【作者】 陈丽；

【导师】 姚开泰； 刘求真；

【作者基本信息】 南方医科大学 ， 病理学与病理生理学， 2009， 硕士

【摘要】 microRNAs(miRNAs)是人类新发现的一类非编码小分子RNA,广泛分布于真核细胞内。miRNAs通过与靶基因互补位点配对结合,在转录后水平负性调控靶基因的表达,参与生长发育、细胞增殖、细胞凋亡、细胞分化等生命过程。成熟的miRNAs长17—24bp,一个miRNA分子能调控多种基因。因而有研究发现,相对于编码蛋白质的mRNA分子来说,一些基因的表达更依赖于miRNAs的调控水平。近年来,有研究发现胚胎干细胞特异性的表达miRNAs,随着胚胎干细胞的分化,这些miRNAs的表达受到抑制,而在成体鼠的成熟器官则检测不到其表达。这些miRNAs包括miR-290、miR-291a-3p、miR-292-3p、miR-293、miR-294和miR-295(miR-290家族),miR-302a、miR-302b、miR-302c和miR-302d(miR-302家族)。MiRBase数据库显示miR-302首先从鼠胚胎干细胞(mESC)克隆而来,基于人类的miR-302和鼠具有同源性,后也被证实从人胚胎干细胞(hESC)克隆出hsa-miR-302。miR-302家族的四个高度同源的成员:miR-302a、miR-302b、miR-302c、miR-302d和miR-367成簇聚集在人类4号染色体上。SHI-LUNG LIN等的研究发现mir-302s高表达于生长缓慢的人胚干细胞,随着细胞的分化和增生其含量迅速减少,因此mir-302s是维持胚胎干细胞自我更新和多潜能性的一个重要因子。当在人肿瘤细胞转入mir-302s(转入后称为mirPS细胞),转入后的mirPS细胞不仅表达胚胎干细胞的表面标志物如Oct3/4、SSEA-3、SSEA-4、Sox2和Nanog等,而且基因组高度去甲基化。微阵列分析进一步发现在基因组表达模式上mirPS细胞和人ES H1和H9细胞有高达86%的相似性。mirPS细胞在体外能分化成不同的组织类型,如神经元、软骨细胞、成纤维细胞和精原细胞样的胚性细胞。miR-302家族的每个成员都能同时调节大约445个基因,并且它们所调节的大部分基因都是相同的。这些基因的功能大多涉及早期胚胎发育相关的信号分子或影响细胞分化的细胞因子。转入miR-302后抑制了这些基因的作用,因而阻止了细胞的发育和分化,这就有可能把细胞重编程为胚胎干细胞样的状态。如miR-302通过减弱SP3、HMG-box、forkhead-box和LIM-homeobox基因家族的作用而发挥其重编程的功能。因而我们有理由相信miR-302是通过减弱胚胎发育相关的基因的作用而不是创造新的信号转导通路来发挥其重编程功能的。另外还有一些mirRNA如mir-93、mir-367、mir-371、mir-372、mir-373和mir-520,它们靶基因的功能也和胚胎发育相关,因而也可能参与重编程。Deborah报道Oct4和Sox2结合于miR-302的启动子区域。在人胚胎干细胞(hESCs),miR-302a的表达依赖于Oct4/Sox2的表达,并且在胚胎发生过程中,Oct4和miR-302a总是相伴随的出现在同一组织和同一发展时期。miR-302a被预测作用于许多与调节细胞周期有关的因子,miR-302a在原发及转化细胞中的表达能增加S期的细胞而减少G1期的细胞。相应的,抑制miR-302a的表达则能使hESCs聚集在G1期。进一步研究发现在hESCs,miR-302a是通过抑制G1期的重要调节蛋白cyclin D1的翻译而起作用的。因此miR-302的活化在翻译水平上抑制了其靶基因cyclin D1的表达,也预示了Oct4/Sox2和细胞周期调节因子在多潜能细胞中存在关联。许多对mir-302的研究都集中在胚胎干细胞或其上下游的靶基因上,我们也只是知道mir-302s高表达于胚胎干细胞,但是如果在鼻咽癌细胞株CNE2中外源性转入mir-302s,会发生怎样的细胞生物学变化,目前不是很清楚。以下我们就会研究mir-302s在鼻咽癌细胞中所起的作用。1 miR-302s前体分子慢病毒表达载体的转染与鉴定miR-302s前体分子慢病毒表达载体经测序鉴定正确后,和包装质粒共转染293FT,48h后收集病毒上清感染CNE2细胞,依据copGFP标记用流式细胞仪分选出稳定表达miR-302s的细胞。采用细胞的原位杂交方法来检测CNE2细胞、CNE2-PMIRNA1和CNE2-PMIRH/302abcd细胞中miR-302a的表达。结果显示miR-302a在CNE2和CNE2-PMIRNA1中为阴性表达,而在转染了miR-302s前体分子的CNE2-PMIRH/302abcd细胞中则呈阳性表达。采用TaqMan荧光定量RT-PCR方法检测miR-302a在三种细胞中的表达,结果显示内参U6Ct值28左右,miR-302a在CNE2-PMIRH/302abcd细胞中有扩增曲线(Ct值35左右)出现,而在50个循环后CNE2和CNE2-PMIRNA1中仍无扩增曲线出现,说明这两个细胞中无miR-302a的表达,初步表明转染及筛选成功,细胞可用于后续功能实验。2 miR-302s过表达对鼻咽癌CNE2细胞生物学特性的影响及干性标志物CD133的检测成功转染的细胞进行了一系列体外功能实验。采用MTT法检测miR-302s过表达后细胞的体外增殖情况,结果显示,与CNE2-PMIRNA和CNE2细胞相比,CNE2-PMIRH/302abcd细胞的增殖速度加快,并且呈时间依赖关系。流式细胞仪检测三组细胞的细胞周期分布发现转染后的CNE2-PMIRH/302abcd细胞S期比例增加,而G1期的细胞比例减少,其差异具有统计学意义(P＜0.05)。综上所述,miR-302s的过表达增强了鼻咽癌细胞的体外增殖能力。采用Transwell小室检测miR-302s过表达后细胞体外迁移运动能力的改变,结果显示,与CNE2-PMIRNA和CNE2细胞相比,CNE2-PMIRH/302abcd细胞穿过聚碳酸酯膜的细胞数增多,差异具有显著性(P＜0.001)。表明miR-302s过表达后增强了鼻咽癌细胞的体外迁移能力。平板克隆实验证实转染mir-302s后的细胞CNE2-PMIRH/302abcd相比CNE2和CNE2-pMIRNA1细胞形成的克隆数增加(P＜0.05)。体内裸鼠成瘤实验变化不是很显著,这可能和裸鼠的例数太少有关,也可能和高浓度细胞的过快生长影响裸鼠的寿命从而影响低浓度细胞的观察有关(本实验中每只裸鼠都注射不同浓度的细胞)。流式细胞仪进一步检测转染前后CD133的表达量,结果显示3种细胞的CD133表达量都恒定维持在0.1%左右,故转染mir-302s后不会影响CD133表面抗原的变化,而其具体作用机制尚需进一步探讨。结论1成功转染并用流式分选出稳定高表达mir-302s的细胞CNE2-PMIRH/302abcd,采用细胞原位杂交及TaqMan荧光定量RT-PCR检测结果显示,插入miRNA前体分子的慢病毒表达载体能成功上调mir-302a的表达,细胞可用于后续功能实验。2通过体内体外功能实验证实,过表达mir-302s后的细胞CNE2-PMIRH/302abcd的增殖能力、迁移和成瘤能力都有所增强。3检测干细胞表面标志物CD133的表达发现CNE2-PMIRH/302abcd中CD133+肿瘤干细胞的量无显著变化,其具体作用机制尚需进一步探讨。更多还原

