人脑胶质瘤中DEC1的表达对烷化剂替莫唑胺化疗敏感性影响的分子机制

发布时间：2018-05-13 00:15

  本文选题：DEC1 + MSP58　； 参考：《第四军医大学》2016年博士论文

【摘要】：神经胶质瘤是人脑最常见的恶性肿瘤,占中枢神经系统肿瘤的半数以上,并且预后极差。其中,多形性胶质母细胞瘤(Glioblastoma multiforme,GBM,WHO IV级)具有低度分化,高度恶性,对放化疗易发生耐受等特点。癌基因胚胎软骨发育基因1(Differentiated embryo chondrocyte expressed gene 1,DEC1)的转录调控功能对肿瘤细胞的发生发展至关重要。该分子的高度表达存在于人类多种恶性肿瘤之中,并且预示肿瘤细胞的恶性分化程度。我们前期研究发现DEC1在胶质瘤中的表达与胶质瘤的病理级别呈显著的相关性,不但可以作为独立的预后因素,而且能够预示高级别胶质瘤患者对烷化剂替莫唑胺(Temozolomide,TMZ)化疗的反应性。然而,DEC1的表达对替莫唑胺细胞毒性影响的具体分子机制却不明确。核微球蛋白58(The nucleolar 58-kD microspherule protein,MSP58)是DEC1的相互作用分子,该分子同样在人类多种肿瘤中存在高表达,是一个癌基因。DEC1发挥多种生物学功能都是通过与MSP58的相互作用而实现的。MLH1(Mut L homolog1,M u t L同源基因)是DNA错配修复系统(Mis-match repair,MMR)的重要调控因子,该分子的表达缺失或功能缺陷能够导致肿瘤细胞对烷化剂细胞毒性产生耐受。转录调控因子DEC1能够结合MLH1启动子区域的E-box序列从而抑制MLH1的表达。msp58和mlh1作为与dec1癌基因功能密切相关的蛋白分子,能否成为dec1影响tmz化疗敏感性的关键所在,为本课题的主要研究内容。第一部分人脑胶质瘤中msp58的表达及其对替莫唑胺化疗敏感性的影响目的:本课题前期研究首次明确了msp58在人脑胶质瘤中的癌基因作用,并且发现dec1的表达对胶质瘤患者替莫唑胺化疗反应性具有重要影响。然而,msp58能否作为胶质瘤患者的预后因素却未见明确报道,并且作为与dec1癌基因功能密切相关的相互作用分子,msp58的表达情况对胶质瘤患者替莫唑胺化疗反应性的影响也未知。方法:我们主要利用免疫组化的方法,在158例原发性胶质瘤样本和63例接受术后tmz化疗、且肿瘤复发的复发性gbm样本中,检测msp58的表达情况,并且进一步与患者临床病理资料、预后、替莫唑胺化疗反应性等方面做相关性分析。肿瘤细胞增殖能力应用核增殖抗原ki-67的免疫组化染色进行评估,并在msp58不同表达水平之间进行统计学分析。结果:msp58的表达水平与原发性胶质瘤患者who级别(p0.001)和kps评分(p=0.003)均显著相关。生存分析表明,msp58的表达水平越高、患者预后越差(p0.001)。多因素cox回归模型结果显示,msp58的表达水平可以作为原发性胶质瘤患者独立的预后因素。另外,随msp58表达水平的不断增高,胶质瘤细胞的增殖能力也在不断的增强(p0.01)。然而,在复发性gbm中,msp58的表达情况与tmz化疗反应性之间并没有表现出显著的统计学差异。结论:msp58的表达水平与胶质瘤患者的who级别和增殖能力具有显著的相关性,说明msp58在神经胶质细胞瘤肿瘤的发生和恶性进展中能够发挥重要的作用。然而,msp58的表达水平在原发性和复发性胶质瘤患者中未见显著差异,说明tmz化疗对胶质瘤患者msp58的表达水平并无明确的影响。另外,msp58的表达与复发性胶质瘤患者tmz化疗反应性之间并未表现出显著的统计学相关性,说明dec1影响tmz细胞毒性的具体分子机制可能并非是通过与msp58的相互作用而实现的。更重要的是,msp58表达水平与胶质瘤患者总生存时间显著相关,说明msp58能够作为一个辅助who分级系统的新型的预后标志,并且这一癌基因很有可能成为神经胶质细胞瘤患者靶向治疗的重要靶点。第二部分人脑胶质瘤中dec1的表达对替莫唑胺化疗敏感性影响的分子机制目的:本课题前期研究发现发现dec1的表达对胶质瘤患者替莫唑胺化疗反应性具有重要影响。mmr是dna损伤修复、调控烷化剂化疗耐受最主要的机制。mlh1作为mmr系统的关键分子,其突变或缺失的细胞系对tmz均具有抵抗性;然而,作为mlh1的转录调控因子,dec1能否通过抑制mlh1的表达水平从而影响tmz发挥细胞毒性却有待验证。方法:首先,在63例接受术后tmz化疗、且肿瘤复发的复发性gbm样本中,我们利用免疫组化的方法检测dec1和mlh1的表达情况;利用tdt介导dutp缺口末端标记法(tunel)评估肿瘤细胞的凋亡情况。然后,以人脑胶质瘤u251和u87细胞为研究对象,通过构建dec1特异性干涉片段慢病毒表达载体的方法,进一步的建立稳定表达dec1-shrna的胶质瘤细胞系。最后,在dec1不同表达背景的胶质瘤细胞中分析tmz细胞毒性的表现差异,并且检测相关蛋白分子的表达水平,以明确dec1对tmz化疗反应性影响的具体分子机制。结果:复发性gbm标本中dec1与mlh1的表达水平之间呈负相关(r=-0.55,p0.001),并且gbm肿瘤细胞的凋亡率随mlh1的表达水平的增加而升高(r=0.29,p0.001)。另外,对dec1特异性干涉片段的测序鉴定结果表明成功的构建了慢病毒表达载体psi-lv-dec1-shrna,并且荧光显微镜下证实获得了稳定表达dec1-shrna的胶质瘤细胞株u251/u87-dec1-shrna。细胞毒性分析结果表明,胶质瘤细胞dec1的表达被干涉后,mlh1的表达显著上调,肿瘤细胞对tmz的敏感性显著增加;相反,在dec1过表达的情况下,胶质瘤细胞mlh1的表达受到了明显的抑制,并且对tmz的敏感性显著下降。结论:mlh1的表达水平与复发性gbm患者tmz的化疗耐受性密切相关。作为mlh1的转录调控基因,dec1影响gbm患者tmz化疗反应性的分子机制正是通过对mlh1的转录调控而实现的。这些结论不但揭示了dec1在tmz化疗耐受过程中发挥的重要作用,更在理论上丰富了TMZ治疗GBM出现化疗耐受的分子机制,为GBM的TMZ化疗提供抗耐药分子靶点,进一步为GBM化疗提供新的治疗策略。
[Abstract]:Neuroglioma is the most common malignant tumor of the human brain, which accounts for more than half of the central nervous system tumors and has a poor prognosis. Among them, Glioblastoma multiforme, GBM, WHO IV are highly differentiated, highly malignant, and easy to tolerate chemotherapy and chemotherapy. The oncogene embryo cartilage development gene 1 (Differentia) The transcriptional regulation function of Ted embryo chondrocyte expressed gene 1, DEC1) is essential for the development of tumor cells. The high expression of this molecule exists in a variety of human malignant tumors and indicates the malignant differentiation of tumor cells. Our previous study found that the expression of DEC1 in glioma and the pathological grade of glioma. There is a significant correlation, not only as an independent prognostic factor, but also to predict the responsiveness of patients