The diffuse idiopathic skeletal hyperostosis related miRNAs analysis
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Abstract:
Objective
To obtain and anlysis the diffuse idiopathic skeletal hyperostosis(DISH)related miRNAs under 3-D adhesion for cell culture.
Methods
From January 2012 to January 2014, 4 ossific ligamenta flava tissues were obtained from DISH patients and 4 normal ligamenta flava tissues were obtained from trauma patients surgically. Fibroblasts were separated by using collagenase technique and then cultured on human acellular amniotic membrane(HAAM). Each sample was identified by immunofluorescence before harvested. Total RNA was extracted and then quantified by microfluidics analysis. The small RNAs(< 300 nt)were isolated by using a YM-100 Microcon centrifugal filter. μParaflo™ MiRNA microarray assay was performed using a service provider to identify miRNAs whose expression was significantly different between the two groups. Part of differential expression miRNAs were verified by qRT-PCR. Targets of miRNAs were obtained using PicTar 2005, miRanda v5, TargetScan 5.1, their function were analyzed by using Gene Ontology. Functional pathway analysis of miRNAs was performed using KEGG Path-way Analysis. TRANSFAC 7.0 public and Patser were used to get the distribution of transcription factor binding sites.
Results
When grown on HAAM, fibroblasts kept their morphology, distributed in the way of cluster, lived in multi-level of HAAM, and established linkage. Collagen I and III were tested positive in normal group cells. Collagen I, II, III and Osteocalcin were tested positive in DISH group cells by immunefluorescence. In total 15 miRNAs showed differential expression, 12 were up-regulated and 3 were down-regulated. The result of qRT-PCR was consistent with MiRNA microarray assay. Totally 67 target genes were predicted which participated in cell differentiation, cell adhesion, mineralization et al, and had function in regulating MAPK, Wnt, TGF-β, Focal adhesion signal pathway et al. In total 10 transcription factors were predicted in differentially expressed miRNAs.
Conclusion
HAAM can provide fibroblasts with 3D adhesion growth, Some differentially expressed miRNAs may participate in the pathogenesis of DISH.
Key words:
MicroRNAs; Ligamentum flavum; Ossification, heterotopic; Microarray analysisKeywords:
KEGG
Objective To explore the miRNA(miRNA) differential expression profile of papillary thyroid carcinoma(PTC),and to provide the evidence that miRNAs are involved in the molecular pathogensis of PTC.Methods Twelve human PTC and control tissues were tested by miRNA array and real-time PCR.mRNA was extracted from the tissues by Trizol reagent.miRNA differential expression profile in PTC and controls were assayed by 875 miRHuman_13.0_091250 miRNA array.Real-time PCR was performed by TaqMan miRNA Assays kits on the tissues of PTC and controls.The relative expression was calculated using the ΔΔCT method and normalized to the expression of U6 snRNA.Results We examined the expression of 875 miRNA species based on version 13.0 of the Sanger miRBase by miRNA microarrays.572 miRNAs were expressed in PTC group,and 388 miRNAs were expressed in control group.Sixty-eight miRNAs were differentially expressed with P0.01.Of these,47 were overexpressed and 21 were underexpressed in PTC.The changes with P0.01 ranged from 1.6-fold to 19.2-fold.Three upregulated(miR-433,miR-221 and miR-21) and three down-regulated miRNA species(miR-138,miR-26a and miR-709) were examined.As anticipated,real-time PCR confirmed the differences in expression of these miRNAs in PTC and control tissues.Conclusion Some miRNAs expressed differently between PTC and control groups,suggesting that they may be involved in the pathogensis of PTC.
MiRBase
Trizol
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To identify pivotal differentially expressed miRNAs and genes and construct their regulatory network in hepatocellular carcinoma.mRNA (GSE101728) and microRNA (GSE108724) microarray datasets were obtained from the NCBI Gene Expression Omnibus (GEO) database. Then, we identified the differentially expressed miRNAs and mRNAs. Sequentially, transcription factor enrichment and gene ontology (GO) enrichment analysis for miRNA were performed. Target genes of these differential miRNAs were obtained using packages in R language (R package multiMiR). After that, downregulated miRNAs were matched with target mRNAs which were upregulated, while upregulated miRNAs were paired with downregulated target mRNA using scripts written in Perl. An miRNA-mRNA network was constructed and visualized in Cytoscape software. For miRNAs in the network, survival analysis was performed. And for genes in the network, we did gene ontology (GO) and KEGG pathway enrichment analysis.A total of 35 miRNAs and 295 mRNAs were involved in the network. These differential genes were enriched in positive regulation of cell-cell adhesion, positive regulation of leukocyte cell-cell adhesion, and so on. Eight differentially expressed miRNAs were found to be associated with the OS of patients with HCC. Among which, miR-425 and miR-324 were upregulated while the other six, including miR-99a, miR-100, miR-125b, miR-145, miR-150, and miR-338, were downregulated.In conclusion, these results can provide a potential research direction for further studies about the mechanisms of how miRNA affects malignant behavior in hepatocellular carcinoma.
