Effect of Glucocorticoids on lncRNA and mRNA Expression Profile of the Human Bone Microcirculatory Endothelial Cells
ABSTRACT: Appropriate gene expression patterns form the basis for bone microcirculatory endothelial cells’ function and bone morphology. Although previous studies have elucidated the impact of hydrocortisone on bone microcirculatory endothelial cells’ specific gene expression, the exact differential transcriptomes and comprehensive gene expression profiles remain unknown. We have investigated the mRNA and lncRNA expression patterns before and after hydrocortisone administration of bone microcirculatory endothelial cells. At mRNAs level totally 518 differentially expressed genes were identified. Furthermore, we identified 73 upregulated and 166 downregulated long non-coding RNAs after administration of hydrocortisone. These RNAs appeared to be highly important to gene co-expression network. Transcriptomic analysis of bone microcirculatory endothelial cells from human samples is highly informative due to their relevance to the large number of expressed genes. Our study provides a very valuable basis for investigation of genes, regulation and their co-expression network contributing to hydrocortisone induced disorders. Two-condition experiment, hydrocortisone treated vs. untreated bone microcirculatory endothelial cells. Biological replicates: 8 control replicates, 8 treated replicates.
Project description:Many studies have demonstrated miRNAs as key regulators of inflammatory responses in endothelial cells (Ecs). However, because of the complexity of inflammatory genes and miRNAs, there would be many undiscovered miRNAs involved in inflammatory responses of ECs. Let-7e is an important member of let-7e family and plays key roles in the regulation of inflammation and endothelial cell proliferation. Furthermore, let-7e expression is significantly increased in many cardiovascular diseases including coronary heart disease. Therefore,we speculated that let-7e might play important roles in the regulation of inflammatory responses in endothelial cells by directly or indirectly targeting certain inflammatory genes. In order to reveal the action of let-7e in vascular endothelial cells, the expression profiles of mRNAs and lncRNAs induced by let-7e in human umbilical vein endothelial cells (HUVECs) were investigated using microarray technology.
Project description:Long noncoding RNAs (lncRNAs) play a key role in regulating immunological functions. Their impact on the chronic inflammatory disease multiple sclerosis (MS), however, remains unknown. We investigated the expression of lncRNAs in peripheral blood mononuclear cells (PBMCs) of patients with MS and try to explain their possible role in the process of MS. we recruited 26 MS patients according to the revised McDonald Criteria. Then we chosen 6 patients for microarray analysis randomly. Microarray assays identified outstanding differences in lncRNA expression, which were verified through real-time PCR. LncRNA functions were annotated for target genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, and regulatory relationships between lncRNAs and target genes were analyzed using the “cis” and “trans” model.
Project description:Appropriate gene expression patterns form the basis for bone microcirculatory endothelial cells’ function and bone morphology. Although previous studies have elucidated the impact of hydrocortisone on bone microcirculatory endothelial cells’ specific gene expression, the exact differential transcriptomes and comprehensive gene expression profiles remain unknown. We have investigated the microRNA expression patterns before and after hydrocortisone administration of bone microcirculatory endothelial cells. Only 5 microRNAs were Benjamini-Hochberg characterized over 368 microRNAs candidates. Transcriptomic analysis of bone microcirculatory endothelial cells from human samples is highly informative due to their relevance to the large number of expressed genes. Our study provides a very valuable basis for investigation of genes, regulation and their co-expression network contributing to hydrocortisone induced disorders. Overall design: Two-condition experiment, hydrocortisone treated vs. untreated bone microcirculatory endothelial cells. Biological replicates: 8 control replicates, 8 treated replicates.
Project description:Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and the second most common form of leukemia in children. Multiple lncRNAs participate in normal and may be implicated in malignant hematopoiesis associated with blood cell cancers, such as leukemia. Currently, the expression profile of lncRNAs in pediatric AML is unclear. In this study, we evaluated the lncRNA expression profile in the tissue of three pediatric AML patients with lncRNA microarray techniques. In order to gain insight into the function of targets of lncRNAs, GO term and KEGG pathway annotation were applied to the target gene pool. qPCR was performed to evaluate the expression patterns of dys-regulated lncRNAs. Bone marrow specimens were obtained at the time of diagnosis during routine clinical assessment of 3 pediatric patients with AML, who presented at the Department of Hematology and Oncology, Children's Hospital of Soochow University between 2000 and 2011. Additionally, bone marrow samples from 3 healthy donors were analyzed as controls. Human LncRNA Array analysis was performed by KangChen Bio-tech, Shanghai P.R. China. Total RNA from each sample was quantified by the NanoDrop ND-1000 and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. For microarray analysis, Agilent Array platform was employed. The sample preparation and microarray hybridization were performed based on the manufacturer’s standard protocols with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3’ bias utilizing a random priming method. The labeled cRNAs were hybridized onto the Human LncRNA Array v2.0 (8 x 60K, Arraystar). After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C. Agilent Feature Extraction software (version 18.104.22.168) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v12.0 software package (Agilent Technologies). After quantile normalization of the raw data, LncRNAs and mRNAs that at least 4 out of 6 samples have flags in Present or Marginal (“All Targets Value”) were chosen for further data analysis. Differentially expressed LncRNAs and mRNAs with statistical significance between the two groups were identified through Volcano Plot filtering. Pathway analysis and GO analysis were applied to determine the roles of these differentially expressed mRNAs played in these biological pathways or GO terms. Finally, Hierarchical Clustering was performed to show the distinguishable LncRNAs and mRNAs expression pattern among samples.
