TRIM24促进胶质细胞瘤的生长和耐药性研究

发布时间：2018-04-27 00:42

本文选题：TRIM24 + 胶质细胞瘤　；参考：《第四军医大学》2014年博士论文

【摘要】：胶质细胞瘤包括星形胶质细胞瘤、少突胶质细胞瘤、室管膜瘤以及混合型胶质细胞瘤等。中枢神经系统最常见的原发恶性肿瘤是恶性胶质细胞瘤，涵盖间变型星形细胞瘤(WHO Ⅲ级)、间变型少突胶质细胞瘤(WHO Ⅲ级)和多形性胶质母细胞瘤(也称胶质母细胞瘤，WHO Ⅳ级)。大多数恶性胶质细胞瘤是胶质母细胞瘤。尽管近年来手术技术、放疗策略和化疗方案取得了重大进展，间变型星形细胞瘤患者的中位总存活期为2-3年，间变型少突胶质细胞瘤患者中位总存活期为3-5年，而胶质母细胞瘤患者中位总存活期仅为12-15个月。因此，恶性胶质细胞瘤成为肿瘤研究领域的迫切课题。肿瘤标志物的鉴定可以提高预后评估的准确度，增加常规治疗的效率，并且开拓了分子靶向疗法。 TRIM24(tripartite motif-containing24)也被称为TIF1(transcription intermediaryfactor1)，是转录中介因子家族的创始成员。TRIM24蛋白的氨基末端有TRIM模体，即RING结构域、B盒和回旋卷曲区，羧基末端有PHD和Bromo结构域。其中，具有泛素-蛋白连接酶E3活性的RING结构域能介导p53的泛素化降解，而PHD和Bromo结构域识别特定修饰的组蛋白分子。此外，TRIM24蛋白中间段的LxxLL序列能够配体依赖性地结合多种细胞核受体，而PxVxL序列可以结合HP1参与染色质结构改造。由于其蛋白结构的多样性，TRIM24与肿瘤的密切关系在近些年被逐渐揭示。对于肝癌，TRIM24是作为抑癌基因；但对于粒细胞白血病和乳腺癌，TRIM24则发挥癌基因功能。比较基因组杂交实验已发现大约1/3的胶质母细胞瘤存在TRIM24基因片段的扩增，尤其是对于50岁以上的患者，提示TRIM24可能参与了胶质细胞瘤的发生和发展。为了阐明它们之间可能存在的关系，我们收集手术标本检测TRIM24表达水平，开展一系列体外和体内实验以研究其细胞分子机理，并探索相关的临床意义。 1. TRIM24在胶质细胞瘤中的过表达呈现病理级别依赖性的特征并且参与胶质母细胞瘤的复发免疫组化方法检测TRIM24在15例正常脑组织、24例毛细胞型星形细胞瘤(WHOⅠ级)、57例弥漫型星形细胞瘤(WHOⅡ级)、63例间变型星形细胞瘤、297例初诊胶质母细胞瘤、48例复发性胶质母细胞瘤以及44例少突胶质细胞瘤中的表达情况。在正常脑胶质组织和少突胶质细胞瘤中，TRIM24免疫染色阴性或仅在少数细胞中显示弱染色；但是在星形细胞瘤中，TRIM24呈现过表达，并且随着病理级别的升高，其表达量也相应增加，只是在毛细胞型星形细胞瘤和弥漫型星形细胞瘤之间未见统计学差异。通过Westernblot检测冷冻保存组织标本中的蛋白含量，进一步验证了免疫组化所观察到的TRIM24表达谱。将取自同一患者的初诊和复发性胶质母细胞瘤标本进行配对分析发现，TRIM24的表达在胶质母细胞瘤复发时有所升高，尤其是对于初诊时表达水平相对较低的病例。这些结果提示，TRIM24可能参与了胶质细胞瘤的发生，并且其表达水平与胶质细胞瘤的恶性程度之间存在着正相关关系。 2. TRIM24表达水平与初诊胶质母细胞瘤患者的预后具有负相关关系随访工作从2009年开始，不断纳入病例并且更新资料，直至2013年结束，共记录了297例初诊胶质母细胞瘤患者的年龄、性别、手术切除程度、KPS评分、化疗状态、总生存期(overall survival, OS)和无进展生存期(progression-free survival，PFS)等数据。采用Kaplan-Meier法绘制TRIM24低表达组和高表达组患者的生存曲线得出：低表达组的中位OS为13.9个月(95%CI，11.5-16.3个月)，而高表达组的中位OS为11.0个月(95%CI，10.6-11.4个月)；低表达组的中位PFS为6.5个月(95%CI，5.5-7.5个月)，而高表达组的中位PFS为5.3个月(95%CI，4.8-5.8个月)。log-rank检验发现低表达组和高表达组的OS和PFS之间均存在显著性差异(P＜0.001)。多因素Cox比例风险回归模型发现，KPS评分、年龄和TRIM24表达水平三者是影响OS的独立预后因素，而KPS评分和TRIM24水平还是PFS的独立预后影响因素。这些结果提示，检测恶性标志物TRIM24的水平，并结合患者临床指标综合考虑，在一定程度上可以提高胶质母细胞瘤患者预后评估的准确度；同时进一步说明了对于高度异质性的单一病理级别的胶质细胞瘤——胶质母细胞瘤，TRIM24水平与肿瘤恶性程度之间仍然存在着正相关关系。 