【Abstract】 microRNAs(miRNAs) are a recently discovered family of small non-coding RNAs, which exist in eukaryotes and regulate gene expression post-transcriptionally by binding to complementary sites on target mRNAs,miRNAs play important roles in regulation of cell proliferation,development,apoptosis and cell differentiation.The mature miRNAs are 17-24 bp single-stranded RNA molecules.Each miRNA seems to be able to regulate multiple genes.It was shown recently that the expression of certain genes is more dependent on the level of regulatory miRNAs than on the level of mRNAs that encode the proteins(Johnson et al.2005).Recently,a set of miRNAs was described to be ESC-specific in mouse,with their expression being repressed during ESC differentiation and undetectable in adult mouse organs.This set of miRNAs consists of miR-290,miR-291a-3p,miR-292-3p, miR-293,miR-294 and miR-295(miR-290 family),and miR-302a,miR-302b, miR-302c and miR-302d(miR-302 family).MiRBase database show that miR-302 was first cloned from mouse embryonic stem cells.Human miR-302 was predicted based on homology with mouse and later confirmed by cloning in human embryonic stem cells,miR-302a is located in a cluster with the related miR-302b,miR-302c,miR-302d,and miR-367 on human chromosome 4.Results of SHI-LUNG LIN found that mir-302s expressed most abtmdantly in slow-growing human ES cells and quickly decreased after cell differentiation and proliferation.Therefore,mir-302s was investigated as one of the key factors essential for maintenance of ES cell self-renewal and pluripotency.When transgenically transfect the mir-302s into several human cancer cell lines,The mir-302-transfected cells(we also called mirPS cells) not only expressed many key ES cell markers,such as Oct3/4,SSEA-3,SSEA-4,Sox2,and Nanog,but also had a highly demethylated genome similar to a reprogrammed zygotic genome.Microarray analyses further revealed that genomewide gene expression pattems between the mirPS and human ES H1 and H9 cells shared over 86%similarity.Using molecular guidance in vitro,these mirPS cells could differentiate into distinct tissue cell types, such as neuron-,chondrocyte-,fibroblast- and spermatogonia-like primordial cells.Each member of the mir-302 family is able to simultaneously regulate over 445 cellular genes and they all share almost the same target genes.Many of the mir-302 targeted genes are active developmental signals involved in initiation or facilitation of lineage-specific cell differentiation during early embryonic development.By suppressing these developmental genes,mir-302s may reprogram cells into a more ES-like state.Alternatively,mir-302s may also attenuate the expression of their predicted target transcription factors,such as SP3,HMG-box,forkhead-box,and LIM-homeobox gene families,to provide the cell reprogramming effect.Thus,the function of mir-302s is more likely to attenuate the global production of developmental signals and/or transcription factors rather than to directly create transcriptional stimulation on certain embryonic signaling pathways.Nevertheless, mir-302s may not be the only miRNA family involved in this mechanism because their target genes are also redundantly silenced by the group of mir-93,mir-367, mir-371,mir-372,mir-373,and mir-520 in human ES cells,as predicted by the TARGETSCAN program.Learning why these target genes must be simultaneously silenced during the reprogramming process of cancer-ES cell transformation may shed light on the mechanism underlying this miRNA-mediated effect on ES cell maintenance and self-renewal.Deborah showed that Oct4 and Sox2 bound to a conserved promoter region of miR-302.The expression of miR-302a is dependent on Oct4/Sox2 in human ESCs (hESCs),and miR-302a is expressed at the same developmental stages and in the