【作者】 舒艺；

【导师】 邹声泉；

【作者基本信息】 华中科技大学 ， 外科学， 2011， 博士

【摘要】 目的采用高通量MeDIP芯片分析胆管癌的甲基化谱,筛选胆管癌相关甲基化肿瘤标志物；采用能较好地模拟肿瘤体内生物学特征的3D细胞培养模型研究肼曲嗪和丙戊酸对2D和3D模型中胆管癌细胞甲基化的影响。方法1.采用MeDIP芯片分析人正常胆管上皮细胞株BEC和人胆管癌上皮细胞株TFK-1之间的差异甲基化位点；采用亚硫酸氢盐测序方法(BSP)验证目的基因的甲基化水平。采用RT-PCR检测甲基化酶抑制剂药物干预后,目的基因在TFK-1中表达的改变。采用免疫荧光检测目的基因在30例人胆管癌组织和9例人正常胆管组织中的表达；2.将单层培养的TFK-1细胞接种于2%poly-HEMA 100ul包被的U型底-96孔板中制备TFK-1 3D肿瘤细胞球。采用WST法和FDA/PI荧光染色法检测表观遗传学药物作用后2D和3D胆管癌肿瘤模型中肿瘤细胞的存活率；采用甲基化特异性PCR检测药物作用前后HOXA5,APC,和E-Cadherin基因甲基化程度的改变；3.利用肼曲嗪和丙戊酸单独或联合干预胆管癌细胞TFK-1, Annexin-FITC和Propidium双染检测药物干预后细胞凋亡率；用RT-PCR和MSP技术检测干预后ASC/TMS1基因甲基化状态的改变和mRNA的转录水平,用RT-PCR检测干预后caspase-1的mRNA转录水平。结果1.分析胆管癌甲基化谱发现胆管癌差异甲基化位点涉及多基因和信号通道改变。证实970个基因启动子区域内的CpG岛表现为高甲基化,其中317个为未知基因,653个已知基因,值得注意的是启动子区域呈现高甲基化改变的基因中,94个属于Homeobox家族。经BSP检测,甲基化程度最高的5个基因分别为HOXA2 (94.29%), HOXA5 (95.38%), HOXA11 (91.67%), HOXB4 (90.56%),和HOXD13 (94.38%)。在药物干预后,上述5个目的基因的表达得到恢复。免疫荧光检测分析证实上述5个目的基因在人正常胆管组织和人胆管癌组织中表达呈明显差异(P<0.05),分别为(66.67-100%正常胆管组,3.33%-10%胆管癌组)。2.3D培养方法形成外观圆形内部紧密的肿瘤小球,球体的平均直径为350-400μm。TFK-1胆管癌细胞3D模型比2D模型对药物治疗抵抗更明显。在2D培养模型组中,肼曲嗪和丙戊酸的IC 50值分别为59.2136μM和5.9214mM,但在3D培养模型组中肼曲嗪和丙戊酸的IC 50值分别为664.1101μM and 66.4110mM是2D模型的11.2155倍。在2D模型中,当药物剂量逐步提高时,药物呈现剂量依赖性地诱导基因去甲基化,但是这种平行的剂量和去甲基化的关系在3D肿瘤小球中并未体现。3.单用肼曲嗪或丙戊酸干预对ASC/TMS1表达无明显恢复,而联用以上两药后ASC/TMS1表达明显增加(P<0.05)。两药合用48h组基因表达量高于合用24h组表达量(P<0.05)；且caspase-1表达也明显增加(P<0.05),胆管癌细胞生长明显受抑制,凋亡率明显增加(49.88±0.044)%。结论1.采用NimbleGen HG18 CpG Promoter芯片分析胆管癌差异甲基化位点,差异位点主要集中在Calcium, MAPK, Wnt, Hedgehog, TGF-beta信号通路,涉及的细胞功能主要为细胞生长、细胞周期、细胞分化、细胞损伤和修复、细胞凋亡、细胞运动,细胞转录和信号通路以及血管生成等。2.胆管癌差异甲基化位点所包含的CpG岛中有94个属于homeobox基因家族,进一步分析结果证实HOXA2, HOXA5, HOXA11, HOXAB4和HOXD13在人正常胆管上皮组织中呈现高表达,在人胆管癌组织中呈现低表达,这为我们发现新的胆管癌肿瘤标记物提供了思路。3.成功建立胆管癌上皮细胞3D模型,立体的3D肿瘤模型比单细胞层的2D模型生物学特点更为复杂,该特点能影响表观遗传学药物的药效。TFK-1胆管癌细胞3D模型比2D模型对去甲基化药物抵抗更明显。与2D肿瘤模型相比,需要更高剂量的肼曲嗪和丙戊酸逆转3D模型中肿瘤细胞的高甲基化状态,同时2D模型所表现的剂量依赖的去甲基化特点在3D模型中并未出现。我们推测当TFK-1细胞从单层2D培养转变为3D培养时,结构发生改变,可能需要细胞-细胞间粘附相关基因的表达增强,导致在不同培养模型中基因对去甲基化药物的反应不同。4.肼曲嗪和丙戊酸联合干预对ASC/TMS1去甲基化有明显的协同作用。两药联用后胆管癌细胞凋亡率的增加可能系因去甲基化后ASC/TMS1基因表达增加,通过caspase-1途径诱导细胞凋亡第一部分MeDIP甲基化芯片分析胆管癌差异甲基化位点目的人胆管癌是一种起源于胆管上皮细胞的恶性肿瘤。甲基化事件在不影响基因遗传密码的情况下影响着基因的表达,诱导基因沉默,是肿瘤重要的发生机制。本文旨在分析人胆管癌细胞株和人正常胆管上皮细胞株间的基因甲基化差异,建立胆管癌相关基因甲基化谱。方法采用全基因组甲基化芯片杂交技术(MeDIP)分析人胆管癌细胞株TFK-1和人正常胆管上皮细胞株BEC之间的差异甲基化位点,应用Molecule Annotation System(MAS)软件分析分析差异位基因的功能和作用。结果与BEC细胞株相比,TFK-1细胞株有2103个CpG岛表现为差异高甲基化,其中531个为未知CpG岛,1672个为已知CpG岛；其中970个高甲基化CpG岛位于基因启动子区域内,317个为未知基因,653个已知基因,包含11个homeobox家族的HOX基因,涉及多个信号通路主要为Calcium, MAPK, Wnt, Hedgehog, TGF-beta信号通路,发生甲基化改变的基因占其所在的信号通路已知基因中的比例为9%-30%,胆管癌差异甲基化位点所涉及的细胞功能主要为细胞生长、细胞周期、细胞分化、细胞损伤和修复、细胞凋亡、细胞运动,细胞转录和信号通路以及血管生成等。结论HOX基因呈现高甲基化改变的基因涉及细胞周期和分化,粘附与转移,信号通路和肿瘤血管生成等多个肿瘤发生机制,可能与胆管癌的发生相关,为寻找特异性胆管癌肿瘤标志物提供了新思路。第二部分DNA甲基化对HOX基因在人胆管癌中表达的影响目的Homeobox基因是转录因子家族中重要的组成成员,在胚胎的发育过程中发挥着重要作用,调控细胞的增殖、分化、凋亡和信号通路。