人工microRNA抑制PAR4表达载体的构建和应用

目的:构建靶向PAR4的人工microRNA表达载体并用于抑制人肠癌细胞SW620中PAR4表达。方法:设计针对人PAR4的人工microRNA序列,利用2次PCR扩增获得目的片段,克隆于pMD-19T载体。经DNA测序证实后,将8个串联连接的PAR4人工microRNA序列亚克隆于真核表达载体pcDNA3.1(+),构建成pcDNA3.1(+)-8xPAR4-microR-NA真核表达载体。用脂质体将表达载体转染SW620细胞,经G418筛选出稳定转染细胞系,Western blot检测PAR4的表达水平。结果:DNA测序结果表明,克隆的针对人PAR4的人工microRNA序列正确。Western blot检测结果显示,稳定转染pcDNA3.1(+)-8xPAR4-microRNA载体的SW620中PAR4的表达较对照组明显受到抑制。结论:成功地构建了靶向PAR4的人工microRNA的表达载体,并能明显抑制靶基因的表达,为进一步研究PAR4的功能及探讨以PAR4为靶点的基因治疗打下了基础。     

Genome‐wide transcriptome analyses reveal p53 inactivation mediated loss of miR‐34a expression in malignant peripheral nerve sheath tumours

Malignant peripheral nerve sheath tumours (MPNSTs) are aggressive soft tissue tumours that occur either sporadically or in patients with neurofibromatosis type 1. The malignant transformation of the benign neurofibroma to MPNST is incompletely understood at the molecular level. We have determined the gene expression signature for benign and malignant PNSTs and found that the major trend in malignant transformation from neurofibroma to MPNST consists of the loss of expression of a large number of genes, rather than widespread increase in gene expression. Relatively few genes are expressed at higher levels in MPNSTs and these include genes involved in cell proliferation and genes implicated in tumour metastasis. In addition, a gene expression signature indicating p53 inactivation is seen in the majority of MPNSTs. Subsequent microRNA profiling of benign and malignant PNSTs indicated a relative down‐regulation of miR‐34a in most MPNSTs compared to neurofibromas. In vitro studies using the cell lines MPNST‐14 (NF1 mutant) and MPNST‐724 (from a non‐NF1 individual) show that exogenous expression of p53 or miR‐34a promotes apoptotic cell death. In addition, exogenous expression of p53 in MPNST cells induces miR‐34a and other miRNAs. Our data show that p53 inactivation and subsequent loss of expression of miR‐34a may significantly contribute to the MPNST development. Collectively, our findings suggest that deregulation of miRNAs has a potential role in the malignant transformation process in peripheral nerve sheath tumours. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Identification of miR‐305, a microRNA that promotes aging,and its target mRNAs in Drosophila

MicroRNAs (miRNAs) are involved in the regulation of important biological processes. Here, we describe a novel Drosophila miRNAs involved in aging. We selected eight Drosophila miRNAs, displaying high homology with seed sequences of aging‐related miRNAs characterized in other species, and investigated whether the over‐expression of these miRNAs affected aging in Drosophila adult flies. The lifespan of adults over‐expressing miR‐305, a miRNA showing high homology with miR‐239 in C. elegans, was significantly shorter. Conversely, a reduction in miR‐305 expression led to a longer lifespan than that in control flies. miR‐305 over‐expression accelerated the impairment of locomotor activity and promoted the age‐dependent accumulation of poly‐ubiquitinated protein aggregates in the muscle, as flies aged. Thus, we show that the ectopic expression of miR‐305 has a deleterious effect on aging in Drosophila. To identify the targets of miR‐305, we performed RNA‐Seq. We discovered several mRNAs encoding antimicrobial peptides and insulin‐like peptides, whose expression changed in adults upon miR‐305 over‐expression. We further confirmed, by qRT‐PCR, that miR‐305 over‐expression significantly decreases the mRNA levels of four antimicrobial peptides. As these mRNAs contain multiple sequences matching the seed sequence of miR‐305, we speculate that a reduction in target mRNA levels, caused by ectopic miRNA expression, promotes aging.     

