Response of quiescent human fibroblasts to miR-22 overexpression
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ABSTRACT: Quiescent human fibroblasts (2091) were transfected with 100nM mature miR-22 RNA duplexes. Cells were collected 24 hours after miR-22 transfection.
Project description:Quiescent human fibroblasts (2091) were transfected with 100nM mature miR-22 RNA duplexes. Cells were collected 24 hours after miR-22 transfection. 4 biological replicates were run, one for each of the 4 dual channel arrays. Each biological replicate included a miR-22 transfection sample and a control transfection sample
Project description:To examine the expression patterns of human miR-22-responsive transcripts in estrogen receptor alpha positive cell line MCF7, we transfected miR-22 duplex or negative control RNA duplex into MCF7 cells. Gene expression patterns were then evaluated using Affymetrix Human Genome U133 Plus 2.0 Array microarrays. Keywords: comparision of expression patterns in MCF7 cells with or without human miR-22 overexpression.
Project description:Quiescent human fibroblasts (2091 and Wi-38) were stimulated with different growth factors and serum. Cells were collected at 6 different time points followed by global transcriptional profiling. Keywords: time course, growth factor response, cell line comparison
Project description:To examine the expression patterns of human miR-22-responsive transcripts in estrogen receptor alpha positive cell line MCF7, we transfected miR-22 duplex or negative control RNA duplex into MCF7 cells. Gene expression patterns were then evaluated using Affymetrix Human Genome U133 Plus 2.0 Array microarrays. Keywords: comparison of expression patterns in MCF7 cells with or without human miR-22 overexpression. Total RNA was collected from two groups MCF7-NC (MCF7 cells transfected with negative control RNA duplex) and MCF7-miR (MCF7 cells transfected with miR-22 duplex) and hybridized to Affymetrix microarrays. Each group has three repeat samples.
Project description:Vitamin D deficiency is associated with high risk of colon cancer and a variety of other diseases. The active vitamin D metabolite 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) regulates gene transcription via its nuclear receptor (VDR), and posttranscriptional regulatory mechanisms of gene expression have also been proposed. We have identified microRNA-22 (miR-22) and several other miRNA species as 1,25(OH)2D3 targets in human colon cancer cells. Remarkably, miR-22 is induced by 1,25(OH)2D3 in a time-, dose-, and VDR-dependent manner. In SW480-ADH and HCT116 cells, miR-22 loss-of-function by transfection of a miR-22 inhibitor (anti-miR-22) suppresses the effect of 1,25(OH)2D3. Additionally, miR-22 inhibition increases cell migration per se and decreases the antimigratory effect of 1,25(OH)2D3 in both cell types. In silico analysis shows a significant overlap between genes suppressed by 1,25(OH)2D3 and miR-22 putative target genes. Consistently, miR-22 inhibition abrogates the reduction by 1,25(OH)2D3–mediated suppression of NELL2, OGN, HNRPH1, and NFAT5 genes. In 39 out of 50 (78%) human colon cancer patients, miR-22 expression was found lower in the tumor than in the matched normal tissue and correlated directly with that of VDR. Our results indicate that miR-22 is induced by 1,25(OH)2D3 in human colon cancer cells and it may contribute to its antitumor action against this neoplasia.
Project description:Transient transfection of miRNA mimics are widely used to alter microRNA expression in cells. The goal of this study is to perform small RNA deep sequencing analysis of HeLa cells transfected with miR-17~92 cluster and address potential source of non-specific effects caused by this approach. HeLa cells were cultured as adherent cells. Equal molar concentration of miRNA mimics of miR-17~92 (100nM, 16.7nM each) were transfected and harvedsted at 6h post-transfection for small RNA deep sequencing analysis.
Project description:To examine the genome-wide gene regulation due to the common seed sequence of the members of miR-302/3272/3273/520 family miRNAs and dsEcad640, we performed microarray profiling of gene expression by the transfection of chemically synthesized dsEcad640, miR-302a,miR-372, miR-373, miR-520c,and miR-520f duplexes into PC-3 cells. Chemically synthesized dsEcad640, miR-302a, miR-372, miR-373, miR-520c,and miR-520f duplexes were transfected into PC-3 cells. At 24 hours after transfection,the total RNA was extracted using the RNeasy Mini Kit (Qiagen). As a control, mock transfected cells treated with transfection reagent without siRNA and miRNA were used.Transfection was carried out four times independently, and the RNA quality was examined using Bioanalyzer (Agilent). Mixed RNA samples (150 ng total) of four experiments were used for the microarray analysis. After cDNA was synthesized with the Agilent One Color Spike Mix Kit, cRNA was prepared using the Quick Amp Labeling Kit (Agilent), labeled with Cy-3, and purified with the RNeasy Mini Kit (Qiagen).The cRNA was hybridized against the Agilent Whole Human Genome Microarray (4 × 44 K multipack format) at 65°C for 17 hours, then washed with Gene Expression Wash Buffer 1 and Wash Buffer 2 (Agilent) in 0.005% Triton X-102. The microarray slide was scanned using the DNA Microarray Scanner (Agilent) and quantified using Feature Extraction Software (Agilent).
Project description:To further development of our gene expression approach to microRNA-26a over-expression, we have employed whole mRNA microarray expression profiling as a discovery platform. Hela cells at 70–80% confluence in 6-well plates were transfected using Lipofectamine 2000 (Invitrogen). Negative control mimics or miR-26a mimics (100nM) were transfected in each well. Cell extracts were prepared 48 h after transfection, total RNA was checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).
Project description:Myc ChIP in HeLa cells and 2091 fibroblasts Keywords: ChIP-chip HeLa cells, quiescent 2091 fibroblasts, and serum stimulated 2091 fibroblasts were used for Myc ChIP reactions.
Project description:We transfected C2C12 cells with 100nM of scrambled siRNA scramble and an siRNA directed against Fndc1. Cells were harvested at 48h post-transfection and RNAseq was performed with polyA selection