same tissues as Oct4 during embryogenesis,miR-302a is predicted to target many cell cycle regulators,and the expression of miR-302a in primary and transformed cell lines promotes an increase in S-phase and a decrease in G1-phase cells. Correspondingly,the inhibition of miR-302 causes hESCs to accumulate in G1 phase. Moreover,they showed that miR-302a repressed the productive translation of an important G1 regulator,cyclin D1,in hESCs.The transcriptional activation of miR-302 and the translational repression of its targets,such as cyclin D1,may provide a link between Oct4/Sox2 and cell cycle regulation in pluripotent cells.So many studies about mir-302 have focused on the ESC or the target genes which upstream or downstream it.We just know mir-302s expressed abundantly in ESC,but when we transfected mir-302s into nasopharyngeal carcinoma cells what would happen,it’s not clear.In this research,the role of mir-302s in NPC will be studied.1 Transfection and identification of the lentiviral expression vector of human miR-302s precursorLentivector-based microRNA precursor constructs were confirmed by DNA sequencing.The viral supernatant was harvested from 293FT cells which were co-transfected with expression vectors and packaging plasmids.CNE2 cells were infected with the viral supernatant and the cells with stable miR-302s expression were separated by fluorescence activated cell sorter(FASC). In situ hybridization was used to detect the expression of miR-302s in CNE2 cells、CNE2-PMIRNA1 and CNE2-PMIRH/302abcd cells.The results indicated there were no positive signals in CNE2 cells,however,strongly positive signals were manifested in the CNE2-PMIRH/302abcd cells transfected with miR-302s precursor.And the TaqMan real-time RT-PCR also confirmed this results.2 Effects of miR-302s overexpression on biological behaviors of CNE2 cellsThe successfully transfected cells were used in vitro experiments.MTT assay showed CNE2-PMIRH/302abcd cells have a significantly enhanced proliferation compared with CNE2 and CNE2- pMIRNA1 cells(P＜0.001 ).However,the cell cycle distribution detected by flow cytometry showed that CNE2-PMIRH/302abcd cells have an increase in S-phase and a decrease in G1-phase cells.These results indicated overexpression of miR-302s partially enhanced proliferatin of CNE2 cells.The results of in vitro motility assay showed that CNE2-PMIRH/302abcd cells had significantly enhanced migration as compared with CNE2 and CNE2-pMIRNA1 cells(P＜0.001).This showed over-expression of miR-302s partially led to a up-regulated migration of CNE2 cells.Flat colony forming experiment proved that CNE2-PMIRH/302abcd cells formed more clonies than CNE2 and CNE2- pMIRNA1 cells(P＜0.05).This also indicates that the cells after transfection with miR-302s had a enhanced tumorigenicity.In vivo experiments don’t have significant changes perhaps correlate with the less cases or we should observe longer time for these mouses.The expression of CD133 remained invariable when transfected the mir-302s also can’t exclude the possibility of dedifferentiation from cancer cells to cancer stem cells. Conclusion1 Successfully transfected and separated the cells which over-expression the miR-302a precursors and detected by in situ hybridization and TaqMan real-time RT-PCR.2 Overexpression of miR-302s in vitro results in an enhanced of cell proliferation and motility in NPC-derived cell lines CNE2.but the expression of CD133 remains invariable which detected by flow cytometry.更多还原