Next-Generation Sequencing Analysis Reveals Differential Expression Profiles of MiRNA-mRNA Target Pairs in KSHV-Infected Cells

Kaposi’s sarcoma associated herpesvirus (KSHV) causes several tumors, including primary effusion lymphoma (PEL) and Kaposi’s sarcoma (KS). Cellular and viral microRNAs (miRNAs) have been shown to play important roles in regulating gene expression. A better knowledge of the miRNA-mediated pathways affected by KSHV infection is therefore important for understanding viral infection and tumor pathogenesis. In this study, we used deep sequencing to analyze miRNA and cellular mRNA expression in a cell line with latent KSHV infection (SLKK) as compared to the uninfected SLK line. This approach revealed 153 differentially expressed human miRNAs, eight of which were independently confirmed by qRT-PCR. KSHV infection led to the dysregulation of ~15% of the human miRNA pool and most of these cellular miRNAs were down-regulated, including nearly all members of the 14q32 miRNA cluster, a genomic locus linked to cancer and that is deleted in a number of PEL cell lines. Furthermore, we identified 48 miRNAs that were associated with a total of 1,117 predicted or experimentally validated target mRNAs; of these mRNAs, a majority (73%) were inversely correlated to expression changes of their respective miRNAs, suggesting miRNA-mediated silencing mechanisms were involved in a number of these alterations. Several dysregulated miRNA-mRNA pairs may facilitate KSHV infection or tumor formation, such as up-regulated miR-708-5p, associated with a decrease in pro-apoptotic caspase-2 and leukemia inhibitory factor LIF, or down-regulated miR-409-5p, associated with an increase in the p53-inhibitor MDM2. Transfection of miRNA mimics provided further evidence that changes in miRNAs are driving some observed mRNA changes. Using filtered datasets, we also identified several canonical pathways that were significantly enriched in differentially expressed miRNA-mRNA pairs, such as the epithelial-to-mesenchymal transition and the interleukin-8 signaling pathways. Overall, our data provide a more detailed understanding of KSHV latency and guide further studies of the biological significance of these changes.     

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。     

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.     

星形细胞瘤生存预后相关miRNA-mRNA调控关系对筛选

利用GEO数据库中的芯片数据,筛选与星形细胞瘤生存预后相关的miRNA-mRNA调控关系对,为后续研究提供理论支持。下载芯片数据利用R语言进行差异表达分析,得到星形细胞瘤较正常组织表达显著改变的miRNA与mRNA;通过miRNA靶基因预测,将靶基因与差异表达mRNA取交集,明确mRNA与miRNA之间的关系;利用GEPIA2.0工具在TCGA数据库中筛选有生存价值的mRNA并验证表达情况,利用OncoLnc工具对相应miRNA进行生存分析。筛选到差异表达的miRNA 90个(表达上调22个,下调68个);差异表达的mRNA 644个(表达下调476个,上调168个);根据miRNA靶基因预测结果,整理出miRNA-mRNA关系对30个,对其中的mRNA与miRNA进行生存分析,共得到7个miRNA-mRNA关系对与LGG患者生存预后明显相关,未筛选到与GBM患者生存预后有关的调控关系对。本研究筛选到的7个miRNA-mRNA调控关系对与LGG患者生存预后显著相关,可为相关研究与治疗提供靶点和参考方向。     

The RNA expression signature of the HepG2 cell line as determined by the integrated analysis of miRNA and mRNA expression profiles

Understanding miRNAs' regulatory networks and target genes could facilitate the development of therapies for human diseases such as cancer. Although much useful gene expression profiling data for tumor cell lines is available, microarray data for miRNAs and mRNAs in the human HepG2 cell line have only been compared with that of other cell lines separately. The relationship between miRNAs and mRNAs in integrated expression profiles for HepG2 cells is still unknown. To explore the miRNA–mRNA correlations in hepatocellular carcinoma (HCC) cells, we performed miRNA and mRNA expression profiling in HepG2 cells and normal liver HL-7702 cells at the genome scale using next-generation sequencing technology. We identified 193 miRNAs that are differentially expressed in these two cell lines. Of these, 89 miRNAs were down-regulated in HepG2 cells compared with HL-7702 cells, while 104 miRNAs were up-regulated. We also observed 3035 mRNAs that are significantly dys-regulated in HepG2 cells. We then performed an integrated analysis of the expression data for differentially expressed miRNAs and mRNAs and found several miRNA–mRNA pairs that are significantly correlated in HepG2 cells. Further analysis suggested that these differentially expressed genes were enriched in four tumorigenesis-related signaling pathways, namely, ErbB, JAK–STAT, mTOR, and WNT, which until now had not been fully reported. Our results could be helpful in understanding the mechanisms of HCC occurrence and development.     

