miRNA-451在胶质瘤细胞增殖—迁移表型转换中的开关作用

发布时间：2018-04-30 00:04

本文选题：胶质瘤 + miR-451　；参考：《天津医科大学》2014年硕士论文

【摘要】：胶质母细胞瘤(glioblastoma, GBM)是侵袭性最强的恶性胶质瘤,也是最常见的原发性中枢神经系统肿瘤,属人类预后极差的肿瘤之一。如果仅进行支持治疗,胶质母细胞瘤病人的中位生存期不足3个月,97%的病人在12月内死亡；对于手术治疗病人,由于肿瘤细胞在脑实质内呈弥漫浸润性生长,常不能实现满意的扩大切除。面对并不乐观的现状,现代医学工作者依然不懈的致力于对胶质母细胞瘤治疗手段的研究。近十余年来,微创理念和影像导引外科等新技术的不断发展,显著提高了恶性脑肿瘤影像学全切除率,并降低了术后致残、致死率,同时各种改良的综合治疗措施和新疗法也为胶质母细胞瘤的治疗带来了新希望。目前,尽管肿瘤切除手术后替莫唑胺(temozolomide,TMZ)联合常规分割照射的标准治疗方案有了值得关注的发展,接受标准方案治疗的新诊断的胶质母细胞瘤患者相对于单纯放疗的患者二年生存率由10.9%提高到27.2%,三年生存率由4.4%提高到16%,但胶质母细胞瘤的侵袭迁移生物学特性依然严重制约着各种治疗手段的进一步提高。肿瘤细胞的增殖和运动密切相关。早在1996年就有研究提出了肿瘤细胞的"go or grow'假说。当肿瘤细胞所处的局部微生态环境不利于肿瘤细胞的增殖时,肿瘤细胞会迁移至适宜肿瘤细胞生存和增殖的环境。在这一过程中,肿瘤细胞在适合的微环境中进行增殖与迁移表型的转换,启动相应的生物学变化。尽管对肿瘤细胞迁移运动和增殖转换的分子调控机制还很不清楚,但肿瘤细胞的运动与增殖表型转换肯定不是受外部因素单独影响的,在细胞增殖和细胞运动两种表型复杂的分子和信号通路调控之间,细胞内应该存在一个表观遗传学的调控开关。微小RNA (microRNA, miRNA, miR)是一种内源性的非编码RNA,对基因表达进行抑制性调控,调节包括增殖,分化,凋亡等多种肿瘤细胞的发生、发展过程。最近研究表明,miR-451在迁移运动的胶质瘤细胞中明显下调,miR-451调节LKB1-AMPK通路,可能使胶质瘤细胞在不同的葡萄糖环境中分别表现出增殖活性和迁移运动活性。本研究主要讨论miR-451对胶质瘤细胞增殖和运动的作用,对胶质瘤细胞增殖相关蛋白mTOR和迁移调控蛋白Rac1活性的影响,以及miR-451调控AMPK, mTOR, Racl蛋白的激活状态的潜在机制。本课题研究分为以下两个部分：第一部分明确miR-451在人脑胶质瘤中的表达情况,观察miRNA-451对胶质瘤细胞增殖和运动的影响。我们收集40例胶质母细胞瘤(WHO IV)标本以及6例颞叶癫痫手术患者的对照脑组织。本课题组运用qRT-PCR检测不同标本中miR-451的表达情况。通过将miR-451类似物和抑制物转染U87、U251、SNB19三个人脑胶质瘤细胞系,探讨miRNA-451对胶质瘤细胞增殖和运动能力的作用。qRT-PCR验证转染效果；MTT实验检测细胞增殖活性；体外划痕实验和transwell迁移实验检测细胞的迁移能力。结果显示：胶质母细胞瘤组织中miR-451的相对表达量是对照脑组织的(33.58±5.19)%；MTT实验表明miR-451对胶质瘤细胞增殖能力呈正性调节作用；划痕实验和transwell迁移实验发现miR-451对胶质瘤细胞运动能力呈负性调节作用。第二部分miR-451对胶质瘤细胞增殖及运动能力作用的潜在机制。1.课题组将miR-451类似物和抑制物转染进入U87、U251、SNB19三个人脑胶质瘤细胞系,运用qRT-PCR验证转染效果；western blot技术检测不同处理组细胞中p-AMPK、AMPK、p-Raptor、Raptor、Racl的表达；GST-pulldown技术检测GTP-Racl表达。结果显示：各组细胞中AMPK、Raptor、Racl的表达与miR-451的表达无明确相关性;p-AMPK、GTP-Racl的表达量与miR-451表达呈负相关,与p-Raptor的表达呈正相关。2.为了进一步验证结论,我们通过RNA干扰技术沉默U87、U251、SNB19三个人脑胶质瘤细胞系中AMPKal的表达,在此基础上转染miR-451类似物和抑制物,qRT-PCR和western blot验证转染效果；MTT实验检测细胞增殖活性；体外划痕实验和transwell迁移实验检测细胞的迁移能力；western blot技术检测不同处理组中p-AMPK、AMPK、p-Raptor、Raptor、Rac1的表达；GST-pulldown技术检测GTP-Racl表达。结果显示：敲低AMPKal后,miR-451对细胞增殖,迁移能力及相关蛋白p-Raptor、GTP-Racl的影响不同程度减小甚至消失。结论： 1.miR-451在胶质母细胞瘤肿瘤组织标本的miR-451的表达量显著低于对照脑组织标本。体外实验证实,miR-451增强胶质瘤细胞的增殖能力,同时抑制胶质瘤细胞的迁移能力。 2.miR-451通过调节AMPK的激活,调控Rac1和mTORC1活性,从而影响胶质瘤细胞的增殖能力和迁移运动能力。
[Abstract]:Glioblastoma (GBM) is the most invasive glioma. It is also one of the most common primary central nervous system tumors. It is one of the most malignant tumors in human prognosis. If only support treatment, the median survival time of the patients with glioblastoma is less than 3 months, and 97% of the patients died in December. Patients, due to the diffuse infiltrating growth of tumor cells in the brain parenchyma, are often unable to achieve satisfactory expanded excision. Facing the unoptimistic situation, modern medical workers are still unremitting efforts to study the treatment of glioblastoma. In the past ten years, new techniques such as minimally invasive ideas and imaging guidance surgery have developed continuously. The total resection rate of malignant brain tumors is improved, and the postoperative disability and mortality rate are reduced. Meanwhile, various improved comprehensive treatments and new treatments have also brought new hope for the treatment of glioblastoma. Currently, the standard treatment regimen of temozolomide, TMZ combined with conventional fractionated irradiation after tumor resection is available. The two year survival rate of the newly diagnosed glioblastoma patients with the standard regimen treatment increased from 10.9% to 27.2%, and the three year survival rate increased from 4.4% to 16%, but the biological characteristics of the invasion and migration of glioblastoma still severely restricted the further improvement of various treatments.
