【作者】 韩坤；

【导师】 余露山；

【作者基本信息】 浙江大学 ， 药学， 2017， 硕士

【摘要】 结直肠癌(colorectal cancer, CRC)是严重危害人类健康的常见消化道恶性肿瘤之一,在结直肠癌的发病机制中,表观遗传学的改变发挥了重要的作用。核苷转运体CNT2 (concentrative nucleoside transporter 2, CNT2/SLC28A2)是表达在肠上皮细胞顶膜侧的摄取转运体,主要负责核苷类物质及其类似物的转运。本研究通过对Oncomine数据库以及结直肠癌患者的结直肠肿瘤组织及配对的正常组织进行分析,发现CNT2基因在CRC肿瘤组织中的表达显著被抑制。转录水平的抑制是CNT2转运体蛋白表达丢失的重要原因之一。表观遗传修饰在基因的转录调控中发挥重要作用。DNA甲基化和组蛋白修饰是目前研究最为深入的表观遗传调控机制。通过对CRC肿瘤组织和配对正常组织中CNT2基因启动子区进行DNA甲基化分析和组蛋白修饰表征后发现,肿瘤组织和配对正常组织中CNT2基因启动子区的DNA甲基化水平无显著性差异,且肿瘤组织中与转录激活相关的组蛋白修饰H3K9Ac、H3K18Ac和H4Ac水平降低,与转录抑制相关的组蛋白修饰H3K9me3水平升高。我们以结直肠癌细胞为模型,进一步揭示组蛋白修饰对CNT2基因转录的影响。研究发现,组蛋白去乙酰化酶抑制剂曲古抑菌素A(Trichostatin A, TSA)可以降低CNT2基因启动子区去乙酰化水平,并且能够诱导结直肠癌细胞中CNT2基因的转录,增强CRC细胞对CNT2转运体底物的摄取。为探究组蛋白修饰在CNT2基因转录调控中的作用,我们考察了组蛋白去乙酰化酶抑制剂对结直肠癌细胞中CNT2基因启动子区域组蛋白修饰的影响。染色质免疫共沉淀实验结果表明,HCT15和HT29细胞经TSA处理后,随着CNT2基因启动子区去乙酰化水平的降低,该基因H3K18Ac和H4Ac水平升高,H3K9me3水平降低。之后我们在组织和细胞水平考察了与组蛋白修饰相关的酶以及转录因子的变化,采用Knock-down实验进一步验证了我们的推测。综合以上结果,我们推测,启动子区组蛋白的修饰改变可能是结直肠癌中CNT2基因转录抑制的因素之一。鉴于CNT2转运体在核苷类抗肿瘤药物的摄取中发挥重要作用,我们推测降低CRC细胞中组蛋白去乙酰化水平诱导CNT2的表达,可能会增强CRC细胞对克拉屈滨的摄取,从而增强对CRC细胞的毒性。体外实验结果表明,TSA或FK228与克拉屈滨合用可以协同增强化学药物对结直肠癌细胞系HCT15和HT29的抗肿瘤效应。因此,本研究对于临床上结直肠癌的联合用药治疗具有积极的借鉴意义。更多还原

【Abstract】 Colorectal cancer (CRC) is one of the most aggressive cancers and a leading cause of cancer deaths worldwide. One of the fundamental processes driving the initiation and progression of CRC is the accumulation of epigenetic changes in colorectal cancer cells. Concentrative nucleoside transporter 2 (CNT2/SLC28A2), an influx transporter expressed in the apical side of intestinal, is mainly responsible for the transport of nucleoside drugs. In our study, using data obtained from the cancer transcriptome database Oncomine, we identified the repression of CNT2 as a potential factor contributing to clinical treatment in CRC. By analyzing CNT2 expression in collected patient tissues, we demonstrated CNT2 repression in CRC tissues at both transcription and protein levels. Transcriptional inactivity of CNT2 contributed to the repression of CNT2 in CRC tissues.Epigenetics plays a significant role in the transcriptional regulation. DNA methylation and histone modification are the most intensely studied epigenetic pathways. DNA methylation and histone modification profiles at promoter region of CNT2 gene in CRC tissues and paired normal tissues were mapped in this study. Epigenetic analysis revealed that the repressed CNT2 promoter in CRC was mainly related to histone modification. There was no significant difference in methylation level of promoter region in colorectal cancer tissues and paired normal tissues. However, in colorectal cancer tissues, transcriptional active histone modifications such as H3K9Ac, H3K18Ac and H4Ac were impaired and transcriptional inhibition histone modifications such as H3K9me3 was increased in the promoter region of CNT2.To determine the effect of histone modification on the transcription of CNT2, CRC cells were used as a model in our study. Treatment with histone deacetylation inhibition trichostatin A (TSA) resulted in acetylation at CNT2 promoter region in 2 different CRC cell lines. Transcription expression of CNT2 were dramatically induced in CRC cell lines. The uptake function to ribavirin was also increased in CRC cells.To investigate the function of histone modifications in the transcriptional regulation of CNT2, differential histone modification patterns were determined after histone deacetylation was inhibited in HCT15 and HT29 cells with the treatment of TSA. H3K18Ac and H4Ac were found to be more enriched, while H3K9me3 level was decreased at the promoter region of CNT2. Then we investigated the changes of histone modification enzymes and transcriptional factors in tissues and cells. It was found that transcription factors, such as HDAC7, SUV39H1, PGClα may play a vital role in the regulation of CNT2 expression. The siRNA transfections were also involved. Based on these data, histone modifications seems to be one of the direct mechanisms for the transcriptional inactivity of CNT2 in CRC tissues and CRC cells.CNT2 is known to transport the nucleoside drugs. We hypothesized that the reactivation of CNT2 expression through histone deacetylation inhibition might enhance the cytotoxicity of cladribine to CRC cells. Results from in vitro experiment showed that combination of TSA/FK228 and cladribine synergied in the cytotoxic response to CRC cell lines. It highlighted the potential of a novel combinational chemotherapy in clinical CRC therapy.更多还原