大熊猫脐带间充质干细胞外泌体的分离鉴定及miRNAs富集分析

大熊猫脐带间充质干细胞(Umbilical cord mesenchymal stem cells, UC-MSCs)通过旁分泌所释放的外泌体在大熊猫保健与疾病治疗方面具有一定的应用前景。本研究旨在建立大熊猫UC-MSCs外泌体分离方法,开展生物学特征分析和分子鉴定,并研究UC-MSCs外泌体中miRNAs的种类与功能。采用超速离心法从大熊猫UCMSCs培养上清中成功分离外泌体,通过透射电子显微镜进行形态学观察,纳米颗粒跟踪分析仪检测粒径大小,蛋白免疫印迹法检测特异性分子标记表达。采用Small RNA测序技术对UC-MSCs外泌体中的miRNAs进行鉴定,并对其靶基因进行了预测与功能分析。结果显示,大熊猫UC-MSCs外泌体呈圆形杯托状结构,直径为(79.15±4.81) nm,外泌体标志蛋白CD81与TSG101呈阳性表达而CALNEXIN呈阴性表达。大熊猫UC-MSCs外泌体中的miRNA主要为miR-148-3p (30.28%)与miR-21-5p (21.72%)。本研究首次从大熊猫UC-MSCs培养上清中分离出外泌体,并对其所含的miRNAs进行富集分析及功能预测,为大熊...     

Epigenomic profiling indicates a role for DNA methylation in the postnatal liver and pancreas development of giant pandas

Giant pandas are unique within Carnivora with a strict bamboo diet. Here, the epigenomic profiles of giant panda liver and pancreas tissues collected from three important feeding stages were investigated using BS-seq. Few differences in DNA methylation profiles were exhibited between no feeding and suckling groups in both tissues. However, we observed a tendency toward a global loss of DNA methylation in the gene-body and promoter region of metabolism-related genes from newborn to adult. Correlation analysis revealed a significant negative correlation between the changes in methylation levels within gene promoters and gene expression. The majority of genes related to nutrition metabolism had lost DNA methylation with increased mRNA expression in adult giant pandas. The few galactose metabolism and unsaturated fatty acid metabolism related genes that were hypomethylated and highly-expressed at early stages of giant panda development may meet the nutritional requirement of this species' highly altricial neonates.     

n-3多不饱和脂肪酸调节饮食诱导肥胖大鼠miRNA表达变化(英文)

目的:研究n-3多不饱和脂肪酸(polyunsaturated fatty acids,PUFA)饮食对饮食诱导肥胖大鼠的miR NA表达影响。方法:将10只饮食诱导肥胖(diet induced obese,DIO)大鼠随机分成两组:n-3PUFA添加组和安慰剂添加组(对照组);每周记录两组老鼠的体重、体长和进食量。对外周血miR NA的表达并进行分析和预测。结果:两组老鼠Lee指数有统计学差异(P0.05);与对照组相比,在n-3组的外周血单核细胞中,29个miR NA上调,31个下调;其中rno-miR-200和rno-miR-211的表达量上调,rno-miR-29b和rno-miR-92b的表达量下调,其靶基因预测结果与神经营养因子,脂肪细胞因子,趋化因子和胰岛素信号通路有关。结论:n-3PUFA能够调节DIO大鼠的miR NA水平,其中有些与脂肪代谢相关。     

Integrated analysis of miRNA and mRNA expression in childhood medulloblastoma compared with neural stem cells

