共查询到17条相似文献,搜索用时 78 毫秒
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本文将Dicer基因的RNA酶III结构域作为靶区,设计并构建了两个抗Dicer基因的小发夹样RNA(shRNA)表达载体,将其转染2215、结肠癌TC细胞和基因组中整合有绿色荧光蛋白基因(GFP)的HepG2A9细胞,通过RT-PCR评价RNA干扰抑制Dicer基因表达的效率;当HepG2A9细胞Dicer基因表达被上述RNA干扰抑制时,再转染抗GFP的shRNA表达载体,通过RT-PCR和荧光显微镜观察GFP表达水平。结果显示,在不同细胞系中,这两个抗Dicer基因shRNA表达载体,均能明显抑制Dicer基因的表达;当Dicer基因受抑时,后续转染抗GFP的shRNA表达载体不能有效抑制GFP的表达。结果表明,抗Dicer基因shRNA表达载体,能够明显抑制Dicer基因的表达;shRNA表达载体的功能发挥需要Dicer酶的直接参与。 相似文献
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MicroRNA的结构、生物合成及功能 总被引:5,自引:0,他引:5
MicroRNA是真核生物中一类长度约为22个核苷酸的参与基因转录后水平调控的非编码小分子RNA。成熟的microRNA是由较长的可折叠形成发夹结构的前体转录物经过Dicer酶或类似Dicer酶的内切核酸酶加工而来。MicroRNA基因存在于基因组的基因间隔区或者内含子当中。这些小分子RNA通过碱基配对与靶mRNA序列的3′非翻译区或编码区结合以调控基因的表达。它们呈现出组织特异性或发育阶段特异性表达特征。MicroRNA具有调节细胞增殖、死亡、神经细胞分化、个体发育等生物学功能。 相似文献
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Dicer蛋白是RNaseⅢ家族中重要的一员,对miRNA或siRNA的产生起着至关重要的作用。Dicer蛋白通常由1个DEXH盒子或H盒子、1个DUF283结构域、1个PAZ结构域、2个RNaseⅢ结构域(RNaseⅢa和RNaseⅢb)和1个dsRNA结合结构域组成。Dicer蛋白的分子结构决定了其在miRNAs合成中发挥着重要作用。Dicer及生成的miRNA与肿瘤又有着密切关系。本文主要针对Dicer及其与肿瘤的关系作简要综述。 相似文献
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Dicer是微小非编码RNA生成的关键内切酶,介导微小RNA(micro RNA,miRNA)和小干扰RNA(small interfering RNA,siRNA)的产生,通过RNA干扰(RNA interference,RNAi)途径实现转录或转录后水平基因调控,在调节细胞增殖、分化、凋亡等方面起重要作用。近年来Dicer基因在生殖领域的研究越来越受关注,最近的研究表明Dicer与男性生精细胞发育、精子形成及成熟、精子活力和形态生成、卵泡发育、排卵及黄体形成、性激素合成、输卵管功能、子宫内膜容受性等方面都有密切关系。繁衍后代需要精子和卵子的共同参与,Dicer可能通过影响精子和卵子的数量或者质量进而导致胚胎发育异常,因此理解Dicer在雄性与雌性生殖的重要调节作用对于理解生殖调节异常相关的疾病如无精子症、复发性流产等的发病机制具有重要的作用。本文对Dicer在雄性生殖道与雌性生殖中的关键作用进行了综述,旨在进一步从分子层面深入理解Dicer与生殖相关疾病的关系。 相似文献
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microRNA异常表达促进癌症的发生发展.本研究通过microRNA表达谱分析2个肝癌细胞和2个正常细胞microRNA的表达,寻找与肝癌相关的microRNA,发现microRNA-215在肝癌细胞中高表达,q RT-PCR验证microRNA-215在肝癌细胞呈显著高表达.进一步研究发现,microRNA-215直接靶向Dicer1基因的3′UTR并抑制Dicer1蛋白表达,Dicer1是microRNA加工成熟过程中必需的蛋白.过表达microRNA-215抑制Dicer1从而促进肝癌细胞迁移和转化,而抑制microRNA-215表达起相反作用.Dicer1抑制后,许多抑癌microRNA表达被抑制,从而促进迁移和转化.相对于癌旁组织,Dicer1在肝癌组织呈明显低表达.本研究揭示,microRNA-215异常活化并抑制Dicer1表达与肝癌发展相关. 相似文献
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吴凌娟陈鲤群 《中国生物化学与分子生物学报》2017,(1):17-21
Dicer蛋白是RNaseⅢ家族中重要的一员,对miRNA或siRNA的产生起着至关重要的作用。Dicer蛋白通常由1个DEXH盒子或H盒子、1个DUF283结构域、1个PAZ结构域、2个RNaseⅢ结构域(RNaseⅢa和RNaseⅢb)和1个dsRNA结合结构域组成。Dicer蛋白的分子结构决定了其在miRNAs合成中发挥着重要作用。Dicer及生成的miRNA与肿瘤又有着密切关系。本文主要针对Dicer及其与肿瘤的关系作简要综述。 相似文献
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本文将Dicer基因的RNA酶Ⅲ结构域作为靶区,设计并构建了两个抗Dicer基因的小发夹样RNA(shRNA)表达载体,将其转染2215、结肠癌TC细胞和基因组中整合有绿色荧光蛋白基因(GFP)的HepG2
A9细胞,通过RT-PCR评价RNA干扰抑制Dicer基因表达的效率;当HepG2 A9细胞Dicer基因表达被上述RNA干扰抑制时,再转染抗GFP的shRNA表达载体,通过RT-PCR和荧光显微镜观察GFP表达水平.结果显示,在不同细胞系中,这两个抗Dicer基因shRNA表达载体,均能明显抑制Dicer基因的表达;当Dicer基因受抑时,后续转染抗GFP的shRNA表达载体不能有效抑制GFP的表达.结果表明,抗Dicer基因shRNA表达载体,能够明显抑制Dicer基因的表达;shRNA表达载体的功能发挥需要Dicer酶的直接参与. 相似文献
