本文選題：BRD4 + JQ1��； 參考：《華中科技大學(xué)》2016年博士論文

【摘要】：甲狀腺癌,是一種內(nèi)分泌系統(tǒng)最為常見的惡性腫瘤,約占人類惡性腫瘤總數(shù)的1%。當(dāng)前,基因異常調(diào)節(jié)在腫瘤發(fā)生發(fā)展中的作用越來越受到研究人員重視,同樣,甲狀腺癌中的基因異常調(diào)節(jié)也對疾病的發(fā)展起到了關(guān)鍵作用。過去的數(shù)十年中,很多研究報(bào)道并證實(shí)了分化型甲狀腺癌(DTC)中經(jīng)典的基因突變,近年來也有研究強(qiáng)調(diào)了DTC中其他表觀遺傳學(xué)調(diào)節(jié)(如對組蛋白的調(diào)節(jié))作為重要的BET家族成員,BRD4在多種細(xì)胞中發(fā)揮染色體重塑、轉(zhuǎn)錄調(diào)節(jié)等作用,其異常調(diào)節(jié)可導(dǎo)致多種疾病(包括腫瘤),但機(jī)制仍不明確。本研究第一部分對BRD4在甲狀腺癌中的表達(dá)水平及抑制BRD4后對甲狀腺癌的生物學(xué)功能產(chǎn)生的影響及機(jī)制進(jìn)行了探討。甲狀腺癌起源于甲狀腺濾泡上皮。由于體檢的增加、診斷影像技術(shù)的進(jìn)步、甚至是環(huán)境的改變,全球甲狀腺微小乳頭狀癌(papillary thyroid microcarcinoma, PTMC)的發(fā)病率持續(xù)上升,成為甲狀腺乳頭狀癌(papillary thyroid carcinoma, PTC)流行的重要原因。PTMC是PTC的特殊亞型,其定義為PTC腫瘤最大直徑≤1cm,常以低度惡性、生長緩慢、低侵襲性及低死亡率為特征,常出現(xiàn)于“正�！奔谞钕倩蚪Y(jié)節(jié)性甲狀腺腫中。目前,PTMC的最佳治療方案仍舊存在爭議。一方面,不同的指南、專家共識及文獻(xiàn)為臨床醫(yī)生提供了不同的意見,導(dǎo)致臨床實(shí)踐中治療方案模棱兩可；而另一方面,越來越多的報(bào)道證實(shí)部分PTMC進(jìn)展可導(dǎo)致遠(yuǎn)處轉(zhuǎn)移及死亡,提示PTMC的治療不能一概而論,在某些PTMC中應(yīng)該采用更激進(jìn)的手段。因此,為了充分研究PTMC患者預(yù)后有關(guān)的臨床病理特征,尚需要進(jìn)行大型、隨機(jī)、對照的臨床試驗(yàn),以幫助了解該疾病的生物學(xué)行為及臨床轉(zhuǎn)歸。術(shù)后刺激性Tg (ps-Tg)在較大TC(1cm)中是預(yù)后較差的預(yù)測因素,而在PTMC中,ps-Tg的臨床意義鮮有報(bào)道。因此本研究第二部分針對本院280例PTMC患者的臨床病理特征進(jìn)行了分析,并重點(diǎn)討論了術(shù)后刺激性Tg在PTMC患者治療及隨訪中的重要意義。本研究分為以下兩個(gè)部分：第一部分：抑制BRD4對甲狀腺癌生物學(xué)行為的影響及機(jī)制研究背景及目的：盡管放射性碘131I內(nèi)照射治療已廣泛應(yīng)用于分化型甲狀腺癌的術(shù)后治療,但仍有部分患者病情進(jìn)展及攝碘能力下降,使其患者無法從該治療中獲益。含溴結(jié)合域蛋白(Bromodomain-containing protein 4, BRD4)是BET家族的重要成員之一,它可以通過與乙�；慕M蛋白結(jié)合而影響下游基因的轉(zhuǎn)錄,這在多種疾病(包括腫瘤)的發(fā)生及發(fā)展中發(fā)揮著重要作用。然而,BRD4在DTC中的表達(dá)水平及生物學(xué)作用并不清楚,且BET抑制劑在DTC治療中的作用尚未見報(bào)道。方法：采用實(shí)時(shí)定量PCR (Real-time PCR, RT-PCR)和western blot分別檢測45對臨床組織標(biāo)本(DTC組織及周圍正常甲狀腺組織配對)及K1、BCPAP、NthyOri3-1細(xì)胞系中BRD4基因的mRNA相對表達(dá)水平和蛋白相對表達(dá)量。JQ1以不同濃度(250 nM,500 nM,1000 nM)處理K1及BCPAP不同時(shí)間(24h,48h,72h)后,用MTT法檢測細(xì)胞活力。JQ1處理K1及BCPAP后G0/G1百分比用流式檢測。以DMSO處理作為對照,分別比較K1及BCPAP細(xì)胞G0/G1細(xì)胞周期百分比。不同濃度JQ1 (250 nM,500 nM,1000 nM)處理K1及BCPAP細(xì)胞后進(jìn)行碘攝取實(shí)驗(yàn)。JQ1、NaClO4阻斷、JQ1+NaClO4阻斷處理后放射性碘孵育不同時(shí)間進(jìn)行碘攝取實(shí)驗(yàn)。