Implicación de los microRNAs en la toxicidad del metotrexato en Leucemia Linfoblástica Aguda infantil

Analisis de la implicacion de los Polimorfismos de LLA en la respuesta al Metotrexato

PLOD3基因3'调控区的人类特异突变改变miR-124a对PLOD3的调控

microRNAs(miRNAs)是一类在转录后水平调控基因表达的不编码蛋白质的小RNA(长度20-24个碱基).其中,miR-124a是一个在哺乳动物中枢神经系统高度表达的miRNA,在神经前体细胞向神经元分化的过程中起着举足轻重的作用.由于miRNAs特异性地识别靶基因的3'端调控区(3'UTR)的靶序列,因此,在人类起源过程中基因3'UTR的单核苷酸序列变异有可能导致miRNA调控的改变.通过靶基因预测和3'UTR区在哺乳动物代表物种间的同源序列比较,我们发现miR-124a的靶基冈中有一个基因(PLOD3)3UTR的靶位点中存在人类特异突变位点.利用体外报告基因系统,发现PLOD3基因3'UTR靶位点中所含的一个人类特异的突变导致miR-124a对PLOD3的调控效率降低.研究表明,miRNAs靶基因3'UTR的序列变异具有功能效应,它有可能足人类中枢神经系统在起源和演化中发挥关键作用的重要遗传机制之一.

一个快速进化的microRNA家族

microRNAs(miRNAs)是长约22nt的非编码RNA,它可通过降解mRNA或抑制其翻译来调控基因.本文简要介绍了miRNAs的研究背景,阐述了通过比较近缘物种中非保守的miRNA基因来研究miRNAs分子进化的理由.我们的研究结果证实miRNA基因会受到达尔文正选择的影响,发生快速的进化.这一结果将为我们了解非蛋白编码基因的功能与进化提供崭新的视角.

Rapid evolution of mammalian X-linked testis microRNAs

Background: MicroRNAs (miRNAs), which are small, non-coding RNAs approximately 21-nucleotides in length, have become a major focus of research in molecular biology. Mammalian miRNAs are proposed to regulate approximately 30% of all protein-coding genes. P

MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant.

The Arabidopsis genome contains a highly complex and abundant population of small RNAs, and many of the endogenous siRNAs are dependent on RNA-Dependent RNA Polymerase 2 (RDR2) for their biogenesis. By analyzing an rdr2 loss-of-function mutant using two different parallel sequencing technologies, MPSS and 454, we characterized the complement of miRNAs expressed in Arabidopsis inflorescence to considerable depth. Nearly all known miRNAs were enriched in this mutant and we identified 13 new miRNAs, all of which were relatively low abundance and constitute new families. Trans-acting siRNAs (ta-siRNAs) were even more highly enriched. Computational and gel blot analyses suggested that the minimal number of miRNAs in Arabidopsis is approximately 155. The size profile of small RNAs in rdr2 reflected enrichment of 21-nt miRNAs and other classes of siRNAs like ta-siRNAs, and a significant reduction in 24-nt heterochromatic siRNAs. Other classes of small RNAs were found to be RDR2-independent, particularly those derived from long inverted repeats and a subset of tandem repeats. The small RNA populations in other Arabidopsis small RNA biogenesis mutants were also examined; a dcl2/3/4 triple mutant showed a similar pattern to rdr2, whereas dcl1-7 and rdr6 showed reductions in miRNAs and ta-siRNAs consistent with their activities in the biogenesis of these types of small RNAs. Deep sequencing of mutants provides a genetic approach for the dissection and characterization of diverse small RNA populations and the identification of low abundance miRNAs.

单细胞真核生物的miRNA系统及其进化意义

miRNA系统在高等多细胞真核生物中得到了广泛深入的研究。近年来,人们在单细胞真核生物上的miRNA研究也取得了重要进展。这不仅丰富了人们对miRNA在整个生物界中的认识,更重要的是对于揭示miRNA这一表达调节系统是如何在生物界中起源进化的问题具有重要意义。该文结合作者在最低等单细胞真核生物——贾第虫上的研究结果,对该领域的研究进展作一概述,并对有关miRNA这一系统的起源进化问题进行了探讨。

Genome-wide computational identification of microRNAs and their targets in the deep-branching eukaryote Giardia lamblia

Using a combined computational program. we identified 50 potential microRNAs (miRNAs) in Giardia lamblia. one of the most primitive unicellular eukaryotes. These miRNAs are unique to G. lamblia and no homologues have been found in other organisms; miRNAs.