The proliferation and movement of tumor cells are closely related. A "go or GROW'hypothesis" was proposed in 1996. When the local microecological environment in the tumor cells is not conducive to the proliferation of tumor cells, the tumor cells migrate to the environment suitable for the survival and proliferation of tumor cells. In this process, the tumor cells are suitable for the tumor cells. In the microenvironment, the transformation of proliferation and migration phenotypes is carried out, and the corresponding biological changes are initiated. Although the molecular regulation mechanism of the migration and proliferation of tumor cells is not clear, the transformation of the tumor cell's movement and proliferation phenotype must not be affected by external factors alone, in cell proliferation and cell movement two phenotypes. Between complex molecular and signal transduction pathways, there should be a regulatory switch in epigenetics. Micro RNA (microRNA, miRNA, miR) is an endogenous non coded RNA that regulates the expression of genes and regulates the occurrence and development of a variety of tumor cells including proliferation, differentiation and apoptosis. Recent studies have shown that MiR-451 is obviously down regulated in the glioma cells of the migratory movement, and miR-451 regulates the LKB1-AMPK pathway, which may show the proliferation and mobility activity of glioma cells in different glucose environments.
The purpose of this study is to discuss the effect of miR-451 on the proliferation and movement of glioma cells, the effect of the proliferation related protein mTOR on glioma cells and the activity of migration regulatory protein Rac1, and the potential mechanism of miR-451 regulation of the activation state of AMPK, mTOR, and Racl proteins.
This research is divided into the following two parts:
The first part is to clarify the expression of miR-451 in human glioma and observe the effect of miRNA-451 on the proliferation and movement of glioma cells. We collect 40 cases of glioblastoma (WHO IV) and 6 cases of temporal lobe epilepsy surgery. The study group used qRT-PCR to detect the expression of miR-451 in different specimens. MiR-451 analogues and inhibitors were transfected into U87, U251, SNB19 three human glioma cell lines to explore the effect of miRNA-451 on the proliferation and movement of glioma cells by.QRT-PCR; MTT test was used to detect cell proliferation activity; in vitro scratch test and Transwell migration test to detect cell migration ability. The results showed: glue The relative expression of miR-451 in the tissue of the blastoma was (33.58 + 5.19)% of the control brain tissue. The MTT experiment showed that miR-451 had a positive regulating effect on the proliferation ability of glioma cells, and the scratch test and Transwell migration test showed that miR-451 had a negative regulating effect on the motor ability of glioma cells.
The potential mechanism of the second part miR-451 on the proliferation and motor ability of glioma cells.1. subjects transfected miR-451 analogues and inhibitors into U87, U251, SNB19 three human glioma cell lines, using qRT-PCR to verify the transfection effect; Western blot technique was used to detect p-AMPK, AMPK, p-Raptor, and expressions in different treatment groups. The expression of AMPK, Raptor, Racl in each group had no definite correlation with the expression of miR-451; the expression of p-AMPK, GTP-Racl was negatively correlated with the expression of miR-451, and was positively correlated with the expression of p-Raptor, and was positively correlated with the expression of p-Raptor. In order to further verify the conclusion, we were silent by RNA interference. The expression of AMPKal in SNB19 three human glioma cell lines, on this basis, transfection of miR-451 analogue and inhibitor, qRT-PCR and Western blot to verify the transfection effect; MTT test was used to detect cell proliferation activity; in vitro scratch test and Transwell migration test to detect cell migration energy; Western blot technique was used to detect p- in different treatment groups. The expression of AMPK, AMPK, p-Raptor, Raptor, Rac1; GST-pulldown technology to detect GTP-Racl expression. The results showed that after knocking low AMPKal, the effect of miR-451 on cell proliferation, mobility and associated protein p-Raptor, GTP-Racl was reduced and even disappeared in varying degrees.
Conclusion:
The expression of 1.miR-451 in the miR-451 of glioblastoma tumor tissue was significantly lower than that of the control brain tissue. In vitro experiments confirmed that miR-451 enhanced the proliferation ability of glioma cells and inhibited the migration ability of glioma cells.
2.miR-451 regulates the activity of Rac1 and mTORC1 by regulating the activation of AMPK, thereby affecting the proliferation and migration ability of glioma cells.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

【引证文献】