Identification of lncRNA–miRNA–mRNA regulatory network associated with epithelial ovarian cancer cisplatin-resistant

To construct a long noncoding RNA (lncRNA)–microRNA (miRNA)–messenger RNA (mRNA) regulatory network related to epithelial ovarian cancer (EOC) cisplatin-resistant, differentially expressed genes (DEGs), differentially expressed lncRNAs (DELs), and differentially expressed miRNAs (DEMs) between MDAH and TOV-112D cells lines were identified. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted to analyze the biological functions of DEGs. Downstream mRNAs or upstream lncRNAs for miRNAs were analyzed at miRTarBase 7.0 or DIANA-LncBase V2, respectively. A total of 485 significant DEGs, 85 DELs, and 5 DEMs were identified. Protein–protein interaction (PPI) network of DEGs contrains 81 nodes and 141 edges was constructed, and 25 hub genes related to EOC cisplatin-resistant were identified. Subsequently, a lncRNA–miRNA–mRNA regulatory network contains 4 lncRNAs, 4 miRNAs, and 35 mRNAs was established. Taken together, our study provided evidence concerning the alteration genes involved in EOC cisplatin-resistant, which will help to unravel the mechanisms underlying drug resistant.     

Bioinformatic analysis and validation of microRNA-508-3p as a protective predictor by targeting NR4A3/MEK axis in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) featured a debilitating progressive disorder. Here, we intend to determine diagnosis-valuable biomarkers for PAH and decode the fundamental mechanisms of the biological function of these markers. Two mRNA microarray profiles (GSE70456 and GSE117261) and two microRNA microarray profiles (GSE55427 and GSE67597) were mined from the Gene Expression Omnibus platform. Then, we identified the differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs), respectively. Besides, we investigated online miRNA prediction tools to screen the target gene of DEMs. In this study, 185 DEGs and three common DEMs were screened as well as 1266 target genes of the three DEMs were identified. Next, 16 overlapping dysregulated genes from 185 DEGs and 1266 target gene were obtained. Meanwhile, we constructed the miRNA gene regulatory network and determined miRNA-508-3p-NR4A3 pair for deeper exploring. Experiment methods verified the functional expression of miR-508-3p in PAH and its signalling cascade. We observed that ectopic miR-508-3p expression promotes proliferation and migration of pulmonary artery smooth muscle cell (PASMC). Bioinformatic, dual-luciferase assay showed NR4A3 represents directly targeted gene of miR-508-3p. Mechanistically, we demonstrated that down-regulation of miR-508-3p advances PASMC proliferation and migration via inducing NR4A3 to activate MAPK/ERK kinase signalling pathway. Altogether, our research provides a promising diagnosis of predictor and therapeutic avenues for patients in PAH.     

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.     

高通量测序技术分析急性髓系白血病血浆外泌体微RNA表达谱差异

急性髓系白血病（acute myeloid leukemia,AML）是一类造血干细胞的恶性克隆性疾病,目前的诊断方法不利于疾病的早期发现,且诊断结果重复性较差。已有大量研究显示,细胞外microRNA（miRNA或miR）富集在外泌体（exosome）中,且受其表面膜的保护而具有很好的稳定性,是理想的分子标志物。目前,多种实体肿瘤均已检测到肿瘤特异性外泌体miRNA(exosomal miRNA)。然而,在AML患者中未见此外泌体miRNA报道。本研究探讨急性髓系白血病血浆外泌体miRNA表达谱差异及新miRNA序列。采用solexa高通量测序技术对7例AML患者（AML组）及7例健康对照者（对照组）血浆外泌体miRNAs进行测序,利用Mireap预测软件进行新miRNAs分析,通过edger差异分析软件筛选组间差异miRNA,获得211个已知的差异表达miRNAs以及2个新miRNAs,选择4个差异表达的miRNAs：miR-155-5p、miR-335-5p、miR-451a及xxx-m0038 5p（新miRNA）,在两组（各23例）的血浆外泌体样本中,进行实时荧光定量PCR（qRT-PCR）验证,验证结果与测序结果一致。对差异表达的外泌体miRNA进行靶基因预测及其GO（Gene Ontology）和信号通路富集分析,发现靶基因聚集的生物学功能多数参与生物进展过程的调控。靶基因主要富集在FoxO、MAPK、Hippo信号通路以及HTLV-I感染等。结果显示,AML患者与健康对照者的血浆外泌体miRNA存在着差异性表达。差异性表达的miRNA特异性很高,对进一步阐明AML白血病发生与发展的分子机制、研发新的无创诊断方法、新的诊断标记物和有效治疗AML的方法具有十分重要和深远的意义。     