大脑表达 miRNA 在大脑进化中的作用及其遗传机制初步研究

人类区别于其它动物的最本质特征是其拥有其他动物所无法比拟的大脑容量及高 级的认知能力。即使与其近亲－非人灵长类相比，人也拥有比非人灵长类大好几倍的脑 容量和更为发达的认知能力。现在一般认为，人类大脑的形成是适应性选择(达尔文正 选择)的结果。但是到目前为止，对人类起源过程中大脑容量增大及认知能力提高的遗 传学机制却知之甚少。以前的研究表明，几个与大脑发育相关的蛋白质编码基因在人类 起源中受到了正向选择。同时，也有证据表明人类大脑的进化也可能是基因表达调控变 化的结果。因此寻找人与非人灵长类大脑表达基因的调控差异或许能够进一步为人与非 人灵长类为何有如此巨大的差异提供分子生物学水平上的解释。 MicroRNA(miRNA)是一类在转录后水平调控基因表达的不编码蛋白质的小 RNA(长度20－24个碱基)。通过调控靶基因的表达，miRNA参与了众多的生理过程。而 很多大脑表达的miRNA的表达量在大脑发育过程中呈显著的变化，这表明miRNA参与 了大脑的发育调控。由此可知，miRNA对其靶基因调控效率的改变很可能引起大脑发育 调控的改变。 本文通过寻找大脑表达的保守microRNA 及对其靶基因的预测，同时用比较基因组 学的方法发现：有很多microRNA 的靶基因3’UTR 的靶位点中存在人类特异突变位点。 我们推测这些人类特异突变位点可能改变microRNA 对其靶基因的表达调控效应，而调 控效应的改变可能在进化过程中对认知能力的提高发挥重要作用。利用体外报告基因系 统，我们发现有几个预测的靶基因是对应miRNA 的真实靶基因。其中，miR-127 的靶 基因（SEMA3F）3’UTR 的靶位点中所含的一个人类特有的突变增强了miR-127 对 SEMA3F 的调控效率。将该位点突变回复为黑猩猩的位点使得miR-127 对SEMA3F 的 调控效率降低到黑猩猩的水平。这表明miR-127 对SEMA3F 的调控效率的改变确实是 由该人类特异突变位点引起的。我们提供了人类特异突变位点能够引起miR-127 对SEMA3F 的调控效率的改变的体外证据，但是体内的调控模式是否如此尚需进一步的工 作。 总之，本文通过体外试验表明，miRNA靶基因3’UTR的序列变异具有功能效应，它 有可能是人类中枢神经系统在起源和演化中发挥关键作用的重要遗传机制之一。

microRNA 在灵长类中的进化及其对靶基因表达变异度的影响

microRNAs（miRNAs）是基因组中广泛编码的一类小RNA 基因，存在于绝大多数多细胞生物中，而且在各种生物学过程中都起着举足轻重的作用。miRNAs 在转录后水平通过与mRNAs 的3’UTRs 序列互补识别靶基因，并引起靶基因的降解或阻遏其翻译。在动物中，一个miRNA 可以调控数百个靶基因的表达。大多数miRNAs 在物种间高度保守，暗示了其功能的重要性。然而，非保守的miRNAs可能对物种特有新功能的产生有贡献。为了回答miRNAs是如何起源，如何进化的问题，我们研究了两个非保守miRNA 家族在灵长类中的进化历史。第一个miRNA 家族位于X 染色体上，在灵长类中的数目比狗或啮齿类中的多。我们比较了这一家族在灵长类主要分支－人、大猿、小猿、旧大陆猴和新大陆猴中的序列情况，发现了这一家族在灵长类中的快速进化。这种快速进化包括频繁的串联重复和碱基替换现象。此外，在人和黑猩猩中还发现了相应进化分支特有的替换，可能会导致分支特有的新miRNAs 的产生。对这一miRNA 家族在不同发育阶段恒河猴睾丸中的表达分析揭示了miRNA 表达变化和雄性性成熟之间的负相关，暗示这一家族在睾丸发育和精子成熟中可能起的调节作用。最后，我们认为，像蛋白编码基因一样，与雄性生殖功能相关的miRNAs 容易受到性选择而发生适应性进化。第二个miRNA 家族是位于19 号染色体上的一个灵长类特有的家族。通过分析和比较这一家族以及其临近区域在9 个不同灵长类物种中的序列，我们发现了 Alu 介导的这一家族的产生和扩张。序列比较表明，物种内和物种间miRNAs 的序列分歧相似；同时，在各个灵长类分支中均存在基因拷贝的获得和丢失，也存在基因的假基因化。由此表明，这一家族在灵长类中经历了典型的“生－死”进化历程，暗示这个家族的miRNA 基因在灵长类的进化中其功能可能发生了多样化，以适应不同灵长类物种在发育过程中的需要。此外，二级结构的保守性和前体miRNAs 区域的低SNP 密度都表明这一家族受到功能性约束。最后，我们进一步分析了这一家族在胎盘和胎儿大脑中的表达，揭示其对灵长类胚胎发育可能的重要性。除了研究miRNAs 在灵长类中的进化，我们还探讨了miRNAs 对基因表达变异度的影响。通过对已发表的193 例人类大脑基因表达谱的分析发现，基因在人群中的表达变异的大小和调控它的miRNA 数目呈正比，这暗示了miRNAs 对基因表达变异度的直接影响。相比于不受miRNA 调控的基因，受到两个以上 miRNA 核心区调控的基因有较高的表达变异度，不受miRNA 类型的影响。同时，我们还证明，人群中靶基因miRNA 识别序列上的变异（SNPs）会进一步导致靶基因表达变异的增加。我们的研究表明miRNAs 是影响人群中基因表达变异度的因素之一。

Udział miRNA w regulacji alternatywnego splicingu u roślin

Wydział Biologii

Effect of microRNA modulation on bioartificial muscle function.

