression levels of miR-21 and miR-146b showed potential to discriminate malignant from benign lesions (91-96% sensitivity and 96-97% specificity). An increased expression of miR-146b due to methylation loss was detected in the TPC1 cell line. The miRNA mimic transfection highlighted putative target mRNAs. CONCLUSIONS:The increased expression of miR-21 and miR-146b due to loss of DNA methylation in PTC resulted in the disruption of the transcription machinery and biological pathways. These miRNAs are potential diagnostic biomarkers, and these findings provide support for future development of targeted therapies.
Project description:Glioblastoma (GBM) has a high rate of local recurrence despite chemoradiotherapy (CRT). Genome-wide expression profiling was performed on patient tumors before and after chemoradiotherapy to identify genes and gene pathways associated with recurrence.Median time to recurrence was 8.9 months with median time to second surgery of 9.6 months. The microRNA (miRNA) analysis identified 9 oncologic and immune-related miRNAs to be differentially expressed, including the hypoxia-related miR-210 and the immune-modulatory miR-146b. More than 1200 differentially-expressed genes were identified with RNA-sequencing (RNA-seq). Gene set enrichment analysis (GSEA) identified p53 signaling, Notch, Wnt, VEGF, and MEK gene sets enriched in recurrent GBM. Consistent with the miRNA profiling data, the miR-146b target gene set from GSEA analysis was also associated with recurrence.Fourteen patients with GBM recurrence after CRT who had available tumor tissue from the initial diagnosis as well as recurrence were selected. Total RNA was isolated from formalin-fixed paraffin-embedded (FFPE) tumor specimens. Genome-wide expression profiling using RT-PCR for miRNA analysis and RNA-seq for messenger RNA (mRNA) analysis were conducted to identify differentially-expressed genes. GSEA was performed on the differential expression data.Genome-wide expression profiling identifies multiple oncologic and immune-related gene sets associated with GBM recurrence. In particular, immune-related miR-146b is upregulated in recurrence and deserves further investigation.
Project description:CONTEXT:A single microRNA gene may give rise to several mature products that differ in length, called isomiRs. IsomiRs are known to be tissue specific and functionally relevant. The microRNA sequence heterogeneity of the thyroid gland has yet to be determined. OBJECTIVE:The objective of the study was to provide a comprehensive view of the microRNA transcriptome in normal thyroid and papillary thyroid carcinoma (PTC). DESIGN:We used next-generation deep sequencing to analyze microRNA length heterogeneity and expression profiles of PTC tumors (n = 14), unaffected tissue adjacent to tumors (n = 14), and control, noncancerous thyroid tissue (n = 14). The results were validated with a microarray on an additional set of 9 PTC tumor/normal tissue pairs. RESULTS:Eighty-nine microRNAs were significantly deregulated in PTC compared with normal thyroid tissue (false discovery rate < 0.05, fold change 0.13-20.7). Top deregulated miRNAs included miR-146b-5p, miR-221-3p, miR-7-3p, miR-551b-3p, miR-486-3p, and miR-144-3p, confirming previous microarray profiling. The expression of miRNAs did not depend on the BRAF mutation status. Interestingly, 85% of the most abundant microRNAs consisted of isoforms that differed from the standard reference sequence deposited in miRBase. Moreover, the reference microRNAs were completely absent in 42.4% and 35.9% of the microRNAs expressed in normal thyroid and PTC tumors, respectively. Numerous isomiRs had altered seed sequences, which led to a different set of target genes. For highly deregulated miR-146b-5p, we detected 6 isoforms (tumor/normal fold change 14.4-28.7, false discovery rate < 0.002) that varied at their 5' ends with a 1-nt difference that created 2 alternative seeds. The target genes for those 2 seeds overlapped in only 13.1% of genes. CONCLUSIONS:Almost all microRNAs exhibit isoforms of variable length and potentially distinct function in thyroid tumorigenesis.