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Strain (injury)
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The microRNA (miRNA) expression profiles and their biological functions in calcium oxalate nephrolithiasis remain unclear. In this study, we investigate the miRNA and mRNA expression profiles of kidney tissues in calcium oxalate stone rats. 16 Sprague Dawley rats were divided into control group and stone-forming group. 24-hour urine samples and kidney tissues were collected for biochemical and histological determination after 4 weeks. MiRNA and mRNA microarray were applied to evaluate the miRNA and mRNA expression profiles. To validate the microarray results, the quantitative real-time PCR (qRT-PCR) was performed. A total of 38 miRNAs and 2728 mRNAs were significantly and differentially expressed in kidney tissues of stone-forming group versus control group. Gene Ontology (GO) analysis revealed that most of the target genes were enriched in terms of oxidation reduction, ion transport, inflammatory response, and response to wounding. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of these targets highlights their critical role in cytokine-cytokine receptor interaction, gap junction, and chemokine signaling pathway. Furthermore, the reliability of the microarray-based results was confirmed by using qRT-PCR determination. The miRNA and mRNA expressions in calcium oxalate stone rat kidneys might provide a basis for further research on urolithiasis mechanism.
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To detect the candidate genes for preeclampsia (PE).The gene expression profiles in preeclamptic and normal placental tissues were analyzed using cDNA microarray approach and the altered expression of important genes were further confirmed by real-time RT-PCR (reverse transcription polymerase chain reaction) technique. Total RNA was extracted from placental tissues of three cases with severe PE and from three cases with normal pregnancy. After scanning, differentially expressed genes were detected by software.In two experiments (the fluorescent labels were exchanged), a total of 111 differentially expressed genes were detected. In placental tissue ofpreeclamptic pregnancy, 68 differentially expressed genes were up-regulated, and 44 differentially expressed genes were down-regulated. Of these genes, 16 highly differentially expressed genes were confirmed by real-time fluorescent quantitative RT-PCR, and the result showed that the ratio of gene expression differences was comparable to that detected by cDNA microarray.The results of bioinformatic analysis showed that encoding products of differentially expressed genes were correlated to infiltration of placenta trophoblastic cells, immunomodulatory factors, pregnancy-associated plasma protein, signal transduction pathway, and cell adhesion. Further studies on the biological function and regulating mechanism of these genes will provide new clues for better understanding of etiology and pathogenesis of PE.
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Objective To screen the osteogenesis-specific miRNAs and forecasting target gene in human adipose-derived stem cells (ADSCs). Methods Three pairs of microRNAs of osteo-differentiated human ADSCs were collected. miRNAs were screened by microarray technique, and analysis with SAM and Cluster software. miRNAs were confirmed by qRT-PCR. Results Total nine different miRNAs were found, among which has-miR-17, has-miR-20a, has-miR-20b, has-miR-106a, and has-miR-199b-5p were up-regulated, while has-miR-125a-5p, has-miR-125b, has-miR-31, and has-miR-193a-3p were down-regulated. Conclusions There was differential miRNAs expression during ADSCs osteo-differentiation and some of the target genes were forcasted.
