人脑胶质瘤MGMT表达及miR-4539对MGMT表达调控作用的研究

发布时间：2018-05-29 00:57

本文选题：胶质瘤 + 替莫唑胺　；参考：《山东大学》2017年硕士论文

【摘要】：脑胶质细胞瘤是由于大脑中的胶质细胞癌变所引起的,在原发性的颅内肿瘤中最为常见,约占颅内所有恶性肿瘤的50%,发病率为3～10/10万,占全身恶性肿瘤的1%～3%,病人死亡率较高。目前胶质瘤主要通过手术切除,随后放射治疗和替莫唑胺(TMZ)化疗联合治疗。TMZ是新一代的烷化剂类抗肿瘤药物,能够穿过血脑屏障并通过产生引发DNA错配修复途径的无效循环的甲基加合物而诱导肿瘤细胞凋亡。目前临床使用TMZ治疗胶质瘤的效果并不能达到令人满意的程度,主要的原因是部分胶质瘤患者对烷化剂化疗药物存在耐药性。近来有研究表明,导致烷化剂类药物耐药主要的原因是DNA修复酶06-甲基鸟嘌呤DNA 甲基转移酶(06-methylguanine-DNA methyltransferase,MGMT)的存在。MGMT通过将烷基从DNA鸟嘌呤的06位置转移到其半胱氨酸残基来逆转DNA烷基化过程,恢复肿瘤细胞的繁殖增生能力,胶质瘤具有升高的MGMT活性时通常显示对TMZ治疗的化学抗性。一些研究已经证实,当MGMT启动子处于甲基化状态时,能够使MGMT基因处于静止状态,进而阻止其所介导的DNA修复功能和从而增强胶质瘤对烷化剂的敏感性。MicroRNA(miRNA)通过与靶基因的非编码区结合而导致靶mRNA的降解或翻译过程的抑制,进而调控基因的表达,参与一系列生物学行为。研究目的:检测MGMT在胶质瘤病例标本中的表达水平,分析MGMT表达水平与胶质瘤级别、胶质瘤大小之间的关系,探寻能够影响MGMT表达水平的MiRNA,并为缓解部分胶质瘤患者对烷化剂类药物耐药寻找新的作用靶点。研究方法:山东大学齐鲁医院随机选择47例于2015年7月至2015年11月接受手术切除的患者神经胶质瘤组织样本。所有样品通过独立的病理学检查鉴定为神经胶质瘤。每例样本分为两部分,一部分用于组织切片进行免疫组织化学染色检测MGMT表达量,另一部分用于提取RNA。根据WHO中枢神经系统肿瘤分级标准(2007年),将取得的胶质细胞瘤标本分为Ⅰ-Ⅳ级,其中低恶性组包含Ⅰ级与Ⅱ级,高恶性组包含Ⅲ级与Ⅳ级。选用患者颅脑磁共振横断位液体衰减反转恢复(fluidattenuated inversion recovery,FLAIR)序列成像测量最大直径。免疫组化中采用H评分法对MGMT表达情况进行定量。使用SPSS18.0统计学软件对数据进行统计分析,MGMT表达阳性率与胶质瘤级别及大小的关系采用χ2检验。miRNA及mRNA表达水平计算采用2^-ΔΔCT方法计算,实验组与对照组两组间的比较采用t-检验法,p0.05为具有显著统计学差异。利用miRNA数据库(TargetScan database;http://www.targetscan.org/vert_61/)找出在 MGMT 3'UTR 存在结合位点的miRNA,并进行细胞水平验证。对胶质瘤组织中MGMT与miRNA的表达量采用Westen blot及Real-Time PCR方法检测,明确两者表达关系。构建MGMT 3'UTR荧光素酶野生型及突变型载体,进行双荧光素酶检测,明确两者调控关系。细胞水平验证,选取MGMT表达相对较高的胶质瘤细胞系转入miRNA mimics及inhibitor,检测MGMT表达变化情况。功能验证,对转入miRNA mimics及inhibitor的细胞进行耐药性实验,利用cck-8,凋亡蛋白表达情况及流式细胞术检测细胞增殖及凋亡情况。探讨miRNA-4539对MGMT的调控作用,及这种作用在胶质瘤中的表达与替莫唑胺(TMZ)耐药之间的关系。结果:1.MGMT在胶质瘤组织中的表达量与胶质瘤病理级别及胶质瘤大小没有相关关系;2.MGMT与MiRNA-4539在胶质瘤组织中表达呈负相关关系;3.MiRNA-4539能够作用于MGMT 3'UTR,抑制其表达;4.在体外细胞水平,转入 MiRNA-4539 mimics 时 MGMT 表达下降,转入 MiRNA-4539 inhibitor 时MGMT表达升高;5.在体外细胞水平,转入MiRNA-4539 mimics时,胶质瘤细胞对TMZ的耐药性减低,而转入MiRNA-4539 inhibitor时则相反,TMZ耐药性增加。结论:MiRNA-4539在体外试验中可以抑制MGMT表达并改善胶质瘤细胞对TMZ的耐药性。
[Abstract]:Glioblastoma is caused by glial carcinogenesis in the brain. It is the most common in the primary intracranial tumor, accounting for about 50% of all the malignant tumors of the brain. The incidence is 3 to 10/10 million, which accounts for 1% to 3% of the malignant tumors of the whole body. The mortality of the patients is high. Amine (TMZ) chemotherapy combined with.TMZ is a new generation of alkylating antitumor drugs, which can pass through the blood brain barrier and induce the apoptosis of tumor cells by producing an ineffective cyclic methyl adduct that causes DNA mismatch repair. The main reason for the clinical use of TMZ in the treatment of gliomas is not satisfactory. Some glioma patients have resistance to alkylating agents. Recent studies have shown that the main cause of drug resistance of alkylating agents is the existence of the DNA repair enzyme 06- methyl guanine DNA methyltransferase (06-methylguanine-DNA methyltransferase, MGMT) in the presence of.MGMT by transferring alkyl from the 06 position of DNA guanine to its half. Cystine residues reversion the DNA alkylation process and restore the proliferative ability of tumor cells. The MGMT activity of glioma usually shows chemical resistance to TMZ treatment. Some studies have shown that when the MGMT promoter is in the methylation state, the MGMT gene can be kept in a state of rest, thereby preventing its mediated DNA repair. Compound function and thus enhance the sensitivity of glioma to alkylating agent.MicroRNA (miRNA) by combining with the non coding region of the target