| Thyroid cancer, the most common seen malignancy of the endocrine system, accounts for almost 1% of the overall malignancies in human. Nowadays, the profound effects of gene dysregulation on tumor genesis and cancer progression are becoming evident and are presently the major subject of research. In thyroid cancer, gene dysregulation have been reported to contribute to tumor progression. In addition to the many classical genetic mutations that have been proved over the last several decades, recent studies highlight the significance of other epigenetic regulators such as histone modifiers that are frequently mutated or deregulated in DTC. As important conserved epigenome readers, the BET family proteins link to chromatin remodeling, and generally act as transcription regulators in a series of cells. The important BET member BRD4 is aberrantly regulated in several cancer cells and tissues with unknown mechanism. In this study, we observed that BRD4 was upregulated in DTC cells and tissues, indicating a pro-oncogenic function of BRD4 in cancer progression. Moreover, we demonstrated that inhibition of BRD4 using JQ1 influences cell proliferation, cell radioiodine uptake and tumor growth in K1 and BCPAP cells and mouse models, suggesting a potential role of BRD4 in regulating DTC biological activity, which has not been reported.Thyroid cancer derives from the follicular epithelium of the thyroid. Due to the developments in physical examinations and diagnostic imaging, the incidence of papillary thyroid microcarcinoma (PTMC) has been gradually increasing worldwide, contributing significantly to the prevalence of papillary thyroid carcinoma (PTC). PTMC is defined as PTC with the largest dimension ?1 cm and with the following characteristics:low malignancy, slow growth, minimal invasiveness, and low mortality. PTMCs are frequently detected in "normal" glands or nodular goiters unexpectedly. In fact, according to recent studies, PTMC is already the most common thyroid malignancy in patients older than 45 years in the US. Currently, the optimal management of PTMC remains controversial for different reasons. On one hand, various guidelines, expert consensuses, and literatures provide different perspectives for clinicians, resulting in ambiguous therapy in clinical practice. On the other hand, increasing reports have demonstrated that PTMC progression could result in distant metastases and death. Therefore, large, randomized, controlled trials aimed at investigating the clinical factors and relative statistics of PTMC should be performed to broaden our understanding of the underlying biology and clinical outcome. Post-surgery stimulated Tg (ps-Tg) is an early predictor of poor prognosis in larger (> 1 cm) TC. In PTMC, however, the clinical significance of ps-Tg has rarely been investigated. Therefore, the cliniclpathological characteristics of 280 PTMC cases were analyzed and discussed in Part two. The role of ps-Tg in treatment and follow-up of PTMC patients was also discussed.Our research is composed of the following two parts:Part one:Effects and mechanisms of BRD4 inhibition on thyroid cancer bio-behaviorBackgrouond and Objective:Although radioiodine 131I treatment on differentiated thyroid cancer is widely used in clinical, many patients still fail to benefit from 131I therapy owing to consecutive tumor progression and decreased ability of radioiodine uptake. Bromodomain-containing protein 4 (BRD4) is an important member of the bromodomain and extra terminal domain family that influences transcription of downstream genes by binding to acetylated histones, which plays an important role in genesis and development of many diseases including cancer. However, expression level and biological function of BRD4 in DTC are still largely unknown, and the effect of BET inhibitor in treatment of DTC has yet to be reported.Methods:Real-time PCR and western blot were applied to determine relative BRD4 gene as well as protein expression level in 45 pairs of DTC specimens and adjacent normal thyroid tissues, K1, BCPAP and Nthy Ori3-1 cell lines, respectively. The cell viability of K1 and BCPAP determined by MTT was performed at designed time intervals (24h,48h and 72h) after JQ1 (250 nM,500 nM,1000 nM) treatment. G0/G1 arrest induced by JQ1 treatment was performed by flow cytometry. Compared with the DMSO treatment, percentages of G0/G1 cell cycle arrest were analyzed for both K1 and BCPAP cells. Iodine uptake assays were examined 60 min after JQl (250 nM,500 nM,1000 nM) treatment for both K1 and BCPAP cell lines. Radioiodine uptake tests after treatments of JQ1, NaClO4 block, or JQ1 along with NaClO4 block were carried out at various time points of radioiodine incubation. K1 and BCPAP were treated with DMSO or different concentrations of JQ1 (250,500 or 1000 nM) for 48h, and the relative mRNA expression levels of BRD4, C-MYC and NIS were analyzed by real time PCR. K1 and BCPAP were treated with JQ1 (500 nM) for indicated time intervals (0,12,24 or 48h) and the mRNA expression levels versus control group were examined. Western blot was used to monitor relative protein expression levels of BRD4, C-MYC, NIS and P21 for different concentrations of JQ1 (250,500 or 1000 nM) or for indicated time intervals (0,12,24 or 48h). Luciferase reporter assay indicated that BRD4 inhibition influenced the C-MYC promoter activity. CHIP experiment was further carried out to explore the potential mechanism.Results:BRD4 was up-regulated in DTC tissues and cell lines by Real-time PCR and western blot analysis. BRD4 inhibition induced cell viability suppression, cell cycle arrest and iodine uptake enhancement. Inhibition of BRD4 suppresses C-MYC transcription and enhances NIS expression in vitro. CHIP experiments demonstrated that BRD4 could be recruited to MYC promoter and JQ1 treatment markly attenuates the recruitment of BRD4 to MYC promoter. JQ1 treatment impairs growth of human papillary thyroid s in vivo.Conclusions:Aberrant expression of BRD4 in thyroid cancer is possibly involved in tumor progression, and JQ1 is potentially an effective chemotherapeutic agent against human thyroid cancer.Part two:Is Papillary Thyroid Microcarcinoma an Indolent Tumor? A Retrospective Single-Center StudyBackground and Objective:Despite its significant contribution to the rapid increase of thyroid cancer worldwide, the clinical importance of papillary thyroid microcarcinoma (PTMC) remains controversial. To date, post-surgery stimulated Tg (ps-Tg)? 10 ?g/L has been demonstrated to be an important predictive value of disease persistence in patients with larger thyroid cancers (> 1 cm), but the role of ps-Tg in PTMC still requires further clarification.Methods:The original data of patients in the retrospective single-center cohort study were obtained from the hospital database between Jan.2003 and Jun.2014. Two hundred and eighty PTMC patients who underwent total thyroidectomy and subsequent radioactive iodine (RAI) therapy were reviewed. The tumor stages were classified according to the TNM (tumor, node, metastasis) staging system and the consensus of the European Thyroid Association risk stratification system. Clinico-histopathological characteristics, ps-Tg levels, and different tumor stages were identified and statistically analyzed.Results:No cancer-specific recurrences or deaths were registered. The incidence of persistent disease was 14.3% after a median 43.0 months of follow-up (range 13-121). High-level ps-Tg (> 10 ?g/L) was negatively correlated with benign thyroid pathology (P=0.022) and positively correlated with high-risk stratification (P-0.047). Univariate analysis demonstrated that male sex (P=0.021), the absence of benign thyroid pathology at diagnosis (P=0.003), lymph node metastasis (P=0.006), and high level ps-Tg (P=0.000) were related to persistent disease. On multivariate analysis, high level ps-Tg (P=0.000) presented as the only independent prognostic variable significantly related to disease persistence. In this series, the overall prognosis for patients with PTMC was favorable with a 100% disease-specific survival rate.Conclusions:PTMC should not be described as an inert tumor in the condition of high level ps-Tg and high-risk stratification. Meanwhile, the administration of aggressive therapy should be recommended once prognostic risk factors coexist with a PTMC patient. |
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