miR-874的过表达促进多形性胶质母细胞瘤对替莫唑胺化疗敏感性的研究

发布时间：2018-04-06 23:18

本文选题：多形性胶质母细胞瘤　切入点：miR-874　出处：《天津医科大学》2017年博士论文

【摘要】：研究目的和内容:多形性胶质母细胞瘤是一种常见的高度恶性的颅内原发性肿瘤。目前,临床对于恶性胶质瘤的治疗主要是手术结合放疗、化疗及其他综合治疗。多形性胶质母细胞瘤呈浸润性生长,手术很难完全切除,术后易复发,且对化疗和放疗往往具有抗性,因而其预后很差,病人的平均生存期一般不超过两年。替莫唑胺(Temozolomide,TMZ)是一种DNA烷化剂,可使O6-甲基鸟嘌呤-DNA烷基转移酶(一种DNA修复蛋白)失活。O6-甲基鸟嘌呤在下一个DNA复制循环中无法配对正确的碱基对,子链DNA形成缺口,最终导致细胞凋亡。研究表明,替莫唑胺能够显著改善胶质瘤患者的预后,已成为胶质瘤化疗的一线药物。但是由于胶质瘤存在于颅内,药物需要穿透血脑屏障,以及现在抗癌药物的耐药性等不利因素,胶质瘤的治疗及预后并不是很理想。所以,进一步探讨胶质瘤的发病机制,找寻新的增强肿瘤化疗敏感性的方法,是十分迫切的。Micro RNAs是一类长度约为21-23个核苷酸的非编码单链RNA分子,它们在胶质瘤的癌变及发生发展过程中起到重要的调控作用。其主要是通过与靶基因的3’端非转录区域(3‘UTR)特异性结合。人类超过1/3的基因都可以被mi RNAs所靶定,这些mi RNAs在细胞凋亡、增殖、分化、生长和代谢中发挥着重要作用。Micro RNA-874(mi R-874)参与了人类许多的肿瘤调控过程。本课题组的前期实验研究也发现在人类胶质瘤细胞中mi R-874表达量降低,并且在体外胶质瘤细胞中过表达mi R-874能够抑制胶质瘤细胞的增殖并且促进凋亡。但是,调控mi R-874在胶质瘤内的表达是否能够促进胶质瘤的化疗增敏性,我们还不是很清楚。所以,在本实验中,我们着重研究上调mi R-874的表达,对于替莫唑胺(TMZ)在多形性胶质母细胞瘤化疗治疗中敏感性的影响,并从分子水平探讨其作用的机制。方法:1.细胞转染及转染效率的检测:首先培养胶质瘤细胞株U87和LN229,采用瞬时转染技术,利用lipofectamine 2000将mi R-874模拟体(mimics)转染至两种胶质瘤细胞内。用lipofectamine 2000将阴性对照序列scramble转染至两种胶质瘤细胞内。2.实验分组及药物处理:分为scramble,TMZ,mi R-874,mi R-874+TMZ治疗组。上调胶质瘤细胞中mi R-874的表达,6小时后更换新鲜完全培养基,同时给予不同剂量替莫唑胺进行作用,化疗24小时、48小时。3.利用MTT方法检测细胞增殖,Annexin V/PI流式染色检测细胞凋亡,JC-1流式染色检测线粒体膜电位变化情况,Transwell实验检测细胞的侵袭、迁移情况,Western blotting检测细胞STAT3、凋亡相关蛋白Bcl-2、Bax以及迁移侵袭相关蛋白MMPs的表达情况。结果:1.转染mi R-874 mimics上调胶质瘤细胞中mi R-874的表达,给予替莫唑胺治疗24、48小时后,MTT结果显示相对于对照组,mi R-874联合TMZ治疗能够明显抑制胶质瘤细胞U87和LN229的增殖活性,差异显著具有统计学意义(P0.05);2.Annexin V/PI及线粒体膜电位流式分析发现,相对于其他三组(scramble,TMZ,mi R-874),mi R-874联合TMZ治疗组的胶质瘤细胞凋亡最为明显,差异显著具有统计学意义(P0.05);3.通过Transwell实验我们发现mi R-874联合TMZ组的胶质瘤细胞的迁移、侵袭能力明显受到抑制,与对照组差异显著具有统计学意义(P0.05);4.Western Blotting结果显示,mi R-874联合TMZ组的p-STAT3/STAT3信号通路受到抑制,抗凋亡蛋白Bcl-2表达水平降低,促凋亡蛋白Bax表达水平上调,迁移侵袭相关蛋白MMP-2、MMP-9的表达水平降低,差异显著具有统计学意义(P0.05)。结论:MTT发现转染mi R-874能够明显增强TMZ抑制胶质瘤细胞生长活性的能力;Annexin V/PI及线粒体膜电位流式分析发现,mi R-874联合TMZ治疗可以显著增强替莫唑胺对恶性胶质瘤细胞的凋亡诱导作用,其机制可能与p-STAT3/STAT3信号通路受到明显抑制,Bcl-2表达降低,Bax表达上调,MMP-2、MMP-9表达降低有关;Transwell实验发现转染mi R-874能明显抑制胶质瘤细胞的迁移、侵袭能力。这些提示:恶性胶质瘤细胞中mi R-874过表达,通过调控p-STAT3/STAT3通路增强胶质瘤细胞对TMZ的化疗敏感性,两者在恶性胶质瘤化疗中有协同作用。通过阐明mi R-874在胶质瘤细胞化疗中的作用及其机制有助于对恶性肿瘤的耐药进一步深入的了解,同时也为肿瘤化疗治疗提供新的线索。
[Abstract]:The research purpose and content: glioblastoma multiforme is a common malignant primary intracranial tumor. At present, the clinical treatment for malignant glioma is mainly surgery combined with radiotherapy, chemotherapy and other comprehensive treatment. Glioblastoma multiforme infiltrative growth, it is difficult to completely surgical resection, surgery after recurrence, and often with resistance to chemotherapy and radiotherapy, the prognosis is very poor, the average survival time of patients is generally not more than two years. Temozolomide (Temozolomide, TMZ) is a DNA alkylating agent, can make O6- methylguanine -DNA alkyl transferase (a DNA repair protein) inactivation of.O6- methylguanine in the next DNA copy the correct base pair not matching cycle, DNA sub chain form a gap, eventually lead to apoptosis. The results show that temozolomide could significantly improve the prognosis of patients with glioma, glioma chemotherapy has become a Line drugs. But due to intracranial glioma in medicine, need to penetrate the blood-brain barrier and anticancer drug resistance now and other unfavorable factors, treatment and prognosis of glioma is not very satisfactory. Therefore, to further explore the pathogenesis of glioma, the method to find the tumor chemotherapy sensitivity enhancement, is very urgent.Micro RNAs is a kind of non encoding single stranded RNA molecules of about 21-23 nucleotides in length, they play an important role in the process of carcinogenesis and development of glioma. It is mainly through the target gene 3 'untranslated regions (3' UTR) specific binding to human 1/3 gene over mi RNAs can be targeted, these mi RNAs in apoptosis, proliferation, differentiation, and plays an important role in the growth and metabolism of.Micro RNA-874 (MI R-874) is involved in many human tumor regulation process. This lesson early