BRD4相关的甲状腺癌分子机制研究及微小乳头状甲状腺癌患者临床转归分析

发布时间：2018-06-11 16:07

本文选题：BRD4 + JQ1　；参考：《华中科技大学》2016年博士论文

【摘要】：甲状腺癌,是一种内分泌系统最为常见的恶性肿瘤,约占人类恶性肿瘤总数的1%。当前,基因异常调节在肿瘤发生发展中的作用越来越受到研究人员重视,同样,甲状腺癌中的基因异常调节也对疾病的发展起到了关键作用。过去的数十年中,很多研究报道并证实了分化型甲状腺癌(DTC)中经典的基因突变,近年来也有研究强调了DTC中其他表观遗传学调节(如对组蛋白的调节)作为重要的BET家族成员,BRD4在多种细胞中发挥染色体重塑、转录调节等作用,其异常调节可导致多种疾病(包括肿瘤),但机制仍不明确。本研究第一部分对BRD4在甲状腺癌中的表达水平及抑制BRD4后对甲状腺癌的生物学功能产生的影响及机制进行了探讨。甲状腺癌起源于甲状腺滤泡上皮。由于体检的增加、诊断影像技术的进步、甚至是环境的改变,全球甲状腺微小乳头状癌(papillary thyroid microcarcinoma, PTMC)的发病率持续上升,成为甲状腺乳头状癌(papillary thyroid carcinoma, PTC)流行的重要原因。PTMC是PTC的特殊亚型,其定义为PTC肿瘤最大直径≤1cm,常以低度恶性、生长缓慢、低侵袭性及低死亡率为特征,常出现于“正常”甲状腺或结节性甲状腺肿中。目前,PTMC的最佳治疗方案仍旧存在争议。一方面,不同的指南、专家共识及文献为临床医生提供了不同的意见,导致临床实践中治疗方案模棱两可；而另一方面,越来越多的报道证实部分PTMC进展可导致远处转移及死亡,提示PTMC的治疗不能一概而论,在某些PTMC中应该采用更激进的手段。因此,为了充分研究PTMC患者预后有关的临床病理特征,尚需要进行大型、随机、对照的临床试验,以帮助了解该疾病的生物学行为及临床转归。术后刺激性Tg (ps-Tg)在较大TC(1cm)中是预后较差的预测因素,而在PTMC中,ps-Tg的临床意义鲜有报道。因此本研究第二部分针对本院280例PTMC患者的临床病理特征进行了分析,并重点讨论了术后刺激性Tg在PTMC患者治疗及随访中的重要意义。本研究分为以下两个部分：第一部分：抑制BRD4对甲状腺癌生物学行为的影响及机制研究背景及目的：尽管放射性碘131I内照射治疗已广泛应用于分化型甲状腺癌的术后治疗,但仍有部分患者病情进展及摄碘能力下降,使其患者无法从该治疗中获益。含溴结合域蛋白(Bromodomain-containing protein 4, BRD4)是BET家族的重要成员之一,它可以通过与乙酰化的组蛋白结合而影响下游基因的转录,这在多种疾病(包括肿瘤)的发生及发展中发挥着重要作用。然而,BRD4在DTC中的表达水平及生物学作用并不清楚,且BET抑制剂在DTC治疗中的作用尚未见报道。方法：采用实时定量PCR (Real-time PCR, RT-PCR)和western blot分别检测45对临床组织标本(DTC组织及周围正常甲状腺组织配对)及K1、BCPAP、NthyOri3-1细胞系中BRD4基因的mRNA相对表达水平和蛋白相对表达量。JQ1以不同浓度(250 nM,500 nM,1000 nM)处理K1及BCPAP不同时间(24h,48h,72h)后,用MTT法检测细胞活力。JQ1处理K1及BCPAP后G0/G1百分比用流式检测。以DMSO处理作为对照,分别比较K1及BCPAP细胞G0/G1细胞周期百分比。不同浓度JQ1 (250 nM,500 nM,1000 nM)处理K1及BCPAP细胞后进行碘摄取实验。JQ1、NaClO4阻断、JQ1+NaClO4阻断处理后放射性碘孵育不同时间进行碘摄取实验。K1、BCPAP以DMSO或不同浓度JQ1 (250 nM,500 nM,1000 nM)处理48h或K1、BCPAP用500 nM JQ1处理不同时间(0h,12h,24h,48h), BRD4、C-MYC、NIS基因的相对表达水平用real-time PCR检测。Western blot用于监测不同浓度JQ1 (250 nM,500 nM,1000 nM)或不同处理时间(Oh,12h,24h,48h) BRD4, C-MYC, NIS及P21的蛋白表达水平。双荧光素酶报告基因检测抑制BRD4时C-MYC启动子的相对活性。CHIP用于进一步研究其机制。结果：Real-time PCR及western blot证实BRD4在DTC标本及细胞系中呈高表达。抑制BRD4导致细胞活性降低、细胞周期停滞、碘摄取增强。体外实验中,抑制BRD4降低C-MYC转录并促进NIS的表达。CHIP实验阐明BRD4可以被招募到MYC启动子,而JQ1治疗可以显著减弱这一过程。体内实验中,JQ1治疗可以抑制荷人乳头状甲状腺癌裸鼠模型肿瘤生长。结论：BRD4在甲状腺癌中的异常表达可能与肿瘤进展有关,JQl在治疗人甲状腺癌方面可能是一有潜力化疗药物。第二部分：280例微小乳头状甲状腺癌患者转归分析的回顾性研究背景及目的：甲状腺微小乳头状癌(papillary thyroid microcarcinoma, PTMC)的发病率快速增长,该疾病的临床意义仍有争议。到目前为止,术后刺激性甲状腺球蛋白(post-surgery stimulated Tg, ps-Tg) ≥10μg/L在较大甲状腺癌(lcm)的患者中是预测患者带病生存的独立危险因素,但是它在PTMC中的作用仍待进一步讨论。方法：本文收集了2003年1月-2014年6月在本院进行双侧甲状腺全切手术及放射性碘-131 (radioactive iodine, RAI)治疗PTMC患者的临床及病理资料。采用TNM肿瘤分期系统及欧洲甲状腺协会(European Thyroid Association, ETA)危险分层系统对PTMC患者进行分期分级,用Kaplan-Meier曲线比较不同危险分期系统无病生存(disease free survival, DFS)率的差异,并采用单／多因素Logistic Regression分析患者预后与各因素之间的关系。结果：随访过程中无患者死亡或复发,8年DFS为76.9%。Kaplan-Meier曲线证实TNM Ⅰ期与Ⅳ期、Ⅲ期与Ⅳ期之间,ETA极低危组与高危组、低危组与高危组,各组之间的DFS差异有统计学意义(P0.05)。而TNM Ⅰ期与Ⅲ期、ETA极低危组与低危组之间的DFS差异无统计学意义(P0.05)。最终40例(14.3%)患者带病生存。单因素回归分析中,男性、无合并甲状腺良性疾病、淋巴结转移(lymph node metastasis, LNM)、术后刺激性甲状腺球蛋白(post-surgery stimulated thyroglobulin, ps-Tg)≥10μg/L4个变量与带病生存有关。而多因素回归分析中,ps-Tg≥10μg/L是唯一预测PTMC患者带病生存的独立危险因素(OR 36.294,P=0.000)。结论：肿瘤直径≤1cm的PTMC并非总是惰性肿瘤。在随访中ps-Tg≥10μg/L患者带病生存风险增高。ETA危险分层系统比TNM分期系统更能有效预测患者带病生存。所以,在ps-Tg≥10μg/L或分级为高危的甲状腺全切术后PTMC患者中,RAI应当推荐应用。
[Abstract]:Thyroid cancer is the most common malignant tumor of the endocrine system, which accounts for about 1%. of the total number of human malignant tumors. The role of gene abnormal regulation in the development of tumor is becoming more and more important. Also, the abnormal regulation of genes in thyroid cancer has also played a key role in the development of disease. Many studies have reported and confirmed the classic gene mutations in differentiated thyroid carcinoma (DTC). In recent years, some studies have emphasized other epigenetic regulation in DTC (such as the regulation of histone) as an important member of the BET family. BRD4 plays the role of dyed weight plasticity and transcriptional regulation in a variety of cells, and its abnormal regulation can lead to much more. Disease (including tumors), but the mechanism is still unclear. The first part of this study discussed the expression level of BRD4 in thyroid carcinoma and the effect of inhibition of BRD4 on the biological function of thyroid carcinoma. Thyroid cancer originated from the thyroid follicle epithelium. Environmental changes, the incidence of papillary thyroid microcarcinoma (PTMC) in the global thyroid papillary carcinoma (microcarcinoma, PTMC) continues to rise. The important cause of the epidemic of thyroid papillary carcinoma (papillary thyroid carcinoma, PTC) is a special subtype of PTC, which is defined as the maximum diameter of PTC tumor less than 1cm, often low malignancy, slow growth, and low growth. Invasiveness and low mortality are characterized by "normal" thyroid or nodular goiter. Currently, the best treatment for PTMC remains controversial. On the one hand, different guidelines, expert consensus and literature provide different opinions to clinicians, leading to ambiguous treatment in clinical practice; on the other hand, More and more reports have confirmed that partial PTMC progress can lead to distant metastasis and death, suggesting that the treatment of PTMC should not be generalized and that more radical means should be used in some PTMC. Therefore, in order to fully study the clinicopathological features of the prognosis of PTMC patients, large, random, controlled clinical trials are needed to help understand this. The biological behavior and clinical outcome of the disease. The postoperative irritant Tg (ps-Tg) is a predictor of poor prognosis in the larger TC (1cm), and in PTMC, the clinical significance of ps-Tg is rarely reported. Therefore, the second part of this study has analyzed the clinicopathological features of 280 cases of PTMC in our hospital, and focused on the postoperative irritant Tg in PTMC patients. This study is divided into two parts: Part I: the first part: the effect of inhibition of BRD4 on the biological behavior of thyroid cancer and its background and purpose: Although radioactive iodine 131I therapy has been widely used in the treatment of differentiated thyroid cancer, there are still some patients' progress. The decline in iodine uptake has made it impossible for the patients to benefit from this treatment. Bromodomain-containing protein 4 (BRD4) is one of the important members of the BET family. It can affect the transcription of the downstream genes through the combination of acetylation histone, which plays an important role in the occurrence and development of various diseases, including tumors. However, the expression level and biological effect of BRD4 in DTC are not clear, and the role of BET inhibitors in the treatment of DTC has not yet been reported. Methods: the real-time quantitative PCR (Real-time PCR, RT-PCR) and Western blot were used to detect 45 pairs of clinical tissue specimens (DTC tissue and peripheral normal thyroid tissue) and K1. The relative expression level of mRNA and the relative expression of BRD4 in the -1 cell line were.JQ1 at different concentrations (250 nM, 500 nM, 1000 nM) to treat K1 and BCPAP at different times (24h, 48h, 72h). The percentage of cell cycle. JQ1 (250 nM, 500 nM, 1000 nM) treated K1 and BCPAP cells after K1 and BCPAP cells were treated with.JQ1, NaClO4 blocking, and JQ1+NaClO4 blocking treatment for iodine uptake experiments at different time, BCPAP was treated with DMSO or different