Circulating miRNA signature in serum of pateints with primary sclerosing cholangitis (PSC), cholangiocarcinoma (CC) and healthy controls.
ABSTRACT: RNA was isolated from serum of pateints with PAS, CC and healthy controls. Global microRNA profiling was done using miRNome microRNA Profiler QuantiMir Human PCR Array (#RA660A-1, version 15; BioCat, Heidelberg, Germany) miRNA arrays were performed on pooled RNA samples from six patients for PSC and CC each and a comparison was performed to healthy control patients. MiRNAs with Ct value above 32 were discarded.
Project description:RNA was isolated from bile of pateints with PAS or CC and global microRNA profiling was done using miRNome microRNA Profiler QuantiMir Human PCR Array (#RA660A-1, version 15; BioCat, Heidelberg, Germany) Three arrays were performed for the PSC group with pooled RNA from n = 5 patients each and two arrays for the CC group with pools of RNA from n = 4 patients each
Project description:Background. Abcb4 (-/-) mice secrete phosphatidylcholine-deficient bile and develop sclerosing cholangitis (SC). The cholangitis involves differential hepatic transcription of genes whose products govern inflammation, activation of hepatic stellate cells and fibrosis. This study was undertaken to test the hypothesis that several genes involved in regulation of tissue inflammation and fibrosis display transcription rates that reflect SC disease activity. Methods. Abcb4 (-/-) mice fed cholic acid (CA) display high SC activity and ursodeoxycholic acid (UDCA) fed mice display low SC activity. Differential hepatic transcription of genes was accordingly measured in abcb4 (-/-) mice maintained on CA- and UDCA-supplemented diets using cDNA microarrays. Abcb4 (+/+) mice served as controls. The differential transcription of selected genes was verified by real time polymerase chain reaction. Liver tissue pathology was quantified by histopathology scoring and immunohistochemistry to visualize bile duct cells and activated hepatic stellate cells. Results. Differential transcription of Ccl2, Ccl20, Cxcl10, Nfκb1, Nfκb2, Tgfβ1, Tgfβ2, Sparc, Ctgf, Lgals3, Elf3, Spp1, Pdgfa, Pdgfrb, Col1a1, Col1a2 and Col4a1 genes paralleled the differing SC activities of cholic acid- and UDCA-fed abcb4 (-/-) mice. Histopathology scores and immunohistochemistry showed greatly enhanced activation of hepatic stellate cells during high SC activity due to CA feeding. Conclusion. Differential transcription of several genes relating to tissue inflammation and hepatic stellate cell activation parallels SC activity in abcb4 (-/-) mice. Data on their differential transcription may be used to gauge SC disease activity. Overall design: To achieve different degrees of disease activity we have fed the mice with either cholic acid (CA) or ursodeoxycholic acid (UDCA) for 9 weeks to increase and decrease disease activity, respectively. Each group uses wild type mice as controls. 5 different mice (livers) in each group was harvested. In 3 hybridizations wild-type livers were marked with Cy3, in 2 hybridizations wild-type livers were marked with Cy5. In addition to these 5 hybridizations one dye swap was performed from a randomly selected liver from the 5 and wild-type marked with Cy5. Thus, a total of 6 hybridizations was used as input for data-processing in each of the two groups. No pooling was done.
Project description:CC-671 has been identified as an inhibitor of Cdc2-like kinase 2 (CLK2) and TTK in direct enzyme assays. CLK2 is a member of the CLK family that phosphorylates serine- and arginine-rich (SR) proteins of the spliceosomal complex as part of a regulatory mechanism for control of pre-mRNA splicing. SR proteins are a family of small nuclear ribonucleoprotein particle (snRNP) splicing factors involved in constitutive and alternative splicing. Monitoring specific phospho-biomarkers of CLK2 demonstrated that CC-671 inhibited phosphorylation of CLK2 substrates in cancer cells with mean IC50 of 549 nM in the triple negative breast cancer (TNBC) line CAL51. In this study, RNA sequencing approach was used to quantify the impact of CC-671 treatment on gene transcription and global alternative splicing in CAL51 cells. Differential exon usage analysis demonstrated that CC-671 changed alternative splicing of many genes. In addition, different sets of genes are impacted by CC-671 at both the alternative splicing and mRNA expression. Genes impacted by alternative splicing shared a set of common pathways with genes altered by mRNA expression. This result indicates that CC-671 regulates transcription via both gene expression and alternative splicing mechanisms. Overall design: Triple negative breast cancer (TNBC) line CAL51 was grown in DMEM medium containing 10% fetal bovine serum, as recommended by vendor. The growing cells were treated by CC-671 in three biological replicates at the following concentrations and time intervals. The treatment time points were 6 hour and 24 hour. Concentration of compounds used was 3 and 10 uM. Six million cells from each treatment were harvested and RNA was isolated by RNeasy kit. Poly-A selection and strand-specific RNA library construction were performed, followed by multiplexing indexed libraries and sequencing on the HiSeq 2500 with 2x100 bp read lengths. A total of 16 samples were included in this experiment, including 4 treatment groups with three biological replicates and 2 vehicle control groups with two biological replicates
Project description:Cumulus cells surrounding mature oocytes that developed to moruale/blastocyst stage on day 5 of IVF cycle were collected and used for gene expression profiling using Affymetrix Human Gene 1.0 ST Arrays in order to determine differences in gene expression between the modified natural and stimulated in vitro fertilization (IVF) procedures. Microarray analysis was made on 8 individual CC samples, derived from 5 patients that first underwent MNIVF, followed by a controlled ovarian hyperstimulation (COH) cycle. As the quality of the isolated RNA derived from 2 of the MNIVF CC samples was not good, these samples were not included in the analysis. Altogether, 3 CC samples from MNIVF and 5 CC samples from COH cycles were included in microarray analysis.
Project description:Temporal lobe epilepsy (TLE) is the most common intractable form of epilepsy in adults and status epilepticus (SE) is the most severe form of seizure that can be non-convulsive and is then difficult to diagnose. Diagnosis of both conditions is principally based on clinical examination and history, often depending on EEG and imaging. A molecular biomarker of these two conditions would be transformational in supporting both diagnoses.Cerebrospinal fluid offers an alternative source of microRNA biomarkers with the advantage of being in closer contact with the target tissue and sites of pathology. The present study indicates cerebrospinal fluid may contain microRNA biomarkers of TLE and SE and offers insights into trafficking mechanisms of biofluid microRNAs that may further enhance diagnostic value. Overall design: microRNA expression in the CSF of 15 patients with headache, 15 pateints with TLE and 15 pateints with SE was measured by TaqMan OpenArray Human MicroRNA Panel.
Project description:Pancreatic Gene Expression from Wild-Type, Stat1-Transgenic, and Stat1-CC-Transgenic Mice. This data was part of the set of data utilized to identify PARP9/DTX3L as a novel antiviral gene. Keywords: transgene state analysis RNA was extracted from the pancreas of 3 mice per genotype (wild-type, Stat1-Transgenic, Stat1-CC-Transgenic). Comparisons were made between Stat1-Transgenic and Stat1-CC-Transgenic mice.