with high grade glioma to Temozolomide (TMZ). However, the specific molecular mechanism of the cytotoxic effect of DEC1 on the cytotoxicity of temozolomide is not clear. Nuclear microglobulin 58 (The nucleolar 58-kD microsphe Rule protein, MSP58) is the interaction molecule of DEC1, which is also highly expressed in a variety of human tumors. It is an important tone of the.MLH1 (Mut L homolog1, M U), which is an oncogene.DEC1 playing a variety of biological functions. The deletion or functional defects of the molecule can lead to the tolerance of the tumor cells to the cytotoxicity of alkylating agents. The transcriptional regulator DEC1 can combine the E-box sequence of the MLH1 promoter region to inhibit the expression of MLH1,.Msp58 and MLH1 as a protein molecule closely related to the function of the DEC1 oncogene, and whether DEC1 affects TMZ The key part of the treatment sensitivity is the main research content of this subject. Part 1 the expression of MSP58 in human glioma and its effect on the chemosensitivity of temozolomide: the first study of this subject was first to clarify the oncogene role of MSP58 in human glioma, and to present the expression of DEC1 for the timozolamine in the patients with glioma. Therapeutic responsiveness has an important impact. However, the prognostic factors of MSP58 as a glioma have not been clearly reported, and as a interacting molecule closely related to the function of the DEC1 oncogene, the expression of MSP58 has no effect on the chemotherapeutic reactivity of temozolomide in glioma patients. Methods the expression of MSP58 was detected in 158 cases of primary glioma samples and 63 patients receiving postoperative TMZ chemotherapy and recurrence of tumor recurrence, and the correlation analysis was done with the patient's clinicopathological data, prognosis, and the reactivity of temozolomide in chemotherapy. The proliferation ability of tumor cells was immune to the use of nuclear proliferation antigen Ki-67. The results were statistically significant correlation between the expression level of MSP58 and the WHO level (p0.001) and KPS score (p=0.003) of the patients with primary glioma. The survival analysis showed that the higher the expression level of MSP58 was, the worse the prognosis of the patients (p0.001). The results of the multiple factor Cox regression model showed that the results of the MSP58 were significant. The expression level of MSP58 can be used as an independent prognostic factor in patients with primary glioma. In addition, the proliferation ability of glioma cells is constantly enhanced with the increasing level of MSP58 expression (P0.01). However, there is no significant statistical difference between the expression of MSP58 and the response of TMZ in recurrent GBM. Conclusion: the expression level of MSP58 has a significant correlation with the WHO level and proliferation ability of glioma patients, indicating that MSP58 plays an important role in the occurrence and malignant progression of glioblastoma tumor. However, the expression level of MSP58 is not significantly different in the patients with primary and recurrent gelatoma, indicating the TMZ chemotherapy. There is no definite effect on the expression level of MSP58 in patients with glioma. In addition, there is no significant statistical correlation between the expression of MSP58 and the response to TMZ chemotherapy in patients with recurrent glioma, indicating that the specific molecular mechanism that DEC1 affects the toxicity of TMZ cells may not be achieved through the interaction