Through bioinformatics predictions, we identified that GTF2I and FAT1 were downregulated in thyroid carcinoma (TC). Further, Pearson's correlation coefficient revealed a positive correlation between GTF2I expression and FAT1 expression. Therefore, we selected them for this present study, where the effects of bone marrow mesenchymal stem cell-derived EVs (BMSDs-EVs) enriched with GTF2I were evaluated on the epithelial-to-mesenchymal transition (EMT) and stemness maintenance in TC. The under-expression of GTF2I and FAT1 was validated in TC cell lines. Ectopically expressed GTF2I and FAT1 were found to augment malignant phenotypes of TC cells, EMT, and stemness maintenance. Mechanistic studies revealed that GTF2I bound to the promoter region of FAT1 and consequently upregulated its expression. MSC-EVs could shuttle GTF2I into TPC-1 cells, where GTF2I inhibited TC malignant phenotypes, EMT, and stemness maintenance by increasing the expression of FAT1 and facilitating the FAT1-mediated CDK4/FOXM1 downregulation. In vivo experiments confirmed that silencing of GTF2I accelerated tumor growth in nude mice. Taken together, our work suggests that GTF2I transferred by MSC-EVs confer antioncogenic effects through the FAT1/CDK4/FOXM1 axis and may be used as a promising biomarker for TC treatment.
Mice ; Animals ; Cell Line, Tumor ; Cell Proliferation ; Mice, Nude ; Epithelial-Mesenchymal Transition ; Thyroid Neoplasms/pathology* ; Extracellular Vesicles/pathology* ; Mesenchymal Stem Cells ; Transcription Factors, TFIII/metabolism* ; Neoplastic Stem Cells/pathology*

Targeted gene therapy has become a promising approach for lung cancer treatment. In our previous work, we reported that the targeted expression of microRNA-7 (miR-7) operated by thyroid transcription factor-1 (TTF-1) promoter inhibited the growth of human lung cancer cells in vitro and in vivo; however, the intervention efficiency needed to be further improved. In this study, we identified the core promoter of TTF-1 (from -1299 bp to -871 bp) by 5' deletion assay and screened out the putative transcription factors nuclear factor-1 (NF-1) and activator protein-1 (AP-1). Further analysis revealed that the expression level of NF-1, but not AP-1, was positively connected with the activation of TTF-1 core promoter in human non-small-cell lung cancer (NSCLC) cells. Moreover, the silencing of NF-1 could reduce the expression level of miR-7 operated by TTF-1 core promoter. Of note, we optimized four distinct sequences to form additional NF-1-binding sites (TGGCA) in the sequence of TTF-1 core promoter (termed as ^optTTF-1 promoter), and verified the binding efficiency of NF-1 on the ^optTTF-1 promoter by electrophoretic mobility shift assay (EMSA). As expected, the ^optTTF-1 promoter could more effectively drive miR-7 expression and inhibit the growth of human NSCLC cells in vitro, accompanied by a reduced transduction of NADH dehydrogenase (ubiquinone) 1α subcomplex 4 (NDUFA4)/protein kinase B (Akt) pathway. Consistently, ^optTTF-1 promoter-driven miR-7 expression could also effectively abrogate the growth and metastasis of tumor cells in a murine xenograft model of human NSCLC. Finally, no significant changes were detected in the biological indicators or the histology of some important tissues and organs, including heart, liver, and spleen. On the whole, our study revealed that the optimized TTF-1 promoter could more effectively operate miR-7 to influence the growth of human NSCLC cells, providing a new basis for the development of microRNA-based targeting gene therapy against clinical lung cancer.
Animals ; Humans ; Mice ; Carcinoma, Non-Small-Cell Lung/therapy* ; Lung Neoplasms/metabolism* ; MicroRNAs/metabolism* ; Nuclear Proteins/metabolism* ; Thyroid Gland/pathology* ; Thyroid Nuclear Factor 1/genetics* ; Transcription Factors/metabolism*