MicroRNA profile analysis of a feline kidney cell line before and after infection with mink enteritis virus

MicroRNAs (miRNAs) are small regulatory RNAs that play a significant role in eukaryotes by targeting mRNAs for cleavage or translational repression. Recent studies have also shown them to be associated with cellular changes following viral infection. Mink enteritis virus (MEV) is one of the most important viral pathogens in the mink industry. To study the involvement of miRNAs in the MEV infection process, we used Illumina's ultrahigh throughput approach to sequencing miRNA libraries from the feline kidney (F81) cell line before and after infection with MEV. Using this bioinformatics approach we identified 196 known mammalian miRNA orthologs belonging to 152 miRNA families in F81 cells. Additionally, 97 miRNA*s of these miRNAs were detected. As well as known miRNAs, 384 and 398 novel miRNA precursor candidates were identified in uninfected and MEV-infected F81 cells respectively that have not been reported in other mammals. In MEV-infected cells 3 miRNAs were significantly down-regulated and 4 up-regulated including 3 significantly. The majority (12 of 16) of randomly selected miRNA expression profiles by qRT-PCR were consistent with those identified by deep sequencing. A total of 88 miRNAs were predicted to target interferon-associated genes; 6 appear to target the 3′UTR of MEV-specific receptor transferring receptor mRNAs; and 8 to target the MEV mRNA coding region. No miRNAs coded by MEV itself were detected.     

High-Throughput MicroRNA and mRNA Sequencing Reveals that MicroRNAs May Be Involved in Pectinesterase-Mediated Cold Resistance in Potato

Since potato cultivars are sensitive to low temperature, cold injury severely affects the geographical distribution and yield of potato. Although some miRNAs have been identified in response to cold stress in plants, there is no report about the role of miRNAs in the response to cold stress in potato. Here, via high throughput sequencing, we described the profiling of cold stress response to miRNA and mRNA in potato. Two small RNA and six mRNA libraries were constructed and sequenced. 296 known and 211 novel miRNAs were identified, in which 34 miRNAs in Cold Group (CG) had the higher expression quantity than which in Normal Group (NG) and 32 in CG had lower expression quantity than which in NG. 3068 differentially expressed genes were detected between NG and CG, in which 1400 genes were up-regulated and 1668 genes were down-regulated. The metabolism pathway of starch and sucrose (ko00500) is the common KEGG pathway in differentially expressed miRNA and mRNA. In this pathway, StuPME21575 and StuPME42971 are pectinesterase which mainly catalyzes the pectin-forming pectate, which are controlled by stu-miR6023 and stu-novel-miR42365. As the potato suffering cold stress, these two miRNAs expression levels became higher, but their target genes expression levels were just opposite and this result is the same with qRT-PCR.     

miR-21-5p is associated with the regulation of estradiol benzoate and oxytocin induced primary dysmenorrhea in rat uterus: a bioinformatic study

To determine whether miRNAs, particularly miR-21-5p, are associated with primary dysmenorrhea (PD) and to elucidate the in-depth molecular mechanisms underlying PD regulation, estradiol benzoate and oxytocin were used to induce PD syndrome in rats followed by miRNA microarray and mRNA profiling of the rat uteruses. Additionally, the expression level of miR-21-5p in the uterus was measured by a real time RT-PCR approach. The differentially expressed mRNAs in the uterus of PD syndrome rats were then bioinformatically analyzed by ClueGo to be enriched in specific biological pathways. The targets of miRNAs in the pathways were predicted by TargetScan. Consequently, the expression levels of 13 miRNAs are significantly changed in the uterus of PD syndrome rats, of which 7 are up-regulated (including miR-21-5p) and 6 are down-regulated. Further, 682 mRNAs were found to be differentially expressed, with 267 and 415 are up- and down-regulated, respectively. These genes are significantly enriched in a series of biological pathways closely relevant to miR-21-5p function and uterine smooth muscle (USM) contraction. Our data, for the first time, disclose that miR-21-5p, as well as some other miRNAs, is associated with PD regulation through the involvement of USM contractability.     