Medulloblastoma (MB) is the most common malignant brain tumor in children and a leading cause of cancer-related mortality and morbidity. Several molecular sub-types of MB have been identified, suggesting they may arise from distinct cells of origin. Data from animal models indicate that some MB sub-types arise from multipotent cerebellar neural stem cells (NSCs). Hence, microRNA (miRNA) expression profiles of primary MB samples were compared to CD133+ NSCs, aiming to identify deregulated miRNAs involved in MB pathogenesis. Expression profiling of 662 miRNAs in primary MB specimens, MB cell lines, and human CD133+ NSCs and CD133- neural progenitor cells was performed by qRT-PCR. Clustering analysis identified two distinct sub-types of MB primary specimens, reminiscent of sub-types obtained from their mRNA profiles. 21 significantly up-regulated and 12 significantly down-regulated miRNAs were identified in MB primary specimens relative to CD133+ NSCs (p<0.01). The majority of up-regulated miRNAs mapped to chromosomal regions 14q32 and 17q. Integration of the predicted targets of deregulated miRNAs with mRNA expression data from the same specimens revealed enrichment of pathways regulating neuronal migration, nervous system development and cell proliferation. Transient over-expression of a down-regulated miRNA, miR-935, resulted in significant down-regulation of three of the seven predicted miR-935 target genes at the mRNA level in a MB cell line, confirming the validity of this approach. This study represents the first integrated analysis of MB miRNA and mRNA expression profiles and is the first to compare MB miRNA expression profiles to those of CD133+ NSCs. We identified several differentially expressed miRNAs that potentially target networks of genes and signaling pathways that may be involved in the transformation of normal NSCs to brain tumor stem cells. Based on this integrative approach, our data provide an important platform for future investigations aimed at characterizing the role of specific miRNAs in MB pathogenesis.     

Integrated analysis of miRNA and mRNA expression profiles in response to gut microbiota depletion in the abdomens of female Bactrocera dorsalis

Insect gut microbiota has been reported to participate in regulating host multiple biological processes including metabolism and reproduction. However, the corresponding molecular mechanisms remain largely unknown. Recent studies suggest that microRNAs (miRNAs) are involved in complex interactions between the gut microbiota and the host. Here, we used next-generation sequencing technology to characterize miRNA and mRNA expression profiles and construct the miRNA–gene regulatory network in response to gut microbiota depletion in the abdomens of female Bactrocera dorsalis. A total of 3016 differentially expressed genes (DEGs) and 18 differentially expressed miRNAs (DEMs) were identified. Based on the integrated analysis of miRNA and mRNA sequencing data, 229 negatively correlated miRNA–gene pairs were identified from the miRNA–mRNA network. Gene ontology enrichment analysis indicated that DEMs could target several genes involved in the metabolic process, oxidation–reduction process, oogenesis, and insulin signaling pathway. Finally, real-time quantitative polymerase chain reaction further verified the accuracy of RNA sequencing results. In conclusion, our study provides the profiles of miRNA and mRNA expressions under antibiotics treatment and provides an insight into the roles of miRNAs and their target genes in the interaction between the gut microbiota and its host.     

Differential expression of microRNAs in decidua-derived mesenchymal stem cells from patients with pre-eclampsia

Background

Mesenchymal stem cells (MSCs) at maternal-fetal interface are considered to play an important role in the pathogenesis of pre-eclampsia (PE). microRNAs (miRNAs) also have an important influence on differentiation, maturation, and functions of MSCs. Our aim in this study was to determine the differential expression of miRNAs in decidua-derived MSCs (dMSCs) from severe PE and normal pregnancies.

Results

miRNA expression profiles in dMSCs from five patients with severe PE and five healthy pregnant women were screened using microarray. Then, bioinformatic analysis of the microarray results was performed. Out of 179 differentially expressed miRNAs, 49 miRNAs had significant (p < 0.05) differential expression of ≥ 2.0-fold changes, including 21 up-regulated and 28 down-regulated. miRNA-Gene-network and miRNA-Gene ontology (GO) -network analyses were performed. Overall, 21 up-regulated and 15 down-regulated miRNAs showed high degrees in these analyses. Moreover, the significantly enriched signaling pathways and GOs were identified. The analyses revealed that pathways associated with cell proliferation, angiogenesis, and immune functions were highly regulated by the differentially expressed miRNAs, including Wnt signaling pathway, mitogen-activated protein kinase signaling pathway, transforming growth factor beta signaling pathway, T-cell receptor signaling pathway, and B cell receptor signaling pathway. Four miRNA predicted target genes, vascular endothelial growth factor A (VEGFA), indoleamine 2,3-dioxygenase, suppression of cytokine signaling 3, and serine/threonine protein phosphatase 2A 55 kDa regulatory subunit B α isoform (PPP2R2A) were all decreased in dMSCs from patients with PE. Furthermore, the physiological roles of miR-16 and miR-136 in the down-regulation of VEGFA and PPP2R2A, respectively, were confirmed through reporter assays.