OBJECTIVE: To assess the value of DNA methylation level of HYAL2 gene as a molecular marker for differential diagnosis of malignant and benign thyroid tumors. METHODS: DNA methylation of HYAL2 gene in tissue specimens of 190 patients with papillary thyroid cancer (PTC) and 190 age- and gender-matched patients with benign thyroid tumors was examined by mass spectrometry, and the protein expression of HYAL2 was detected immunohistochemically for another 55 pairs of patients. Logistic regression analysis was performed to calculate the odds ratio (OR) and evaluate the correlation of per 10% reduction in DNA methylation with PTC. Receiver operating characteristic (ROC) curve analysis was performed and the area under curve (AUC) was calculated to assess the predictive value of alterations in HYAL2 methylation. RESULTS: Hypomethylation of HYAL2_CpG_3 was significantly correlated with early-stage PTC (OR=1.51, P=0.001), even in stage I cancer (OR=1.42, P=0.007). Age-stratified analysis revealed a significantly stronger correlation between increased HYAL2_CpG_ 3 methylation and early-stage PTC in patients below 50 years than in those older than 50 years (OR: 1.89 vs 1.37, P < 0.05); ROC analysis also showed a larger AUC of 0.787 in younger patients. The results of immunohistochemistry showed that patients with PTC had significantly higher protein expressions of HYAL2 than patients with benign tumors. CONCLUSION The alterations of DNA methylation level of HYAL2 gene is significantly correlated with early-stage PTC, suggesting the value of DNA methylation level as a potential biomarker for differentiation of malignant from benign thyroid tumors.
Adenoma, Oxyphilic/genetics* ; Biomarkers, Tumor/metabolism* ; Cell Adhesion Molecules/metabolism* ; DNA Methylation ; GPI-Linked Proteins/metabolism* ; Humans ; Hyaluronoglucosaminidase/metabolism* ; Immunohistochemistry ; Middle Aged ; Thyroid Cancer, Papillary/pathology* ; Thyroid Neoplasms/pathology*

To investigate the effect of serine/threonine-protein kinase B-Raf (BRAF)-activated long-chain non-coding RNA (lncRNA-BANCR) on apoptosis and autophagy in thyroid carcinoma cells and the underlying mechanisms.
 Methods: RT-PCR was used to detect the expression of lncRNA-BANCR in thyroid carcinoma and normal thyroid tissues. The association between lncRNA-BANCR and clinicopathological data was analyzed in patients with thyroid cancer. Cell counting kit-8 (CCK-8) was used to detect the effect of lncRNA-BANCR on the proliferation of thyroid cancer cells. The effect of lncRNA-BANCR on the apoptosis of thyroid carcinoma cells was detected by flow cytometry. Transwell invasion assay was used to detect the effect of lncRNA-BANCR on the invasive ability of thyroid cancer cells. Western blot was used to detect the changes of autophagy proteins LC3-I and LC3-II after the lncRNA-BANCR expression was suppressed.
 Results: Compared with normal thyroid tissues, the expression level of lncRNA-BANCR in thyroid carcinoma tissues was elevated (P<0.05). The expression of lncRNA-BANCR was positively related to the pathological stage of thyroid carcinoma and the lymph node metastasis. Inhibition of lncRNA-BANCR expression attenuated the proliferation and invasion ability of thyroid cancer cells (both P<0.05); but the apoptosis was enhanced (P<0.05); the expression levels of autophagy protein LC3-I and LC3-II were also increased (P<0.05).
 Conclusion: The expression level of lncRNA-BANCR affects the proliferation, invasion and apoptosis of thyroid cancer cells through modulation of autophagy behavior.
Apoptosis ; Autophagy ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Neoplasm Invasiveness ; Proto-Oncogene Proteins B-raf ; metabolism ; RNA, Long Noncoding ; metabolism ; Serine ; metabolism ; Threonine ; metabolism ; Thyroid Gland ; cytology ; metabolism ; Thyroid Neoplasms ; metabolism ; pathology

BACKGROUNDThe Notch-regulated ankyrin repeat protein (NRARP) is recently found to promote proliferation of breast cancer cells. The role of NRARP in carcinogenesis deserves extensive investigations. This study attempted to investigate the expression of NRARP in thyroid cancer tissues and assess the influence of NRARP on cell proliferation, apoptosis, cell cycle, and invasion in thyroid cancer.

