【作者】 邱湘宁；

【导师】 陆前进； Peter A Jones；

【作者基本信息】 中南大学 ， 皮肤病与性病学， 2010， 博士

【摘要】 研究背景DNA甲基化是指由DNA甲基转移酶DNMTs催化,在胞嘧啶的第5位碳原子上加上一甲基基团,使之变成5-甲基胞嘧啶(5-mC)的化学修饰过程,通常发生在双核甘酸CpG中的胞嘧啶,是表观遗传重要的调控机制之一。人类的CpG以两种形式存在,一种是分散于DNA中的CpG位点,另一种是CpG位点富集的CpG岛(CpG Island), CpG岛主要分布在基因中的启动子上。在正常组织里,70%-90%的散在的CpG位点是甲基化的,而CpG岛则是非甲基化的。肿瘤的DNA甲基化改变表现为总体的甲基化水平降低与启动子区CpG岛的甲基化水平升高。抑癌基因与修复基因的甲基化导致抑癌基因沉默与修复基因失活,造成肿瘤抑制丧失与基因损伤增加；而总体低甲基化使反转录转座子、癌基因活化,使染色体不稳定。5-氮杂胞苷(5-Azacytidine,5-Aza-CR)与5-氮杂脱氧胞苷(5-Aza-2’-deoxycytidine,5-Aza-CdR)是两种常用的核苷类似物DNA甲基化抑制剂,均已被FDA批准用于骨髓增生异常综合症(myelodysplastic syndrome,MDS)的治疗。5-Aza-CR和5-Aza-CdR在细胞内的代谢有一定差别,5-Aza-CdR的代谢产物以磷酸盐的形式与DNA掺合；而5-Aza-CR大部分代谢产物与RNA结合,少部分代谢产物与DNA掺合。大量的临床试验表明5-Aza-CR和5-Aza-CdR均能缓解MDS病情,但近期的两个临床三期试验结果显示该两种药物在延长MDS患者的生存期上存在差异,5-Aza-CR能够明显延长生存时间,而5-Aza-CdR不能。为了探讨这种差异是否有可能是因为5-Aza-CR除了具有去DNA甲基化作用外,还能与RNA结合而干扰RNA结构和／或功能所致,本研究使用高通量筛选技术来检测这两种药物在使用等毒性剂量处理MDS/AML细胞系(P39细胞系)和实体肿瘤细胞系(T24细胞系)后对信使RNA (Message RNA, mRNA).微小RNA (microRNA, miRNA)和长链非编码RNA (Longer non-coding RNA, ncRNA)表达的影响。目的：比较等毒性剂量5-Aza-CR和5-Aza-CdR对肿瘤细胞系中多种RNA表达的影响,探讨5-Aza-CR和5-Aza-CdR在细胞内代谢上的差别是否可以影响这两种药物调节编码和非编码RNA表达的能力。方法：使用等毒性剂量处理血液系统肿瘤细胞系(MDS/AML, P39细胞系)和实体肿瘤细胞系(膀胱移形细胞癌,T24细胞系)。药物处理24小时后(Day1)更换新鲜培养液,于药物处理后48小时(Day 2)及8天(Day 8)两个时间点收集细胞。使用高通量筛选技术(microarray)来检测信使RNA (Message RNA, mRNA)、微小RNA(MicroRNA, miRNA)和长链非编码RNA (Longer non-coding RNA, ncRNA)表达,采用Limma进行差异表达分析。实时定量RT-PCR检测XAGE1D的mRNA表达,甲基化敏感性单核苷酸引物延伸(MS-SNuPE)检测XAGE1D基因启动子的甲基化状态。结果：两种药物均能影响mRNA、miRNA和ncRNA的表达。总体而言,5-aza-CdR能比5-aza-CR诱导更多的RNA表达上调,尤其在Day 8时这种差异更明显；Day 2时,5-aza-CR能诱导更多的RNA表达下调。在Day 8时,大部分被5-aza-CR上调的RNA也能被5-aza-CdR诱导表达上调,两种药物共同上调的基因中的一部分属于肿瘤-睾丸抗原基因(CTAs)。在CTAs表达上调的同时,CTAs转录调节因子CTCFL/BORIS表达也上调。另外,在Day 8时,两种药物均能上调白介素和干扰素信号通路中的多个基因的表达,显示该两种药物具有诱导炎症和／或免疫反应的作用。两种药物存在不同的即时效应(Day 2):5-aza-CR能显著下调非编码小RNA的表达；在T24细胞中,5-aza-CR能显著上调tRNA合成酶和参与氨基酸代谢的一些基因的表达。结论：5-aza-CR和5-aza-CdR这两种药物调节RNA表达的即时效应存在显著性差异,然而其持续效应的主要模式和可能的可遗传的改变是重叠的。目的：研究miR-886转录本是否是vault RNA2以及DNA甲基化抑制剂5-Aza-CR和5-Aza-CdR能否调节其表达。方法：通过cDNA末端快速扩增技术(Rapid amplification of cDNA ends, RACE)确定miR-886转录本的转录起始位点和3’末端。使用stem-loop RT-PCR检测成熟的miR-886在P39细胞系中各药物处理组中及其他四种细胞系(LD419、UROtsa、T24和UMUC3)中的表达,甲基化敏感性单核苷酸引物延伸(MS-SNuPE)检测miR-886转录本基因启动子的甲基化状态。采用连续两次转染的方法将Drosha siRNA转染至T24细胞,在第二次转染后72小时收集细胞,采用western blot及实时定量RT-PCR检测Drosha的表达,使用stem-loop RT-PCR检测成熟的miR-886及miR-21的表达。使用RNA聚合酶Ⅱ抑制剂a-amanitin处理T24细胞,分别于24小时、48小时收集细胞,采用RT-PCR来检测miR-886转录本的表达。实时定量RT-PCR及Northern blot检测miR-886转录本(vault RNA2)表达。结果：miR-886的转录本较一般的pri-miRNA短,其序列与miRbase预测的miRNA前体序列存在差异,可以直接形成茎环结构。无miR-886表达的P39和UMUC3细胞中,miR-886基因启动子处于高甲基化状态,而有miR-886表达的LD419、UROtsa以及T24细胞中,miR-886基因启动子处于低甲基化状态,并且DNA甲基化抑制剂5-Aza-CR和5-Aza-CdR能重新激活miR-886的表达。miR-886的转录本由RNA聚合酶Ⅲ催化转录,而且成熟miR-886的生成不依赖核糖核酸酶ⅢDrosha的加工,证实了miR-886转录本不是miRNA的前体。Northern blot的结果表明miR-886的转录本是vault RNA2,而且vault RNA2能产生miR-886或类似于miRNA的小分子RNA,并且5-Aza-CR和5-Aza-CdR在UMUC3和P39细胞中能重新激活vault RNA 2的表达。结论：miR-886转录本是vault RNA 2,而且vault RNA 2能产生小分子RNA,即曾被注释为miR-886-3p和miR-886-5p的小分子RNA,并且首次发现DNA甲基化调控vault RNA 2的表达。更多还原