Conclusions

These findings suggest that miRNAs in dMSCs may be important regulatory molecules in the development of PE.     

Integrated miRNA-mRNA Analysis Revealing the Potential Roles of miRNAs in Chordomas

Introduction

Emerging evidence suggests that microRNAs (miRNAs) are crucially involved in tumorigenesis and that paired expression profiles of miRNAs and mRNAs can be used to identify functional miRNA-target relationships with high precision. However, no studies have applied integrated analysis to miRNA and mRNA profiles in chordomas. The purpose of this study was to provide insights into the pathogenesis of chordomas by using this integrated analysis method.

Methods

Differentially expressed miRNAs and mRNAs of chordomas (n = 3) and notochord tissues (n = 3) were analyzed by using microarrays with hierarchical clustering analysis. Subsequently, the target genes of the differentially expressed miRNAs were predicted and overlapped with the differentially expressed mRNAs. Then, GO and pathway analyses were performed for the intersecting genes.

Results

The microarray analysis indicated that 33 miRNAs and 2,791 mRNAs were significantly dysregulated between the two groups. Among the 2,791 mRNAs, 911 overlapped with putative miRNA target genes. A pathway analysis showed that the MAPK pathway was consistently enriched in the chordoma tissue and that miR-149-3p, miR-663a, miR-1908, miR-2861 and miR-3185 likely play important roles in the regulation of MAPK pathways. Furthermore, the Notch signaling pathway and the loss of the calcification or ossification capacity of the notochord may also be involved in chordoma pathogenesis.

Conclusion

This study provides an integrated dataset of the miRNA and mRNA profiles in chordomas, and the results demonstrate that not only the MAPK pathway and its related miRNAs but also the Notch pathway may be involved in chordoma development. The occurrence of chordoma may be associated with dysfunctional calcification or ossification of the notochord.     

Comprehensive miRNome and in silico analyses identify the Wnt signaling pathway to be altered in the diabetic liver

Aberrant microRNA expression patterns underlie the pathogenesis of diverse diseases, however in a disease as complex as diabetes where the liver exhibits deregulations of normal metabolic processes, the status and role of microRNAs are not yet completely understood. In a step towards unraveling this correlation, we assessed the global microRNA expression profiles in the control and diabetic (db/db) mice liver. These db/db mice were on a C57BLKS/J background and they exhibit diabetic phenotypes that are remarkably similar to those in humans. microRNA microarray profiling revealed 11 miRNAs to be up-regulated and 2 to be down-regulated in the db/db mice liver. Predicted targets of these differentially expressed microRNAs were retrieved from miRanda and TargetScan and the maximum number of commonly predicted targets mapped onto the Wnt signaling pathway that is otherwise conventionally associated with organogenesis and development. Towards validation of this prediction, we found that major components of the Wnt signaling pathway are inhibited in the db/db mice liver. A significant number of these down-regulated genes of the Wnt signaling pathway are predicted targets to the up-regulated miRNAs and specifically our results show that miR-34a and miR-22 decreased the protein levels of their targets. Overexpression of miR-34a and miR-22 and also inhibition of Wnt signaling using specific inhibitors led to increased lipid accumulation in HepG2 cells. Our data suggest that the Wnt signaling pathway could contribute towards the deregulated hepatic behavior in these animals and an altered hepatic miRNA signature could be playing a regulatory role herein.     