现已证实在肿瘤的生长过程中homeobox (HOX)基因启动子区域高甲基化可导致其表达的降低,并和肿瘤的临床预后密切相关。本部分旨在分析HOX基因在胆管癌中甲基化水平及甲基化水平的改变对HOX基因在人胆管癌中表达的影响。方法采用亚硫酸氢盐测序方法检测差异HOX基因的甲基化水平。采用RT-PCR检测甲基化酶抑制剂药物干预后,目的基因在TFK-1中表达的改变。采用免疫荧光检测目的基因在30例人胆管癌组织和9例人正常胆管组织中的表达。结果我们分析胆管癌甲基化谱发现胆管癌差异甲基化位点涉及多基因和信号通道改变。值得注意的是启动子区域呈现高甲基化改变的基因中,94个属于Homeobox家族。经BSP检测,甲基化程度最高的5个基因分别为HOXA2 (94.29%), HOXA5 (95.38%), HOXA11 (91.67%), HOXB4 (90.56%),和HOXD13 (94.38%)。在药物干预后,上述5个目的基因的表达得到恢复。免疫荧光检测分析证实上述5个目的基因在人正常胆管组织和人胆管癌组织中表达呈明显差异,分别为(66.67-100%正常胆管组,3.33%-10%胆管癌组)。结论HOXA2, HOXA5, HOXA11, HOXB4和HOXD13在胆管癌细胞株TFK-1中呈现为DNA高甲基化,给予去甲基化药物作用后,呈现为DNA低甲基化。上述HOX基因在人正常胆管上皮组织中呈现高表达,在人胆管癌组织中呈现低表达。上述研究证实DNA甲基化是诱导HOXA2, HOXA5, HOXA11, HOXB4和HOXD13在肿瘤组织中表达降低的重要原因,这为我们发现新的胆管癌肿瘤标记物提供了思路。第三部分肼曲嗪和丙戊酸对2D和3D模型中胆管癌细胞甲基化的影响目的现已广泛被证实表观遗传学改变可以影响胆管癌相关基因的表达,但是关于表观遗传学药物治疗胆管癌的体内研究却进展缓慢。多细胞肿瘤小球能较好地模拟肿瘤在体内的生物学特征,因此3D细胞培养已经广泛应用于系统性药效毒理学研究。方法本研究采用将单层培养的TFK-1细胞接种于2% poly -HEMA 100ul包被的U型底-96孔板中制备TFK-1 3D肿瘤细胞球.采用WST法和FDA/PI荧光染色法检测表观遗传学药物作用后2D和3D胆管癌肿瘤模型中肿瘤细胞的存活率,采用甲基化特异性PCR检测药物作用前后APC, E-Cadherin和HOXA5基因甲基化程度的改变。结果3D培养方法形成外观圆形、内部紧密的肿瘤小球,球体的平均直径为350-400μm。TFK-1胆管癌细胞3D模型比2D模型对药物治疗抵抗更明显。在2D培养模型组中,肼曲嗪和丙戊酸的IC 50值分别为59.2136μM和5.9214mM,但在3D培养模型组中肼曲嗪和丙戊酸的IC 50值分别为664.1101μM and 66.4110mM是2D模型的11.2155。在2D模型中,当药物剂量逐步提高时,药物呈现剂量依赖性地诱导基因去甲基化,但是这种平行的剂量和去甲基化的关系在3D肿瘤小球中并未体现。结论成功建立胆管癌上皮细胞3D模型,立体的3D肿瘤模型比单细胞层的2D模型生物学特点更为复杂,该特点能影响表观遗传学药物的药效。TFK-1胆管癌细胞3D模型比2D模型对去甲基化药物抵抗更明显。与2D肿瘤模型相比,需要更高剂量的肼曲嗪和丙戊酸逆转3D模型中肿瘤细胞的高甲基化状态,同时2D模型所表现的剂量依赖的去甲基化特点在3D模型中并未出现。我们推测当TFK-1细胞从单层2D培养转变为3D培养时,结构发生改变,可能需要细胞-细胞间粘附相关基因的表达增强,导致在不同培养模型中基因对去甲基化药物的反应不同。第四部分肼曲嗪和丙戊酸对胆管癌ASC/TMS1基因甲基化的影响目的研究甲基化酶抑制剂肼曲嗪和组蛋白脱乙酰化酶抑制剂丙戊酸联合干预对胆管癌细胞TFK-1ASC/TMS1基因甲基化调控的影响,并探讨caspase-1介导的细胞凋亡与ASC/TMS1甲基化的关系。方法利用肼曲嗪和丙戊酸单独或联合干预胆管癌细胞TFK-1, Annexin-FITC和Propidium双染检测干预后细胞凋亡率；用RT-PCR和MSP技术检测干预后ASC/TMS1基因甲基化状态的改变和mRNA的转录水平,用RT-PCR检测干预后caspase-1的mRNA转录水平。结果单用肼曲嗪或丙戊酸干预对ASC/TMS1表达无明显恢复,而联用以上两药后ASC/TMS1表达明显增加(P<0.05)。两药合用48h组基因表达量高于合用24h组表达量(P<0.05)；且caspase-1表达也明显增加(P<0.05),胆管癌细胞生长明显受抑制,凋亡率明显增加(49.88±0.044)%。结论肼曲嗪和丙戊酸联合干预对ASC/TMS1去甲基化有明显的协同作用。两药联用后胆管癌细胞凋亡率的增加可能是因为去甲基化后ASC/TMS1基因表达增加,通过caspase-1途径诱导细胞凋亡。更多还原

【Abstract】 PurposeOur aims were to investigate the methylation profile of cholangiocarcinoma to screen methylation biomarker of cholangiocarcinoma by high-throughout MeDIP microarray, establish 3D model of human cholangiocarcinoma epithelial cell line TFK-1 and identify the differential effect of hydralazine and valproic acid between 2D and 3D cultured models.Methods1. In an effort to identify new cancer-specific methylation markers, we employed a high-throughput MeDIP microarray chip to explore the methylation profile differences between TFK-1 and BEC and utilized and NimbleScanTM2.2 and SignaMap software to analyze the