Project description:Studies have demonstrated an association of the BRAF(V600E) mutation and microRNA (miR) expression with aggressive clinicopathologic features in papillary thyroid cancer (PTC). Analysis of BRAF(V600E) mutations with miR expression data may improve perioperative decision making for patients with PTC, specifically in identifying patients harboring central lymph node metastases (CLNM).Between January 2012 and June 2013, 237 consecutive patients underwent total thyroidectomy and prophylactic central lymph node dissection (CLND) at four endocrine surgery centers. All tumors were tested for the presence of the BRAF(V600E) mutation and miR-21, miR-146b-3p, miR-146b-5p, miR-204, miR-221, miR-222, and miR-375 expression. Bivariate and multivariable analyses were performed to examine associations between molecular markers and aggressive clinicopathologic features of PTC.Multivariable logistic regression analysis of all clinicopathologic features found miR-146b-3p and miR-146b-5p to be independent predictors of CLNM, while the presence of BRAF(V600E) almost reached significance. Multivariable logistic regression analysis limited to only predictors available preoperatively (molecular markers, age, sex, and tumor size) found miR-146b-3p, miR-146b-5p, miR-222, and BRAF(V600E) mutation to predict CLNM independently. While BRAF(V600E) was found to be associated with CLNM (48% mutated in node-positive cases vs. 28% mutated in node-negative cases), its positive and negative predictive values (48% and 72%, respectively) limit its clinical utility as a stand-alone marker. In the subgroup analysis focusing on only classical variant of PTC cases (CVPTC), undergoing prophylactic lymph node dissection, multivariable logistic regression analysis found only miR-146b-5p and miR-222 to be independent predictors of CLNM, while BRAF(V600E) was not significantly associated with CLNM.In the patients undergoing prophylactic CLNDs, miR-146b-3p, miR-146b-5p, and miR-222 were found to be predictive of CLNM preoperatively. However, there was significant overlap in expression of these miRs in the two outcome groups. The BRAF(V600E) mutation, while being a marker of CLNM when considering only preoperative variables among all histological subtypes, is likely not a useful stand-alone marker clinically because the difference between node-positive and node-negative cases was small. Furthermore, it lost significance when examining only CVPTC. Overall, our results speak to the concept and interpretation of statistical significance versus actual applicability of molecular markers, raising questions about their clinical usefulness as individual prognostic markers.
Project description:Multiple sclerosis (MS) is a debilitating autoimmune disease affecting over 2.3 million people worldwide, and it is characterized by inflammation and demyelination of nerve cells. The currently available biomarkers for the diagnosis and management of MS have inherent limitations, therefore, additional new biomarkers are needed. We studied the microRNA (miRNA) profile of splenocytes of mice having experimental autoimmune encephalomyelitis (EAE), a model of human MS. A miRNA-microarray analysis revealed increased expression of nine miRNAs (let-7e, miR-23b, miR-31, miR-99b, miR-125a, miR-146b, miR-155, miR-193b, and miR-221) following EAE development. Interestingly, serum levels of miR-99b, miR-125a, and miR-146b were significantly higher in EAE mice compared to normal mice. Bioinformatics analysis revealed the experimentally validated as well as predicted gene targets of specific miRNAs that are important for disease progression in MS. Specifically, we observed inverse correlation in the levels of miR-99b versus LIF, and between miR-125a versus BDNF and LIF. Our results suggest that above-mentioned miRNAs may play a crucial role in the pathogenesis of MS, and that miR-99b, miR-125a, and miR-146b in particular may serve as useful biomarkers for disease activity.
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: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:Background. Papillary thyroid carcinoma (PTC) is the commonest thyroid malignancy originating from the follicle cells in the thyroid. Despite a good overall prognosis, certain high-risk cases as in those with lymph node metastasis (LNM) have progressive disease and poorer prognosis. MicroRNAs are a class of non-protein-coding, 19-24 nucleotides single-stranded RNAs which regulate gene expression and these molecules have been shown to play a role in LNM. The integrated analysis of miRNAs and gene expression profiles together with transcription factors (TFs) has been shown to improve the identification of functional miRNA-target gene-TF relationships, providing a more complete view of molecular events underlying metastasis process. Objectives. We reanalyzed The Cancer Genome Atlas (TCGA) datasets on PTC to identify differentially expressed miRNAs/genes in PTC patients with LNM-positive (LNM-P) versus lymph node negative (LNN) PTC patients and to investigate the miRNA-gene-TF regulatory circuit that regulate LNM in PTC. Results. PTC patients with LNM (PTC LNM-P) have a significantly shorter disease-free survival rate compared to PTC patients without LNM (PTC LNN) (Log-rank Mantel Cox test, p = 0.0049). We identified 181 significantly differentially expressed miRNAs in PTC LNM-P versus PTC LNN; 110 were upregulated and 71 were downregulated. The five topmost deregulated miRNAs were hsa-miR-146b, hsa-miR-375, hsa-miR-31, hsa-miR-7-2 and hsa-miR-204. In addition, 395 miRNAs were differentially expressed between PTC LNM-P and normal thyroid while 400 miRNAs were differentially expressed between PTC LNN and normal thyroid. We found four significant enrichment pathways potentially involved in metastasis to the lymph nodes, namely oxidative phosphorylation (OxPhos), cell adhesion molecules (CAMs), leukocyte transendothelial migration and cytokine-cytokine receptor interaction. OxPhos was the most significantly perturbed pathway (p = 4.70E-06) involving downregulation of 90 OxPhos-related genes. Significant interaction of hsa-miR-301b with HLF, HIF and REL/NFkB transcription factors were identified exclusively in PTC LNM-P versus PTC LNN. Conclusion. We found evidence of five miRNAs differentially expressed in PTC LNM-P. Alteration in OxPhos pathway could be the central event in metastasis to the lymph node in PTC. We postulate that hsa-miR-301b might be involved in regulating LNM in PTC via interactions with HLF, HIF and REL/NFkB. To the best of our knowledge, the roles of these TFs have been studied in PTC but the precise role of this miRNA with these TFs in LNM in PTC has not been investigated.