坐骨神经损伤早期背根神经节组织microRNA的表达变化

目的研究大鼠坐骨神经损伤以后短时间背根神经节组织中microRNA(miRNA)的表达变化。方法采用miRNA芯片检测神经损伤0 h(对照组)、3 h和9 h(实验组)背根神经节组织miRNA的表达情况,应用real time Taqman PCR对芯片结果进行验证。结果损伤3 h实验组与对照组比较发生上调和下调1.3倍以上的miRNA分别有4个和2个,损伤9h实验组与对照组比较发生上调和下调1.3倍以上的miRNA分别有7个和5个,real time Taqman PCR检测miR-188和miR-500的表达变化与芯片一致。结论大鼠坐骨神经损伤3h和9h后背根神经节组织中miRNA的表达谱发生改变。     

Identification of microRNA expression signature for the diagnosis and prognosis of cervical squamous cell carcinoma

Cervical cancer is the fourth leading cause of cancer death among women globally. The prognosis of cervical cancer patients differs considerably, and clinical outcomes are difficult to predict. Given the significant roles of miRNAs in human cancers, identification of novel and reliable miRNA biomarkers is important for targeted cervical cancer therapy. In the present study, we aimed to reveal biological significance of miR-200a, miR-423, miR-34a, miR-193a, and miR-455 for the prognosis and diagnosis of cervical cancer and their association with the clinical outcomes of patients. Distinct expression profiles of miRNAs in formalin-fixed paraffin-embedded tissue samples of patients and healthy controls were evaluated using qRT-PCR. We identified miR-200a, miR-455, and miR-34a were significantly downregulated in cervical squamous cell carcinoma tissues compared to normal cervix tissue from healthy controls. Both miR-455 and miR-34a confer a promising diagnostic factor for the cervical cancer while miR-200a showed no significance in ROC analysis. Notably, low expression of miR-34a was markedly associated with the poor overall survival of cervical cancer patients as revealed by Kaplan-Meier survival analysis. Also, univariate and multivariate analysis indicated miR-34a expression as an independent prognostic factor. Consequently, our results underline the importance of distinct expression miRNAs in cervical squamous cell carcinoma.     

Altered patterns of gene expression in endothelial cells in scleroderma

Introduction Scleroderma (systemic sclerosis, SSc) presents clinically as fibrosis of the skin but also involves fibrosis of blood vessels and internal organs, causing damage and complications that in the most severe forms lead to organ failure and death. The earliest detectable structural feature of SSc pathology appears to be endothelial cell damage. This leads to an inflammatory response with adhesion of leucocytes to the blood vessel walls, emigration and accumulation in the tissue. Paracrine factors secreted from activated endothelial cells have been implicated in the consequential fibroblast dysfunction and excessive deposition of extracellular matrix in lesional tissues ( Denton et al. 1996 ). Activated lesional fibroblasts, which maintain their variant phenotype in culture for several passages, in turn, act upon the endothelial cells causing a ‘cross-talk’ which perpetuates the SSc phenotype of both endothelial cells and fibroblasts. The aim of this work was to use an in vitro approach to identify the altered pattern of gene expression in endothelial cells induced by co-culture with SSc lesional fibroblasts, which may reflect the phenotypic changes in endothelial cells in SSc. Materials and methods Human dermal microvascular endothelial cells (HMEC-1, Ribeiro et al. 1995 ) were co-cultured with dermal fibroblasts obtained from biopsies of lesional areas of the skin of patients with scleroderma or normal demal fibroblasts. Co-cultures were carried out in six-well transwell tissue culture plates. All cells were grown to confluence then fibroblasts with their conditioned medium were co-cultured with HMEC-1 for 2, 4, 6, 24 or 48 h. Total RNA was extracted from HMEC-1, reverse transcribed, and expressed genes were identified using Atlas™ Nylon cDNA Expression Arrays (human broad range 1.2) (Clontech). Arrays were imaged using a Typhoon 9210 phosphorimager (Molecular Dynamics), and images were analysed using the AtlasImage™ 2.0 software. Selected mRNA levels were subsequently measured by real-time PCR using a LightCycler™ (Roche). Results The human broad range 1.2 array has 1176 cDNAs plus nine housekeeping cDNAs and negative control cDNAs. The overall pattern of gene expression in HMEC-1 co-cultured with normal dermal fibroblasts (control) and HMEC-1 co-cultured with lesional SSc fibroblasts (diseased) was similar. In total, 32–49% of cDNAs were detectably expressed of which between 5 and 10% of cDNAs were apparently differentially expressed in diseased relative to control. Ratios of diseased: normal ranged from 11.7 to 0.09. Six genes of potential interest were selected from the 6-h array and measured by real-time PCR. Array results (at 6 h) showed that the leucocyte function-associated molecule 1 alpha chain and caspase 10 were up-regulated, and MMP-11 was down-regulated; connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1) and endothelin-2 (ET-2) were expressed at similar levels in diseased and normal samples. When measured by real-time PCR at all co-culture time points, these genes were found to be either unchanged or down-regulated in HMEC-1 co-cultured with SSc fibroblasts. Discussion We have identified the pattern of expression of 1176 cDNAs in HMEC-1 co-cultured with normal fibroblasts or with those from patients with SSc. These contain candidate genes which may be responsible for early vascular abnormalities in SSc. In future experiments, we will study the role of these candidate genes using in vitro models of the pathology of SSc.     