3. TRIM24在体外和体内条件下促进胶质细胞瘤生长为了研究TRIM24促进胶质细胞瘤恶性进展的机理，我们构建了2种TRIM24干涉逆转录病毒、1种阴性对照逆转录病毒、过表达人TRIM24基因慢病毒以及阴性对照慢病毒，并将这些病毒感染人胶质母细胞瘤细胞系，从而检测TRIM24表达水平改变对胶质细胞瘤生长的影响。细胞增殖曲线显示，干涉TRIM24表达延长了T98和U87细胞的倍增时间。流式细胞仪测定细胞周期分布发现，TRIM24干涉的T98细胞的G2/M期比例减少，而过表达TRIM24的U251细胞的G2/M期比例增加。软琼脂克隆形成实验得出，TRIM24干涉的T98和U87细胞形成的克隆不仅数量减少，，而且体积缩小。体内实验发现，稳定干涉TRIM24表达的T98细胞丧失了裸鼠皮下成瘤能力，而阴性对照细胞仍然具有成瘤性。这些结果表明，TRIM24能够促进胶质细胞瘤的生长，所以针对TRIM24的靶向治疗有望起到干扰胶质细胞瘤进一步发展的作用。 4. TRIM24通过激活PI3K/Akt信号转导促进胶质细胞瘤生长由于PI3K/Akt和Raf/MEK/ERK信号转导途径是与胶质细胞瘤生长相关的主要通路，故采用Western blot检测TRIM24干涉对Akt和ERK活化程度的影响，并进行TRIM24表达恢复实验。干涉TRIM24表达引起p-Akt水平降低，同时p-ERK水平升高，反映了由于p-Akt减少后对Raf/MEK/ERK通路的抑制作用得到缓解。当TRIM24干涉的细胞感染过表达TRIM24的慢病毒时，TRIM24含量有一定程度的恢复，原先降低的p-Akt水平有所上升，而原先升高的p-ERK水平则有所回落。MTT实验发现，与TRIM24低表达细胞相比，TRIM24高表达细胞的增殖对PI3K抑制剂LY294002和针对PIK3CA的siRNA更加敏感。Real-time RT-PCR和Western blot检测发现，当干涉TRIM24表达时，PIK3CA表达水平下调。ChIP实验显示，TRIM24蛋白能够结合T98和U87细胞中PIK3CA基因的启动子。野生型TRIM24能够在一定程度上恢复TRIM24干涉细胞的PIK3CA和p-Akt水平，而PHD-Bromo段缺失的TRIM24突变体则未见效果。这些结果说明，TRIM24对胶质细胞瘤生长的促进作用是由PI3K/Akt信号通路所介导。 5. TRIM24促进胶质细胞瘤的耐药性除了肿瘤生长，耐药是恶性胶质细胞瘤进展和复发的重要因素。为了研究TRIM24是否影响细胞对替莫唑胺的反应性，我们将TRIM24干涉和阴性对照细胞分别暴露于替莫唑胺作用之下。MTT实验发现，干涉TRIM24表达可以增加替莫唑胺对T98和U87细胞的毒性。平板克隆形成实验显示，TRIM24干涉削弱了胶质瘤细胞在替莫唑胺作用下形成克隆的能力。将TRIM24干涉的T98细胞暴露于替莫唑胺1周后，可见明显的caspase-7剪切活化体、TUNEL标记信号以及Annexin V着色，而对照组细胞却很少有凋亡发生。体内实验同样发现TRIM24干涉能够提高替莫唑胺的治疗效果。而且，在临床背景下，TRIM24低表达的胶质母细胞瘤患者能够从化疗中获益，而TRIM24高表达组患者的预后不会因为化疗而有所改变。这些结果说明，TRIM24可以促进胶质细胞瘤的耐药性，将TRIM24干涉和化疗联合应用有望提高胶质母细胞瘤的治疗效果。 6. TRIM24通过PI3K/Akt/NF-κB信号转导调节耐药基因MGMT的表达由于DNA修复酶MGMT在替莫唑胺耐药中发挥着关键作用，所以我们检测了TRIM24干涉对MGMT的mRNA和蛋白水平的影响。Western blot实验发现，TRIM24干涉导致T98细胞的MGMT蛋白含量减少。Real-time RT-PCR实验显示，TRIM24干涉是在转录水平上调节MGMT的表达。虽然ChIP实验未见TRIM24结合MGMT基因启动子，但是双荧光素酶报告基因检测发现，TRIM24干涉引起NF-κB转录活性降低，即与抑制PI3K/Akt信号转导的结果类似。TRIM24干涉对MGMT表达的影响可以被重新引入的TRIM24所逆转，但是如果同时用siRNA干涉NF-κB亚单位p65的编码基因RelA的表达，MGMT水平的恢复则受到阻断。这些结果说明，TRIM24对MGMT表达的调控作用是(或者至少部分是)由PI3K/Akt/NF-κB途径所介导的。