gene to lead to the degradation of target mRNA or the inhibition of translation process, and then regulate gene expression and participate in a series of biological behavior. Objective: to detect the expression level of MGMT in the specimens of glioma and analyze MGMT The relationship between the level of expression and glioma size, the size of glioma, the exploration of MiRNA that can affect the MGMT expression level, and to find new targets for alleviating the drug resistance of alkylating agents in patients with partial glioma. A randomized selection of 47 cases of surgical excision from July 2015 to November 2015 in Qilu Hospital of Shandong University. All samples were identified as gliomas by independent pathological examination. Each sample was divided into two parts. Part of the sample was used to detect MGMT expression by immunohistochemical staining in tissue sections, and the other was used to extract RNA. according to the WHO central deity system tumor grading standard (2007). The glioma specimens were divided into grade I and IV, of which the low malignant group included grade I and grade II, and the high malignant group consisted of grade III and IV. The maximum diameter was measured by the fluidattenuated inversion recovery, FLAIR sequence imaging in the patients with craniocerebral magnetic resonance transverse declines. The H score was used in the immunohistochemical method for the expression of MGMT. SPSS18.0 statistics software was used to analyze the data. The relationship between the positive rate of MGMT expression and the grade and size of glioma was calculated by the 2^- Delta Delta CT method using the chi 2 test,.MiRNA and mRNA, and the comparison between the experimental group and the control group was compared with the t- test, and P0.05 had significant statistical difference. MiRN was used in miRN. The A database (TargetScan database; http://www.targetscan.org/vert_61/) identified the miRNA of the presence of the binding site of MGMT 3'UTR and tested the cell level. The expression of MGMT and miRNA in glioma tissues was detected by Westen blot and Real-Time methods. The mutant vector was tested by double luciferase, and the regulation relationship was clear. The cell level was verified by selecting the relatively high MGMT expression of glioma cell lines into miRNA mimics and inhibitor to detect the changes of MGMT expression. Expression and flow cytometry to detect cell proliferation and apoptosis. The role of miRNA-4539 in the regulation of MGMT and the relationship between the expression of this effect in glioma and the resistance to temozolomide (TMZ). Results: the expression of 1.MGMT in glioma tissues is not related to the level of gliomatosis and the size of glioma; 2.MGMT The expression of MiRNA-4539 was negatively correlated with the expression of MiRNA-4539 in glioma tissue, and 3.MiRNA-4539 could act on MGMT 3'UTR and inhibit its expression. The expression of MGMT decreased at the level of cell at MiRNA-4539 mimics in vitro, and the expression of MGMT increased at MiRNA-4539 inhibitor; 5. the glioma cells were transferred to MiRNA-4539 mimics, at the cell level in vitro. Resistance to TMZ decreased, while MiRNA-4539 inhibitor was reversed and TMZ resistance increased. Conclusion: MiRNA-4539 can inhibit the expression of MGMT and improve the drug resistance of glioma cells to TMZ in vitro.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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