参与调控意大利蜜蜂工蜂中肠基因表达的东方蜜蜂微孢子虫miRNA的组学解析及其调控网络

【目的】本研究结合前期已获得的miRNA和mRNA组学数据对东方蜜蜂微孢子虫Nosema ceranae的差异表达miRNA(differentially expressed miRNA, DEmiRNA)靶向意大利蜜蜂Apis mellifera ligustica工蜂中肠的mRNA和差异表达mRNA(differentially expressed mRNA, DEmRNA)进行生物信息学预测、数据库注释和调控网络分析,旨在解析东方蜜蜂微孢子虫对意大利蜜蜂工蜂中肠的基因表达调控。【方法】比较东方蜜蜂微孢子虫感染7 d(AmT1)和10 d(AmT2)的意大利蜜蜂工蜂中肠和未感染中肠(分别为AmCK1和AmCK2)的mRNA数据,筛选意大利蜜蜂工蜂的显著性DEmRNA;通过比较侵染AmT1和AmT2的东方蜜蜂微孢子虫(分别为NcT1和NcT2)和东方蜜蜂微孢子虫纯净孢子(NcCK)的miRNA数据筛选出东方蜜蜂微孢子虫的DEmiRNA。利用TargetFinder软件预测东方蜜蜂微孢子虫DEmiRNA靶向的意大利蜜蜂工蜂中肠DEmRNA。利用相关生物信息学工具对上述意大利蜜蜂工蜂中肠靶DEmRNA进行GO和KEGG数据库注释。根据KEGG数据库注释信息筛选出意大利蜜蜂工蜂中肠免疫防御和能量代谢通路相关DEmRNA,并构建和分析上述DEmRNA与相应的东方蜜蜂微孢子虫DEmiRNA之间的调控网络。【结果】NcCK vs NcT1比较组中东方蜜蜂微孢子虫的77条显著上调miRNA和52条显著下调miRNA可分别靶向AmCK1 vs AmT1比较组中意大利蜜蜂工蜂中肠的118条显著下调mRNA和135条显著上调mRNA,这些mRNA可分别注释到31和25个GO条目,以及113和107条KEGG通路。NcCK vs NcT2比较组中东方蜜蜂微孢子虫的52条显著上调miRNA和49条显著下调miRNA可分别靶向AmCK2 vs AmT2比较组中意大利蜜蜂工蜂中肠的97条显著下调mRNA和210条显著上调mRNA,这些mRNA可分别注释到27和30个GO条目以及97和127条KEGG通路。NcCK vs NcT1和NcCK vs NcT2比较组中的11条共同显著上调miRNA和19条共同显著下调miRNA分别靶向AmCK1 vs AmT1和AmCK2 vs AmT2比较组的6条共同显著下调和14条共同显著上调mRNA,可分别注释到7和10个GO条目以及0和9条KEGG通路。NcCK vs NcT1和NcCK vs NcT2比较组中东方蜜蜂微孢子虫的DEmiRNA可靶向AmCK1 vs AmT1和AmCK2 vs AmT2比较组中意大利蜜蜂工蜂中肠的氧化磷酸化和硫代谢等能量代谢通路相关DEmRNA,以及胞吞作用、黑色素生成、溶酶体、自噬、Toll样受体信号通路、细胞凋亡、Ras信号通路、泛素介导的蛋白水解和MAPK信号通路等免疫防御通路相关DEmRNA。进一步分析发现,miR-216-x, miR-5119-y, bantam-y和miR-8-y在NcCK vs NcT1和NcCK vs NcT2比较组中皆显著上调表达,且靶向意大利蜜蜂工蜂中肠的溶酶体、黑色素生成、泛素介导的蛋白水解、MAPK信号通路及Ras信号通路等免疫防御通路相关的显著下调mRNA。【结论】在侵染过程中,东方蜜蜂微孢子虫的DEmiRNA对意大利蜜蜂工蜂的基因表达具有广泛的潜在影响,东方蜜蜂微孢子虫可能通过上调部分miRNA跨界调控意大利蜜蜂工蜂的免疫防御以促进侵染,通过下调部分miRNA跨界调控意大利蜜蜂工蜂的能量代谢以加强能量窃取并促进增殖。     

Growth Hormone-Regulated mRNAs and miRNAs in Chicken Hepatocytes

Growth hormone (GH) is a key regulatory factor in animal growth, development and metabolism. Based on the expression level of the GH receptor, the chicken liver is a major target organ of GH, but the biological effects of GH on the chicken liver are not fully understood. In this work we identified mRNAs and miRNAs that are regulated by GH in primary hepatocytes from female chickens through RNA-seq, and analyzed the functional relevance of these mRNAs and miRNAs through GO enrichment analysis and miRNA target prediction. A total of 164 mRNAs were found to be differentially expressed between GH-treated and control chicken hepatocytes, of which 112 were up-regulated and 52 were down-regulated by GH. A total of 225 chicken miRNAs were identified by the RNA-Seq analysis. Among these miRNAs 16 were up-regulated and 1 miRNA was down-regulated by GH. The GH-regulated mRNAs were mainly involved in growth and metabolism. Most of the GH-upregulated or GH-downregulated miRNAs were predicted to target the GH-downregulated or GH-upregulated mRNAs, respectively, involved in lipid metabolism. This study reveals that GH regulates the expression of many mRNAs involved in metabolism in female chicken hepatocytes, which suggests that GH plays an important role in regulating liver metabolism in female chickens. The results of this study also support the hypothesis that GH regulates lipid metabolism in chicken liver in part by regulating the expression of miRNAs that target the mRNAs involved in lipid metabolism.     

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.