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目的:微小RNA(microRNAs,miRNAs)在胆固醇的合成,代谢和转运中起着重要作用,而mi RNAs在胆固醇代谢物胆酸的代谢和转运中的作用尚不清楚。Dicer基因是miRNAs生成过程的关键酶。本课题使用肝脏特异的Dicer1基因敲除小鼠,考察肝脏Dicer1基因敲除对C57BL/6小鼠肝脏胆酸代谢和转运的影响。方法:使用白蛋白启动子驱动的Cre重组酶和Loxp系统(Alb-Cre/Loxp)在小鼠肝脏中特异的敲除Dicer1基因;分别收集3~12周龄的小鼠血液和肝脏组织,使用Cobas生化仪检测小鼠血液和肝脏中总胆酸含量;利用实时定量PCR的方法分析肝脏中胆汁酸代谢转运相关基因的表达。结果:实验发现,肝脏Dicer基因敲除后,胆酸在血液和肝脏中明显蓄积,弥漫性肝细胞轻微空泡化,偶见单个肝细胞坏死。检测胆酸代谢和转运相关基因的表达发现,胆酸合成相关基因的表达有轻度升高,但缺乏统计学差异;在肝脏细胞血管侧的胆酸摄取转运体中,Oatp1a1在Dicer1敲除小鼠肝脏中明显下调,Ntcp和Oatp1b2则无明显改变;而肝细胞血管侧胆酸外排转运体的表达均有显著升高,胆管侧的外排转运体中Abcb11表达有明显增加。结论:Dicer基因敲除后,胆酸在血液和肝脏中明显蓄积,肝脏和血液中胆酸总量显著增加。血液中胆酸的蓄积可能与肝脏细胞血管侧摄取转运体的低表达和血管侧外排转运体的高表达有关;而肝脏中胆酸的蓄积可能部分来自于轻度升高的胆酸合成酶,胆酸在肝细胞内运输途径的紊乱可能与肝脏和血液中胆酸总量的显著增加相关。 相似文献
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RNA干扰(RNAi)是由双链RNA触发的在mRNA水平进行的特异靶序列的基因沉默现象,广泛存在于动物、植物和病毒中,主要包括小干扰RNA(siRNA)及微小RNA(miRNA)两种作用途径。人工miRNA(amiRNA)是将天然miRNA的成熟序列替换成人工设计的靶向其他感兴趣基因的反义序列,通过天然miRNA的生成和作用途径达到RNAi的效果,具有干扰效果明显、作用迅速、毒性低等优点,拥有广阔的应用前景。我们对基于amiRNA的基因沉默技术进行了较为系统的介绍和总结,梳理了该技术的优缺点和适用范围,并展望了其进一步发展的方向和应用前景。 相似文献
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Fumitaka Mizoguchi Yayoi Izu Tadayoshi Hayata Hiroaki Hemmi Kazuhisa Nakashima Takashi Nakamura Shigeaki Kato Nobuyuki Miyasaka Yoichi Ezura Masaki Noda 《Journal of cellular biochemistry》2010,109(5):866-875
Osteoclasts are unique cells that resorb bone, and are involved in not only bone remodeling but also pathological bone loss such as osteoporosis and rheumatoid arthritis. The regulation of osteoclasts is based on a number of molecules but full details of these molecules have not yet been understood. MicroRNAs are produced by Dicer cleavage an emerging regulatory system for cell and tissue function. Here, we examine the effects of Dicer deficiency in osteoclasts on osteoclastic activity and bone mass in vivo. We specifically knocked out Dicer in osteoclasts by crossing Dicer flox mice with cathepsin K‐Cre knock‐in mice. Dicer deficiency in osteoclasts decreased the number of osteoclasts (N.Oc/BS) and osteoclast surface (Oc.S/BS) in vivo. Intrinsically, Dicer deficiency in osteoclasts suppressed the levels of TRAP positive multinucleated cell development in culture and also reduced NFATc1 and TRAP gene expression. MicroRNA analysis indicated that expression of miR‐155 was suppressed by RANKL treatment in Dicer deficient cells. Dicer deficiency in osteoclasts suppressed osteoblastic activity in vivo including mineral apposition rate (MAR) and bone formation rate (BFR) and also suppressed expression of genes encoding type I collagen, osteocalcin, Runx2, and Efnb2 in vivo. Dicer deficiency in osteoclasts increased the levels of bone mass indicating that the Dicer deficiency‐induced osteoclastic suppression was dominant over Dicer deficiency‐induced osteoblastic suppression. On the other hand, conditional Dicer deletion in osteoblasts by using 2.3 kb type I collagen‐Cre did not affect bone mass. These results indicate that Dicer in osteoclasts controls activity of bone resorption in vivo. J. Cell. Biochem. 109: 866–875, 2010. © 2009 Wiley‐Liss, Inc. 相似文献
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Ricardo Cortez Cardoso Penha Romina Sepe Marco De Martino Francesco Esposito Simona Pellecchia Maddalena Raia 《Cell cycle (Georgetown, Tex.)》2017,16(23):2282-2289
DICER1 plays a central role in the biogenesis of microRNAs and it is important for normal development. Altered microRNA expression and DICER1 dysregulation have been described in several types of tumors, including thyroid carcinomas. Recently, our group identified a new somatic mutation (c.5438A>G; E1813G) within DICER1 gene of an unknown function. Herein, we show that DICER1 is overexpressed, at mRNA level, in a significant-relative number of papillary (70%) and anaplastic (42%) thyroid carcinoma samples, whereas is drastically downregulated in all the analyzed human thyroid carcinoma cell lines (TPC-1, BCPAP, FRO and 8505c) in comparison with normal thyroid tissue samples. Conversely, DICER1 is downregulated, at protein level, in PTC in comparison with normal thyroid tissues. Our data also reveals that DICER1 overexpression positively regulates thyroid cell proliferation, whereas its silencing impairs thyroid cell differentiation. The expression of DICER1 gene mutation (c.5438A>G; E1813G) negatively affects the microRNA machinery and cell proliferation as well as upregulates DICER1 protein levels of thyroid cells but has no impact on thyroid differentiation. In conclusion, DICER1 protein is downregulated in papillary thyroid carcinomas and affects thyroid proliferation and differentiation, while DICER1 gene mutation (c.5438A>G; E1813G) compromises the DICER1 wild-type-mediated microRNA processing and cell proliferation. 相似文献
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Tomasz Jan Nowakowski Karolina Sandra MysiakTimothy O‘Leary Vassiliki FotakiThomas Pratt David Jonathan Price 《Developmental biology》2013
