miR-128、miR-149和miR-181靶向抑制Rap1B影响脑胶质瘤细胞增殖、侵袭及化疗敏感性的机制研究

发布时间：2018-06-07 20:32

  本文选题：脑胶质瘤 + 替莫唑胺　； 参考：《中南大学》2014年博士论文

【摘要】：[研究背景] 脑胶质瘤是最常见的颅内肿瘤之一,侵袭性生长的生物学特性使其治疗效果不尽理想。胶质瘤的形成可能是一个多因素参与、多步骤累积的病理过程。因此,从发生发展机制入手,探寻脑胶质瘤早期诊断和治疗的分子靶点依然是目前神经外科领域面临的重要课题之一 miRNA是一类内源性的非编码RNA,其异常表达不仅与脑胶质瘤的发生发展密切相关,而且也为探寻脑胶质瘤新的诊断和治疗方法提供了全新的思路。我们前期构建了WHO不同分化级别的脑胶质瘤的差异miRNA表达谱和差异mRNA表达谱,并通过整合分析发现miR-107、miR-124、miR-149等可以调控多个靶基因的表达,而Rap1B等基因又可受miR-128a/128b、miR-149、miR-181b等多个miRNA调控。通过芯片数据进行Pearson相关性分析发现,Rap1B与miR-128a/b、miR-149和miR-181b呈明显负相关,提示Rap1B可能受这些miRNA分子直接调控,参与脑胶质瘤的发生和发展。 本研究拟在此基础上进一步证实Rap1B是miR-128、miR-149及miR-181的直接调控靶基因,并证实它们在不同WHO分化级别的星形胶质细胞瘤中的表达变化,进而阐明miR-128、miR-149及miR-181通过靶向Rap1B对脑胶质瘤细胞增殖和侵袭的影响以及增强替莫唑胺(temozolomide, TMZ)对脑胶质瘤细胞化疗敏感性的作用机制。 [Rap1B是miR-128、miR-149和miR-181a/b/c/d共同作用靶基因] 通过targetScan、Picta和miRDB等软件分析,均预测到Rap1B是miR-128、miR-149和miR-181a/b/c/d的共同靶基因。通过分别构建含Rap1B和miR-128、miR-149和miR-181a/b/c/d不同结合位点的荧光素酶报告基因载体及突变体,荧光素酶活性分析、Real-time PCR和Western blotting等实验证实了Rap1B是miR-128、miR-149和miR-181a,miR-181b, miR-181c和miR-181d的共同作用靶基因,miR-128、miR-149和miR-181家族各成员可以在转录及转录后水平调控Rap1B的表达。 [miR-128、miR-149和miR-181a/b/c/d及Rap1B在WHO不同分化级别星形胶质细胞瘤中的表达及相关性分析] 我们收集了WHO不同分化级别的星形胶质细胞瘤组织及正常对照脑组织标本,通过Real-time PCR检测了miR-128、miR-149和miR-181a/b/c/d及靶基因Rap1B在星形胶质细胞瘤中的表达。我们的研究结果表明,miR-128和miR-181家族各分子的表达下调均与星形胶质瘤细胞瘤的发生密切相关,而miR-149的表达下调和Rap1B的表达上调与星形胶质细胞瘤的发生及恶性级别的进展密切相关。Pearson相关性分析证实了miR-128、miR-149和miR-181a/b/c/d与Rap1B在星形胶质细胞瘤中的表达呈负相关,这种负相关与WHO的分化级别进展无关。 [miR-128、miR-149和miR-181a/b/c/d通过Rap1B对脑胶质瘤细胞增殖和侵袭的影响] 研究表明,异常表达的miRNA分子通常可以发挥癌基因或者是抑癌基因的作用,参与肿瘤的发生发展。我们通过细胞克隆形成实验、MTT和Transwell实验分别检测了转染miR-128、miR-149和miR-181a/b/c/d等miRNAs mimics或干扰脑胶质瘤细胞中内源性Rap1B的表达后对脑胶质瘤细胞克隆形成能力、增殖和侵袭能力的影响。结果表明,miR-128、miR-149和miR-181a/b/c/d的过表达和Rap1B的敲除可以抑制脑胶质瘤细胞的增殖和侵袭能力,说明,miR-128、miR-149和miR-181a/b/c/d等多个miRNAs均可能作为抑瘤性miRNAs,通过作用其共同的靶基因Rap1B影响脑胶质瘤细胞的侵袭性生物学行为。 [miR-128、miR-149和miR-181a/b/c/d增强脑胶质瘤细胞化疗敏感性的作用机制研究] TMZ是一种新型的脑胶质瘤化疗药物,主要用于恶性脑胶质瘤或者是复发性脑胶质瘤的治疗。尽管TMZ对恶性脑胶质瘤的有效治疗率达45%,但仍有相当部分脑胶质瘤对TMZ治疗疗效欠佳或逐渐出现继发性耐药现象。现有的研究表明,miRNA作为潜在的脑胶质瘤治疗靶点,可增强TMZ对脑胶质瘤细胞的化疗敏感性。本课题进一步的研究表明,miR-128、miR-149和]miR-181a/b/c/d的过表达均可以增加TMZ对脑胶质瘤细胞的化疗敏感性。