幽门螺杆菌感染细胞模型的建立及感染相关microRNAs的筛选鉴定

目的：建立稳定的幽门螺杆菌（H.pylori）感染人胃上皮细胞模型;筛选并鉴定H.pylori感染相关microRNAs（miRNAs）的表达,为深入研究感染相关miRNAs的调控作用机制奠定基础。方法：将H.pylori标准株按MOI=100：1感染人胃上皮细胞,通过检测炎性细胞因子及炎症反应关键酶的表达综合评价感染模型;采用博奥公司miRNAs V3.0芯片分析细胞感染前后miRNAs表达谱变化,运用实时定量PCR技术和Northern杂交对表达显著差异的miRNAs进行分析鉴定。结果：H.pylori感染细胞24 h后,细胞分泌促炎细胞因子IL-8显著升高（P〈0.01）;启动炎症反应的关键酶COX-2的表达明显增加。芯片数据显示：H.pylori感染引起超过2倍显著差异表达的miRNAs包括：表达上调的PREDICTED-MIR191、miR-155、miR-92b、miR-30b、miR-146a、miR-16等,和表达降低的miR-181b、miR-324。实时定量PCR和Northern杂交结果显示感染相关miR-155和miR-146a在H.pylori感染细胞模型中表达均显著增加（P〈0.01）。结论：miR-155和miR-146a在感染细胞模型中的表达增加提示二者可能参与H.pylori感染的免疫调控过程。     

The Hsa-miR-27a rs895819 (A > G) polymorphism and cancer susceptibility

Published data on the association between the rs895819 (A > G) polymorphism in the terminal loop of pre-miR-27a and cancer risk is inconclusive. Therefore, we conducted a meta-analysis to estimate the association between this polymorphism and cancer. The PubMed, Web of science, and Embase databases were searched for articles on the hsa-miR-27a rs895819 polymorphism and cancer risk published up to November 24, 2012. The genotype data obtained in the searches were pooled in our meta-analysis, and pooled odds ratio (OR) with 95% confidence interval (CI) was used to assess the association. Seven studies with a total of 3849 cases and 4781 controls were eligible for analysis. Overall, we found no significant associations between the hsa-miR-27a rs895819 (A > G) polymorphism and cancer susceptibility (homozygote model: OR = 0.88, 95% CI: 0.68–1.14; heterozygote model: OR = 0.96, 95% CI: 0.79–1.17; dominant model: OR = 0.94, 95% CI: 0.79–1.12; recessive model: OR = 0.88, 95% CI: 0.69–1.12). In the subgroup analysis by ethnicity, we found that the rs895819 AG genotype was associated with a decreased risk of cancer in white individuals (dominant model: OR = 0.85, 95% CI: 0.76–0.94; heterozygote model: OR = 0.84, 95% CI: 0.75–0.94). This meta-analysis indicated that the hsa-miR-27a rs895819 polymorphism did not correlate with overall cancer risk in the general population. However, the rs895819 AG genotype may protect against the development of cancer in white individuals. Larger, better studies of homogeneous cancer patients are needed to further assess the correlation between this polymorphism and cancer risk.     

An efficient method to enhance gene silencing by using precursor microRNA designed small hairpin RNAs

Gene silencing can be mediated by small interfering RNA (siRNA) and microRNA (miRNA). To investigate the potential application of using a precursor microRNA (pre-miRNA) backbone for gene silencing, we studied the inhibition efficiency of exogenous GFP and endogenous GAPDH by conventional shRNA- and pre-miRNA-designed hairpins, respectively. In this study, the conventional shRNA-, pre-miRNA-30-, and pre-miRNA-155-designed hairpins targeting either GFP or GAPDH were transfected into the HEK293 cells that were mediated by the pSilencer-4.1-neo vector, which carries a modified RNA polymerase II-type CMV promoter. Comparisons with conventional GFP shRNA showed that GFP levels were reduced markedly by pre-miRNA-30- and pre-miRNA-155-designed GFP shRNAs by fluorescence microscopy. The consistent results from semi-quantitative RT-PCR and Western blot analysis revealed that pre-miRNA-30- and pre-miRNA-155-designed GFP shRNAs could suppress GFP expression significantly. As for endogenous GAPDH, the results from semi-quantitative RT-PCR and Western blot analysis showed that pre-miRNA-30- and pre-miRNA-155-designed GAPDH shRNAs could suppress GAPDH expression even more efficiently than conventional GAPDH shRNA. Together, this study confirmed the efficiency of gene silencing mediated by pre-miRNA-30- and pre-miRNA-155-designed shRNAs, demonstrating that pre-miRNA-designed hairpins are a good strategy for gene silencing.     

中枢神经系统弥漫大B 细胞淋巴瘤相关microRNAs 的研究进展

中枢神经系统(central nervous system,CNS)复发是弥漫大B细胞淋巴瘤(diffuse large B-cell lymphoma,DLBCL)的一种不常见的严重的并发症,新诊断的患者2年内易于发生,最常见于非霍奇金淋巴瘤弥漫大B细胞型(non-Hodgkin diffuse large B-cell lymphoma-,NHL-DLBCL),目前,对于初始治疗后出现中枢复发其发病机制并不清楚。microRNA(miRNA)是一类新发现的非编码小分子RNA,通过抑制靶基因翻译或降解靶miRNA调控基因表达,参与细胞分化、增殖、调亡等生命活动。miRNA在淋巴瘤的发生发展中有重要作用。近年来大量研究已证实miRNA与肿瘤的组织来源、进展、转移预后与耐药都密切相关,既可作为抑癌基因,也可作为癌基因。淋巴瘤是一种血液免疫系统肿瘤,与淋巴瘤相关的miRNA已成为当前研究热点之一。microRNAs的功能紊乱如何导致DLBCL发生的机制目前还没有得到很好的证明,但DLBCL患者中464种miRNAs显示microRNA(包括miRNA-17-92簇)预测淋巴瘤的准确率达95%,为淋巴瘤研究提供了新依据。     