[Abstract]:Glioblastoma includes astrocytoma, oligodendroglioma, ependymoma, and mixed glioblastoma. The most common primary malignant tumor of the central nervous system is malignant glioblastoma, which covers the variant type astrocytoma (WHO grade III), oligodendroglioma (WHO grade III) and glioblastoma. (also known as glioblastoma, WHO IV). Most of the malignant glioblastomas are glioblastoma. Although significant progress has been made in surgical techniques, radiotherapy strategies and chemotherapy schemes in recent years, the median survival time of patients with mesenchymal astrocytoma is 2-3 years, and the median survival time of the patients with oligodendroglioma is 3-5 years, and the glue is in glue. The median survival period of the patients with blastoma is only 12-15 months. Therefore, malignant glioblastoma has become an urgent subject in the field of cancer research. Identification of tumor markers can improve the accuracy of prognosis assessment, increase the efficiency of conventional therapy, and develop molecular targeting therapy.
TRIM24 (tripartite motif-containing24), also known as TIF1 (transcription intermediaryfactor1), is the original member of the transcriptional mediator family, the amino terminal of the amino terminal of the.TRIM24 protein has TRIM modules, namely the RING domain, B box and cyclotron curling area, and the carboxyl terminal has PHD and Bromo domains. The domain can mediate the ubiquitination of p53, while the PHD and Bromo domains identify the specific modified histone molecules. In addition, the LxxLL sequence of the intermediate segment of the TRIM24 protein can be ligands dependent on a variety of nuclear receptors, and the PxVxL sequence can be combined with HP1 to modify the chromatin structure. Because of its protein structure diversity, TRIM24 and swelling The close relationship between tumors has been gradually revealed in recent years. For liver cancer, TRIM24 is a tumor suppressor gene, but for granulocytic leukemia and breast cancer, TRIM24 plays the oncogene function.