Radial glia of the mouse cerebral cortex emerge from neuroepithelial stem cells around embryonic day 11 and produce excitatory cortical neurons until a few days before birth. The molecular mechanisms that regulate the end of cortical neurogenesis remain largely unknown. Here we investigated if the Dicer-dependent microRNA (miRNA) pathway is involved. By electroporating a cre-recombinase expression vector into the cortex of E13.5 embryos carrying a conditional allele of Dicer1, we induced mosaic recombination causing Dicer1 deletion and reporter activation in a subset of radial glia. We analysed the long-term fates of their progeny. We found that mutant radial glia produced abnormally large numbers of Cux1-positive neurons, many of which populated the superficial cortical layers. Injections of the S-phase marker bromodeoxyuridine between postnatal days 3 and 14 showed that much of this population was generated postnatally. Our findings suggest a role for Dicer-dependent processes in limiting the timespan of cortical neurogenesis. 相似文献
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The ribonuclease III enzymes Drosha and Dicer are renowned for their central roles in the biogenesis of microRNAs (miRNAs). For many years, this has overshadowed the true versatility and importance of these enzymes in the processing of other RNA substrates. For example, Drosha also recognizes and cleaves messenger RNAs (mRNAs), and potentially ribosomal RNA. The cleavage of mRNAs occurs via recognition of secondary stem-loop structures similar to miRNA precursors, and is an important mechanism of repressing gene expression, particularly in progenitor/stem cell populations. On the other hand, Dicer also has critical roles in genome regulation and surveillance. These include the production of endogenous small interfering RNAs from many sources, and the degradation of potentially harmful short interspersed element and viral RNAs. These findings have sparked a renewed interest in these enzymes, and their diverse functions in biology. 相似文献
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为了研究Dicer及其所产生的miRNAs在血管平滑肌细胞(VSMC)中的作用,本研究采用条件性基因打靶法敲除了小鼠VSMC中的Dicer外显子23,通过采用连续性剖检妊娠小鼠法、病理组织学、免疫荧光、PCR、Western blot和实时PCR等技术对条件性敲除Dicer(Dicer c KO)胚胎的血管病变和VSMC中的Dicer、miRNAs和信号转导通路蛋白变化进行了详细研究.结果发现,在培育条件性敲除Dicer小鼠的过程可产生三种不同基因型小鼠,即野生型、杂合型和纯合型(Dicer c KO)小鼠.其中野生型和杂合型小鼠出生后无明显临床异常,而Dicer c KO小鼠却死于腹中而不能出生.Dicer c KO胚胎在胚胎发育的第12.5天(E12.5)就出现发育迟滞变化,在E14.5,皮肤、骨骼肌和肝脏的血管极度扩张、血液淤滞和广泛的弥漫性出血,在E15.5死亡.Dicer c KO胚胎血管壁的病变于E13.5即出现,主要表现为血管中膜的VSMC排列不整,增生减少;E14.5血管壁变薄、塌陷,管腔不规则,细胞增生明显减少;E15.5血管壁的结构完全破坏,细胞增生停止,血管壁的屏障作用破坏,通透性增强,向外渗血.在胚胎发育的E14.5,VSMC标志性基因的表达明显下调,VSMC中大部分受检miRNAs的表达也明显降低,磷酸化的信号转导通路蛋白,即细胞外信号调节激酶和蛋白激酶明显衰减.研究证明,Dicer是血管发育所必需的基因,它可通过控制mi RNA产生和成熟来调节VSMC标志性基因的表达,借以促进VSMC的增殖与分化,保障血管壁结构的完整. 相似文献