Rap1B作为miR-128、miR-149和miR-181a/b/c/d的直接调控靶基因,是RAS样小GTP结合蛋白家族成员Rap1的亚基,Rap1和Rho家族GTPases成员Racl、 RhoA和CDC42一样,均可以参与调控细胞骨架重塑、细胞的粘附和侵袭等多种生物学行为。我们的研究也进一步证实了TMZ可以通过上调miR-128、miR-149和miR-181家族分子的表达,靶向抑制Rap1B的表达,从而影响细胞粘附和细胞骨架相关蛋白CDC42、RhoA和N-cadherin的表达,调控细胞骨架蛋白F-actin的表达和分布,抑制脑胶质瘤细胞的侵袭性增殖。同时,]miR-128、miR-149和miR-181家族分子也可以通过靶向Rap1B介导的细胞骨架蛋白的重塑增强替莫唑胺对脑胶质瘤细胞的化疗敏感性。
[Abstract]:[research background]
Glioma is one of the most common intracranial tumors. The biological characteristics of invasive growth are not ideal for the treatment of glioma. The formation of glioma may be a multi factor participation and multi step cumulative pathological process. Therefore, the molecular target of the early diagnosis and treatment of glioma is still the present God from the development mechanism. One of the most important subjects in the field of surgery
MiRNA is a class of endogenous non coding RNA. Its abnormal expression is not only closely related to the development of glioma, but also provides a new way of thinking for the exploration of new diagnosis and treatment of glioma. We constructed the differential miRNA expression profiles and differential mRNA expression profiles of different differentiated gliomas of WHO. The integration analysis found that miR-107, miR-124, miR-149 and so on can regulate the expression of multiple target genes, while Rap1B and other genes can be regulated by miR-128a/128b, miR-149, miR-181b and so on. The Pearson correlation analysis of the chip data shows that Rap1B is negatively correlated with miR-128a/b, miR-149 and miR-181b, suggesting that these genes may be affected by these genes. Direct regulation of molecules is involved in the occurrence and development of gliomas.
On this basis, we further confirm that Rap1B is a direct target gene for miR-128, miR-149 and miR-181, and confirms their expression in astrocytoma at different WHO differentiation levels, and then elucidates the effect of miR-128, miR-149 and miR-181 on the proliferation and invasion of brain gelatoma cells through target Rap1B, and the enhancement of the enhancement. The mechanism of temozolomide (TMZ) on chemosensitivity of glioma cells.