MicroRNA expression profile in RAW264.7 cells in response to Brucella melitensis infection

MicroRNA (miRNA) is small non-coding RNA with approximate 22 nt in length. Recent studies indicate that miRNAs play significant roles in pathogen-host interactions. Brucella organisms are Gram-negative facultative intracellular bacteria that cause Brucellosis. Brucella strains infect macrophages and establish chronic infection by altering host life activities including apoptosis and autophagy. Here, we report a comprehensive analysis of miRNA expression profiles in mock- and Brucella-infected RAW264.7 cells using high-throughput sequencing approach. In total, 344 unique miRNAs were co-expressed in the two libraries, in which 57 miRNAs were differentially expressed. Eight differentially expressed miRNAs with high abundance were subjected to further analysis. The GO enrichment analysis suggests that the putative target genes of these differentially expressed miRNAs are involved in apoptosis, autophagy and immune response. In particular, a total of 25 target genes are involved in regulating apoptosis and autophagy, indicating that these miRNAs may play important regulatory roles in the Brucella-host interactions. Furthermore, the interactions of miR-1981 and its target genes, Bcl-2 and Bid, were validated by luciferase assay. The results show that miR-1981 mimic up-regulated the luciferase activity of psiCHECK-2 Bcl-2 3' UTR, but the luciferase activity of psiCHECK-2 Bid 3' UTR was not changed significantly. Taken together, these data provide valuable framework on Brucella induced miRNA expression in RAW264.7 cells, and suggest that Brucella may establish chronic infection by regulating miRNA expression profile.     

Comparative profile of exosomal microRNAs in postmenopausal women with various bone mineral densities by small RNA sequencing

To explore the role of plasma miRNAs in exosomes in early postmenopausal women. Small RNA sequencing was implemented to clarify the expression of miRNA in plasma exosomes obtained from 15 postmenopausal women, divided into groups of osteoporosis, osteopenia, and normal bone mass based on bone mineral density. Differentially expressed miRNAs (DEMs) were identified by comparing miRNA expression profiles. Five putative miRNAs, miR-224-3p, miR-25-5p, miR-302a-3p, miR-642a-3p, and miR-766-5p were confirmed by real-time PCR; miRNA target genes were obtained from 4 databases: miRWalk, miRDB, RNA22, and TargetScan. The miRNA-mRNA- Kyoto Encyclopedia of Genes and Genomes (KEGG) networks were analyzed, and the DEMs' potential role was investigated by gene ontology terms and KEGG pathway annotation. The results suggest that characterizing plasma exosomal miRNA profiles of early postmenopausal women by small RNA sequencing could identify novel exo-miRNAs involved in bone remodeling, and miR-642a-3p maybe contribute to the prediction and diagnosis of early postmenopausal osteoporosis.     

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。     

Comparison of microRNAs in adipose and muscle tissue from seven indigenous Chinese breeds and Yorkshire pigs

Elucidation of the pig microRNAome is essential for interpreting functional elements of the genome and understanding the genetic architecture of complex traits. Here, we extracted small RNAs from skeletal muscle and adipose tissue, and we compared their expression levels between one Western breed (Yorkshire) and seven indigenous Chinese breeds. We detected the expression of 172 known porcine microRNAs (miRNAs) and 181 novel miRNAs. Differential expression analysis found 92 and 12 differentially expressed miRNAs in adipose and muscle tissue respectively. We found that different Chinese breeds shared common directional miRNA expression changes compared to Yorkshire pigs. Some miRNAs differentially expressed across multiple Chinese breeds, including ssc‐miR‐129‐5p, ssc‐miR‐30 and ssc‐miR‐150, are involved in adipose tissue function. Functional enrichment analysis revealed that the target genes of the differentially expressed miRNAs are associated mainly with signaling pathways rather than metabolic and biosynthetic processes. The miRNA–target gene and miRNA–phenotypic traits networks identified many hub miRNAs that regulate a large number of target genes or phenotypic traits. Specifically, we found that intramuscular fat content is regulated by the greatest number of miRNAs in muscle tissue. This study provides valuable new candidate miRNAs that will aid in the improvement of meat quality and production.     

