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.     

The microRNA expression profile in porcine skeletal muscle is changed by constant heat stress

Heat stress has profound effects on animal performance and muscle function, and microRNAs (miRNAs) play a critical role in muscle development and stress responses. To characterize the changes in miRNAs in skeletal muscle responding to heat stress, the miRNA expression profiles of longissimus dorsi muscles of pigs raised under constant heat stress (30 °C; n = 8) or control temperature (22 °C; n = 8) for 21 days were analyzed by Illumina deep sequencing. A total of 58 differentially expressed miRNAs were identified with 30 down‐regulated and 28 up‐regulated, and 63 differentially expressed target genes were predicted by miRNA–mRNA joint analysis. GO and KEGG analyses showed that the genes regulated by differentially expressed miRNAs were enriched in glucose metabolism, cytoskeletal structure and function and stress response. Real‐time PCR showed that the mRNA levels of PDK4, HSP90 and DES were significantly increased, whereas those of SCD and LDHA significantly decreased by heat exposure. The protein levels of CALM1, DES and HIF1α were also significantly increased by constant heat. These results demonstrated that the change in miRNA expression in porcine longissimus dorsi muscle underlies the changes in muscle structure and metabolism in porcine skeletal muscle affected by constant heat stress.     

Identification and bioinformatics analysis of microRNAs associated with stress and immune response in serum of heat-stressed and normal Holstein cows

MicroRNAs (miRNAs) are small single-stranded non-coding RNAs that have an important regulatory function in animal growth and developmental processes. However, the differential expression of miRNA and the role of these miRNAs in heat-stressed Holstein cows are still unknown. In this study, the profile of differentially expressed miRNAs and the target genes analysis in the serum of heat-stressed and normal Holstein cows were investigated by a Solexa deep-sequencing approach and bioinformatics. The data identified 52 differentially expressed miRNAs in 486 known miRNAs which were changed significantly between heat-stressed and normal Holstein cows (fold change >2, P < 0.001). Target genes analysis showed that at least 7 miRNAs (miR-19a, miR-19b, miR-146a, miR-30a-5p, miR-345-3p, miR-199a-3p, and miR-1246) were involved in the response to stress, oxidative stress, development of the immune system, and immune response among the identified 52 differentially expressed miRNAs. Five miRNAs (miR-27b, miR-181a, miR-181b, miR-26a, and miR-146b) were involved in stress and immune responses and the expression of five miRNAs was striking (P < 0.001). In addition, RT-qPCR and deep-sequencing methods showed that 8 miRNAs among the 12 selected miRNAs (miR-19a, miR-19b, miR-27b, miR-30a-5p, miR-181a, miR-181b, miR-345-3p, and miR-1246) were highly expressed in the serum of heat-stressed Holstein cows. GO and KEGG pathway analysis showed that these differentially expressed miRNAs were involved in a pathway that may differentially regulate the expression of stress response and immune response genes. Our study provides an overview of miRNAs expression profile and the interaction between miRNAs and their target genes, which will lead to further understanding of the important roles of miRNAs in heat-stressed Holstein cows.     

Analysis of miRNA-seq combined with gene expression profile reveals the complexity of salinity stress response in <Emphasis Type="Italic">Oryza sativa</Emphasis>

High salinity is a major abiotic stressor that affects crop productivity and quality. While proper seedling growth is critical for crop reproduction under high salinity stress. Nowadays, genes/miRNAs expression is used for studying salinity stress response in rice seedlings. However, analysis of miRNA combined with gene expression is rare. To this end, we used miRNA-seq and gene expression profile to ascertain 6335 genes (3276 genes up-regulated, 3059 genes down-regulated) and 126 miRNAs (47 miRNAs up-regulated, 79 miRNAs down-regulated) that respond to salinity stress in rice seedlings. We then used these 126 miRNAs (including the novel miRNA osa-Chr12_1506) to identify 121 differentially expressed predicted target genes. In addition, we identified 34 miRNA-target RNA pairs, consisting of 9 differentially expressed miRNAs with complementary expression patterns. Combined with previous studies, we proposed a simple model for the molecular mechanism of a 12-h salinity stress response in rice seedlings. The findings lead to a deeper understanding of the function of miRNAs and genes that respond to salinity, and contributed to the elucidation of the complex mechanisms activated by salinity stress.     