with MSP58. More importantly, it is The expression level of MSP58 is significantly associated with the total survival time of the patients with glioma, indicating that MSP58 can be a new prognostic marker for a adjuvant who grading system, and this oncogene is very likely to be an important target for targeting treatment of glioblastoma patients. The expression of DEC1 in second human gliomas is treated with temozolomide chemotherapy The molecular mechanism of sensitivity influence: the previous study found that the expression of DEC1 has an important effect on the reactivity of timozolamine in the patients with glioma..mmr is the DNA damage repair, and the most important mechanism for regulating the tolerance of alkylating agents,.Mlh1, is the key molecule of the MMR system, and the mutation or missing cell lines are all against TMZ. Resistance; however, as a transcriptional regulator of MLH1, whether DEC1 can affect the cytotoxicity of TMZ by inhibiting the expression level of MLH1 remains to be verified. First, in 63 cases of postoperative TMZ chemotherapy and recurrent GBM samples, we used immunohistochemical method to detect the expression of DEC1 and MLH1; DUTP nick end labeling (TUNEL) is used to evaluate the apoptosis of tumor cells. Then, the human glioma U251 and U87 cells are used as the research object. By constructing the DEC1 specific interference fragment Lentivirus Expression Vector, the glioma cell lines that express dec1-shrna are further established. Finally, the colloid expressing the background in DEC1 is different. The difference in the expression of TMZ cytotoxicity was analyzed in the tumor cells and the expression level of the related protein molecules was detected to determine the specific molecular mechanism of the effect of DEC1 on TMZ chemotherapy reactivity. Results: there was a negative correlation between the expression level of DEC1 and MLH1 in the recurrent GBM specimens (r=-0.55, p0.001), and the apoptosis rate of the GBM tumor cells was expressed with the expression of MLH1. In addition, r=0.29, p0.001. In addition, the sequencing of DEC1 specific interference fragments showed that the Lentivirus Expression Vector psi-lv-dec1-shrna was successfully constructed, and the cytotoxicity analysis of the glioma cell line u87-dec1-shrna. was confirmed by the fluorescence microscope, and the results of the cytotoxicity analysis of the glioma cell line of the glioma cell line showed that the glioma was glioma. After the expression of DEC1 was interfered, the expression of MLH1 was significantly up-regulated and the sensitivity of the tumor cells to TMZ increased significantly. On the contrary, the expression of MLH1 in the glioma cells was significantly inhibited and the sensitivity to TMZ decreased significantly in the case of DEC1 overexpression. Conclusion: the expression level of MLH1 is closely related to the tolerance of TMZ in recurrent GBM patients. As a transcriptional regulation gene of MLH1, the molecular mechanism that DEC1 affects the responsiveness of TMZ chemotherapy in GBM patients is achieved through the transcription regulation of MLH1. These conclusions not only reveal the important role of DEC1 in the TMZ chemotherapy tolerance, but also theoretically enrich the molecular mechanism of the chemotherapy tolerance in TMZ treatment GBM, for GBM. TMZ chemotherapy provides a target for anti drug resistance, and further provides a new therapeutic strategy for GBM chemotherapy.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.4

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