MicroRNA profiling using microarrays of two human metformin-treated non-small cell lung cancer cell-lines
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ABSTRACT: To examine the effect of metformin on lung cancer biology, human lung H226 and H1299 squamous cell carcinoma cell-lines were grown in RPMI-1640 medium with 10% v/v fetal bovine serum and with or without 15 uM metformin hydrochloride for 7-8 days. Medium (with any metformin) was replaced every two days. Paired cultures with and without metformin were grown and maintained in parallel. Three separate paired cultures, all seeded with same stock of frozen cells, were grown.
Project description:To examine the effect of metformin on lung cancer biology, human lung A549 adenocarcinoma, H460 large cell carcinoma, and H226 and H1299 squamous cell carcinoma cell-lines were grown in RPMI-1640 medium with 10% v/v fetal bovine serum and with or without 15 uM metformin hydrochloride for 7-8 days. Medium (with any metformin) was replaced every two days. Paired cultures with and without metformin were grown and maintained in parallel. Three separate paired cultures, all seeded with same stock of frozen cells, were grown. Cells, within 15%-95% confluence range, were harvested by scraping. Total RNA from cells was prepared using Norgen Biotek® Total RNA Isolation kit (with on-column DNAse I treatment). All RNA integrity number (RIN) values were greater than 8.2.
Project description:Sepsis induces systemic stress by augmenting inflammatory and pro-coagulant responses resulting in microvascular dysfunction and end organ failure, events modulated by the Protein C pathway. MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional regulation of gene transcription yet their role in sepsis remains poorly defined. We hypothesized that aPC selectively alters the expression of specific miRNAs implicated in protection of hepatic function during septic shock. Male Sprague-Dawley rats underwent sham surgery or cecal ligation and puncture (CLP). Twenty-four later, animals were randomized and treated with aPC (1mg/kg) or vehicle (0.9% (w/v) saline) via an indwelling venous catheter at 12 hour intervals for 24 hours. Gene array was performed on hepatic RNA to determine miRNA expression, and predicted mRNA targets determined using a bioinformatics approach. Of 351 rat miRNAs examined by microarray hybridization, 17 were highly expressed during sepsis and restored to basal levels after aPC treatment. In silico analysis identified 9 miRNAs significantly regulating target genes of the focal adhesion pathway. These data suggest aPC treatment coordinates beneficial cytoprotective effects during sepsis by modulating miRNA expression. While translational effects remain to be fully elucidated in a clinical setting, we demonstrate herein the potential experimental and computational benefits for use of microRNA analysis in sepsis. 12 samples were analyzed. Microarray experiments were performed, in which liver tissue was harvested from variuous groups (Sham+Vehicle, Sham+aPC, CLP+Vehicle, CLP+aPC; n=3/group) and pooled.
Project description:Advances in pluripotent stem cell and reprogramming technologies have given hope of generating hematopoietic stem cells (HSC) in culture. To succeed, greater understanding of the self-renewing HSC during human development is required. We discovered that glycophosphatidylinositol-anchored surface protein GPI-80 (Vanin 2) defines a distinct subpopulation of human fetal hematopoietic stem/progenitor cells (HSPC) with self-renewal ability. CD34+CD90+CD38-GPI-80+ HSPC were the sole population that maintained proliferative potential and undifferentiated state in bone marrow stroma co-culture, and engrafted in immunodeficient mice. GPI-80 expression also enabled tracking of HSC migration between human fetal hematopoietic niches. The most highly enriched surface protein in GPI-80+ HSPC as compared to their progeny was Integrin alpha-M (ITGAM), which in leukocytes cooperates with GPI-80 to support migration. Knockdown of either GPI-80 or ITGAM was sufficient to perturb undifferentiated HSPC in stroma co-culture. These findings indicate that human fetal HSC utilize common mechanisms with leukocytes for cell-cell interactions governing HSC self-renewal. We used microarrays to identify genes enriched in the GPI-80+ hematopoietic stem and progenitor population in fetal liver. RNA was extracted from enriched fetal liver hematopoietic stem and progenitor cells, and downstream progenitors, for comparison based on Affymetrix arrays.