[Rap1B is miR-128, miR-149 and miR-181a/b/c/d target genes together]
Through software analysis such as targetScan, Picta and miRDB, it is predicted that Rap1B is the common target gene of miR-128, miR-149 and miR-181a/b/c/d. By constructing the luciferase reporter gene carrier and mutant containing the different binding sites of Rap1B and miR-128, miR-149 and miR-181a/b/c/d, the luciferase activity analysis. The experiment confirmed that Rap1B is the common target gene of miR-128, miR-149 and miR-181a, miR-181b, miR-181c and miR-181d, and the members of miR-128, miR-149 and miR-181 family can regulate the Rap1B expression at transcriptional and post transcriptional levels.
Expression and correlation analysis of [miR-128, miR-149, miR-181a/b/c/d and Rap1B in different astrocytomas of WHO
We collected WHO different grade astrocytoma tissue and normal control brain tissue specimens. The expression of miR-128, miR-149, miR-181a/b/c/d and target gene Rap1B in astrocytoma was detected by Real-time PCR. Our results showed that the expression of each molecule in miR-128 and miR-181 family was down to star. The occurrence of glioma cell tumor is closely related, and the down regulation of miR-149 expression and the up regulation of Rap1B expression are closely related to the occurrence of astrocytoma and the progression of malignant grade,.Pearson correlation analysis confirms that miR-128, miR-149 and miR-181a/b/c/d and Rap1B are negatively correlated with the expression of Rap1B in astrocytoma, and this negative correlation and WHO The progression of differentiation is not related.
Effects of [miR-128, miR-149 and miR-181a/b/c/d on proliferation and invasion of glioma cells through Rap1B]
Studies have shown that abnormal expression of miRNA molecules can usually play the role of oncogene or tumor suppressor gene and participate in the development of tumor. We have developed cell clones and MTT and Transwell experiments to detect miRNAs mimics, such as miR-128, miR-149 and miR-181a/b/c/d, or interfere with endogenous Rap1B in glioma cells. The results showed that the overexpression of miR-128, miR-149 and miR-181a/b/c/d and the knockout of Rap1B could inhibit the proliferation and invasion of glioma cells, indicating that the multiple miRNAs of miR-128, miR-149 and miR-181a/ b/c/d may be used as tumor suppressor miRNAs. The interaction of its common target gene Rap1B affects invasive biological behavior of glioma cells.
Mechanisms of [miR-128, miR-149 and miR-181a/b/c/d enhancing chemosensitivity of glioma cells
TMZ is a new type of chemotherapeutic drug for glioma, which is mainly used for the treatment of malignant glioma or recurrent glioma. Although the effective rate of treatment for malignant glioma by TMZ is 45%, there are still a considerable number of gliomas that have poor curative effect on TMZ treatment or secondary drug resistance. The present study shows that miRNA is used as a method. The potential therapeutic target of glioma can enhance the chemosensitivity of TMZ to glioma cells. Further studies in this topic suggest that the overexpression of miR-128, miR-149 and]miR-181a/b/c/d can increase the chemosensitivity of TMZ to glioma cells,.Rap1B as a direct target gene for miR-128, miR-149 and miR-181a/b/c/d, and is RAS. The subunit of Rap1, a member of the small GTP binding protein family, Rap1 and the GTPases members of the Rho family, Racl, RhoA, and CDC42, are all involved in the regulation of cytoskeleton remodeling, cell adhesion and invasion. Our study also further confirmed that TMZ can be targeted by up regulation of miR-128, miR-149, and miR-181 family molecules. Inhibits the expression of Rap1B, which affects the expression of cell adhesion and cytoskeleton related proteins CDC42, RhoA and N-cadherin, regulates the expression and distribution of cytoskeleton F-actin, and inhibits the invasive proliferation of glioma cells. At the same time, the]miR-128, miR-149 and miR-181 family components can also be mediated by the target Rap1B mediated cytoskeleton protein. Remodeling enhances the chemosensitivity of temozolomide to glioma cells.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

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