Altered microRNA expression levels in mononuclear cells of patients with pulmonary and pleural tuberculosis and their relation with components of the immune response

Different lines of evidence demonstrate that microRNAs (miRNAs) play an important role in host-pathogen interactions. In this study we investigated the expression patterns of several miRNAs, most of them involved in regulating inflammatory responses, in patients with tuberculosis (TB). In order to understand the events occurring at the site of infection, we employed mononuclear cells obtained from both peripheral blood (PBMC) and pleural fluids (PFMC) of patients. Interestingly, we found that the miRNA signature of each compartment is different, with a strong down-regulation in PFMCs of miR-223, miR-144* and miR-421. In addition, we observed that miR-146a expression is also down-regulated in tuberculosis patients, both in PBMCs and PFMCs while miR-424 levels are elevated only in the peripheral compartments. We also showed that systemic expression of these miRNAs changes upon specific treatment and is associated with IL-6 levels, a cytokine playing a substantial role in TB immunopathology. Present results contribute to a better knowledge of the host responses in TB pathogenesis, pointing out the role of miRNAs in this disease.     

Altered microRNA expression levels in mononuclear cells of patients with pulmonary and pleural tuberculosis and their relation with components of the immune response

Different lines of evidence demonstrate that microRNAs (miRNAs) play an important role in host–pathogen interactions. In this study we investigated the expression patterns of several miRNAs, most of them involved in regulating inflammatory responses, in patients with tuberculosis (TB). In order to understand the events occurring at the site of infection, we employed mononuclear cells obtained from both peripheral blood (PBMC) and pleural fluids (PFMC) of patients. Interestingly, we found that the miRNA signature of each compartment is different, with a strong down-regulation in PFMCs of miR-223, miR-144* and miR-421. In addition, we observed that miR-146a expression is also down-regulated in tuberculosis patients, both in PBMCs and PFMCs while miR-424 levels are elevated only in the peripheral compartments. We also showed that systemic expression of these miRNAs changes upon specific treatment and is associated with IL-6 levels, a cytokine playing a substantial role in TB immunopathology. Present results contribute to a better knowledge of the host responses in TB pathogenesis, pointing out the role of miRNAs in this disease.     

The microRNA expression signature on modified titanium implant surfaces influences genetic mechanisms leading to osteogenic differentiation

Topographically and chemically modified titanium implants are recognized to have improved osteogenic properties; however, the molecular regulation of this process remains unknown. This study aimed to determine the microRNA profile and the potential regulation of osteogenic differentiation following early exposure of osteoprogenitor cells to sand-blasted, large-grit acid-etched (SLA) and hydrophilic SLA (modSLA) surfaces. Firstly, the osteogenic characteristics of the primary osteoprogenitor cells were confirmed using ALP activity and Alizarin Red S staining. The effect of smooth (SMO), SLA and modSLA surfaces on the TGF-β/BMP (BMP2, BMP6, ACVR1) and non-canonical WNT/Ca(2+) (WNT5A, FZD6) pathways, as well as the integrins ITGB1 and ITGA2, was determined. It was revealed that the modified titanium surfaces could induce the activation of TGF-β/BMP and non-canonical WNT/Ca(2+) signaling genes. The expression pattern of microRNAs (miRNAs) related to cell differentiation was evaluated. Statistical analysis of the differentially regulated miRNAs indicated that 35 and 32 miRNAs were down-regulated on the modSLA and SLA surfaces respectively, when compared with the smooth surface (SMO). Thirty-one miRNAs that were down-regulated were common to both modSLA and SLA. There were 10 miRNAs up-regulated on modSLA and nine on SLA surfaces, amongst which eight were the same as observed on modSLA. TargetScan predictions for the down-regulated miRNAs revealed genes of the TGF-β/BMP and non-canonical Ca(2+) pathways as targets. This study demonstrated that modified titanium implant surfaces induce differential regulation of miRNAs, which potentially regulate the TGF-β/BMP and WNT/Ca(2+) pathways during osteogenic differentiation on modified titanium implant surfaces.     