methylation level and function of interesting genes. Validation of methylation level of candidate genes has been performed by bisulfite sequence-PCR. Furthermore, expression of target genes was investigated after the treatment with DNA demethylation agent. Expression of candidate genes was also observed by immunofluorescence in 30 chlangiocarcinoma tissues and 9 normal bile duct tissues.2.3D cultured cells were established in culture plates coated with poly-HEMA on a gyratory shaker.Viability of 2D and 3D cultured cells was examined by WST-1 viability assay and FDA/PI staining. Methylation status of promoters regarding 3 tumor suppressor genes APC, E-Cadherin, and HOXA5 was investigated by methylation-specific PCR.3. Apoptosis was detected by mixed dye including both Annexin-FITC and Propidium with flow cytometry technique, and changes of methylation and transcription of mRNA were explored by RT-PCR and MSP techniques after the intervention of hydralazine and valproic acid either alone or combined for 24 hours and 48 hours.Results1. Compared to BEC cell line,2103 CpG islands were hypermethylation, including 1672 known genes and 531 unknown genes, and 970 hypermethylated CpG islands located in the promoter area, including 317 unknown genes and 653 known genes. Interestingly,94 genes with hypermethylated CpG islands in promoter region were Homeobox genes. The top 5 hypermethylated genes validated by BSP were HOXA2 (94.29%), HOXA5 (95.38%), HOXA11 (91.67%), HOXB4 (90.56%) and HOXD13 (94.38%). Expression of these genes was reactivated with 5’-aza-2’-deoxycytidine. Significant expression differences were detectable between normal bile duct and cholangiocarcinoma tissues (66.67-100% normal vs 3.33%-10% cancer).2. The average diameters of TFK-1 spheroids were in the range of 350-400μm. WST-1 results demonstrated that TFK-1 spheroid cells were more resistant to the epigenetic drugs and 11.22fold higher IC 50 values of hydralazine and valproic acid than did the same cells growing in monolayer culture. FDA/PI staining indicated that death rate was presented in dose-dependent behavior. Higher dose of epigenetic drugs were needed to reverse hypermethylation status in 3D cells compared to 2D cells, while parallel dose-dependent characteristic existing in 2D cells didn’t present in 3D cells.3. The transcription o f mRNA of TMS1/ASC gene and caspase-1 re-expressed again after the combined intervention o f hydralazine and valproic acid, which was higher than that of the cells treated with either hydralazine or valproic acid alone (P<0.05). The demethylation effect of 48 h by combind intervention treatment was better than that o f 24 h (P<0.05). The growth of the QBC 939 cell