【作者】 李一明；

【导师】 卢亦成；

【作者基本信息】 第二军医大学 ， 神经外科， 2009， 博士

【摘要】 microRNA是一类18-26 nt长度,通过与靶基因3’非编码区的序列特异性结合的方式对基因进行转录后水平负性调控的小分子非编码RNA。据估计,人类至少有30%的基因受到miRNA分子的调控。miRNA参与多种生理和病理过程,包括生长发育、细胞增殖、细胞死亡、应激反应、凋亡、脂肪代谢、细胞分化、大脑发展等。多项研究证明,miRNA和恶性肿瘤之间的关系密切。miRNA在多种不同的恶性肿瘤中扮演着癌基因或抑癌基因。miR-21在包括胶质瘤在内的多种肿瘤中高表达,这提示miR-21可能在肿瘤发生中扮演癌基因的角色。作为癌基因簇miR-17-92的成员之一,miR-21参与细胞增殖、凋亡、细胞迁移以及对化疗药物的耐受性增等。研究表明,利用互补寡核苷酸抑制恶性胶质瘤细胞的内源性miR-21的功能将导致caspase的活性增加,并导致细胞活性减低。此外,抑制miR-21的功能可以协同增强恶性神经胶质瘤细胞对细胞毒素NPC-S-TRAIL的敏感性。目前,关于miR-21是否与胶质瘤对化疗药物耐药有关尚不明确。基于此,我们进一步研究miR-21能否在胶质瘤细胞中介导对化疗药物VM-26的化疗药物耐药,并通过基因芯片技术寻找并确定miR-21特异性的靶基因。为了达到这个目的,首先,我们利用反义寡核苷酸来封闭人脑胶质瘤细胞系U373MG细胞中内源性miR-21的功能,而后利用MTT实验检测细胞生长活性的变化。MTT实验检测表明,封闭miR-21的功能后,U373MG细胞表现出生长减缓和活性减低,表明miR-21在U373MG细胞中具有促进恶性表型的作用。随后,为了研究miR-21是否参与介导胶质瘤对化疗药物耐药,我们把miR-21ASO和control ASO转染U373MG细胞后,用不同剂量的VM-26作用U373MG细胞。MTT实验表明,当封闭U373MG细胞中miR-21的功能后,VM-26对其生长的抑制作用明显增强,且呈现剂量依赖性关系。也就是说,miR-21在胶质瘤中可能起到减低其对化疗药物VM-26敏感性的作用。miR-21对细胞表型的作用,通常是通过负性调控其特定的靶基因,影响基因功能来实现的。因此,要阐明miR-21在胶质瘤化疗药物耐药中的作用机制,就需要寻找miR-21发挥作用的靶基因。一方面,利用几种基于miRNA与靶基因作用方式算法的生物信息学数据库,比如:MIRANDA,TARGESCAN和PICTAR,对miR-21的靶基因进行了预测;另一方面,利用7267个基因的基因芯片技术,分析抑制miR-21功能后U373MG细胞的mRNA表达情况。我们综合生物信息数据库预测结果和基因芯片数据,缩小靶基因的搜寻范围,确定LRRFIP 1(leucine rich repeatinteracting protein 1)基因为miR-21候选的功能性靶基因。为了对靶基因的正确性进行验证,我们先利用RT-PCR和Westernblot技术检测miR-21功能被抑制后LRRFIP1的mRNA水平和蛋白水平的变化。实验表明,封闭miR-21的功能后,LRRFIP1基因在mRNA水平和蛋白水平上都明显上调,为了进一步验证miR-21与LRRFIP1的作用关系,我们采用了绿色荧光蛋白(EGFP)报告载体实验,我们将预测到的LRRFIP1基因3’非编码区与miR-21互补结合的一段序列克隆插入报告基因EGFP的下游,构建荧光报告载体,转染U373MG细胞,同时封闭miR-21的功能,检测发现miR-21的功能被封闭后,EGFP的荧光强度明显升高。而我们将报告载体中miR-21的结合位点进行定点突变后,这种miR-21变化导致的荧光强度变化不复存在。以上实验证明,LRRFIP1是miR-21的直接作用靶基因,受到miR-21的特异性调控。LRRFIP1(也称TRAF作用蛋白,TRIP)是肿瘤坏死因子受体超家族的成员之一,包含一个RING手指序列和一个扩展的coiled-coil区域。TRIP与肿瘤坏死因子受体相关因子组成的信号复合体相互作用,抑制TRAF2介导的NF-κB活化途径,减低其对细胞凋亡的抑制作用。NF-κB的活化是肿瘤拮抗化疗药物的主要机制之一。此外,抑制NF-κB的活化后可增强肿瘤坏死因子α(TNFα)或某些化疗药物对细胞凋亡的诱导效应。本实验中,我们发现抑制miR-21的功能可增加靶基因LRRFIP1的上调,从而抑制NF-κB的活化,导致胶质瘤细胞对化疗药物VM-26敏感性增强。本实验首次分析了miR-21与恶性胶质瘤化疗药物耐药之间的关系,机制可能是miR-21通过抑制LRRFIP1基因,从而激活NF-κB通路来降低化疗药物的毒性作用。我们希望通过对miR-21的功能的研究为恶性胶质瘤的治疗提供了新的思路和线索。但由于细胞和分子水平的调控作用是复杂的,miRNA的作用方式和作用范围也是广泛多样的,因此我们期待miRNA在肿瘤耐药中的作用方式和作用机制在以后的研究中得到进一步的阐述。更多还原