MicroRNA与变应性鼻炎

在变应性鼻炎(allergic rhinitis,AR)发病的免疫细胞分化及免疫应答过程中,micro RNA(miRNA)发挥着非常重要的调控作用。本文旨在对AR中miRNA研究进行回顾,以期为AR的有效治疗提供新的视角。首先,变应性鼻炎患者鼻粘膜中miR-7和miRPlus-E1194等差异表达。其次,在T辅助细胞发育、分化与活化过程中,miR-181a、miR-155、miR-21、miR-1、miR-31、miR-223、miR-139-3p、miR-126、Let-7家族成员会发生显著上调或下降,而且某些miRNA的相关作用靶点也得到验证。再次,嗜酸粒细胞定向分化祖细胞向成熟嗜酸粒细胞的分化过程中,miR-21和miR-223都有所上调。最后,在肥大细胞激活和脱颗粒过程中,miR-221和miR-222以及其他miRNA显著上调。以上miRNA参与了T辅助细胞分化与活化、嗜酸粒细胞发育、肥大细胞脱颗粒等AR病理进程的各个环节,而且,miRNA还会参与前期的抗原提呈等环节。可见miRNA对AR调控呈现出复杂性和多重性。     

SR 59230A对心衰大鼠心脏MicroRNAs表达的影响

目的：观察β3肾上腺素受体（β3-AR）对心衰大鼠心脏MicroRNAs表达的影响及可能的作用机制。方法：大鼠冠脉左前降支结扎造成心衰模型,假手术大鼠只穿线不结扎。造模成功大鼠再随机分为：心衰组（CHF control group）和心衰+SR 59230A组（CHF+SR group）;假手术大鼠也随机分为假手术组（Sham group）和假手术+SR 59230A组（Sham+SR group）。Sham+SR组和CHF+SR组每天两次腹腔注射SR （85 mmol/L,1 ml）,连续注射7周。结果：①miScript miRNA PCR Arrays显示,在体阻断β3-AR后,假手术组与心衰组有18种MicroRNAs共同表达下调;经文献比对,与NF-κB相关的MicroRNAs有6种,分别为miR-125b-5p,miR-143-3p,miR-145-5p,miR-26a-5p,miR-30a-5p和miR-320-5p。②大鼠心脏组织切片观察到NF-κB在心衰大鼠心肌细胞核与细胞质中均有分布,而p53在心肌细胞质分布较多,NF-κB和p53表达明显高于假手术组（P<0.05）。阻断β3-AR后,心衰组心脏NF-κB和p53表达显著减少（P<0.05）,而假手术组NF-κB和p53表达略增加（P<0.05）。③Western blot结果发现心衰大鼠NF-κB p65表达高于假手术组（P<0.05）,给予β3-AR阻断剂后,心衰组心脏NF-κB p65和p53-Phospho-Serine 15表达均下降（P<0.05）,而假手术组心脏阻断β3-AR后,NF-κB、p53和p53-Phospho-Serine 15表达均增加（P<0.05）。结论：阻断β3肾上腺素受体有利于缓解心衰大鼠心脏的损伤;β3-AR可引起MicroRNAs表达变化且与NF-κB信号通路有关。     

BmNPV-miR-415 up-regulates the expression of TOR2 via Bmo-miR-5738

MicroRNAs (miRNAs) have emerged as key players in host–pathogen interaction and many virus-encoded miRNAs have been identified (computationally and/or experimentally) in a variety of organisms. A novel Bombyx mori nucleopolyhedrosis virus (BmNPV)-encoded miRNA miR-415 was previously identified through high-throughput sequencing. In this study, a BmNPV-miR-415 expression vector was constructed and transfected into BmN cells. The differentially expressed protein target of rapamycin isoform 2 (TOR2) was observed through two-dimensional gel electrophoresis and mass spectrometry. Results showed that TOR2 is not directly a target gene of BmNPV-miR-415, but its expression is up-regulated by BmNPV-miR-415 via Bmo-miR-5738, which could be induced by BmNPV.     

Calculation of folding energies of single-stranded nucleic acid sequences: conceptual issues

The stability of a folded single-stranded nucleic acid depends on the composition and order of its constituent bases and may be assessed by taking into account the pairing energies of its constituent dinucleotides. To assess the possible biological significance of a computed structure, Maizel and coworkers in the 1980s compared the energy of folding of a natural single-stranded RNA sequence with the energies of several versions of the same sequence produced by shuffling base order. However, in the 2000s many took as self-evident the view that shuffling at the mononucleotide level (single bases) was conceptual wrong and should be replaced by shuffling at the level of dinucleotides (retaining pairs of adjacent bases). Folding energies then became indistinguishable from those of corresponding shuffled sequences and doubt was cast on the importance of secondary structures. Nevertheless, some continued productively to employ the single base shuffling approach, the justification for which is the topic of this paper. Because dinucleotide pairing energies are needed to calculate structure, it does not follow that shuffling should not disrupt dinucleotides. Base shuffling allows determination of the relative contributions of base composition and base order to total folding energy. The potential for secondary structure arises from pressures acting at both DNA and RNA levels, and is abundant throughout genomes-with a probable primary role in recombination. Within a gene the potential can often be accommodated, and base order and composition work together (values have the same negative sign) in contributing to total folding energy. But sometimes protein-coding pressure on base order conflicts with the pressure for secondary structure and the values have opposite signs. Total folding energy can be deemed of potential biological significance when the average of several readings is significantly less than zero.