line was inhibited, and flow cytometry showed marked increase of apoptosis (49.88±0.044)%. Conclusion1. According to the results of data on MeDIP-chip (NimbleScan v2.5; Roche-NimbleGen), differentially methylated DNA concentrated on the Calcium, MAPK, Wnt, Hedgehog, TGF-beta signaling pathway and are involved in growth of cells, DNA synthesis and repair, cell differentiation,cell apoptosis, cell transcription factor, cell migration and adhesion and angiogenesis.2.94 genes with hypermethylated CpG islands in promoter region were Homeobox genes. Demonstrated that HOXA2 (94.29%), HOXA5 (95.38%), HOXA11 (91.67%), HOXB4 (90.56%) and HOXD13 (94.38%) expressed in normal but low expressed in cholangiocarcinoma, which provided us a new strategies to discover new biomarker of cholangiocarcinoma.3. The unique characteristics of spheroid culture, affecting the consequences of epigenetic therapy, are more complex in 3D spheroid cells than that of monolayer culture. Higher dose of epigenetic drugs were needed to reverse hypermethylation status in 3D cells compared to 2D cells, while parallel dose-dependent characteristic existing in 2D cells didn’t present in 3D cells. We speculated that the novel structural changes between cells from 2D monolayer to 3D spheroid culture might require re-expression of cell-cell adhesion genes to maintain the formation of 3D spheroids.4. TMS1 /ASC gene and caspase-1 may re-express after the synergistical intervention of hydralazine and valproic acid, and the effect is more obvious as the treatment time is extended. The apoptosis of TFK-1 cell line is increased, which may be indued by caspase-1 passway. PartⅠIdentification of methylation profile of cholangiocarcinoma with MeDIP microarrayPurpose Cholangiocarcinoma is a malignant cancer arising from the neoplastic transformation of cholangiocytes. It has been identified that methylation events will change the gene expression patterns without causing the changes in the nucleotide sequence of the genetic code, which is a main mechanism of tumorigenesis. The object of this study is to explore methylation profile differences between human cholangiocarcinoma cell line TFK-1 and biliary epithelial cell line BEC.Methods In an effort to identify new cancer-specific methylation markers, we employed a high-throughput MeDIP microarray chip to explore the methylation profile differences between TFK-1 and BEC and utilized and NimbleScanTM2.2 and SignaMap software to analyze the methylation level and function of interesting genes.Results Compared to BEC cell line,2103 CpG islands were hypermethylation, including 1672 known genes and 531 unknown genes, and 970 hypermethylated CpG islands located in the promoter