【Abstract】 MicroRNA are a class of 18-26nt noncoding RNAs that negatively regulate the expression of target genes by interacting with complementary sites in the 3’ untranslated region(3’UTR).This relatively small number of regulatory RNA molecules is predicted to regulate the expression of at least 30%of human transcripts,including those involved in cancer.Several research groups have provided evidences that miRNAs control a wide array of biological processes including early development,cell proliferation and cell death,stress reaction,apoptosis and fat metabolis,cell differentiation,and brain development.The close relationship between miRNAs and cancer was also been demonstrated.Many miRNAs have been classified to oncomiRs and suppressor genes in specific cancers.Mounting evidence indicates that MicroRNA-21(miR-21) is overexpressed in many cancers including glioblastoma and might play a fundamental role as oncogene in tumorigenesis.As a members of the miR-17-92 cluster,miR-21 was shown to be associated with cell proliferation,apotosis,migration and drug resistant and contributes to tumor resistance to chemotherapy.Such as,The introduction of complementary oligonucleotides into glioma cells with high miR-21 levels leads to reduction of glioma cell viability associated with an increase in activity of caspases.The synergistic cytotoxicity of miR-21 knockdown and S-TRAIL in glioblastoma therapy has been found.These facts lead us to ask Whether miR-21 is also involved in the resistance to chemotherapeutic agents in treatment of glioblastoma.We investigated whether miR-21 mediated chemoresistance to the chemotherapeutic agent VM-26 in glioblastoma cells and sought to identify the candidate target genes for miR-21 by gene expression profiling.In order to examine the effect of miR-21 on glioblastoma cell proliferation.U373MG cells were transfected with miR-21ASO or control ASO and cell viability was detected by MTT assay.In U373MG cells,miR-21 ASO showed a significant antiproliferation effect compared with the control group,suggesting that miR-21 could accelerate glioblastoma cell proliferation.To explore the role of miR-21 in chemoresisitance of glioblastoma,U373MG cells were transfected with either miR-21 ASO or control ASO,and were subsequently incubated with different doses of VM-26.Then the cells were examined for viability levels by MTT assay.The result indicated that knockdown of miR-21 enhanced VM26-induced cell viability reduction in U373MG cells,and the sensitization effect of miR-21 inhibition showed a drug dose-dependent manner,suggesting that miR-21 could suppress chemo-sensitivity of glioblastoma。miR-21 usually affect cell phenotype through negatively regulating the expression of specific target genes.To explore the mechanism involving in miR-21-mediated drug resistance in glioblastoma,We selected combined strategies to identify new target gene which may participate in miR-21-mediated drug resistance.Frist step was searched candidate target genes of miR-21 through several bioinformatic database.Such as MIRANDA,TARGESCAN and PICTAR.Second,the 7267-gene microarray was applied to investigate the gene expression profile of miR-21 ASO treated U373MG cells comparing with control cells.Combining target prediction bioinformatics with expression profiling.LRRFIPl,which was up-regulated in miR-21 ASO treated cells,was predicted as a candidate target gene of miR-21.To determine the effect of miR-21 on LRRFIP1 mRNA and protein expression level, U373MG cells were transfected with miR-21 ASO or control ASO,and the mRNA level and protein level of LRRFIP1 was detected.The result indicated that suppression of miR-21 significantly increased expression of LRRFIP1 in both mRNA level and protein level.To confirm the specificity of this regulation,U373MG cells were transfected with EGFP report vector along with either miR-21 ASO or control ASO,the expression level of EGFP is obviously increased when endogenous miR-21 was blocked.However,when the reporter vector containing a mutational miR-21 binding site was used in the fluorescent reporter assay,the miR-21 ASO mediated fluorescent increase could no longer be detected. It is revealed that LRRFIP1 was directly regulated by miR-21.LRRFIP,also known as TRAF-interacting protein(TRIP),is a signaling component of the TNFR(tumor necrosis factor receptor) superfamily and contains a RING finger motif and an extended coiled-coil domain.TRIP interacts with the receptor/TRAF(TNF receptor-associated factor) signaling complex,and inhibits the TRAF2-mediated NF-κB activation.Activation of NF-κB is required for cell activation and also for protection against apoptosis and is a principal mechanism of tumor chemoresistance,which is primarily mediated by its antiapoptotic activity.Constitutive NF-κB activation has been described in various tumors,where it has been implicated in conferring chemoresistance. Additionally,inhibition of NF-κB signaling has been reported to sensitize tumor cells to apoptosis induced by TNF-αor anticancer agents.Here we find that suppression of miR-21 could up-regulate the expression of its target gene LRRFIP1,which inhibits NF-κB activation and leads to enhancement of VM26-mediated cell death in glioblastoma cells. This research is the first to show the chemosensitivity associated miR-21 in glioblastoma.MiR-21 contributes to VM-26 drug resistance through excessive depression of LRRFIP1 gene,leading to reduction of the cytotoxicity of chemotherapy drugs through activation of the NF-κB pathway.Our findings suggest that miR-21 represents a promising target for therapeutic manipulation to increase the efficacy of chemotherapeutic agents in treating glioblastoma.Since the partern of the interaction between cells and moleculars is complex and The express and fuction models of miRNA is diverse.more detail works are needed to evaluate the mechanisms miRNA-mediated drug resistance.更多还原