K1、BCPAP以DMSO或不同濃度JQ1 (250 nM,500 nM,1000 nM)處理48h或K1、BCPAP用500 nM JQ1處理不同時(shí)間(0h,12h,24h,48h), BRD4、C-MYC、NIS基因的相對表達(dá)水平用real-time PCR檢測。Western blot用于監(jiān)測不同濃度JQ1 (250 nM,500 nM,1000 nM)或不同處理時(shí)間(Oh,12h,24h,48h) BRD4, C-MYC, NIS及P21的蛋白表達(dá)水平。雙熒光素酶報(bào)告基因檢測抑制BRD4時(shí)C-MYC啟動子的相對活性。CHIP用于進(jìn)一步研究其機(jī)制。結(jié)果：Real-time PCR及western blot證實(shí)BRD4在DTC標(biāo)本及細(xì)胞系中呈高表達(dá)。抑制BRD4導(dǎo)致細(xì)胞活性降低、細(xì)胞周期停滯、碘攝取增強(qiáng)。體外實(shí)驗(yàn)中,抑制BRD4降低C-MYC轉(zhuǎn)錄并促進(jìn)NIS的表達(dá)。CHIP實(shí)驗(yàn)闡明BRD4可以被招募到MYC啟動子,而JQ1治療可以顯著減弱這一過程。體內(nèi)實(shí)驗(yàn)中,JQ1治療可以抑制荷人乳頭狀甲狀腺癌裸鼠模型腫瘤生長。結(jié)論：BRD4在甲狀腺癌中的異常表達(dá)可能與腫瘤進(jìn)展有關(guān),JQl在治療人甲狀腺癌方面可能是一有潛力化療藥物。第二部分：280例微小乳頭狀甲狀腺癌患者轉(zhuǎn)歸分析的回顧性研究背景及目的：甲狀腺微小乳頭狀癌(papillary thyroid microcarcinoma, PTMC)的發(fā)病率快速增長,該疾病的臨床意義仍有爭議。到目前為止,術(shù)后刺激性甲狀腺球蛋白(post-surgery stimulated Tg, ps-Tg) ≥10μg/L在較大甲狀腺癌(lcm)的患者中是預(yù)測患者帶病生存的獨(dú)立危險(xiǎn)因素,但是它在PTMC中的作用仍待進(jìn)一步討論。方法：本文收集了2003年1月-2014年6月在本院進(jìn)行雙側(cè)甲狀腺全切手術(shù)及放射性碘-131 (radioactive iodine, RAI)治療PTMC患者的臨床及病理資料。采用TNM腫瘤分期系統(tǒng)及歐洲甲狀腺協(xié)會(European Thyroid Association, ETA)危險(xiǎn)分層系統(tǒng)對PTMC患者進(jìn)行分期分級,用Kaplan-Meier曲線比較不同危險(xiǎn)分期系統(tǒng)無病生存(disease free survival, DFS)率的差異,并采用單／多因素Logistic Regression分析患者預(yù)后與各因素之間的關(guān)系。結(jié)果：隨訪過程中無患者死亡或復(fù)發(fā),8年DFS為76.9%。Kaplan-Meier曲線證實(shí)TNM Ⅰ期與Ⅳ期、Ⅲ期與Ⅳ期之間,ETA極低危組與高危組、低危組與高危組,各組之間的DFS差異有統(tǒng)計(jì)學(xué)意義(P0.05)。而TNM Ⅰ期與Ⅲ期、ETA極低危組與低危組之間的DFS差異無統(tǒng)計(jì)學(xué)意義(P0.05)。最終40例(14.3%)患者帶病生存。單因素回歸分析中,男性、無合并甲狀腺良性疾病、淋巴結(jié)轉(zhuǎn)移(lymph node metastasis, LNM)、術(shù)后刺激性甲狀腺球蛋白(post-surgery stimulated thyroglobulin, ps-Tg)≥10μg/L4個(gè)變量與帶病生存有關(guān)。而多因素回歸分析中,ps-Tg≥10μg/L是唯一預(yù)測PTMC患者帶病生存的獨(dú)立危險(xiǎn)因素(OR 36.294,P=0.000)。結(jié)論：腫瘤直徑≤1cm的PTMC并非總是惰性腫瘤。在隨訪中ps-Tg≥10μg/L患者帶病生存風(fēng)險(xiǎn)增高。ETA危險(xiǎn)分層系統(tǒng)比TNM分期系統(tǒng)更能有效預(yù)測患者帶病生存。所以,在ps-Tg≥10μg/L或分級為高危的甲狀腺全切術(shù)后PTMC患者中,RAI應(yīng)當(dāng)推薦應(yīng)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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R736.1

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