【Abstract】 Backgroud DNA methylation occurs almost exclusively on a cytosine in a CpG dinucleotide, and is achieved by the addition of a methyl group to the 5 position of a cytosine ring mediated by DNA methyl transferases (DNMTs). The CpG sites are asymmetrically distributed into CpG poor regions and dense regions called "CpG islands", which are often located in the promoter regions of approximately half of all protein-coding genes. CpG islands normally remain unmethylated, while the most CpG poor regions are normally methylated. DNA methylation dysregulation in cancer includes abnormal global DNA hypomethylation particularly in CpG poor region and repeat regions, which induces oncogene activation and chromosomal instability, and promoter hypermethylation of CpG islands, which leads to the silencing of some tumor suppressor genes.5-Azacytidine (5-Aza-CR) and 5-Aza-2’-deoxycytidine (5-Aza-CdR) are two well-known DNA methylation inhibitors, and they have been approved by the Food and Drug Administration for the treatment of myelodysplastic syndrome. Both drugs are thought to exert their effects after incorporation into DNA and RNA by covalent binding of DNA methyltransferase (DNMT). While 5-aza-CdR is only incorporated into only DNA,5-aza-CR is incorporated into both DNA and RNA.5-aza-CR is the first drug to show a survival benefit in patients with myelodysplastic syndrome. Surprisingly, the deoxyribonucleoside analog 5-aza-CdR did not have a similar positive effect on survival in a large clinical trial. Here, we have analyzed whether this difference in nucleic acid incorporation may influence the capacity of these drugs to regulate the expression of coding and non-coding RNA, and could be one possible explanation for the differences in treatment outcome.Objective To investigate whether the different metabolism mechanisms of 5-Aza-CR and 5-Aza-CdR influence the capacity of these drugs to regulate the expression of coding and non-coding RNA. Methods A hematopoietic (P39; MDS/AML) and a solid (T24; transitional cell carcinoma) cancer cell line were treated with equitoxic doses of 5-aza-CR and 5-aza-CdR. The medium was changed after 24 hours, and cells were collected 1 day (Day 2) and 7 days (Day 8) after the drug had been removed. High-throughput screening approaches, including mRNA array, miRNA array and ncRNA array, were utilized to examine the expression of mRNA, miRNA and longer non-coding RNA (ncRNA). Differential expression analysis was done using Limma. Real-time RT-PCR was used to detect the expression of XAGE1D. DNA methylation status of XAGE1D was measured by Methylation-Specific Single Nucleotide Primer Extension (Ms-SNuPE).Results Both drugs effect the expression of all RNA species:mRNA, microRNA and long non-coding RNA. In general,5-aza-CdR treatment upregulated considerably more RNAs than 5-aza-CR, particulaly on day 8, and more RNAs were downregulated on day 2 by 5-aza-CR. A large proportion of RNAs upregulated on day 8 after 5-aza-CR treatment were also upregulated by 5-aza-CdR. Among those upregulated genes, many are belonged to the group of cancer testis antigens (CTAs). The upregulation of CTAs coincided with upregulation of CTCFL (BORIS), which may act as a