Response of quiescent human fibroblasts to miR-22 overexpression
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Vishy Iyer
PROVIDER: E-GEOD-42788 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Quiescent human fibroblasts (2091) were transfected with 100nM mature miR-22 RNA duplexes. Cells were collected 24 hours after miR-22 transfection.
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2014-07-31 | E-GEOD-45817 | biostudies-arrayexpress
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2012-05-31 | E-GEOD-34482 | biostudies-arrayexpress
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).
2011-10-13 | E-GEOD-32999 | biostudies-arrayexpress
Project description:Gene expression profile following transfection with miR-503, miR-103, or miR-494 mature duplex Examination of mRNA levels in HeLa cells following transfection of miR-503, miR-103, or miR-494 mature duplex, control siRNA against GFP, or mock transfection (lipofectamine 2000 alone)
2015-02-04 | E-GEOD-64364 | biostudies-arrayexpress
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2014-09-09 | E-GEOD-59458 | biostudies-arrayexpress
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2012-03-26 | E-GEOD-34242 | biostudies-arrayexpress
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.
2012-12-01 | E-GEOD-17508 | biostudies-arrayexpress
Project description:B-cell acute lymphoblastic leukemia (B-ALL) is often associated with chromosomal translocations leading to the deregulation of proto-oncogenes. MicroRNAs can also be affected by chromosomal alterations and thus contribute to carcinogenesis. The microRNA miR-125b-1 is over-expressed in B-ALL cases with the t(11;14)(q24;q34) translocation, therefore we sought to determine the role of this microRNA in B-cell fate. We used murine pre-BI cells alongside murine and human leukemic B-cell lines to show that miR-125b expression enhances proliferation by targeting Bright/ARID3a, an activator of immunoglobulin heavy-chain transcription. Accordingly, this target gene was down-regulated in B-ALL patients with the t(11;14)(q24;q34) translocation. Repression of Bright/ARID3A blocked differentiation and conferred a survival advantage to Ba/F3 cells under IL3 starvation. In addition, over-expression of miR-125b protected pre-BI and leukemic B-cell lines from apoptosis through blockade of caspase activation via a mechanism that was independent of p53 and BAK1. In summary, miR-125b can act as an oncogene in B-ALL by targeting ARID3a and mediating its repression, thus leading to a blockage in differentiation, increased proliferation and inhibition of apoptosis. To identify specific targets of mir-125b, we overexpressed miR-125b as well as a control miRNA in 70Z/3 and 18-81 pre-B leukaemia cells. Chemically synthesized miRNA duplexes, called pre-miR-125b and miR-Neg, were purchased from Ambion. The cells were transfected with pre-miRNA at a final concentration of 10 nM using Lipofectamin RNAi MAX (Invitrogen) according to the manufacturer’s instructions. The medium was replaced 8 hours after transfection. RNA samples were harvested at 48 hours post-transfection. 2 independent experiments were carried out for a total of 8 samples labeled with Cy3 dye (one color design).
2012-06-20 | E-GEOD-30647 | biostudies-arrayexpress
Project description:Presence of BM-infiltrating neuroblastoma (NB) cells occurs in about 50% of patients affected by this peculiar pediatric cancer. Children aged more than 18 months at diagnosis with metastatic NB are high-risk patients with 25-30% overall survival at 5 years, despite multimodal aggressive therapy. Little is known about the metastatic process in NB patients. Hereby, we focused on miRNA expression profiles of BM-infiltrating as compared to those of primary NB tumors. For this purpose, we analyzed 22 BM-infiltrating NB cells, 22 primary NB tumors, and 4 paired BM-infiltrating and primary tumor specimens, all from patients with metastatic disease and aged > 18 months at diagnosis. Statistical analysis indicated that in human BM-infiltrating cells miR-659-3p was down-modulated as compared to primary tumors, in all sets of samples. Functional studies in HTLA and SH-SY5Y NB cell lines by miR-659-3p mimic and inhibitors demonstrated that miR-659-3p regulates the expression of the transcription factor CNOT1, that in turn regulates the expression of AU-rich element (ARE)-containing target genes AKT3, BCL2, THSB2 and CYR61, all belonging to the focal adhesion pathway. Indeed, CNOT1 expression was found significantly higher, whereas that of the ARE-containing target genes was significantly lower in BM-infiltrating cells than in primary tumors. Our findings about a role of the focal adhesion pathway, regulated by miR-659-3p through CNOT1, in NB metastatic process are intriguing for the development of new therapeutic strategies aimed to interrupt the metastatic process. To evaluate the effect of miR-659-3p up- and down-regulation on gene expression, HTLA-230 and SH-SY5Y cells were transfected with specific miR-659-3p mirVana™ mimic and inhibitor, respectively (Ambion, Life Technologies, Carlsbad, CA, USA, catalog# MC and MH 11582). Samples transfected with mirVana™ miRNA Mimic Negative Control #1 and mirVana™ miRNA Inhibitor Negative Control #1 were used as reference conditions. Transfection was performed at 20 nM miRNA concentration in OptiMEM© medium (Sigma-Aldrich), using Lipofectamin RNAiMAX© (Life Technologies), according to manufacturer’s protocol. Cells were then cultured for 48 hours, checked for viability and processed for total mRNA and miRNA extraction, as described below. Transfection experiments were performed twice with similar results. HTLA-230 (kindly donated by Dr E. Bogenman, Los Angeles, USA) and SH-SY5Y (purchased from Banca Biologica and Cell Factory, Genoa, Italy) NB cell lines were cultured in RPMI 1640 medium supplemented with 10% heat inactivated FCS, 50 mg/ml streptomycin, 50 mg/ml penicillin and 2 mM glutamine (Sigma-Aldrich, Milan, Italy). The HTLA-230 cells present MYCN amplification and SH-SY5Y cells have normal MYCN status. Both cell lines were checked for MYCN amplification, morphology, proliferation rate and mycoplasma contamination, after thawing and within four passages in culture. To evaluate the effect of miR-659-3p up- and down-regulation on gene expression, 1x106 HTLA-230 and SH-SY5Y cells were transfected with specific miR-659-3p mirVana™ mimic and inhibitor, respectively (Ambion, Life Technologies, Carlsbad, CA, USA, catalog# MC and MH 11582). Samples transfected with mirVana™ miRNA Mimic Negative Control #1 and mirVana™ miRNA Inhibitor Negative Control #1 were used as reference conditions. Transfection was performed at 20 nM miRNA concentration in OptiMEM© medium (Sigma-Aldrich), using Lipofectamin RNAiMAX© (Life Technologies), according to manufacturer’s protocol. Cells were then cultured for 48 hours, checked for viability and processed for total mRNA and miRNA extraction, as described below. Transfection experiments were performed twice with similar results.
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