Comparative genomic hybridization has found that approximately 1/3 glioblastoma exists TRIM24 gene fragment amplification, especially for patients over 50 years old, suggesting that TRIM24 may be involved in the occurrence and development of glioblastoma. In order to clarify the possible relationship between them, we collect surgical specimens to detect the level of TRIM24 expression and develop A series of in vitro and in vivo experiments were carried out to study the cellular molecular mechanism and explore the clinical relevance.
1. the overexpression of TRIM24 in glioblastoma is pathological grade dependent and participates in the recurrence of glioblastoma.
Immunohistochemical method was used to detect TRIM24 in 15 normal brain tissues, 24 cases of hair cell type astrocytoma (WHO grade I), 57 cases of diffuse astrocytoma (WHO class II), 63 cases of Intervariant astrocytoma, 297 newly diagnosed glioblastoma, 48 recurrent glioblastoma, and 44 oligodendroglioma. In both qualitative and oligodendrogliomas, TRIM24 immunostaining is negative or only a few cells show weak staining. But in astrocytoma, TRIM24 is overexpressed, and its expression increases with the rise of pathological grade, only between the hair cell type astrocytoma and the diffuse astrocytoma. The protein content in the frozen tissue specimens was detected by Westernblot, and the TRIM24 expression profile observed by immunohistochemistry was further verified. The paired analysis of the primary and recurrent glioblastoma specimens from the same patient showed that the expression of TRIM24 increased in the recurrence of glioblastoma, especially in the case of recurrent glioblastoma. These results suggest that TRIM24 may be involved in the occurrence of glioma and that there is a positive correlation between the level of the expression and the malignancy of glioma.
2. the level of TRIM24 expression is negatively correlated with the prognosis of patients with newly diagnosed glioblastoma.
The follow-up work began in 2009 and continued to be included in cases and updated until the end of 2013. A total of 297 patients with newly diagnosed glioblastoma were recorded, including age, sex, surgical excision, KPS, chemotherapy, total survival (overall survival, OS), and progression free survival (progression-free survival, PFS). The an-Meier method was used to draw the survival curve of TRIM24 low expression group and high expression group. The median OS of the low expression group was 13.9 months (95%CI, 11.5-16.3 month), while the middle OS in the high expression group was 11 months (95%CI, 10.6-11.4 month), and the median PFS in the low expression group was 6.5 months (95%CI, 5.5-7.5 month), while the median PFS in the high expression group was 5.3. There was a significant difference between the OS and PFS in the low expression group and the high expression group (P < 0.001). The multiple factor Cox proportional risk regression model found that KPS score, age and TRIM24 expression level three were independent preconditioning factors affecting OS, while KPS score and TRIM24 level were the independent prognostic factors, and the independent prognosis was also found to be an independent prognosis. These results suggest that the detection of the level of the malignant marker TRIM24 and the combination of the patient's clinical indicators can improve the accuracy of the prognosis evaluation of the patients with glioblastoma to a certain extent, and further explain the single pathological grade of glioblastoma, glioblastoma, TRI, which is highly heterogeneous. There is still a positive correlation between the level of M24 and malignancy of tumor.
3. TRIM24 promotes glioma growth in vitro and in vivo.
In order to study the mechanism of TRIM24 to promote the malignant progression of glioblastoma, we constructed 2 kinds of TRIM24 interfering retroviruses, 1 negative control retroviruses, TRIM24 gene lentivirus and negative control lentivirus, and the virus infection of human glioblastoma cell lines, so as to detect the change of TRIM24 expression level to glue The cell proliferation curve showed that interferometric TRIM24 expression extended the doubling time of T98 and U87 cells. Flow cytometry detected cell cycle distribution, the G2/M phase ratio of TRIM24 interfered T98 cells decreased, while the G2/M phase of U251 cells over expressed TRIM24 increased. The soft agar clone formation experiment obtained, TRIM24, TRIM24. The clones formed by interfered T98 and U87 cells are not only reduced in quantity but also in size. In vivo experiments have found that T98 cells that stably interfere with TRIM24 expression have lost the ability to tumour subcutaneously in nude mice, while negative control cells still have tumorigenicity. These results suggest that TRIM24 can promote the growth of glioma, so the targeting of TRIM24 is targeted. Treatment is expected to play a role in further development of glioma.