METHODSThirty-four cases with thyroid cancer were collected from the Department of General Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine between 2011 and 2012. Immunohistochemistry was used to detect the level of NRARP in cancer tissues. Lentivirus carrying NRARP-shRNA (Lenti-NRARP-shRNA) was applied to down-regulate NRARP expression. Cell viability was tested after treatment with Lenti-NRARP-shRNA using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis and cell cycle distribution were determined by flow cytometry. Cell invasion was tested using Transwell invasion assay. In addition, expressions of several cell cycle-associated and apoptosis-associated proteins were examined using Western blotting after transfection. Student's t-test, one-way analysis of variance (ANOVA), or Kaplan-Meier were used to analyze the differences between two group or three groups.

RESULTSNRARP was highly expressed in thyroid cancer tissues. Lenti-NRARP-shRNA showed significantly inhibitory activities against cell growth at a multiplicity of infection of 10 or higher (P < 0.05). Lenti-NRARP-shRNA-induced G1 arrest (BHT101: 72.57% ± 5.32%; 8305C: 75.45% ± 5.26%) by promoting p21 expression, induced apoptosis by promoting bax expression and suppressing bcl-2 expression, and inhibited cell invasion by suppressing matrix metalloproteinase-9 expression.

CONCLUSIONDownregulation of NRARP expression exerts significant antitumor activities against cell growth and invasion of thyroid cancer, that suggests a potential role of NRARP in thyroid cancer targeted therapy.

Adult ; Aged ; Animals ; Apoptosis ; genetics ; physiology ; Cell Cycle ; genetics ; physiology ; Cell Line, Tumor ; Cell Proliferation ; genetics ; physiology ; Cell Survival ; genetics ; physiology ; Female ; Humans ; In Vitro Techniques ; Kaplan-Meier Estimate ; Male ; Mice ; Mice, Nude ; Middle Aged ; Neoplasm Proteins ; genetics ; metabolism ; Proteins ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Thyroid Neoplasms ; genetics ; metabolism ; mortality ; pathology

BACKGROUNDBrain acid soluble protein 1 (BASP1) is identified as a novel potential tumor suppressor in several cancers. However, its role in thyroid cancer has not been investigated yet. In the present study, the antitumor activities of BASP1 against the growth and migration of thyroid cancer cells were evaluated.

METHODSBASP1 expression in thyroid cancer tissues and normal tissues were examined by immunohistochemical staining and the association between its expression and prognosis was analyzed. pcDNA-BASP1 carrying full length of BASP1 cDNA was constructed to restore the expression of BASP1 in thyroid cancer cell lines (BHT-101 and KMH-2). The cell proliferation in vitro and in vivo was evaluated by WST-1 assay and xenograft tumor models, respectively. Cell cycle distribution after transfection was analyzed using flow cytometry. Cell apoptosis after transfection was examined by annexin V/propidium iodide assay. The migration was examined using transwell assay.

RESULTSBASP1 expression was abundant in normal tissues while it is significantly decreased in cancer tissues (P = 0.000). pcDNA-BASP1 restored the expression of BASP1 and significantly inhibited the growth of BHT-101 and KMH-2 cells as well as xenograft tumors in nude mice (P = 0.000). pcDNA-BASP1 induced G1 arrest and apoptosis in BHT-101 and KMH-2 cells. In addition, pcDNA-BASP1 significantly inhibited the cell migration.

