5-羟甲基胞嘧啶在无功能垂体腺瘤中的变化及其调节机制研究

发布时间：2018-08-24 20:07

【摘要】：背景 表观遗传修饰的改变在肿瘤的发生中发挥重要的作用。5-羟甲基胞嘧啶(5hmC)与DNA的去甲基化有关,是DNA分子表观遗传调控的重要方式之一,其在多种肿瘤,如肾癌、乳腺癌、胶质瘤等中的总体水平和调控模式均发生了明显变化。染色体10/11易位家族蛋白2(TET2)是一种双氧酶,可催化5-甲基胞嘧啶(5mC)生成5hmC,继续催化生成5-甲酰胞嘧啶(5fC)和5-羧基胞嘧啶(5caC)。TET2突变的发生在血液系统疾病中常见且影响5hmC水平,而TET2蛋白的表达量和定位情况也与5hmC的水平高低直接相关。研究证实表观遗传学改变也参与了垂体腺瘤的发生,但5hmC是否参与其中尚不清楚。无功能垂体腺瘤(NFPAs)中5hmC的水平及改变机制,尚无相关研究证实。NFPAs作为一种发病率高、危害严重、治疗困难的肿瘤,有必要对其发病机制进行进一步的研究。目的 本研究旨在检测NFPAs全基因组胞嘧啶修饰的变化,分析5hmC生成的催化酶TET2编码基因的变异、蛋白表达及定位等对5hmC水平的影响,初步探索5hmC水平变化的机制。研究内容包括3个部分。第一,收集样本及临床资料并分组,测定NFPAs和正常垂体全基因组5hmC总体水平；第二,对所有NFPAs样本进行TET2外显子部分区域测序,检测有无变异；第三,进行NFPAs和正常垂体的TET2免疫组化染色。方法 选取NFPAs 57例(57例均进行超高效液相色谱-电喷雾串联质谱(UPLC-ESI-MS/MS)分析和TET2外显子部分区域测序,26例进行免疫组化染色),正常垂体6例(5例进行UPLC-ESI-MS/MS分析,1例进行免疫组化染色),收集并整理临床资料,NFPAs分为侵袭性和非侵袭性两组,由北京协和医院神经外科两位经验丰富的医生根据影像学资料和手术术野所见分别独立判断。通过质谱检测的方法比较NFPAs和正常垂体全基因组5hmC的总体水平,通过PCR扩增TET2编码区进行测序寻找变异,通过免疫组化染色分析TET2蛋白的表达和定位,表达水平以软件分析的H-score值表示。结果NFPAs中5hmC的总体水平明显低于正常垂体(0.38‰(0.13～1.23‰)vs2.47‰(0.82-2.86‰),P0.0001),5caC的总体水平则明显升高(0.20%o(0.04～0.24%o) vs 0.16‰(0.14-0.19‰), P=0.005)。 5hmC水平低的病例,肿瘤直径更大(34.6±12.7mm vs 27.4±8.5mm, P=0.023), Ki-67指数更高(≤3%/3%的例数分别为9/5 vs 16/0,P=0.014)。低5hmC组的年龄、性别、病程、侵袭性、P53及Knosp分级与高5hmC组无明显差异。非侵袭组相比侵袭组,5hmC水平无明显差异(0.44%o (0.15～1.04%o) vs 0.35%o (0.13～1.23%o), P=0.30)。测序检测到TET2 c.86CG、c.5162TG和c.5284AG(正链)3个单核苷酸多态性(SNP)位点,对应有TET2 p.P29R、p.L1721W和p.I1762V 3个位点氨基酸改变。其中TET2 R29组相比P29组5hmC水平明显降低(0.25‰(0.13～0.80‰)vs 0.46%o (0.16-1.23‰), P=0.013), TET2 p.L1721W、p.l1762V对5hmC水平无明显影响。NFPAs中5hmC水平和TET2总表达量、细胞核表达量呈中度正相关(r=0.461,P=0.018; r=0.458, P=0.019)。免疫组化样本按质谱检测的5hmC水平高低分成两组,高5hmC组相比低5hmC组TET2总表达量和细胞核表达量明显升高(192.78±79.87 vs 129.58±60.18,P=0.032；121.49±49.21 vs 80.07±36.68,P=0.023)。非侵袭组相比侵袭组,TET2表达量和定位无明显差异。结论1、NFPAs存在全基因组5hmC的缺失,5hmC水平越低预示着肿瘤直径越大、Ki-67指数越高,提示DNA羟甲基化修饰的总量降低可能参与NFPAs的肿瘤发生,并且和肿瘤的大小、Ki-67指数相关。2、存在TET2 p.P29R变异的样本5hmC的水平明显降低,TET2 p.P29R变异影响5hmC水平的机制尚不清楚。随着TET2总表达量、细胞核定位的增多5hmC总体水平升高,提示5hmC水平的变化可能受到TET2表达量和亚细胞定位的影响。
[Abstract]:Background Epigenetic modification plays an important role in tumorigenesis. 5-hydroxymethyl cytosine (5hmC) is related to DNA demethylation and is one of the important ways of DNA molecular epigenetic regulation. It has been found that the overall level and regulation pattern of 5-hydroxymethyl cytosine (5hmC) in various tumors, such as renal cancer, breast cancer, glioma, etc. have changed significantly. Body 10/11 translocation family protein 2 (TET2) is a hydrogen peroxidase that catalyzes 5-methyl-cytosine (5mC) to form 5-hmC and continues to catalyze the formation of 5-formyl-cytosine (5fC) and 5-carboxy-cytosine (5caC). Mutations in TET2 are common in hematological diseases and affect the level of 5-hmC. The expression and localization of TET2 protein are also directly related to the level of 5-hmC. Studies have confirmed that epigenetic alterations are also involved in the development of pituitary adenomas, but it is not clear whether 5hmC is involved in the development of pituitary adenomas. Objective To detect the changes of cytosine modification in the whole genome of NFPAs, analyze the effect of the mutation of TET2 gene, protein expression and localization on the 5hmC level, and explore the mechanism of the changes in the 5hmC level. Group A, 5 hmC genome level of NFPAs and normal pituitary were measured; secondly, some regions of exon TET2 were sequenced in all samples of