Infection with street strain rabies virus induces modulation of the microRNA profile of the mouse brain

ABSTRACT: BACKGROUND: Rabies virus (RABV) causes a fatal infection of the central nervous systems (CNS) of warm-blooded animals. Once the clinical symptoms develop, rabies is almost invariably fatal. The mechanism of RABV pathogenesis remains poorly understood. Recent studies have shown that microRNA (miRNA) plays an important role in the pathogenesis of viral infections. Our recent findings have revealed that infection with laboratory-fixed rabies virus strain can induce modulation of the microRNA profile of mouse brains. However, no previous report has evaluated the miRNA expression profile of mouse brains infected with RABV street strain. RESULTS: The results of microarray analysis show that miRNA expression becomes modulated in the brains of mice infected with street RABV. Quantitative real-time PCR assay of the differentially expressed miRNAs confirmed the results of microarray assay. Functional analysis showed the differentially expressed miRNAs to be involved in many immune-related signaling pathways, such as the Jak-STAT signaling pathway, the MAPK signaling pathway, cytokine-cytokine receptor interactions, and Fc gamma R-mediated phagocytosis. The predicted expression levels of the target genes of these modulated miRNAs were found to be correlated with gene expression as measured by DNA microarray and qRT-PCR. CONCLUSION: RABV causes significant changes in the miRNA expression profiles of infected mouse brains. Predicted target genes of the differentially expression miRNAs are associated with host immune response, which may provide important information for investigation of RABV pathogenesis and therapeutic method.     

Expression profiles of microRNAs in oxidized low-density lipoprotein-stimulated RAW 264.7 cells

Macrophage-derived foam cells were one of the hallmarks of atherosclerosis, and microRNAs played an important role in the formation of foam cells. In order to explore the roles of miRNA in the formation of foam cells, we investigated miRNA expression profiles in foam cells through high-throughput sequencing technology. A total of 84 miRNAs were differentially expressed between RAW 264.7 macrophages and foam cells induced by ox-LDL. Thirty miRNAs were upregulated and 54 miRNAs were downregulated. GO terms and KEGG pathways analysis revealed that the target genes of most of DE miRNAs were mainly enriched in “cell differentiation,” “endocytosis,” “MAPK signaling pathway,” and “FoxO signaling pathway.” The target genes of some DE miRNAs were enriched in “Insulin signaling pathway,” “Hippo signaling pathway,” “TNF signaling pathway,” “NF-kappa B signaling pathway,” and “cell death.” Using bioinformatics analyses and dual-luciferase reporter assays, we found that miR-28a-5p and miR-30c-1-3p directly inhibited LRAD3 and LOX-1 mRNA expression through targeting the 3’UTR of LRAD3 and LOX-1 mRNA, respectively. Our study indicates that miRNAs are extensively involved in the formation of foam cells, and provides a valuable resource for further study the role of miRNAs in atherosclerosis.     

Coordinated miRNA/mRNA expression profiles for understanding breed-specific metabolic characters of liver between Erhualian and large white pigs

MicroRNAs (miRNAs) are involved in the regulation of various metabolic processes in the liver, yet little is known on the breed-specific expression profiles of miRNAs in coordination with those of mRNAs. Here we used two breeds of male newborn piglets with distinct metabolic characteristics, Large White (LW) and Erhualian (EHL), to delineate the hepatic expression profiles of mRNA with microarray and miRNAs with both deep sequencing and microarray, and to analyze the functional relevance of integrated miRNA and mRNA expression in relation to the physiological and biochemical parameters. EHL had significantly lower body weight and liver weight at birth, but showed elevated serum levels of total cholesterol (TCH), high-density lipoprotein cholesterol (HDLC) and low-density lipoprotein cholesterol (LDLC), as well as higher liver content of cholesterol. Higher serum cortisol and lower serum insulin and leptin were also observed in EHL piglets. Compared to LW, 30 up-regulated and 18 down-regulated miRNAs were identified in the liver of EHL, together with 298 up-regulated and 510 down-regulated mRNAs (FDR<10%). RT-PCR validation of some differentially expressed miRNAs (DEMs) further confirmed the high-throughput data analysis. Using a target prediction algorithm, we found significant correlation between the up-regulated miRNAs and down-regulated mRNAs. Moreover, differentially expressed genes (DEGs), which are involved in proteolysis, were predicted to be mediated by DEMs. These findings provide new information on the miRNA and mRNA profiles in porcine liver, which would shed light on the molecular mechanisms underlying the breed-specific traits in the pig, and may serve as a basis for further investigation into the biological functions of miRNAs in porcine liver.     

