MicroRNA expression in microvesicles (MVs) derived from endothelial progenitor cells (EPCs)
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Vincenzo Cantaluppi
PROVIDER: E-MEXP-2956 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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2012-07-31 | E-GEOD-31956 | biostudies-arrayexpress
Project description:To investigate the regulatory mechanisms governing the malignant signature of different gliomas we analyzed microRNA expression profiles in human tumor samples of world health organization (WHO) grade I (benign tumors), II (low grade tumors) and IV (high grade tumors) and from primary cultures obtained from tumor samples of grade II and IV. Patients This study included tumor samples histologically verified as astrocytic gliomas obtained from patients who had undergone craniotomy for microsurgical tumor removal. According to the revised WHO classification, tumors were diagnosed as: grade I or pilocytic astrocytomas; grade II or diffuse fibrillary astrocytomas; grade IV or glioblastoma multiforme. Primary cell cultures from grade II and grade IV gliomas were also obtained and miRNA expression in these cultures were analyzed RNA extraction Total RNA, including small RNA, was isolated from tissue samples using the mirVanaTM miRNA Isolation Kit (Ambion) following the standard protocol. The quantity and quality of the purified RNA was evaluated by spectrophotometric analysis and electrophoresis on denaturing gel of acrylamide. Multiplex Real-Time Quantitative Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) The miRNAs were first converted to cDNA using Multiplex RT for TaqMan Array Human MicroRNA Panel. The RT Master mix included 100 mM each of dNTPs , 50 U/ml MultiScrabe reverse transcriptase (Applied Biosystems), 20 U/M-BM-5l RNase inhibitor (Applied Biosystems) and 10X RT Buffer. The 10 M-BM-5l reactions, including 7 M-BM-5l of RT master mix, 2 M-BM-5l of purified microRNA and 1 M-BM-5l of Multiplex RT Human primer pool (Applied Biosystem), were incubated in ice for 5 min and then in a thermal cycler for 30 min at 16M-BM-0C, 30 min at 42M-BM-0C, 5 min at 85M-BM-0C, and then hold at 4M-BM-0C. miRNA levels were normalized to the expression of small nucleolar RNAs, RNU44, RNU48 and RNU6B. All reverse transcriptase reactions, including no-template controls and RT controls, were run in duplicate. Real-time PCR was performed using a standard TaqMan PCR kit procedure on an M-bM-^@M-^\Real Time Fast 7900 HTM-bM-^@M-^] PCR System (Applied Biosystems). The 100 M-BM-5l PCR included 50 M-BM-5l RT product (before diluited 1:60) and 50 M-BM-5l TaqMan Universal PCR Master Mix (2X) (Applied Biosystems). The total volume were loaded into Card TaqMan Low Density Array Human MicroRNA Panel (Applied Biosystem) including a total of 384 human microRNAs publicated on databases www.sanger.ac.uk. The reaction cards was runned at 50M-BM-0C for 2 min and 95M-BM-0C for 10 min, followed by 40 cycles of 97M-BM-0C for 30s and 59,7M-BM-0C for 1 min. All reactions were run in triplicate. Analysis of data was performed using the SDS 2.3 software using the 2-M-bM-^HM-^FM-bM-^HM-^FCt (relative quantitative) method . The M-bM-^HM-^FCt of every miRNA was determined in relation to the endogenous control RNA U6 that was invariably expressed in all samples. The M-bM-^HM-^FM-bM-^HM-^FCt value was determined in relation to the calibrator, namely the normal brain tissue. Resulting data were grouped according to the tumor grading e selectioned using a cut-off value of 3. Results were expressed as M-bM-^@M-^\fold changeM-bM-^@M-^] over normal brain tissue. We analyzed two samples of grade I, two samples of grade II, two samples of grade IV gliomas. Four samples form norma brain were used as norma control. Primary cell cultures form grade II and grade IV samples were used for the analysis. All reverse transcriptase reactions, including no-template controls and RT controls, were run in triplicate.
2013-09-18 | E-GEOD-50924 | biostudies-arrayexpress
Project description:The study describes miRNA expression in intact duodenum following chronic delta 9 tetrahydrocannabinol (M-NM-^T9-THC) administration to SIV-infected rhesus macaques. Chronic M-NM-^T9-THC administration to uninfected macaques significantly and positively modulated intestinal miRNA expression by increasing the total number of differentially expressed miRNAs from 14 to 60 days post infection (DPI). At 60DPI, ~28% of miRNAs showed decreased expression in VEH/SIV compared to none in the THC/SIV group. Furthermore, compared to the VEH/SIV group, THC selectively upregulated the expression of miR-10a, miR-24, miR-99b, miR-145, miR-149 and miR-187 previously shown to target proinflammatory molecules. NOX4, a potent reactive oxygen species generator was confirmed as a direct miR-99b target. A significant increase in NOX4+ crypt epithelial cells was detected in VEH/SIV compared to the THC/SIV group. We speculate that miR-99b-mediated NOX4 downregulation may protect the intestinal epithelium from oxidative stress-induced damage. Twelve age and weight matched male Indian rhesus macaques were randomly divided into 4 groups. Group 1 (n=1) received vehicle (1:1:18 of emulphor : alcohol : saline) and no infection. Group 2 (THC only, n=3) animals received twice daily intramuscular injections of M-NM-^T9-THC and no infection. Group-3 THC/SIV, (n=4) animals received twice daily injections of vehicle and were infected intravenously with 100TCID50 of SIVmac251. Group-4 (VEH/SIV, n=4) animals received twice daily injections of M-NM-^T9-THC similar to group 1 for four weeks prior to SIV infection. Duodenal pinch biopsies were collected before infection and thereafter at 14 and 30 days post infection. All animals were necropsied at 60 days post SIV infection. ~100 ng of total RNA was first reverse transcribed and preamplified according to the manufacturerM-bM-^@M-^Ys recommendation. microRNA expression profiling was performed using TaqMan M-BM-.OpenArrayM-BM-. Human microRNA panels. Data analysis was performed using ExpressionSuiteM-BM-. software. Data was normalized to three endogenous controls (RNU44, RNU48 and snoU6). Delta CT values were calculated by subtracting individual miRNA CT values from an average of all three endogenous controls. Comparisons were made between preinfection and all three treatment groups at 14, 30 and 60 DPI. To determine the effect of chronic THC treatment during SIV infection, comparisons were also made between VEH/SIV and THC/SIV at all three time points.
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