Comprehensive analysis of lncRNA–miRNA–mRNA during proliferative phase of rat liver regeneration

This study aims to reveal the regulatory mechanism of lncRNAs–miRNAs–mRNAs network during the proliferative phase of liver regeneration (LR). High-throughput sequencing technology was performed, and a total of 1,738 differentially expressed lncRNAs (DE lncRNAs), 167 known differentially expressed miRNAs (DE miRNAs), and 2,727 differentially expressed mRNAs were identified. Then, the target DE lncRNAs and DE mRNAs regulated by the same miRNAs were screened and a ceRNA regulatory network containing 32 miRNAs, 107 lncRNAs, and 270 mRNAs was constructed. Insulin signaling pathway, pyrimidine metabolism, axon guidance, carbohydrate digestion and absorption, and pyruvate metabolism were significantly enriched in the network. Through literature review and the regulatory relationship between lncRNAs and miRNAs, nine core lncRNAs were identified, which might play important roles during the proliferative phase of rat LR. This study analyzed lncRNA–miRNA–mRNA regulatory network for the first time during the proliferative phase of rat LR, providing clues for exploring the mechanism of LR and the treatment of liver diseases.     

Parallel mRNA and MicroRNA Profiling of HEV71-Infected Human Neuroblastoma Cells Reveal the Up-Regulation of miR-1246 in Association with DLG3 Repression

Human enterovirus 71 (HEV71) has emerged as the leading cause of viral encephalitis in children in most Asian countries. The roles of host miRNAs in the neurological pathogenesis of HEV71 infection remain unknown. In the present study, comprehensive miRNA expression profiling in HEV71-infected human neuroblastoma SH-SY5Y cells was performed using the Affymetrix Gene Chip microarray assay and was validated using real-time RT-PCR. Among the 69 differentially expressed miRNAs, miR-1246 was specifically induced by HEV71 infection in human neuroblastoma cells, but inhibition of miR-1246 failed to affect HEV71 replication. Parallel mRNA and microRNA profiling based on the 35 K Human Genome Array identified 182 differentially regulated genes. Target prediction of miR-1246 and network modeling revealed 14 potential target genes involved in cell death and cell signaling. Finally, a combined analysis of the results from mRNA profiling and miR-1246 target predication led to the identification of disc-large homolog 3 (DLG3), which is associated with neurological disorders, for further validation. Sequence alignment and luciferase reporter assay showed that miR-1246 directly bound with the 3′-UTR of DLG3 gene. Down-regulation of miR-1246 induced significant changes in DLG3 expression levels in HEV71-infected SHSY5Y cells. Together, these results suggested that miR-1246 might play a role in neurological pathogenesis of HEV71 by regulating DLG3 gene in infected cells. These findings provide new information on the miRNA and mRNA profiles of HEV71-infected neuroblastoma cells. The biological significance of miR-1246 and DLG3 during the course of HEV71 infection deserves further investigation.     

Integrated analysis of microRNA expression and mRNA transcriptome in lungs of avian influenza virus infected broilers

ABSTRACT: BACKGROUND: Avian influenza virus (AIV) outbreaks are worldwide threats to both poultry and humans. Our previous study suggested microRNAs (miRNAs) play significant roles in the regulation of host response to AIV infection in layer chickens. The objective of this study was to test the hypothesis if genetic background play essential role in the miRNA regulation of AIV infection in chickens and if miRNAs that were differentially expressed in layer with AIV infection would be modulated the same way in broiler chickens. Furthermore, by integrating with parallel mRNA expression profiling, potential molecular mechanisms of host response to AIV infection can be further exploited. RESULTS: Total RNA isolated from the lungs of non-infected and low pathogenic H5N3 infected broilers at four days post-infection were used for both miRNA deep sequencing and mRNA microarray analyses. A total of 2.6M and 3.3M filtered high quality reads were obtained from infected and non-infected chickens by Solexa GA-I Sequencer, respectively. A total of 271 miRNAs in miRBase 16.0 were identified and one potential novel miRNA was discovered. There were 121 miRNAs differentially expressed at the 5% false discovery rate by Fisher's exact test. More miRNAs were highly expressed in infected lungs (108) than in non-infected lungs (13), which was opposite to the findings in layer chickens. This result suggested that a different regulatory mechanism of host response to AIV infection mediated by miRNAs might exist in broiler chickens. Analysis using the chicken 44K Agilent microarray indicated that 508 mRNAs (347 down-regulated) were differentially expressed following AIV infection. CONCLUSION: A comprehensive analysis combining both miRNA and targeted mRNA gene expression suggests that gga-miR-34a, 122-1, 122-2, 146a, 155, 206, 1719, 1594, 1599 and 451, and MX1, IL-8, IRF-7, TNFRS19 are strong candidate miRNAs or genes involved in regulating the host response to AIV infection in the lungs of broiler chickens. Further miRNA or gene specific knock-down assay is warranted to elucidate underlying mechanism of AIV infection regulation in the chicken.