area, including 317 unknown genes and 653 known genes which contains 11 HOX genes.Conclusion By using MeDIP screen we identified multiple hypermethylated genes involving cell cycle, differentiation, adhesion and metastasis, and tumor angiogenesis, which distribute to the mechanism of tumor. These data suggested new different methylated genes may work as new target genes for the search of possible specific cholangiocarcinoma marker. PartⅡEffects of DNA methylation on Hox gene expression in cholangiocarcinomaPurpose Homeobox genes are members of a transcription factor family and play a crucial role in embryonic development and in the control of cell differentiation proliferation, apoptosis and signaling path. Numerous examples of aberrant Hox gene expression induced by hypermethylation of promoter region have been found in cancer. The purpose was to investigate effect of DNA methylation on HOX expression in cholangiocarcinoma.Methods Validation of methylation level of candidate genes has been performed by bisulfite sequence-PCR. Furthermore, expression of target genes was investigated after the treatment with DNA demethylation agent. Expression of candidate genes was also observed by immunofluorescence in 30 chlangiocarcinoma tissues and 9 normal bile duct tissues.Results We identified methylation profile of cholangiocarcinoma with MeDIP microarray, relating to different gene functions and signaling pathways. Interestingly,94 genes with hypermethylated CpG islands in promoter region were Homeobox genes. The top 5 hypermethylated genes validated by BSP were HOXA2 (94.29%), HOXA5 (95.38%), HOXA11 (91.67%), HOXB4 (90.56%) and HOXD13 (94.38%). Expression of these genes was reactivated with 5’-aza-2’-deoxycytidine. Significant expression differences were detectable between normal bile duct and cholangiocarcinoma tissues (66.67-100% normal vs 3.33%-10% cancer).Conclusion Our research demonstrated methylation profile of cholangiocarcinoma for the first time with high throughput MeDIP chips. These findings supported that HOXA2, HOXA5, HOXA11, HOXAB4 and HOXD13 may work as targets for diagnosis screening and therapeutic intervention. PartⅢComparison of the effect of epigenetic therapy in 2D and 3D cholangiocarcinoma modelsPurpose Epigenetic modifications affect gene expression pattern of cholangiocarcinoma, but very little data of epigenetic therapy of cholangiocarcinoma exists in vivo. Since multicellular tumor spheroids can mimic biological characteristics of tumor,3D spheroid model have been widely applied in systemic drug screening. Our aims were to establish 3D model of human cholangiocarcinoma epithelial cell line TFK-1 and identify the differential effect of epigenetic