Altered microRNA expression profile is linked to T-cell exhaustion-related pathways in pediatric patients with acute lymphoblastic leukemia

BackgroundAlthough the phenotype and functions of exhausted T cells in several cancers have been identified, the involved molecular mechanisms remain to be further elucidated. In this regard, we have recently reported that the immunoregulatory cells, including myeloid-derived suppressor cells (MDSCs) and regulatory T cells (T_regs), share common dysregulated miRNAs that target specific immunosuppressive pathways in patients with in acute lymphoblastic leukemia (ALL). Aim: In this study, we aimed to further explore whether similar dysregulation in miRNA expression is linked to T cell exhaustion and dysfunctionality in B cell ALL patients.MethodsPeripheral blood samples from pediatric patients with ALL were recruited before and after induction chemotherapy as well as from healthy donors. Affymetrix microarray platform was used for miRNA profiling, and qRT-PCR was used to validate the expression of certain miRNAs that are related to T cell exhaustion. Bioinformatics analysis was performed to explore whether the dysregulated miRNAs were linked to T-cell exhaustion related pathways.ResultsA total of 516 miRNAs were dysregulated in ALL patients as compared to the healthy donor. Furthermore, among the total analyzed miRNAs, 10 were found to be linked to the key genes implicated in three exhaustion-related pathways; TGF-β, FOXO, and MAPK, as revealed by miR-pathway analysis. Moreover, qRT-PCR analysis showed similar expression pattern to those obtained by microarray analysis.ConclusionOur pilot study suggests the implication of certain miRNAs in T cell exhaustion pathways via targeting the specific key genes in those pathways.     

免疫性血小板减少症患者外周血Treg 细胞microRNA 分析

目的:分析ITP 患者和健康对照组Treg 细胞中miRNA 的表达谱的差异,探讨ITP 的发病机制。方法:抽取ITP 患者组和健康对照组外周静脉血,应用流式细胞仪分选Treg 细胞,提取每组患者Treg 细胞中的RNA,采用Solexa 深度测序法对其miRNA 表达谱进行分析。对比ITP 组和正常对照组芯片结果,找出差异表达的miRNA,并对其进行富集性分析,找寻异常信号通路。结果:我们筛查到ITP 患者组Treg 细胞中存在多个miRNA 表达异常,其中有显著差异者为miR-1976, miR-548ae-5p、miR-5096-p3、miR-548am-5p、PC-3p-63471、PC-3p-96627。通过富集性分析发现ErbB 和TGF鄄茁两个异常信号通路。结论:ITP 患者外周血Treg 细胞内miRNA 表达谱存在差异表达,这些差异表达的miRNA 可能影响了Treg 细胞发挥正常的免疫调节功能,是ITP 发生的机制之一。     

Gene expression signature of parathion-transformed human breast epithelial cells

Environmental substances seem to be involved in the etiology of breast cancers. Many studies have found an association between human cancer and exposure to agricultural pesticides such as the organophosphorous pesticides. Parathion is a cholinesterase inhibitor that induces the hydrolysis of body choline esters, including acetylcholine at cholinergic synapses. The primary target of action in insects is the nervous system whereby pesticides inhibit the release of the enzyme acetylcholinesterase at the synaptic junction. Atropine is a parasympatholytic alkaloid used as an antidote to acetylcholinesterase inhibitors. The aim of this study was to determine the effect of parathion and atropine on cell transformation of human breast epithelial cells in vitro. These studies showed that parathion alone was able to induce malignant transformation of an immortalized human breast epithelial cell line, MCF-10F as indicated by increased cell proliferation, anchorage independency and invasive capabilities. There was also an increase in c-kit, Trio, Rho-A, Rac-3, EGFR, Notch-4, Dvl-2, Ezrin, beta catenin and mutant p53 protein expression in the parathion-treated cells. However, atropine significantly inhibited this increase. In a human cell cycle array of 96 genes, 13 of them were altered by parathion treatment. Among the genes affected were the cyclins, such as cyclin D3, the cyclin-dependent kinases (CDKs) such as CDK41 and the minichromosome maintenance deficient (MCM) MCM2 and MCM3. It is suggested that parathion influences human breast epithelial cell transformation and is an initiator factor in the transformation process in breast cancer.     