NFPAs, and the mutations were detected; thirdly, TET2 immunohistochemical staining was performed in NFPAs and normal pituitary. Methods 57 cases of NFPAs (57 cases) were selected for ultrahigh performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-M). S/MS analysis and TET2 exon partial region sequencing, 26 cases of immunohistochemical staining, 6 cases of normal pituitary gland (5 cases of UPLC-ESI-MS/MS analysis, 1 case of immunohistochemical staining), collection and collation of clinical data, NFPAs were divided into two groups: invasive and non-invasive, by Beijing Union Medical College Hospital neurosurgery two experienced doctors according to imaging. The total levels of NFPAs and 5 hmC of normal pituitary genome were compared by mass spectrometry. The TET2 coding region was amplified by PCR and sequenced. The expression and localization of TET2 protein were analyzed by immunohistochemical staining. The expression level was expressed by H-score value of software analysis. The overall level of 5 hmC in NFPAs was significantly lower than that in normal pituitary (0.38 8240 (0.13-1.23 82) vs 2.47 82 (0.82-2.86 82), P 0.0001, 0.82-2.86 82), 0.82-2.86 82), while the overall level of 5 caC was significantly higher (0.20% o (0.20% o (0.04-0.24% o) vs 0.16 82 (0.14-0.14-0.19 82), P = 0.005) in patients with lower level of normal pitpituitary (0.38 82 82 (0.13-1.13-1.23 82) vs 2.47 82 (0.47 023), higher Ki-67 index There was no significant difference in age, sex, course of disease, invasiveness, P53 and Knosp grade between the low 5hmC group and the high 5hmC group. There was no significant difference in 5hmC level between the non-invasive group and the invasive group (0.44% o (0.15-1.04% o) vs 0.35% o (0.13-1.23% o), P = 0.30). There were three single nucleotide polymorphisms (SNP) loci in the positive strand, corresponding to three amino acid changes at TET2 p.P29R, p.L1721W and p.I1762V. The levels of 5 hmC in TET2 R29 group were significantly lower than those in P29 group (0.25 (0.13-0.80) vs 0.46% o (0.16-1.23), P = 0.013, TET2 p.L1721W, p.l1762V) and TET2 had no significant effect on 5 hmC levels. Total expression of TET2 and nuclear expression were moderately positively correlated (r = 0.461, P = 0.018; r = 0.458, P = 0.019). Immunohistochemical samples were divided into two groups according to the level of 5 hmC detected by mass spectrometry. Total expression of TET2 and nuclear expression in 5 hmC group were significantly higher than those in 5 hmC group (192.78 [79.87] vs 129.58 [60.18], P = 0.032; 121.49 [49.21] vs 80.07 [36.68], P = 0.032, respectively. There was no significant difference in the expression and localization of TET2 between the non-invasive group and the invasive group. Conclusion 1. There was a deletion of 5hmC in the whole genome in NFPAs. The lower the 5hmC level indicates that the larger the tumor diameter, the higher the Ki-67 index, suggesting that the decrease of DNA hydroxymethylation may be involved in the tumorigenesis of NFPAs, and is related to the tumor size and Ki-67 index. With the total expression of TET2, the overall level of 5 hmC increased, suggesting that the change of 5 hmC level may be affected by the expression of TET2 and subcellular localization.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R736.4

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