miRNAs及其靶基因的识别

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。据推测,大约1%的人类已知基因编码miRNAs,而miRNAs可调控人类基因组中10%~30%的基因。本文就如何识别miRNAs及其靶基因作一简要介绍。     

The regulation of non-coding RNA expression in the liver of mice fed DDC

Mallory-Denk bodies (MDBs) are found in the liver of patients with alcoholic and chronic nonalcoholic liver disease, and hepatocellular carcinoma (HCC). Diethyl 1,4-dihydro-2,4,6,-trimethyl-3,5-pyridinedicarboxylate (DDC) is used as a model to induce the formation of MDBs in mouse liver. Previous studies in this laboratory showed that DDC induced epigenetic modifications in DNA and histones. The combination of these modifications changes the phenotype of the MDB forming hepatocytes, as indicated by the marker FAT10. These epigenetic modifications are partially prevented by adding to the diet S-adenosylmethionine (SAMe) or betaine, both methyl donors. The expression of three imprinted ncRNA genes was found to change in MDB forming hepatocytes, which is the subject of this report. NcRNA expression was quantitated by real-time PCR and RNA FISH in liver sections. Microarray analysis showed that the expression of three ncRNAs was regulated by DDC: up regulation of H19, antisense Igf2r (AIR), and down regulation of GTL2 (also called MEG3). S-adenosylmethionine (SAMe) feeding prevented these changes. Betaine, another methyl group donor, prevented only H19 and AIR up regulation induced by DDC, on microarrays. The results of the SAMe and betaine groups were confirmed by real-time PCR, except for AIR expression. After 1 month of drug withdrawal, the expression of the three ncRNAs tended toward control levels of expression. Liver tumors that developed also showed up regulation of H19 and AIR. The RNA FISH approach showed that the MDB forming cells' phenotype changed the level of expression of AIR, H19 and GTL2, compared to the surrounding cells. Furthermore, over expression of H19 and AIR was demonstrated in tumors formed in mice withdrawn for 9 months. The dysregulation of ncRNA in MDB forming liver cells has been observed for the first time in drug-primed mice associated with liver preneoplastic foci and tumors.     

关于强心苷类药物与无义介导的mRNA降解在抗肿瘤作用中的研究

强心苷是一类具有强心作用的甾体苷类化合物,其可抑制心肌细胞膜上的Na+/K+ ATP酶,通过降低质膜两侧Na+的浓度差以减小Na+-Ca2+交换的驱动力,使胞质内Ca2+浓度增加,从而产生强心效应。强心苷与细胞膜上Na+/K+ ATP酶特异性结合,一方面可抑制其离子通道作用,另一方面在较低浓度下可调控下游信号通路,近来研究表明,其可能具有抗肿瘤效应。无义介导的mRNA降解(nonsense-mediated mRNA decay,NMD)是近年发现的RNA监控机制,被认为与肿瘤的发生发展有关。本文旨在探讨强心苷、NMD与肿瘤之间的相互关系,为肿瘤的治疗提供新思路。     

以腺相关病毒为载体的小干扰RNA对肝星状细胞中金属蛋白酶组织抑制因子的抑制作用

目的 观察以腺相关病毒（AAV）为载体含有针对大鼠金属蛋白酶组织抑制因子（TIMP）-1具有较强抑制作用的小干扰RNA（siRNA）感染大鼠星状细胞系HSC-T6后TIMP-1的表达抑制作用。方法 针对大鼠TIMP-1mRNA基因序列挑选一段22bp片段，在体外构建为短发夹siRNA（short hairpin siRNA，shRNA）表达载体后，将其包装为重组AAV并感染大鼠肝星状细胞系HSC-T6后，于感染后30d及90d应用荧光定量PCR方法及Westem blot方法分别检测TIMP-1 mRNA及蛋白质表达情况，同时通过PCR技术以感染后细胞的基因组DNA为模板扩增外源基因验证其长效表达。结果 经PCR、酶切及序列测定证实含有siRNA-TIMP-1基因的重组AAV载体质粒已成功克隆。将重组质粒包装成病毒后感染HSC-T6细胞，与对照组细胞相比，感染后30d及90d细胞TIMP-1 mRNA水平明显降低（P〈0．01），感染后30d TIMP-1蛋白表达水平较对照组细胞相比下降约60％，而感染后90d，TIMP-1蛋白表达几乎下降90％。PCR结果显示在重组病毒感染后90d细胞基因组DNA中仍可扩增出外源基因，证实外源基因可长期表达。结论 重组病毒rAAV／siRNA-TIMP-1／neo可长期有效地抑制TIMP-1基因的表达。     

Aggregates of Small Nuclear Ribonucleic Acids (snRNAs) in Alzheimer's Disease

We recently discovered that protein components of the ribonucleic acid (RNA) spliceosome form cytoplasmic aggregates in Alzheimer's disease (AD) brain, resulting in widespread changes in RNA splicing. However, the involvement of small nuclear RNAs (snRNAs), also key components of the spliceosome complex, in the pathology of AD remains unknown. Using immunohistochemical staining of post‐mortem human brain and spinal cord, we identified cytoplasmic tangle‐shaped aggregates of snRNA in both sporadic and familial AD cases but not in aged controls or other neurodegenerative disorders. Immunofluorescence using antibodies reactive with the 2,2,7‐trimethylguanosine cap of snRNAs and transmission electron microscopy demonstrated snRNA localization with tau and paired helical filaments, the main component of neurofibrillary tangles. Quantitative real‐time polymerase chain reaction (PCR) showed U1 snRNA accumulation in the insoluble fraction of AD brains whereas other U snRNAs were not enriched. In combination with our previous results, these findings demonstrate that aggregates of U1 snRNA and U1 small nuclear ribonucleoproteins represent a new pathological hallmark of AD.     

