【作者】 陈美华；

【导师】 陈瑞川； 刘敏；

【作者基本信息】 厦门大学 ， 生物学， 2018， 硕士

【摘要】 RNA聚合酶Ⅱ在启动子附近的转录暂停与释放,是真核生物调控基因转录,快速响应刺激的一种手段。我们实验室研究发现,BRD4参与这个过程。JQ1是一种抑制BRD4的小分子抑制剂,目前还未用于临床。阿霉素(doxorubicin)是一种化疗药物,能够用于治疗多种实体瘤和血液肿瘤,它的副作用主要是会产生心肌毒性。在本实验室的前期研究中,我们发现阿霉素能够活化BRD4,我们认为这是细胞应对阿霉素损伤的一种自我保护机制。由此我们推测:抑制BRD4的活化会增加阿霉素诱导的细胞凋亡。本文以JQ1作为抑制BRD4的主要手段,以人结肠癌细胞HCT116、人非小细胞肺癌细胞A549和人黑色素瘤细胞M14为实验模型,运用流式细胞术、蛋白质免疫印迹分析、实时定量PCR、染色质免疫共沉淀等实验技术,探究JQ1能否增敏阿霉素诱导细胞凋亡的问题。经过实验,我们发现JQ1能够增敏阿霉素诱导HCT116等多种实体瘤细胞的凋亡,且增敏效果显著。接着,我们又对JQ1增敏阿霉素诱导细胞凋亡的机理进行研究。实验结果显示JQ1增敏阿霉素诱导细胞凋亡是通过调控抗凋亡和促凋亡基因的表达来实现的,并且这个过程依赖于P53。阿霉素通过活化P53,上调促凋亡基因PUMA的表达;JQ1通过抑制抗凋亡基因BCL2和BCL-XL的表达来增强阿霉素诱导的细胞凋亡。其中,JQ1通过抑制BRD4来抑制BCL-XL的转录表达,但是对BCL2的抑制并不是通过BRD4。对JQ1增敏阿霉素诱导细胞凋亡的机理研究,有助于为临床癌症治疗提供新的用药策略和理论依据。此外,我们的实验结果显示JQ1和阿霉素的联合使用具有高杀伤力、低剂量、广谱性等诸多优点,预示了这对药物组合良好的临床应用潜力。更多还原

【Abstract】 RNA polymerase II promoter-proximal pausing and release,is a way to regulate eukaryotic genes transcription and rapidly response to stimulation.Through previous research our laboratory study found that BRD4 is involved in this process.JQ1 is a kind of small molecule inhibitor that inhibits BRD4 function,but it has not yet been utilized in clinical use.Doxorubicin is a chemotherapy drug that is widely used to treat multiple types of solid tumors and hematologic malignancies.The most dangerous side effect caused by doxorubicin is its cardiotoxicity.In the previous research done by our laboratory,we found that doxorubicin activates BRD4,which we consider to be a self-protection mechanism for cells to respond to doxorubicin damage.Therefore,we hypothesized that inhibition of BRD4 activation would increase the apoptosis induced by doxorubicin.Using JQ1 as means to suppress BRD4,with human colorectal cancer cells HCT116,human non-small-cell lung cancer cells A549 and human melanoma cells M14 as experimental models,using experiment techniques such as flow cytometry,western blot,quantitative real time PCR and chromatin immunoprecipitation assay,we explored if JQ1 can increase the sensitivity of apoptosis induced by doxorubicin.Through a series of experiments,we found that JQ1 can increase the sensitivity of doxorubicin induced apoptosis of HCT116,A549 and M14,and the increase in sensitivity was highly significant.Then,we further studied the mechanism of this increased sensitivity towards doxorubicin by JQ1.The experimental results showed that the increased cellular apoptosis was achieved by deregulated expression of anti-apoptotic and pro-apoptotic genes,and the process was dependent on tumor suppressor protein P53.In detail,doxorubicin increased the expression of pro-apoptotic gene PUMA by activating P53.JQ1 enhances doxorubicin induced apoptosis by inhibiting the expression of anti-apoptotic genes BCL2 and BCL-XL,the suppression of BCL-XL is achieved through the inhibition of BRD4 function,but the inhibition of BCL2 is not achieved via BRD4.The study on the mechanism of increased doxorubicin induced apoptosis by JQ1 is helpful in providing new strategies and theoretical basis for clinical cancer treatment.In addition,our experimental results show that combination of JQ1 and doxorubicin has many advantages,including the high tumor cell lethality,the significant lower dose of medication,the wide range of drug killing,and so on.These superiorities indicate the combination of JQ1 and doxorubicin has good potential in clinical application.更多还原