4. TRIM24 promotes glioma growth by activating PI3K/Akt signal transduction.
As PI3K/Akt and Raf/MEK/ERK signal transduction pathways are the main pathways associated with the growth of glioblastoma, Western blot is used to detect the effect of TRIM24 interference on the activation of Akt and ERK and to restore the TRIM24 expression. The interference TRIM24 expression causes p-Akt level to decrease, and the p-ERK level increases at the same time, reflecting the decrease of p-Akt. The inhibition of the Raf/MEK/ERK pathway was alleviated. When the TRIM24 interfered cells infected the TRIM24's lentivirus, the TRIM24 content was restored to a certain extent, and the previously reduced p-Akt level had increased, while the previously elevated p-ERK level was reduced to the.MTT experiment, and the TRIM24 high expression cells were compared with the TRIM24 low expression cells. The proliferation of the PI3K inhibitor LY294002 and the siRNA for the PIK3CA were more sensitive to.Real-time RT-PCR and Western blot detection. When the interference TRIM24 expression, PIK3CA expression level down.ChIP experiment showed that the TRIM24 protein could combine the promoter and the promoter of the gene. The PIK3CA and p-Akt levels of the cells were interfered, while the TRIM24 mutant of the PHD-Bromo segment was not effective. These results suggest that the promotion of TRIM24 on the growth of glioma is mediated by the PI3K/Akt signaling pathway.
5. TRIM24 to promote the drug resistance of glioma
In addition to tumor growth, drug resistance is an important factor in the progression and recurrence of malignant glioblastoma. In order to study whether TRIM24 affects the reactivity of cells to temozolomide, we found that TRIM24 interference and negative control cells were exposed to the action of temozolomide, respectively, in.MTT experiment. The interference of TRIM24 expression could increase the effect of temozolomide on T98 and U87. TRIM24 interference weakened the ability of glioma cells to form clones under the action of temozolomide. The TRIM24 interfered T98 cells were exposed to temozolomide for 1 weeks, and obvious caspase-7 shear activator, TUNEL marker signal and Annexin V coloring were seen, while few cells in the control group were apoptotic. In vivo experiments also found that TRIM24 interference could improve the therapeutic effect of temozolomide. In the clinical context, patients with TRIM24 low expression of glioblastoma could benefit from chemotherapy, while the prognosis of the TRIM24 high expression group was not altered by chemotherapy. These results suggest that TRIM24 can promote glioblastoma. The combination of TRIM24 interference and chemotherapy is expected to improve the therapeutic effect of glioblastoma.
6. TRIM24 regulates the expression of drug resistance gene MGMT through PI3K/Akt/NF- kappa B signal transduction.
As DNA repair enzyme MGMT plays a key role in the drug resistance of temozolomide, we detected the effect of TRIM24 interference on the mRNA and protein levels of MGMT. The.Western blot experiment found that TRIM24 interference resulted in the decrease of MGMT protein content in T98 cells and.Real-time RT-PCR experiment showed that the interference was at the transcriptional level. Although the ChIP experiment did not see the TRIM24 binding MGMT gene promoter, the double luciferase reporter gene detection found that the TRIM24 interference caused the decrease of NF- kappa B transcriptional activity, that is, the effect of the inhibition of PI3K/Akt signal transduction is similar to that the effect of.TRIM24 interference on MGMT expression can be reversed by the reintroduced TRIM24, but if siRNA interferes with N The expression of the encoding gene RelA of the F- kappa B subunit p65 and the recovery of the MGMT level were blocked. These results suggest that the regulation of TRIM24 on MGMT expression is (or at least partly) mediated by the PI3K/Akt/NF- kappa B pathway.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

【共引文献】