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miR-9靶向CBX7影响神经胶质瘤细胞的增殖
【作者】 晁腾飞；
【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2008， 博士
【摘要】 MicroRNAs是一类长约22个核苷酸、进化上高度保守的内源性非编码RNA,通过转录后水平调节靶基因发挥作用。许多miRNAs被认为在肿瘤发生发展中起到了非常重要的作用,其中部分miRNAs在肿瘤中的作用被看作是原癌miRNA基因(OncomiRs)。神经胶质细胞瘤是中枢神经系统中最常见的肿瘤,起源于神经前体细胞和/或分化的星形胶质细胞等。miR-9是一个在神经系统发育过程中高表达,但在出生和成年后维持相对低表达的miRNA,已有文献报道,它在两种神经胶质瘤(神经母细胞瘤和少突胶质瘤)中异常高表达,但有关miR-9在神经胶质瘤中的具体功能及其分子机制尚不清楚。本论文进行了miR-9在神经胶质瘤中的功能探索并寻找miR-9发挥作用的靶基因。我们首先利用Real-time PCR的方法检测了2例正常人脑组织、9例神经胶质瘤组织(Ⅱ级、Ⅲ级、Ⅳ级各3例)和3株神经胶质瘤细胞系(A172、T98G和U87MG)中miR-9的表达情况。结果发现,miR-9在神经胶质瘤组织和细胞中明显上调。后续研究以miR-9表达量最高的T98G细胞系为模型,通过转染2’-O-Me-miR-9敲低内源性miR-9的表达,然后再用MTT和流式细胞术分析miR-9对细胞增殖的影响。结果发现,T98G细胞中miR-9敲低后,G1期细胞百分比减少,细胞增殖加快。由于miRNAs往往通过调控其靶基因发挥作用,为了进一步阐述miR-9在神经胶质瘤中发挥功能的分子机制,我们结合生物信息学和生物学实验寻找了miR-9发挥作用的靶基因:首先对已有的miRNA靶基因预测算法(miRanda)进行了基于机器学习法的改良,荧光素酶报告基因实验表明,改良后算法能提高miRanda预测的有效性,缩减了候选靶基因的范围;随后,我们用改良后算法对miR-9的靶基因进行了筛选,筛选后结果和文献分析提示,CBX7是可能miR-9的潜在靶基因之一。随后在293ET细胞中,我们在过表达miR-9的基础上,进行了荧光素酶和EGFP两种报告基因实验。结果发现,miR-9能特异性的通过CBX7 3’UTR上的预测位点抑制报告基因的活性,证明CBX7的确是miR-9的靶基因。为了进一步验证miR-9对神经胶质瘤中内源性CBX7的调控作用,我们首先利用Western Blot和RT-PCR的方法检测了2例人正常脑组织、9例神经胶质细胞瘤组织和3种胶质瘤细胞系中CBX7的表达。结果发现,CBX7蛋白在神经胶质瘤组织和细胞系中明显下调或缺失,而其mRNA水平与正常脑组织的差异不明显。随后,在T98G细胞中敲低内源性miR-9的表达后,发现miR-9敲低能显著的上调含CBX7-3’UTR的荧光素酶报告基因的活性,并使细胞中内源性CBX7的蛋白水平明显上调,而mRNA不发生明显的变化。最后,我们在T98G细胞中过表达CBX7蛋白,通过MTT和流式细胞术分析CBX7过表达对肿瘤细胞增殖的影响。结果发现,T98G细胞中CBX7过表达后,G1期细胞减少,细胞增殖加快,与前面miR-9敲低的结果一致。综合以上研究结果,我们初步证明:在人神经胶质细胞瘤中,异常高表达的miR-9通过转录后水平抑制其靶基因CBX7的表达,导致肿瘤中CBX7的表达下调甚或缺失,并可能由此使细胞阻滞于G1期,影响胶质瘤细胞的增殖。
【Abstract】 MicroRNAs (miRNAs) are evolutionarily conserved, endogenous, small, noncoding RNA molecules of about 22 nucleotides in length that function as posttranscriptional gene regulators. Some of them are deemed to play a crucial role in the initiation and progression of human cancers, and those with the role in cancer are designated as oncogenic miRNAs (OncomiRs). Human gliomas are the most common tumor in the central nervous system, which arise from neural progenitor cells and/or differentiated astrocytes. MiR-9 is enriched in developmental brain followed by down-regulation and a steady-state expression level after birth. Previous data identified that miR-9 was up-regulated in human glioblastomas and oligodendrogliomas. However, the roles of miR-9 in gliomagenesis and the molecular mechanism are unclear. This PhD dissertation mainly focused on the studies of biological function of miR-9 and the interaction between miR-9 and its target genes in gliomagenesis.Firstly, we detected the expression of miR-9 in two human normal brain tissues, nine glioma tissues and three glioma cell lines by Real-time PCR. The results indicated that miR-9 was significantly elevated in the glioma tissues and cell lines. Then, we knocked down the expression of miR-9 in T98G cells by transfection of 2’-O-Me-miR-9 and detected the effects of miR-9 knock-down on the cell growth. From the results of Methyl Thiazolyl Tetrazolium (MTT) assay and flow cytometry, we found that the cell number in G1 phase was remarkably reduced and survival cells increased after the transfection.To elucidate the mechanism of miR-9 function in glioma, we looked for the target genes of miR-9 with the application of bioinformatics tools. We developed an ensemble machine learning algorithm which helps to improve the miRanda prediction of miRNA targets.After the classification of miRanda prediction of miR-9 targets by this modified algorithm, we chose CBX7 as the candidate target in glioma and confirmed that miR-9 could inhibit the report gene expression by the miR-9 binding element in CBX7-3’UTR with Luciferase and EGFP report gene assays in 293ET cells.Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western Blot were further applied to detect the expression pattern of CBX7 in those tissues and cell lines. CBX7 protein was abrogated or markedly reduced in glioma tissues and cell lines, while there was no apparent difference in the CBX7 mRNA level between normal and tumor tissues. After miR-9 knock-down in T98G cells, the luciferase activity of CBX7-3’UTR was increased by 1.8 fold, the protein level of endogenous CBX7 was significantly increased, while there was no change of CBX7 mRNA.Finally, we over-expressed the CBX7 in T98G cells, then MTT assay and flow cytometry were applied to detect the effect of CBX7 overexpression in T98G cells. The results demonstrated that the number of T98G cells in G1 phase was decreased and survival cells increased after CBX7 overexpression, which were in accord with the results of miR-9 knock-down.Taken together, these data revealed that elevated expression of miR-9 in human gliomas regulates T98G cells growth by inhibiting its target gene CBX7 at posttranscriptional level.