Project description:The aim of this study is to investigate the alterations in gene expression in Porphyromonas gingivalis W83 after inoculation in rat oral cavity. P.gingivalis W83 inoculation in rat oral cavity caused inflammatory responses in gingival tissues and destroyed host alveolar bone. Microarray analysis revealed that 42 genes were upregulated, and 22 genes were downregulated in the detected 1786 genes in the inoculated P.gingivalis W83. Products of these upregulated and downregulated genes are mainly related to transposon functions, cell transmembrane transportation, protein and nucleic acid metabolism, energy metabolism, cell division and bacterial pathogenicity.P.gingivalis W83 has a pathogenic effect on host oral cavity. Meanwhile, inflammatory oral environment alters P.gingivalis W83 gene expression profile. These changes in gene expression may limit the proliferation and weaken the pathogenicity of P.gingivalis W83, and favor themselves to adapt local environment for survival. 3 samples were picked up from wild strain P.gingivalis W83 and inoculated P.gingivalis W83, respectively. The total RNA was extracted and labeled with Klenow, and then hybridism with P.gingivalis W83 chip. The commercial GeneChip P.gingivalis W83 Genome Array used here was provided by CapitalBio Corporation (http://www.capitalbio.com/en/index.asp, Beijing, China), a service provider authorized by Roche NimbleGen (Wisconsin, USA). Five replicates of the genome were included per chip. An average of 19 different 60-base oligonucleotides (60-mer probes) represented each gene in the genome. Array hybridization, washing, scanning and data analysis were performed at the CapitalBio Corporation, Beijing, China and carried out according to the NimbleGen’s Expression user’s guide. The arrays were scanned using MS200 scanner (NimbleGen), and NimbleScan software (NimbleGen) was used to extract fluorescent intensity raw data from the scanned images. The expression data of probes were normalized using quantile normalization and expression data of genes were generated using the Robust Multichip Average (RMA) algorithm. In a comparison analysis, two class unpaired method in the Significant Analysis of Microarray software (SAM, version 3.02) was performed to identify significantly differentially expressed genes between TEST and CONTROL groups. Genes were determined to be significantly differentially expressed with a selection threshold of false discovery rate, FDR<5% and fold change＞2.0 in the SAM output result.
Project description:Investigation of gene expression level changes in pancreatic and liver tissues of diabetic db/db mice supplemented with selenate, compared to the diabetic db/db mice administered placebo. Fasting blood glucose levels increased continuously in diabetic db/db mice administered placebo (DMCtrl) but decreased gradually in selenate-supplemented diabetic db/db mice (DMSe) and approached normal values when the experiment ended. The size of pancreatic islets increased, causing the plasma insulin concentration to double in DMSe mice compared with that in DMCtrl mice. Two six chip studies using total RNA respectively isolated from pancreatic and liver tissues of three selenate-supplemented diabetic db/db mice, and three diabetic db/db mice administered placebo.
Project description:Investigation of whole genome gene expression level changes in miR-139-5p mimic-treated EC109 cells, compared to the scrambled negative controls. A four chip study using total RNA recovered from two separate cultures of miR-139-5p mimic-transfected EC109 cells and two separate cultures of scrambled negative control-transfected EC109 cells. Each chip measures the expression level of 44,049 genes from human esophageal cancer cell EC109 with three 60-mer probe pairs per gene.
Project description:To study the difference of gene expression pattern of whole blood between klinefelter’s syndrome and normal individuals and discovered the disease-related genes and biological pathway. 717 differentially expressed genes (480 up regulated, 237 down regulated) were identified between whole blood samples of the klinefelter’s patients and normal volunteers. Function enrichment analysis identified a number of genes involved in immune response, metabolism etc. Three genes with significant down-regulation in Klinefelter’s patients were also confirmed by qRT-PCR.Using the whole blood as material in patients with klinefelter’s syndrome, the differentially expressed genes was identified. It is noticed that genes involved in metabolism pathways were significantly decreased. These observations may suggest aberrant of metabolism may be important for developing the klinefelter’s syndrome. In addition, our results also show that no X-linked genes are overexpression in the peripheral blood cells of KS patients. In the present study, we examined gene expression in whole blood of 5 klinefelter’s patients and 5 healthy control volunteers. The RNA of 10 samples was isolated and Gene expression was detected by gene microarray. The different expressed genes were analyzed by SAM software.
Project description:We used Illumina sequencing of poly-A selected RNA of Leishmania mexicana (WHO strain MNYC/BZ/62/M379) culture-adapted promastigotes (PRO), axenic amastigotes (AXA) and intracellular amastigotes (AMA) in mouse bone marrow derived macrophages (BMDM), 24h after infection, to map 5' and 3' ends of Leishmania transcripts and determine transcript abundances. The AMA samples were prepared from total RNA of infected macrophages thus containing a mixture of leishmanial and murine RNA transcripts. We also sequenced poly-A selected RNA from uninfected BMDMs. Three biological replicates per sample.