肝癌耐药细胞的形态学及微小RNA表达谱分析

目的 比较3株肝癌耐药细胞(Huh-7/ADM、Huh-7/CBP、Huh-7/MMC)与亲本肝癌细胞Huh-7的形态学差异;分析这3株肝癌耐药细胞及Huh-7的miRNA表达谱,筛选3株耐药细胞中异常表达的微小RNA(miRNA).方法 光学显微镜下观察各细胞株细胞形态学特点、透射电镜下观察各细胞株细胞内超微结构特点.miRNA芯片检测各细胞株miRNA表达谱,real-time定量PCR(qPCR)法验证芯片结果.结果 镜下见肝癌耐药细胞比亲本肝癌细胞Huh-7更具多形性,核异型性亦增加;细胞周边微绒毛减少;胞质增多,胞质内细胞器含量增加,并出现脂滴、糖原等成分.miRNA表达谱分析显示:与Huh-7相比,有32个miRNA在3种肝癌耐药细胞株中表达同时上调,有22个miRNA在3种耐药细胞株中表达同时下调.real-time qPCR检测证实miR-15a、miR-16、miR-27b、miR-30b、miR-146a、miR-146b-5p、miR-181a、miR-181d、miR-194在各肝癌耐药细胞中表达同时上调.结论 耐药细胞异型性增加,筛选出的肝癌耐药细胞中异常表达miRNA,可为研究肝癌多药耐药与miRNA的相关性提供依据.     

LaCrosse virus gene expression in mammalian and mosquito cells

LaCrosse virus infection of mammalian BHK cells is highly cytopathic, whereas that of mosquito C6/36 cells is asymptomatic and persistent. When the individual mRNAs and their genome segments are followed in parallel infections, cytopathic effects were found to correlate with the rate of synthesis, but not the accumulation, of the viral RNAs. The change from the acute to the persistent phase of the infection in C6/36 cells was found to take place at 24 hr p.i., at which time genome and N protein synthesis was severely reduced, even though mRNA levels remained high. When the persistent infection was followed for 72 days, the total amounts of genomes and their relative proportions were found to fluctuate greatly, whereas mRNA levels were either severely reduced or undetectable. DI genomes could not be detected during this time. The self-limiting nature of the mosquito cell infection appears to be due the translational control of N protein synthesis.     

人成纤维细胞生长因子13基因在酵母细胞中的表达

目的：用酵母细胞表达重组人成纤维细胞生长因子（ｈＦＧＦ１３）蛋白并检测其活性。方法：通过ＲＴ－ＰＣＲ从流产胎儿脑组织中钓取ｈＦＧＦ１３ｃＤＮＡ,将其克隆入ｐＹＥＸ４Ｔ－１载体质粒。在酏母细胞表达ＧＳＴ＿ｈＦＧＦ１３融合蛋白。以细胞增殖实验测定酵母表达蛋白粗提物的活性。结果：ＧＳＴ－ｈＦＧＦ１３融合刷拓酵发母细胞中得到表达;ＧＳＴ－ｈＦＧＦ１３融合蛋白细胞ＮＩＨ３Ｔ３细胞的增殖,而且此活性能够被ＦＧ     

Essential and overlapping functions for mammalian Argonautes in microRNA silencing

MicroRNA (miRNA) silencing fine-tunes protein output and regulates diverse biological processes. Argonaute (Ago) proteins are the core effectors of the miRNA pathway. In lower organisms, multiple Agos have evolved specialized functions for distinct RNA silencing pathways. However, the roles of mammalian Agos have not been well characterized. Here we show that mouse embryonic stem (ES) cells deficient for Ago1–4 are completely defective in miRNA silencing and undergo apoptosis. In miRNA silencing–defective ES cells, the proapoptotic protein Bim, a miRNA target, is increased, and up-regulation of Bim is sufficient to induce ES cell apoptosis. Expression of activated Akt inhibits Bim expression and partially rescues the growth defect in Ago-deficient ES cells. Furthermore, reintroduction of any single Ago into Ago-deficient cells is able to rescue the endogenous miRNA silencing defect and apoptosis. Consistent with this, each Ago is functionally equivalent with bulged miRNA duplexes for translational repression, whereas Ago1 and Ago2 appear to be more effective at utilizing perfectly matched siRNAs. Thus, our results demonstrate that mammalian Agos all contribute to miRNA silencing, and individual Agos have largely overlapping functions in this process.     

An HDAC inhibitor increases AcMNPV gene expression in mammalian cells

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is used as a safer viral vector in mammalian cells with potential applications in gene therapy. However, the mechanism for the insusceptibility of mammalian cells to proliferative infection by entomopathogenic viruses is not well understood. Here, we studied the significance of epigenetic modifications such as histone acetylation, histone methylation and HP1 accumulation for AcMNPV gene expression in mammalian BHK cells. Real-time PCR and chromatin immunoprecipitation with sodium butyrate revealed an important relationship between viral gene expression and histone acetylation, with implications for a mechanism of suppression of AcMNPV gene expression in BHK cells.