CONCLUSIONSDownregulation of BASP1 expression may play a role in the tumorigenesis of thyroid cancer. Restoration of BASP1 expression exerted extensive antitumor activities against growth and migration of thyroid cancer cells, which suggested that BASP1 gene might act as a potential therapeutic agent for the treatment of thyroid cancer.

Aged ; Animals ; Apoptosis ; genetics ; physiology ; Calmodulin-Binding Proteins ; genetics ; metabolism ; Cell Cycle ; genetics ; physiology ; Cell Line, Tumor ; Cell Movement ; genetics ; physiology ; Cell Proliferation ; genetics ; physiology ; Cytoskeletal Proteins ; genetics ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; genetics ; physiology ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mice, Nude ; Middle Aged ; Nerve Tissue Proteins ; genetics ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Thyroid Neoplasms ; metabolism ; pathology ; Xenograft Model Antitumor Assays

OBJECTIVETo study the expression of EpCAM and E-cadherin in papillary thyroid carcinoma and to analyze its correlation with various clinicopathologic parameters.

METHODSImmunohistochemical study for EpCAM and E-cadherin was carried out in 91 cases of papillary thyroid carcinoma. Twenty-four cases of papillary hyperplasia of thyroid were used as controls.

RESULTSIn all of the 24 cases of papillary hyperplasia, EpCAM was located on the cell membrane, while in the 91 cases of papillary thyroid carcinoma studied, EpCAM was located within the cytoplasm, with 36.3% (33/91) showing nuclear localization as well. In all the papillary hyperplasia cases studied, E-cadherin showed membranous expression. E-cadherin expression was reduced in 84.6% (77/91) of papillary thyroid carcinoma, as compared with the surrounding native thyroid parenchyma. Amongst the 33 cases of papillary thyroid carcinoma which showed nuclear localization of EpCAM, 30 cases also showed reduced E-cadherin expression. There was a positive correlation between nuclear expression of EpCAM and loss of E-cadherin expression (P = 0.000; Spearman correlation coefficient = 0.857). Nuclear expression of EpCAM correlated with follicular variant of papillary thyroid carcinoma and presence of extrathyroidal extension ( P = 0.037 and 0.033, respectively). Loss of E-cadherin expression correlated with age of patients and presence of lymph node metastasis (P = 0.018 and 0.010, respectively).

CONCLUSIONSE-cadherin expression is reduced in papillary thyroid carcinoma, as compared with native thyroid parenchyma and papillary hyperplasia. Papillary thyroid carcinoma shows loss of EpCAM membranous expression and increased cytoplasmic/nuclear accumulation. Detection of these two markers may provide a valuable reference in defining the biologic behaviors of papillary thyroid carcinoma, including extrathyroidal extension and lymph node metastasis.

Antigens, Neoplasm ; metabolism ; Cadherins ; metabolism ; Carcinoma, Papillary ; metabolism ; secondary ; Cell Adhesion Molecules ; metabolism ; Cell Membrane ; metabolism ; Cytoplasm ; metabolism ; Epithelial Cell Adhesion Molecule ; Humans ; Lymphatic Metastasis ; Neoplasm Proteins ; metabolism ; Thyroid Neoplasms ; metabolism ; pathology

Carrier Proteins ; metabolism ; Humans ; Microfilament Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; Thyroid Neoplasms ; metabolism ; pathology

OBJECTIVETo investigate the relationship between ALDH1A1 expression and lymph node metastasis (LNM) in papillary thyroid carcinoma (PTC).

METHODSOne hundred and fifty-three paraffin-embedded specimens of PTC treated in the Beijing Tongren Hospital of Capital Medical University were selected from January 2006 to December 2013. The expression of ALDH1A1 was detected in both tumor tissues and adjacent non-tumor tissues by immunohistochemistry and several clinicopathological parameters (size, bilaterality, multifocality, tumor border and extrathyroidal extensions) were assessed by HE staining. The correlation of ALDH1A1 expression with LNM was analyzed.