Integrating miRNA and mRNA expression profiles in response to heat stress-induced injury in rat small intestine

The molecular mechanisms underlying the pathophysiology of heat stress in the small intestine remain undefined. Furthermore, little information is available concerning changes in microRNA (miRNA) expression following heat stress. The present study sought to evaluate miRNA and mRNA expression profiles in the rat small intestine in response to heat stress. Male Sprague-Dawley rats were subjected to 2?h of heat stress daily for ten consecutive days. Rats were sacrificed at specific time points immediately following heat treatment, and morphological changes in the small intestine were determined. The miRNA and mRNA expression profiles from sample of small intestine were evaluated by microarray analysis. Heat stress caused pronounced morphological damage in the rat small intestine, most severe within the jejunum after 3?days of heat treatment. A mRNA microarray analysis found 270 genes to be up-regulated and 122 genes down-regulated (P?≤?0.01, ≥2.0-fold change) in the jejunum after heat treatment. A miRNA microarray analysis found 18 miRNAs to be up-regulated and 11 down-regulated in the jejunum after heat treatment (P?≤?0.05). Subsequent bioinformatic analyses of the differentially expressed mRNAs and miRNAs were carried out to integrate miRNA and mRNA expression and revealed that alterations in mRNA following heat stress were negatively correlated with miRNA expression. These findings significantly advance our understanding of the regulatory mechanisms underlying the pathophysiology of heat stress-induced injury in the small intestine, specifically with regard to miRNAs.     

Regulation of miR319 during cold stress in sugarcane

MicroRNAs (miRNAs) are part of a novel mechanism of gene regulation that is active in plants under abiotic stress conditions. In the present study, 12 miRNAs were analysed to identify miRNAs differentially expressed in sugarcane subjected to cold stress (4 °C). The expression of miRNAs assayed by stem–loop RT‐PCR showed that miR319 is up‐regulated in sugarcane plantlets exposed to 4 °C for 24 h. The induction of miR319 expression during cold stress was observed in both roots and shoots. Sugarcane miR319 was also regulated by treatment with abscisic acid. Putative targets of this miRNA were identified and their expression levels were decreased in sugarcane plantlets exposed to cold. The cleavage sites of two targets were mapped using a 5′ RACE PCR assay confirming the regulation of these genes by miR319. When sugarcane cultivars contrasting in cold tolerance were subjected to 4 °C, we observed up‐regulation of miR319 and down‐regulation of the targets in both varieties; however, the changes in expression were delayed in the cold‐tolerant cultivar. These results suggest that differences in timing and levels of the expression of miR319 and its targets could be tested as markers for selection of cold‐tolerant sugarcane cultivars.     

MicroRNA-663 upregulated by oscillatory shear stress plays a role in inflammatory response of endothelial cells

The mechanisms by which oscillatory shear stress (OS) induces, while high laminar shear stress (LS) prevents, atherosclerosis are still unclear. Here, we examined the hypothesis that OS induces inflammatory response, a critical atherogenic event, in endothelial cells by a microRNA (miRNA)-dependent mechanism. By miRNA microarray analysis using total RNA from human umbilical vein endothelial cells (HUVECs) that were exposed to OS or LS for 24 h, we identified 21 miRNAs that were differentially expressed. Of the 21 miRNAs, 13 were further examined by quantitative PCR, which validated the result for 10 miRNAs. Treatment of HUVECs with the miR-663 antagonist (miR-663-locked nucleic acids) blocked OS-induced monocyte adhesion, but not apoptosis. In contrast, overexpression of miR-663 increased monocyte adhesion in LS-exposed cells. Subsequent mRNA expression microarray study using HUVECs treated with miR-663-locked nucleic acids and OS revealed 32 up- and 3 downregulated genes, 6 of which are known to be involved in inflammatory response. In summary, we identified 10 OS-sensitive miRNAs, including miR-663, which plays a key role in OS-induced inflammatory responses by mediating the expression of inflammatory gene network in HUVECs. These OS-sensitive miRNAs may mediate atherosclerosis induced by disturbed flow.     