drugs between 2D and 3D cultured models.Methods 3D cultured cells were established in culture plates coated with poly-HEMA on a gyratory shaker.Viability of 2D and 3D cultured cells was examined by WST-1 viability assay and FDA/PI staining. Methylation status of promoters regarding 3 tumor suppressor genes APC, E-Cadherin, and HOXA5 was investigated by methylation-specific PCR.Results The average diameters of TFK-1 spheroids were in the range of 350-400μm. WST-1 results demonstrated that TFK-1 spheroid cells were more resistant to the epigenetic drugs and 11.22fold higher IC 50 values of hydralazine and valproic acid than did the same cells growing in monolayer culture. FDA/PI staining indicated that death rate was presented in dose-dependent behavior. Higher dose of epigenetic drugs were needed to reverse hypermethylation status in 3D cells compared to 2D cells, while parallel dose-dependent characteristic existing in 2D cells didn’t present in 3D cells.Conclusions The unique characteristics of spheroid culture, affecting the consequences of epigenetic therapy, are more complex in 3D spheroid cells than that of monolayer culture and 3D spheroid is a promising model for epigenetic therapy.PartⅣEffects of hydralazine and valproic acid on the methylation of ASC /TMS1 of human cholangiocarcinoma cell linePurpose To investigate changes of methylation status of ASC/TMS1 in QBC 939 cell line of cholangiocarcinoma before and after combined DNA methylation and histone deacetylase inhibitors treatment, and the correlation of the apoptosis which is induced by caspase-1, and methylation status of ASC/TMS1.Methods Apoptosis was detected by mixed dye including both Annexin-FITC and Propidium with flow cytometry technique, and changes of methylation and transcription of mRNA were explored by RT-PCR and MSP techniques after the intervention of hydralazine and valproic acid either alone or combined for 24 hours and 48 hours.Results The transcription o f mRNA of TMS1/ASC gene and caspase-1 re-expressed again after the combined intervention o f hydralazine and valproic acid, which was higher than that of the cells treated with either hydralazine or valproic acid alone (P<0.05). The demethylation effect of 48 h by combind intervention treatment was better than that o f 24 h (P<0.05). The growth of the QBC 939 cell line was inhibited, and flow cytometry showed marked increase of apoptosis (49.88±0.044)%.Conclusions TMS1 /ASC gene and caspase-1 may re-express after the synergistical intervention of hydralazine and valproic acid, and the effect is more obvious as the treatment time is extended. The apoptosis of TFK-1 cell line is increased, which may be indued by caspase-1 passway.更多还原