Project description:Advances in pluripotent stem cell and reprogramming technologies have given hope of generating hematopoietic stem cells (HSC) in culture. To succeed, greater understanding of the self-renewing HSC during human development is required. We discovered that glycophosphatidylinositol-anchored surface protein GPI-80 (Vanin 2) defines a distinct subpopulation of human fetal hematopoietic stem/progenitor cells (HSPC) with self-renewal ability. CD34+CD90+CD38-GPI-80+ HSPC were the sole population that maintained proliferative potential and undifferentiated state in bone marrow stroma co-culture, and engrafted in immunodeficient mice. GPI-80 expression also enabled tracking of HSC migration between human fetal hematopoietic niches. The most highly enriched surface protein in GPI-80+ HSPC as compared to their progeny was Integrin alpha-M (ITGAM), which in leukocytes cooperates with GPI-80 to support migration. Knockdown of either GPI-80 or ITGAM was sufficient to perturb undifferentiated HSPC in stroma co-culture. These findings indicate that human fetal HSC utilize common mechanisms with leukocytes for cell-cell interactions governing HSC self-renewal. We used microarrays to identify genes enriched in the CD90+ hematopoietic stem and progenitor population in fetal liver. RNA was extracted from enriched fetal liver hematopoietic stem and progenitor cells, and downstream progenitors, for comparison based on Affymetrix arrays.
Project description:H226 cells transfected with DDX56 siRNA and control siRNA were incubated for 48 hours and total RNA isolated and used for the microarray analysis using Affymetrix Human U133 Plus 2.0 Arrays. Slides were scanned by GeneChip® Scanner 3000 (Cat#00-00212, Affymetrix, Santa Clara, CA, US) and Command Console Software 4.0 (Affymetrix, Santa Clara, CA, US) with default settings.
Project description:AoSMC and FB were cultured and exposed to transforming growth factor beta1 (TGFb1) prior to the exon array analysis The objective was to study the TGFb responsivness, at the gene expression level, in AoSMC and FB form BAV and TAV patients 54 samples in total were generated from biopsies obtained from 23 different individuals. We performed paired analyses to compare the un-treated (c) versus the TGFb1 (t1) treated cells, for each patient
Project description:To develop and validate novel multigene signatures to facilitate individualized treatment of TNBC patients By integrating expression profiles of messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs). We analysed 165 TNBC samples and 33 paired normal breast tissues using transcriptome microarrays. Tumor-specific mRNAs and lncRNAs were identified and correlated with patientsâ?? recurrence-free survival (RFS). Using Cox regression model, we built two multigene signatures incorporating mRNAs and lncRNAs. The prognostic and predictive accuracy of the signatures were tested in a training set of 165 TNBC patients and validated in another 101 TNBC patients.
Project description:The aim of the dataset was to study on a genome-wide level the effect of GTPase of the human immune associated protein 4 (GIMAP4) knockdown on the gene expression of resting T cells and immediately after T cell activation and Th1(Act+IL12) polarizing conditions of human cord blood-derived CD4+ T cells. Total RNA from GIMAP4 siRNA-treated cord blood CD4+ T cells 48 hours after siRNA treatment, before TCR activation and corresponding cells cultured for 2h in TCR activating (antiCD3+antiCD28) plus IL-12 conditions was compared to total RNA from nonspecific control siRNA-treated cells. Samples from 3 biological replicates were analysed.
Project description:In this study we showed that rat XEN cells grown in the presence of a GSK3 inhibitor exhibited enhanced formation of cell contacts and decreased motility. In contrast, treatment with forskolin induced the PE formation and epithelial-mesenchymal transition (EMT) in rat XEN cells. Using microarray and real-time PCR assays, we found that VE versus PE formation of rat XEN cells was correlated with change in expression levels of VE or PE marker genes. Similar to forskolin, EMT was prompted upon treatment of rat XEN cells with recombinant parathyroid hormone related peptide (PTHRP), an activator of the cAMP pathway in vivo. Taken together, our data suggest that rat XEN cells are PrE-like cells. The activation of Wnt pathway in rat XEN cells leads to the acquisition of VE characteristics, whereas the activation of the PTHRP/cAMP pathway leads to EMT and the formation of PE. Rat XEN cells were cultured in four different conditions with 3 parallels for each condition: (1-3) without treatments control, (4-6) 2 days treated with CHIR alone, (7-9) 1 day treated with CHIR and 1 day with both CHIR and forskolin, and (10-12) 1 day treated with forskolin alone. In all samples on DAY1 cells were plated, at DAY2 cells were cultured in usual conditions (DMEM F12 medium and 10% Fetal Bovine Serum, both from Sigma) and inhibitor of GSK3 kinase 3 μM CHIR99021 (Axon Medchem) was added in samples 4-9. On DAY3 all cells were cultured in medium with 0.1% serum to exclude the influence of serum and in CONTROL (1-3) cells were cultured without experimental treatments, in CHIR (4-6) cells were further cultured with 3 μM CHIR99021, in CHIR plus FORSKOLIN cells were cultured with 3 μM CHIR99021 and 10μM Forskolin (Sigma), in FORSKOLIN cells were cultured for 1 day with 10 μM Forskolin. On DAY4 RNA was isolated.