Implication of membrane localization of target mRNA in the action of a small RNA: mechanism of post-transcriptional regulation of glucose transporter in Escherichia coli

Accumulation of phosphosugars such as glucose-6-phosphate causes a rapid degradation of ptsG mRNA encoding the major glucose transporter IICB(Glc) in an RNase E/degradosome-dependent manner. The destabilization of ptsG mRNA is caused by a small antisense RNA (SgrS) that is induced by phosphosugar stress. In this study, we analyzed a series of ptsG-crp translational fusions to identify the mRNA region required for the rapid degradation of ptsG mRNA. We found that the ptsG-crp mRNA is destabilized in response to phosphosugar stress when it contains the 5' portion of ptsG mRNA corresponding up to the first two transmembrane domains (TM1 and TM2) of IICB(Glc). The destabilization of ptsG-crp mRNA was largely eliminated by frameshift mutations in the transmembrane region. The IICB(Glc)-CRP fusion proteins containing more than two transmembrane domains were localized at the membrane. The efficient destabilization of ptsG-crp mRNA was restored when TM1 and TM2 of IICB(Glc) were replaced by part of the LacY transmembrane region. We conclude that the membrane-targeting property of IICB(Glc) protein rather than the particular nucleotide or amino acid sequence is required for the efficient degradation of ptsG mRNA in response to metabolic stress. The stimulation of ptsG-crp mRNA degradation was completely eliminated when either the hfq or sgrS gene is inactivated. The efficient mRNA destabilization was observed in the absence of membrane localization when translation was reduced by introducing a mutation in the ribosome-binding site in the cytoplasmic ptsG-crp mRNA. Taken together, we conclude that mRNA localization to the inner membrane coupled with the membrane insertion of nascent peptide mediates the Hfq/SgrS-dependent ptsG mRNA destabilization presumably by reducing second rounds of translation.     

Two‐dimensional spectroscopic imaging with combined free induction decay and long‐TE acquisition (FID echo spectroscopic imaging,FIDESI) for the detection of intramyocellular lipids in calf muscle at 7 T

The aim of this study was to introduce a two‐dimensional chemical shift imaging (2D CSI) sequence, with simultaneous acquisition of free induction decay (FID) and long TEs, for the detection and quantification of intramyocellular lipids (IMCLs) in the calf at 7 T. The feasibility of the new 2D CSI sequence, which acquires FID (acquisition delay, 1.3 ms) and an echo (long TE) in one measurement, was evaluated in phantoms and volunteers (n = 5): TR/TE*/TE = 800/1.3/156 ms; 48 × 48 matrix; field of view, 200 × 200 × 20 mm³; Hamming filter; no water suppression; measurement time, 22 min 2 s. The IMCL concentration and subcutaneous lipid contamination were assessed. Spectra in the tibialis anterior (TA), gastrocnemius (GM) and soleus (SOL) muscles were analyzed. The water signal from the FID acquisition was used as an internal concentration reference. In the spectra from subcutaneous adipose tissue (SUB) and bone marrow (BM), an unsaturation index (UI) of the vinyl‐H (5.3 ppm) to methyl‐CH₃ ratio, and a polyunsaturation index (pUI) of the diallylic‐H (2.77 ppm) to ‐CH₃ ratio, were calculated. Long‐TE spectra from muscles showed a simplified spectral pattern with well‐separated IMCL for several muscle groups in the same scan. The IMCL to water ratio was largest in SOL (0.66% ± 0.23%), and lower in GM (0.37% ± 0.14%) and TA (0.36% ± 0.12%). UI and pUI for SUB were 0.65 ± 0.06 and 0.18 ± 0.04, respectively, and for BM were 0.60 ± 0.16 and 0.18 ± 0.08, respectively. The new sequence, with the proposed name ‘free induction decay echo spectroscopic imaging’ (FIDESI), provides information on both specific lipid resonances and water signal from different tissues in the calf, with high spectral and spatial resolution, as well as minimal voxel bleeding and subcutaneous lipid contamination, in clinically acceptable measurement times. Copyright © 2014 John Wiley & Sons, Ltd.     

Attenuation of SARS coronavirus by a short hairpin RNA expression plasmid targeting RNA-dependent RNA polymerase

Severe acute respiratory syndrome (SARS) is a highly contagious and sometimes a lethal disease, which spread over five continents in 2002-2003. Laboratory analysis showed that the etiologic agent for SARS is a new type of coronavirus. Currently, there is no specific treatment for this disease. RNA interference (RNAi) is a recently discovered antiviral mechanism in plant and animal cells that induces a specific degradation of double-stranded RNA. Here, we provide evidences that RNAi targeting at coronavirus RNA-dependent RNA polymerase (RDRP) using short hairpin RNA (shRNA) expression plasmids can specifically inhibit expression of extraneous coronavirus RDRP in 293 and HeLa cells. Moreover, this construct significantly reduced the plaque formation of SARS coronaviruses in Vero-E6 cells. The data may suggest a new approach for treatment of SARS patients.     

聚乳酸－乙醇酸共聚物合成与降解

本文对聚乳酸-乙醇酸(PLGA)的合成制备的多种方法进行了阐述,对聚乳酸-乙醇酸(PLGA)的降解性能和降解机理进行了概述.