concentrations (250, 500, 1000). The relative expression levels of 0h, 12h, 24h, 48h, BRD4, C-MYC, NIS were detected by real-time PCR for.Western blot on the monitoring of protein expression levels in different concentrations of JQ1 (250, 500, 1000). The relative activity of.CHIP was used to further study its mechanism. Results: Real-time PCR and Western blot confirmed that BRD4 was highly expressed in DTC specimens and cell lines. Inhibition of BRD4 resulted in reduced cell activity, stagnation of cell cycle, and enhanced iodine uptake. In vitro experiments, inhibition of BRD4 reduced C-MYC transcriptional records and NIS expression.CHIP experiments clarified JQ1 therapy can significantly reduce the process of MYC promoter. In vivo, JQ1 therapy can inhibit the growth of nude mouse model tumor bearing human papillary thyroid carcinoma. Conclusion: abnormal expression of BRD4 in thyroid cancer may be associated with tumor progression. JQl may be a potential chemotherapeutic drug in the treatment of human thyroid adenocarcinoma. The second part: the retrospective study background and purpose of the analysis on the outcome of 280 cases of small papillary thyroid carcinoma: the rapid growth of papillary thyroid microcarcinoma (PTMC), the clinical significance of the disease is still controversial. So far, postoperative stimulant thyroid globulin (post-surgery stimu) Lated Tg, ps-Tg) > 10 mu g/L is an independent risk factor for predicting patient's disease survival in patients with larger thyroid cancer (LCM), but its role in PTMC remains to be further discussed. Methods: This article collected bilateral total thyroidectomy and radioactive iodine -131 (radioactive iodine, RAI) in our hospital in June January 2003. The clinical and pathological data of PTMC patients were treated with the TNM tumor staging system and the European Thyroid Association (ETA) risk stratification system for PTMC patients, and the Kaplan-Meier curve was used to compare the difference between the disease free survival (disease free survival, DFS) rate of different risk staging systems and the use of single / more. Factor Logistic Regression analysis of the relationship between the patient's prognosis and the factors. Results: no patients died or relapse during the follow-up. 8 years DFS was 76.9%.Kaplan-Meier curve confirmed TNM I and IV, between stage III and IV, ETA extremely low risk group and high risk group, low risk group and high risk group, the difference of DFS between the groups was statistically significant (P0.0) 5). There was no significant difference in DFS between TNM stage I and stage III, ETA extremely low risk group and low risk group (P0.05). Finally, 40 cases (14.3%) had the disease. In single factor regression analysis, male, no benign thyroid disease, lymph node metastasis (lymph node metastasis, LNM), and postoperative irritant thyroglobulin (post-surgery stimulated). Thyroglobulin, ps-Tg) more than 10 g/L4 variables were associated with the survival of the disease. And in multivariate regression analysis, ps-Tg > 10 mu g/L was the only independent risk factor for predicting the survival of PTMC patients (OR 36.294, P=0.000). Conclusion: PTMC is not always inert in the tumor diameter less than 1cm. In the follow-up, the risk of survival in g/L patients with ps-Tg > 10 mu is increased. The high.ETA risk stratification system is more effective than the TNM staging system to predict patient's survival. Therefore, RAI should be recommended in PTMC patients with ps-Tg > 10 g/L or high risk of total thyroidectomy.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R736.1

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