家蚕5龄幼虫精巢和卵巢microRNA芯片及转录组比较分析

【目的】本研究旨在通过对家蚕Bombyx mori 5龄幼虫精巢和卵巢组织微小RNA (microRNA, miRNA)基因芯片及转录组进行分析,找到参与家蚕性腺发育相关的miRNA分子及可能的靶基因。【方法】采用新一代高通量测序平台对家蚕5龄幼虫精巢和卵巢(分别定义为Test和Control)进行miRNA基因芯片检测及转录组测序分析,根据P＜0.05且log₂(fold change, FC)≥2的标准,通过比较筛选出Test vs Control的差异表达miRNA;根据q≤0.05且|log₂(fold change)|≥1的标准,通过比较筛选出Test vs Control的差异表达基因 (differentially expressed genes, DEGs);随机选取8个上调和12个下调差异表达miRNA,对其表达及其预测的5个靶基因进行qRT-PCR验证;对DEGs以及差异表达miRNA的靶基因进行KEGG通路富集分析。【结果】从精巢和卵巢样本中(Test vs Control)分别鉴定出68个差异表达miRNA和3 991个DEGs,其中上调和下调miRNA分别为36和32个,上调和下调DEGs分别为2 033和1 958个。差异表达miRNA的qRT PCR验证结果均与芯片数据一致。KEGG通路富集分析结果显示DEGs在新陈代谢及核糖体的信号通路显著富集。对差异表达miRNA在DEGs中的可能靶基因进行预测,结果找到了4组表达趋势相反的miRNA与靶基因：分别是bmo-miR-2774a与LOC101745556;bmo-miR-92b与LOC101735954以及bmo-miR-3266与LOC733130和LOC778467;1组表达趋势一致的miRNA与靶基因：bmo-miR-3321与LOC101744895。5个靶基因的qRT-PCR验证结果与转录组测序结果一致。【结论】本研究获得了家蚕5龄幼虫精巢和卵巢转录组及miRNA芯片数据,筛选并验证了4组差异表达和1组一致表达miRNA及潜在靶基因,为探究家蚕精巢和卵巢发育差异奠定了基础。     

A densely interconnected genome-wide network of microRNAs and oncogenic pathways revealed using gene expression signatures

MicroRNAs (miRNAs) are important components of cellular signaling pathways, acting either as pathway regulators or pathway targets. Currently, only a limited number of miRNAs have been functionally linked to specific signaling pathways. Here, we explored if gene expression signatures could be used to represent miRNA activities and integrated with genomic signatures of oncogenic pathway activity to identify connections between miRNAs and oncogenic pathways on a high-throughput, genome-wide scale. Mapping >300 gene expression signatures to >700 primary tumor profiles, we constructed a genome-wide miRNA-pathway network predicting the associations of 276 human miRNAs to 26 oncogenic pathways. The miRNA-pathway network confirmed a host of previously reported miRNA/pathway associations and uncovered several novel associations that were subsequently experimentally validated. Globally, the miRNA-pathway network demonstrates a small-world, but not scale-free, organization characterized by multiple distinct, tightly knit modules each exhibiting a high density of connections. However, unlike genetic or metabolic networks typified by only a few highly connected nodes ("hubs"), most nodes in the miRNA-pathway network are highly connected. Sequence-based computational analysis confirmed that highly-interconnected miRNAs are likely to be regulated by common pathways to target similar sets of downstream genes, suggesting a pervasive and high level of functional redundancy among coexpressed miRNAs. We conclude that gene expression signatures can be used as surrogates of miRNA activity. Our strategy facilitates the task of discovering novel miRNA-pathway connections, since gene expression data for multiple normal and disease conditions are abundantly available.     

感染GCRV的草鱼miRNA测序与比较分析

为探索基于高通量筛选抗性主效miRNA功能分子的方法应用于抗性草鱼(Ctenopharyngodon idellus)的选育,研究借助高通量测序鉴定分析感染GCRV前后的草鱼头肾组织中miRNAs.测序结果共鉴定出821个成熟miRNAs,其中118个在GCRV攻毒组特异表达,82个为未攻毒组特有;差异分析结果显示,G...