transcriptional regulator of CTAs. On day 8, multiple genes from the interleukin-and interferon pathways were upregulated by both drugs, indicating a role of induction of an inflammatory and/or immunological response. A more detailed analysis immediately after treatment revealed diverse effects of each drug:On day 2, 5-aza-CR led to a remarkable down regulation of small non-coding RNAs, and significant upregulation of tRNA synthetases and certain genes involved in amino acid metabolism was observed in T24 cells.Conclusion Significant differences exist in the immediate action of the two drugs, however the dominant pattern of the lasting, and possible heritable changes, is overlapping.Objective To explore whether miR-886 transcript is vault RNA 2. And to study whether its expression can be regulated by 5-Aza-CR and 5-Aza-CdR.Methods Rapid amplification of cDNA ends (RACE) was used to find the transcription start site and 3’end of mir-886 transcript. The expression of mature miR-886 was detected by stem-loop RT-PCR in P39, LD419, UROtsa, T24, and UMUC3. DNA methylation status of potential promoter of miR-886 transcript (vault RNA2) was measured by Methylation-Specific Single Nucleotide Primer Extension (Ms-SNuPE). T24 cells were transfected twice with Drosha siRNAs, and collected at 72hr after the second transfection. Expression of Drosha mRNA was detected by real-time RT-PCR, and western blot was used for protein expression. Stem-loop RT-PCR was used to measure the expression of mature miR-886 and miR-21. T24 cells were treated with a-amanitin, and harvested at 24 hr and 48hr after treatment. The expression of miR-886 transcript was detected by RT-PCR. Real-time RT-PCR and Northern blot were used to measure the expression of miR-886 transcript.Results Although miR-886 transcript is shorter than predicted pri-miRNA and the transcription product has the appropriate size to form a pre-miRNA hairpin, miR-886 transcript is transcribed by RNA polymerase III and not by RNA polymerase II and mature mir-886 production is independent of Drosha. These results showed miR-886 transcipt is neither pri-miRNA nor pre-miRNA. Futhermore, Northern blot results indicated that instead of miR-886 vault RNA 2 is the major products which can produce miR-886 or miRNA-like small RNA. In general, Vault RNA 2 is expressed in normal cell lines such as LD419, UROtsa. It was silenced by its promoter methylation in UMUC3 and P39 cancer cell lines. The expression of vault RNA2 can be restored by 5-aza-CR and 5-aza-CdR treatment accompanied by reduction of methylation in P39 and UMUC3 cancer cell lines,Conclusion miR-886 transcript is vault RNA 2, which can produce small RNA. miR-886-3p and miR-886-5p. Our study has revealed for the first time that DNA methylation can silence vault RNA2 expression. DNA methylation inhibitors. 5-aza-CR and 5-aza-CdR, can restore the expression of methylation silenced vault RNA 2 in cancer cell lines. Although vault RNA 2 is commonly silenced in cancer cell lines, the role of vault RAN2 during the tumirgenesis is still under investigation.更多还原