The type 1 diabetes resistance locus B10 Idd9.3 mediates impaired B‐cell lymphopoiesis and implicates microRNA‐34a in diabetes protection

NOD.B10 Idd9.3 mice are congenic for the insulin‐dependent diabetes (Idd) Idd9.3 locus, which confers significant type 1 diabetes (T1D) protection and encodes 19 genes, including microRNA (miR)‐34a, from T1D‐resistant C57BL/10 mice. B cells have been shown to play a critical role in the priming of autoantigen‐specific CD4⁺ T cells in T1D pathogenesis in non‐obese diabetic (NOD) mice. We show that early B‐cell development is impaired in NOD.B10 Idd9.3 mice, resulting in the profound reduction of transitional and mature splenic B cells as compared with NOD mice. Molecular analysis revealed that miR‐34a expression was significantly higher in B‐cell progenitors and marginal zone B cells from NOD.B10 Idd9.3 mice than in NOD mice. Furthermore, miR‐34a expression in these cell populations inversely correlated with levels of Foxp1, an essential regulator of B‐cell lymphopoiesis, which is directly repressed by miR‐34a. In addition, we show that islet‐specific CD4⁺ T cells proliferated inefficiently when primed by NOD.B10 Idd9.3 B cells in vitro or in response to endogenous autoantigen in NOD.B10 Idd9.3 mice. Thus, Idd9.3‐encoded miR‐34a is a likely candidate in negatively regulating B‐cell lymphopoiesis, which may contribute to inefficient expansion of islet‐specific CD4⁺ T cells and to T1D protection in NOD.B10 Idd9.3 mice.     

microRNA作为脓毒症早期诊断生物标志物的研究进展

microRNA(miRNA)是一类长度约为22 nt的内源性非编码RNA,其表达异常可能导致炎性反应失衡。在脓毒症期间,miR-146a、miR-223和miR-150表达水平降低,而miR-15a和miR-16表达增高。脓毒症患者外周血中miRNA表达水平的异常与患者病情密切相关,因此,miRNA能否作为脓毒症早期诊断的生物标志物备受关注,并已初步取得可喜成果。     

In utero exposure to second‐hand smoke activates pro‐asthmatic and oncogenic miRNAs in adult asthmatic mice

Exposures to environmental pollutants contribute to dysregulated microRNA (miRNA) expression profiles, which have been implicated in various diseases. Previously, we reported aggravated asthmatic responses in ovalbumin (OVA)‐challenged adult mice that had been exposed in utero to second‐hand smoke (SHS). Whether in utero SHS exposure dysregulates miRNA expression patterns in the adult asthma model has not been investigated. Pregnant BALB/c mice were exposed (days 6–19 of pregnancy) to SHS (10 mg/m³) or HEPA‐filtered air. All offspring were sensitized and challenged with OVA (19–23 weeks) before sacrifice. RNA samples extracted from lung homogenates, were subjected to RNA sequencing (RNA‐seq). RNA‐seq identified nine miRNAs that were most significantly up‐regulated by in utero SHS exposure. Among these nine, miR‐155‐5p, miR‐21‐3p, and miR‐18a‐5p were also highly correlated with pro‐asthmatic Th2 cytokine levels in bronchoalveolar lavage fluid. Further analysis indicated that these up‐regulated miRNAs shared common chromosome locations, particularly Chr 11C, with pro‐asthmatic genes. These three miRNAs have also been characterized as oncogenic miRNAs (oncomirs). We cross‐referenced miRNA‐mRNA expression profiles and identified 16 tumor suppressor genes that were down‐regulated in the in utero‐exposed offspring and that are predicted targets of the up‐regulated oncomirs. In conclusion, in utero SHS exposure activates pro‐asthmatic genes and miRNAs, which colocalize at specific chromosome locations, in OVA‐challenged adult mice. The oncogenic characteristics of the miRNAs and putative miRNA‐mRNA regulatory networks suggest that the synergistic effect of in utero SHS exposure and certain adult irritants may promote an oncogenic milieu in mouse lungs via inhibition of miRNA‐regulated tumor suppressor genes. Environ. Mol. Mutagen. 57:190–199, 2016. © 2016 Wiley Periodicals, Inc.