Double-stranded microRNA mimics can induce length- and passenger strand-dependent effects in a cell type-specific manner (RNA 2015), Exp 2
ABSTRACT: Experiment 2 - MiRNA mimics have a length and passenger strand specific effect Wildtype and mutant variants of miR-155 and negative control miRNA mimics were transfected to address the phenotype Cells were transfected with mimics and samples were collected in duplicate (except 23mer miR-155 wild type mimic and 23mer negative control mimic)
Project description:Experiment 1 - miR-155 and miR-199 Phenotype miR-155 and miR-199 as 23mer miRNA mimics induced a double-stranded RNA response Cells were transfected with miR-155, miR-199 23 mers, negative control or nothing (empty); samples collected in triplicate
Project description:Mouse peritoneal macrophages were transfected with 80-120 nM miRIDIAN miRNA mimics (miR-mimic-33/miR-mimic-33*) or with 80-120 nM miRIDIAN miRNA inhibitors (anti-miR-33 ASO/anti-miR-33*ASO) Control samples were treated with an equal concentration of a non-targeting control mimics sequence (control mimic) or inhibitor negative control sequence (control aso), to control for non-specific effects in miRNA experiments. Overall design: qPCR gene expression profiling. Peritoneal macrophages from 2-4 donors were used and treated separately as indicated in the summary. Equal amount total RNA from each donor was pooled prior to gene expression analysis.
Project description:Analysis of changes in gene expression after transfection with miR-509-3p mimic in A549 cells Total RNA was obtained from A549 cells transfected with miR-509-3p and negative control mimics, and gene expression was compared using microarrays.
Project description:Microarry analysis of mouse gene expression profile after transfected with miR-27a mimics (27a-7) and mimic NC (NC-9) Goal was to determine the effects of miR-27a transfection on global gene expression. Two-condition experiment, 27a-7 vs.NC-9.
Project description:To clarify the gene expression profile in MLE-12 cells transfected with microRNA mimics upon influenza virus infection, we transfected microRNA mimics (mmu-miR-483-3p or Negative control miRNA) into MLE-12 cells and infected A/Puerto Rico/8/1934 (PR8) strain at an MOI of 2 at 24 hours post transfection. RNA was isolated from cells at 12 hours post infection. We found that miR-483-3p transfection down-regulated the genes involved in the innate immunity regulation upon influenza virus infection. Overall design: Each group had three biological replication. MLE-12 cells transfected with control miRNA mimic was used as control. After isolation of total RNA, Gene expression microarray was performed.
Project description:Patients with advanced colorectal cancer (CRC) are commonly treated with systemic combination therapy but suffer eventually from drug resistance. MicroRNAs (miRNAs) are suggested to play a role in treatment resistance of CRC. We studied whether restoring downregulated miR-195-5p and 497-5p sensitize CRC cells to currently used chemotherapeutics 5-fluorouracil, oxaliplatin and irinotecan. Sensitivity to 5-FU, oxaliplatin and irinotecan before and after transfection with miR-195-5p and miR-497-5p mimics was analyzed in CRC cell lines HCT116, RKO, DLD-1 and SW480. Mass spectrometry based proteomic analysis of transfected and wild-type cells was used to identify targets involved in sensitivity to chemotherapy. Proteomic analysis revealed 181 proteins with significantly altered expression after transfection with miR-195-5p mimic in HCT116 and RKO, including 118 downregulated and 63 upregulated proteins. After transfection with miR-497-5p mimic, 130 proteins were significantly downregulated and 102 were upregulated in HCT116 and RKO (P<0.05 and FC<-3 or FC>3). CHUK and LUZP1 were coinciding downregulated proteins in sensitized CRC cells after transfection with either mimic. Resistance mechanisms of these two proteins may be related to nuclear factor kappa-B signaling and G1 cell cycle arrest, respectively. Restoring miR-195-5p and miR-497-5p expression enhanced sensitivity to chemotherapy, mainly oxaliplatin, in CRC cells and could be a promising treatment strategy for patients with mCRC. Proteomics revealed potential targets of these miRNAs involved in sensitivity to chemotherapy.
Project description:We investigated functions of miRNAs at the level of the whole transcriptome of primary neurons. We transfected mouse E17.5 forebrain primary neuronal cultures (at four to six days of in vitro development) with miRNA mimics and inhibitors. After approximately 48 h post transfection we profiled the effect of these transfections on gene expression with Illumina mRNA microarrays. Cultures were transfected with mimics and inhibitors of five mouse miRNAs (mmu-miR-124, mmu-miR-434-3p, mmu-miR-143, mmu-miR-145 and mmu-miR-25) and with a mimic of a non-mouse miRNA (cel-miR-67). Direct widespread inhibition of gene expression by the perturbed miRNAs was evident when gene expression in cultures transfected with miRNA mimics was compared to those transfected with the inhibitors (or to matched mock transfected cultures): 3-prime UTRs of downregulated transcripts were significantly enriched in seed matching sites for the perturbed miRNAs. Interestingly, analysis of differential gene expression in mock transfected cells (identified through comparison of mock transfected cultures with matched untreated cultures) revealed that genes inhibited by miRNAs were enriched in genes upregulated in mock transfected cultures. This inhibition was the most efficient by the two neuronal miRNAs under investigation (mmu-miR-124 and mmu-miR-434-3p). To investigate if miRNA mediated inhibition of stress induced genes (i.e. stress associated with transfections) was also the case in other stresses, we profiled gene expression changes triggered by chronic neuronal depolarisation. For this, we treated the cultures with KCl (15 mM, 48 h) and compared them to matched untreated cultures. We found that genes upregulated by KCl had a significant intersection with those upregulated by the mock transfection. Moreover, we also found that genes upregulated by KCl had a significant intersection with genes inhibited by mmu-miR-124 and mmu-miR-434-3p. Therefore we concluded that neuronal miRNAs stabilise the neuronal transcriptome by inhibiting stress inducible genes.
Project description:RAW264.7 mouse macrophages were transfected with negative control and miR-342-3p mimics and subjected to microarray analysis 18 hours after the transfection. We used microarray to obtain global mRNA expression data of negative control and miR-342-3p mimics-transfected RAW264.7 cells. Overall design: RAW264.7 mouse macrophages were transfected with negative control and mir-342-3p mimics. The global mRNA expression patterns of negative control and miR-342-3p mimics-transfected RAW264.7 cells were analyzed on Affymetrix microarrays.