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。     

The p53-signaling pathway and colorectal cancer: Interactions between downstream p53 target genes and miRNAs

IntroductionWe examined expression of genes in the p53-signaling pathway. We determine if genes that have significantly different expression in carcinoma tissue compared to normal mucosa also have significantly differentially expressed miRNAs. We utilize a sample of 217 CRC cases.MethodsWe focused on fold change (FC) > 1.50 or <0.67 for genes and miRNAs, that were statistically significant after adjustment for multiple comparisons. We evaluated the linear association between the differential expression of miRNA and mRNA. miRNA:mRNA seed-region matches also were determined.ResultsEleven dysregulated genes were associated with 37 dysregulated miRNAs; all were down-stream from the TP53 gene. MiR-150-5p (HR = 0.82) and miR-196b-5p (HR 0.73) significantly reduced the likelihood of dying from CRC when miRNA expression increased in rectal tumors.ConclusionsOur data suggest that activation of p53 from cellular stress, could target downstream genes that in turn could influence cell cycle arrest, apoptosis, and angiogenesis through mRNA:miRNA interactions.     

High-throughput miRNA sequencing and identification of a novel ICE1-targeting miRNA in response to low temperature stress in Eucalyptus camaldulensis

• MicroRNAs (miRNAs) play a crucial role in the growth, development, morphogenesis, signal transduction, and stress response in plants. The ICE (Inducer of CBF expression)-CBF (C-repeat binding factor)-COR (Cold-regulated gene) regulatory cascade is an important signalling pathway in plant response to low temperature stress, and it remains unknown whether this pathway is regulated by miRNAs.
• In this study, high-throughput sequencing was employed for predicting and identifying the miRNAs that were likely to target the ICE-CBF-COR pathway in Eucalyptus camaldulensis. A novel ICE1-targeting miRNA, eca-novel-miR-259-5p (nov-miR259), was further analysed.
• A total of 392 conserved miRNAs and 97 novel miRNAs were predicted, including 80 differentially expressed miRNAs. Of these, 30 miRNAs were predicted to be associated with the ICE-CBF-COR pathway. The full-length of mature nov-miR259 was 22 bp and its precursor gene was 60 bp in length, with a typical hairpin structure. The RNA ligase-mediated 5′ amplification of cDNA ends (5′-RLM-RACE) and Agrobacterium-mediated tobacco transient expression assays demonstrated that nov-miR259 could cleave EcaICE1 in vivo. Moreover, qRT-PCR and Pearson's correlation analysis further revealed that the expression levels of nov-miR259 were almost significantly negatively correlated with those of its target gene, EcaICE1, and the other genes in the ICE-CBF-COR pathway.
• We first identified the nov-miR259 as a novel ICE1-targeting miRNA, and the nov-miR259-ICE1 module may be involved in regulating the cold stress response in E. camaldulensis.

     

Expression of microRNAs associated with oxidative stress in the hippocampus of piglets

Oxidative stress is associated with human diseases and the developmental retardation of animals. The hippocampus is particularly vulnerable to oxidative stress. MicroRNAs (miRNAs), expressed largely in the mammalian brain, are emerging as robust players and have been implicated in many cellular processes. The present study investigated the sub-tissue specificity of miRNA expression in the dorsal hippocampus (DH) and ventral hippocampus (VH) and evaluated the effects of oxidative stress induced by iron dextran (FeDex) treatment on miRNA expression in the DH and VH of pigs using RNA-sequencing technology and bioinformatics, respectively. The results demonstrated that the injection of FeDex significantly increased the levels of several markers of oxidative stress in serum of Rongchang piglets, which indicated that oxidative stress was successfully induced. Sub-tissue specificity was displayed with 54 differentially expressed miRNAs between the VH and DH. The induced oxidative stress emphasized 59 and 46 differentially expressed miRNAs in the DH and VH, respectively. GO and KEGG pathway analyses revealed that the predicted targets of these differentially expressed miRNAs were involved in the pathways that regulate the expression of genes associated with nervous system development, immune response and oxidative stress, which not only revealed the ability of miRNAs to influence complex gene networks in the DH and VH but also further corroborated the successful induction of oxidative stress. Collectively, the results of this study provide a valuable basis for future studies aimed at contributions of miRNAs induced by oxidative stress in growth retardation and neurodegenerative diseases of animals and human.     

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.