RESULTSIn 153 cases of PTC, there were 82 cases with LNM, 126 cases with high ALDH1A1 expression in tumor tissues, and 112 cases with high ALDH1A1 expression in adjacent non-tumor tissues. On univariate analysis, patient age < 45 years, tumor size of 10 mm or more, invasive tumor border, and high ALDH1A1 expression in tumor tissues predicted LNM in PTC (P < 0.05), whereas gender, bilaterality, multifocality, extra-thyroidal extensions and high ALDH1A1 expression in adjacent non-tumor border did not (P > 0.05). On multivariate analysis, invasive tumor border, high ALDH1A1 expression in tumor tissues were found to be independent predictive factors for LNM in PTC (P < 0.05). After a follow-up of 42 months (median time), four patients developed locoregional recurrences, but no distance recurrence or disease related death were seen in 82 patients of follow up. The estimated 5-year locoregional recurrence was 4.88%. Of these four logcoregional recurrences, three involved lymph nodes and one involved the remaining thyroid. The ALDH1A1 expression in tumor tissues was high in all of recurrence cases.

CONCLUSIONHigh ALDH1A1 expression in tumor tissues is correlated with lymph node metastasis in PTC and may be used as an independent predictive factor of LNM, and may improve treatment and follow-up strategies for PTC.

Aldehyde Dehydrogenase ; metabolism ; Carcinoma ; metabolism ; pathology ; Carcinoma, Papillary ; Humans ; Immunohistochemistry ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; Multivariate Analysis ; Neoplasm Recurrence, Local ; Risk Factors ; Thyroid Neoplasms ; metabolism ; pathology

PURPOSE: Ultrasound (US) and US-guided fine needle aspiration biopsies (FNAB) are considered the modalities of choice for assessing lymph nodes suspected of containing metastases, but the sensitivity of FNAB varies and is specific to the operator. We analyzed the risk of FNAB providing false negative results of lateral neck node metastasis, and evaluated diagnostic accuracy of FNAB, in patients with papillary thyroid cancer. MATERIALS AND METHODS: FNAB was performed in 242 patients suspected of having lateral neck node metastasis on preoperative imaging. Thyroglobulin in the fine-needle aspirate washout (FNA wash-out Tg) and computed tomography enhancement (Hounsfield units) were measured. Patients with negative results on FNAB were examined by intraoperative frozen section. The false negative and true negative groups were compared. RESULTS: Of the 242 patients, 130 were confirmed as having lateral neck node metastases. In 74 patients, the metastasis was identified by FNAB. False positive results were observed in 2 patients (0.8%) and false negatives in 58 (44.6%). Risk analysis showed that patient age <45 years (p=0.006), tumor size >1 cm (p=0.008) and elevated FNA wash-out Tg (p=0.004) were significantly associated with false negative results on FNAB. The accuracy of FNAB increased significantly when combined with FNA wash-out Tg (p=0.003). CONCLUSION: To reduce the false negative rate of FNAB, patient age (<45 years), tumor size (>1 cm) and FNA wash-out Tg (>34.8 ng/mL) should be considered in preoperative planning. Accuracy may be improved by combining the results of FNAB and FNA wash-out Tg.
Adolescent ; Adult ; Aged ; Biopsy, Fine-Needle ; Carcinoma/*diagnosis/*pathology/radiography/surgery ; False Negative Reactions ; Female ; Humans ; Lymph Nodes/*pathology/radiography ; Lymphatic Metastasis/*pathology/radiography ; Male ; Middle Aged ; Multivariate Analysis ; Preoperative Care ; Risk Factors ; Sensitivity and Specificity ; Thyroglobulin/metabolism ; Thyroid Gland/*pathology ; Thyroid Neoplasms/*diagnosis/*pathology/radiography/surgery ; Tomography, X-Ray Computed ; Young Adult