Cellular therapies have recently employed the use of small RNA molecules, particularly microRNAs (miRNAs), to regulate various cellular processes that may be altered in disease states. In this study, we examined the effect of transient muscle-specific miRNA inhibition on the function of three-dimensional skeletal muscle cultures, or bioartificial muscles (BAMs). Skeletal myoblast differentiation in vitro is enhanced by inhibiting a proliferation-promoting miRNA (miR-133) expressed in muscle tissues. As assessed by functional force measurements in response to electrical stimulation at frequencies ranging from 0 to 20 Hz, peak forces exhibited by BAMs with miR-133 inhibition (anti-miR-133) were on average 20% higher than the corresponding negative control, although dynamic responses to electrical stimulation in miRNA-transfected BAMs and negative controls were similar to nontransfected controls. Immunostaining for alpha-actinin and myosin also showed more distinct striations and myofiber organization in anti-miR-133 BAMs, and fiber diameters were significantly larger in these BAMs over both the nontransfected and negative controls. Compared to the negative control, anti-miR-133 BAMs exhibited more intense nuclear staining for Mef2, a key myogenic differentiation marker. To our knowledge, this study is the first to demonstrate that miRNA mediation has functional effects on tissue-engineered constructs.

Plasma miRNAs as early biomarkers for detecting hepatocellular carcinoma.

The early detection of hepatocellular carcinoma (HCC) presents a challenge because of the lack of specific biomarkers. Serum/plasma microRNAs (miRNAs) can discriminate HCC patients from controls. We aimed to identify and evaluate HCC-associated plasma miRNAs originating from the liver as early biomarkers for detecting HCC. In this multicenter three-phase study, we first performed screening using both plasma (HCC before and after liver transplantation or liver hepatectomy) and tissue samples (HCC, para-carcinoma and cirrhotic tissues). Then, we evaluated the diagnostic potential of the miRNAs in two case-control studies (training and validation sets). Finally, we used two prospective cohorts to test the potential of the identified miRNAs for the early detection of HCC. During the screening phase, we identified ten miRNAs, eight of which (miR-20a-5p, miR-25-3p, miR-30a-5p, miR-92a-3p, miR-132-3p, miR-185-5p, miR-320a and miR-324-3p) were significantly overexpressed in the HBV-positive HCC patients compared with the HBV-positive cancer-free controls in both the training and validation sets, with a sensitivity of 0.866 and specificity of 0.646. Furthermore, we assessed the potential for early HCC detection of these eight newly identified miRNAs and three previously reported miRNAs (miR-192-5p, miR-21-5p and miR-375) in two prospective cohorts. Our meta-analysis revealed that four miRNAs (miR-20a-5p, miR-320a, miR-324-3p and miR-375) could be used as preclinical biomarkers (pmeta  < 0.05) for HCC. The expression profile of the eight-miRNA panel can be used to discriminate HCC patients from cancer-free controls, and the four-miRNA panel (alone or combined with AFP) could be a blood-based early detection biomarker for HCC screening.

Polymorphisms at the microRNA binding-site of the stem cell marker gene CD133 modify susceptibility to and survival of gastric cancer.

CD133 is one of the most common stem cell markers, and functional single nucleotide polymorphisms (SNPs) of CD133 may modulate its gene functions and thus cancer risk and patient survival. We hypothesized that potentially functional CD133 SNPs are associated with gastric cancer (GC) risk and survival. To test this hypothesis, we conducted a case-control study of 371 GC patients and 313 cancer-free controls frequency-matched by age, sex, and ethnicity. We genotyped four selected, potentially functional CD133 SNPs (rs2240688A>C, rs7686732C>G, rs10022537T>A, and rs3130C>T) and used logistic regression analysis for associations of these SNPs with GC risk and Cox hazards regression analysis for survival. We found that compared with the miRNA binding site rs2240688 AA genotype, AC + CC genotypes were associated with significantly increased GC risk (adjusted OR = 1.52, 95% CI = 1.09-2.13); for another miRNA binding site rs3130C>T SNP, the TT genotype was associated with significantly reduced GC risk (adjusted OR = 0.68, 95% CI = 0.48-0.97), compared with CC + CT genotypes. In all patients, the risk rs3130 TT variant genotype was significantly associated with overall survival (OS) (adjusted P(trend) = 0.016 and 0.007 under additive and recessive models, respectively). These findings suggest that these two CD133 miRNA binding site variants, rs2240688 and rs3130, may be potential biomarkers for genetic susceptibility to GC and possible predictors for survival in GC patients but require further validation by larger studies.