problem sets The study also found that the decrease of MI R-874 expression in human glioma cells and glioma cells in vitro, overexpression of MI in R-874 can inhibit the proliferation of glioma cells and promote apoptosis. However, whether the regulation of MI expression in R-874 glioma can promote glioma chemotherapy sensitization, we are not very sure. So, in this paper, we mainly study the upregulation of MI R-874 expression, for temozolomide (TMZ) sensitivity in glioblastoma chemotherapy, and to explore its mechanism at molecular level. Methods: to detect 1. cell transfection and transfection efficiency: first training U87 and the LN229 glioma cell line by transfection technique, using Lipofectamine 2000 mi R-874 model (mimics) was transfected into two glioma cells by Lipofectamine 2000. The negative control sequence were transfected by scramble two Glioma cell.2. in experimental groups and drug treatment: scramble, TMZ, MI, R-874, MI, R-874+TMZ group. Expression of glioma cells in MI R-874, 6 hours after the replacement of fresh complete medium, and given different doses of temozolomide chemotherapy effect, 24 hours, 48 hours by.3. MTT cell proliferation detection method, Annexin V/PI flow cytometry staining to detect cell apoptosis, JC-1 flow cytometry staining was used to detect the changes of mitochondrial membrane potential, Transwell assay, cell invasion, migration, Western blotting detection of cell STAT3, apoptosis related protein Bcl-2, Bax expression and invasion related protein MMPs. Results: the expression of 1. gene mi R-874 mimics mi R-874 up-regulated in glioma cells, given temozolomide therapy after 24,48 hours, MTT results showed that compared with the control group, MI R-874 combined with TMZ therapy can significantly inhibit glioma cells U87 and LN229 proliferation activity, with significant difference (P0.05); V/PI and mitochondrial membrane potential of 2.Annexin flow cytometry analysis showed that compared with other three groups (scramble, TMZ, MI, R-874) apoptosis of glioma cells combined with TMZ treatment group mi R-874 was the most obvious, significant difference was statistically significant (P0.05) 3.; through the Transwell experiment we found that the migration of MI R-874 combined with TMZ group of glioma cells, the invasion ability was significantly inhibited, and the control group with significant difference (P0.05); 4.Western Blotting showed that p-STAT3/STAT3 signaling pathway mi R-874 combined with TMZ group was inhibited, the anti apoptotic protein Bcl-2 expression level decreased, apoptosis Bax protein expression level increased, the migration and invasion related protein MMP-2, MMP-9 expression level was decreased, the difference was statistically significant (P0.05). Conclusion: MTT R-874 can increase the transfection of MI found Strong ability to inhibit the growth of glioma cells TMZ activity; V/PI and mitochondrial membrane potential of Annexin flow cytometry analysis showed that MI R-874 combined with TMZ therapy can significantly enhance temozolomide on apoptosis of malignant glioma cells induced by hypoxia and its mechanism may be related to p-STAT3/STAT3 signaling pathway was inhibited, Bcl-2 expression decreased, increased expression of Bax MMP-2 the expression of MMP-9, decrease; Transwell experiments indicated that transfection of MI R-874 could significantly inhibit the migration of glioma cells invasion. These suggest that over expression of malignant glioma cells mi R-874 enhances chemosensitivity of glioma cells to TMZ by regulation of p-STAT3 /STAT3 pathway, both have synergistic effect in chemotherapy of malignant glioma through. Clarify mi R-874 in glioma cells in the chemotherapy effect and mechanism contributes to the resistance of malignant tumor and further in-depth understanding, but also for the treatment of cancer chemotherapy The therapy provides a new clue.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.4

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