Alterations in miRNA levels in the dentate gyrus in epileptic rats
Ontology highlight
ABSTRACT:
ORGANISM(S): Rattus norvegicus
SUBMITTER: Katarzyna Lukasiuk
PROVIDER: E-GEOD-49850 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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2013-08-14 | E-GEOD-49851 | biostudies-arrayexpress
Project description:Sprouty proteins are evolutionarily conserved modulators of mitogen-activated protein kinase pathway. Sprouty2 appears to function as a tumor suppressor in cancers, whereas we reported earlier that Sprouty2 functions as an oncogene in colorectal cancer. To further understand the oncogenic potential of Sprouty2 in the colon, microRNA expression profile of colon cancer cells was investigated. Sprouty2 suppression in HCT116 colon cancer cells significantly increased MicroRNA 194-5p. Sprouty2 dependent regulation of microRNA194-5p and its biological targets were studied further for their tumor suppressive actions in reducing epithelial-mesenchymal transition in colorectal cancer. Sprouty2 knockdown was performed by infecting HCT116 cells with three different lentivirus expressing shRNAs against human Sprouty2 mRNA and a control non targeted non-silencing shRNA (Sprouty2 MISSION shRNA Lentiviral Transduction Particles; TRCN 0000007522, TRCN 0000231589, TRCN 0000231588 and a non-targeted shRNA control from Sigma) following lentiviral transduction protocols provided by Sigma. Due to the random integration of the lentivirus into the host genome, varying levels of Sprouty2 gene knockdown was expected in puromycin resistant colonies. Three colonies in triplicate that demonstrated highest to lowest level of Sprouty2 suppression, as assessed by western blotting, were selected. RNA samples from these colonies and one from non-targeted shRNA expressing colony were prepared for microRNA expression profile analysis. Pooled RNA samples from each group were shipped to Exiqon for microRNA profiling based on miRCURY LNATM array technology.
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Project description:The study aimed to identify miRNAs expression profiles associated with growth and regression of dominant-size follicles in bovine. Follicles were collected from abattoir ovaries and their status (healthy/atretic) was assessed by measuring steroid levels and aromatase expression. Total RNA was isolated from whole follicles at different developmental stages. An heterologous microarray (Exiqon, Denmark) approach followed by RT-qPCR validation (Qiagen, UK) was used to identify and compare miRNA profiles between large healthy follicles (diameter, 13M-bM-^@M-^S16 mm, n=6) and each of small (4M-bM-^@M-^S8 mm, n=6 pools of follicles) and large atretic folllicles (13-16 mm, n=6). RNA from the above groups was hybridized to the miRCURY LNAM-bM-^DM-" microRNA Hi-Power Labeling Kit,Hy3M-bM-^DM-"/Hy5M-bM-^DM-" (Exiqon) and hybridized on the miRCURY LNAM-bM-^DM-" microRNA Array (6th gen). A total of 17 and 57 microRNAs were differentially expressed (> 2 fold, adj. P-value < 0.05) between Large Healthy and each of Small and Large Atretic follicles, respectively, a fraction of which corresponded to registered bovine miRNA sequences. A subset of 5 bovine miRNAs (miR-144, miR-202,vmiR-451, miR-652, miR-873) were confirmed by qPCR to be upregulated in Large Healthy follicles, were enriched in mural granulosa cells and their predicted targets mapped to genes involved in follicular cell proliferation and differentiation, suggesting an involvemet of this subset of microRNAs in ovarian follicle development. Six biological replicates per developmental stage (total of 18 samples) were used in a double dye microRNA microarray experiment. Samples were distributed among slides so that each experimental group was represented at least once in each slide. For each gene, mean normalized intensities (n= 6 biological replicates/group) were compared between follicle stages (SF vs LHF and LHF vs LAF).
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Project description:Results Platelets in non-diabetic patients demonstrated miRNA expression profiles comparable to previously published data. The miRNA expression profiles of platelets in diabetics were similar. Statistical analysis unveiled only three miRNAs (miR-377-5p, miR-628-3p, miR-3137) with high reselection probabilities in resampling techniques, corresponding to signatures with only modest discriminatory performance. Functional annotation of predicted targets for these miRNAs pointed towards an influence of diabetes mellitus on mRNA processing. Conclusions/interpretation We did not find any major differences in platelet miRNA profiles between diabetics and non-diabetics. Minor differences pertained to miRNAs associated with mRNA processing. Thus, previously described differences in plasma miRNAs between diabetic and nondiabetic patients cannot be explained by plain changes in the platelet miRNA profile. Platelet miRNA profiles were assessed in clinically stable diabetic and nondiabetic patients (each n=30). Platelet miRNA was isolated from leucocyte-depleted platelet-rich plasma, and miRNA profiling was performed using LNA micro-array technology (miRBase 18.0, containing 1,917 human miRNAs). Effects of diabetes mellitus were explored by univariate statistical tests for each miRNA, adjusted for potential confounders, and by developing a multivariable signature, which was evaluated by resampling techniques. Platelet phenotype was assessed by light transmission aggregometry and impedance aggregometry.
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