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Background: Primary Sjogren’s syndrome (pSS) is a chronic autimmune disease mainly characterized by the inflammation of exocrine glands. There two key insights into pSS pathogenesis, which included “IFN signature” and hyperactivity of B cells. mRNA and microRNA (miRNA) are very important to control the gene expression. Objectives: In this research, we analyzed the differentially expressed genes (DEG) and miRNA of B cells in pSS patients by RNA-sequencing. And we aim to preliminarily screen out some special miRNAs and target gene loci that may be involved in transcription regulation of B cells of pSS. Methods: Peripheral blood samples from 3 pSS patients and 3 age-matched healthy controls (HC) were collected. CD19 + B cells were sorted by Magnetic cell sorting method. Total RNA was extracted and cDNA of transcriptome or miRNA analysis were prepared and RNA-sequencing was performed to screen the DEG and miRNA. The GO Terms was used to uncover the biological function of DEGs, and the KEGG pathway enrichment was used to find out the related signal pathway. The mRNA-miRNA conjoint analysis was also performed. Results: There were a total of 73 significantly DEGs in B cells of pSS patients compared to HC, including 51 upregulated DEGs (such as IFI44L , IFI44 , IFIT1 , IFITM1 , IFIT3 , IFIT2 , IRF7 , IFI6 and ISG15 )and 22 downregulated DEGs (such as ESR2 and EGR1 ). GO Terms and KEGG pathway analyses showed that most of the upregulated DEGs were enriched in IFN signaling and IFN regulatory pathway, and also showed the relationship with microbial infection, such as influenza A virus, hepatitis C virus, measles and herpes simplex virus. There were five significantly differentially expressed miRNAs, including hsa-miR-4485-3p, hsa-miR-144-5p, hsa-miR-144-3p, hsa-miR-451a, hsa-miR-4732-3p. GO Terms and KEGG pathway analyses showed that most of the target genes which regulated by those miRNAs were enrichment on herpes simplex virus and TGF-β signaling pathway. DEG and differentially expressed miRNAs conjoint analysis showed that the target DEGs which regulated by those miRNAs participated in cytoskeleton formation and modification of DNA or RNA, such as RASD2 , CKAP4 , SPARS2L METTL . Conclusion: There were 51 upregulated DEGs and 22 downregulated DEGs in B cells of pSS patients. GO Terms and KEGG pathway analyses showed that most of the upregulated DEGs were enriched in IFN related signaling pathway, and also showed the significant relationship with microbial infection. Conjoint analysis showed that the target DEGs which regulated by differentially expressed miRNAs participated in cytoskeleton formation and modification of DNA or RNA. There maybe more than one regulatory methods lead to DEGs in B cells of pSS patients. Disclosure of Interests: None declared
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Objective.The physiological and pathogenetic role of microRNAs (miRNAs) in the maintenance of joint homeostasis and in the development of arthritis is recently being elucidated.In this study, we attempted to identify differentially expressed miRNAs in human osteoarthritis (OA) chondrocytes in response to interleukin (IL)-1β.In addition, simultaneous profiling of miRNA and mRNA expression was performed to get an integrated analysis of miRNA and mRNA expression.Methods.Monolayer cultured chondrocytes obtained from knee cartilages of OA patients were stimulated with IL-1β for 4 hours and RNA was isolated.One microgram of total RNA was polyadenylated and converted to cDNA and miRNA microarray was performed.Seven hundred thirty five oligos were used, corresponding to 470 well-annotated human miRNA sequences and 265 potential miRNAs that were identified recently.mRNA microarray was performed simultaneously using the RNA samples that were used for miRNA array.Both sequence and expression information was used to identify regulatory relationship between miRNA and mRNA pairs.Results.Expression profiling of miRNA extracted from IL-1β treated chondrocytes identified 25 miRNA which showed differential expression.We also identified 7190 mRNAs differentially regulated by IL-1β treatment.Among the 25 miRNAs differentially regulated, 13 miRNAs had targets searched by MiRANDA scheme.By combining target search and miRNA-mRNA pairing, we could identify 1043 miRNA-mRNA target pairs.MiR-200a was found to be expressed in human OA and normal cartilages, with downregulation in OA lesion cartilages. Conclusion.It is suggested that miRNA may play a role in the regulation of cartilage degradation in OA.
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Objective To compare differential gene expression profiles between lung adenocarcinoma stem cells and normal lung stem cells.Methods Lung adenocarcinoma and normal lung stem cells were obtained by FACS sorting,and the total RNA was isolated from the cells with Trizol,then the RNA was amplified and dyed with Cy3.The fluorescence was detected and analyzed with Agilent feature extraction;the differentially expressed genes were selected(fold changes over 2.0 as the cut-off value) and GO analysis was performed as well as pathway analysis.qRT-PCR was performed to confirme the microarray results.Results Among 41 000 genes examined,5 798 genes were differently expressed by the stem cells derived from lung adenocarcinoma and normal lung.Agilent feature extraction showed obviously different expression profiles between the two stem cells.Gene Ontology(GO) analysis showed that 41 percent differentially expressed genes were related to biological regulation within Biological process,and cellular component organization and biogenesis,intracellular signaling cascade,cell differentiation,and cell cycle were also as a portion of Biological process.In Molecular function,72 percent differentially expressed genes grouped to binding,and others such as transferase activity,kinase activity,and enzyme regulator activity were commonly represented.Within Cellular component,membrane was 13 percent of this term,and others including non-membrane-bounded organelle,extracellular region,cell fraction,and macromolecular complex assembly distributed evenly.There were 14 differentially expressed genes associated with wnt signal pathway and 11 grouped to oncogenes.The results of qRT-PCR and microarrays showed no significant difference(P0.05).Conclusion Our findings suggest that patters of gene expression in the lung adenocarcinoma stem cell and normal lung stem cell are obviously different.
Cell Sorting
Gene chip analysis
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