A Novel Orthotopic Mouse Model of Head and Neck Cancer and Lymph Node Metastasis
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ABSTRACT: Analysis of the genes involved in the metastasis of head and neck squamous cell carcinoma (HNSCC). We hypothesized that there would be overexpression of certain genes in the highly metastatic USC-HN1-GFP-G1 and USC-HN1-GFP-G2 cell lines compared to the parental USC-HN1-GFP cell line. We used the USC-HN1 cell line stably transfected with H2B-GFP and developed an immortal cell line (USC-HN1-GFP), which was injected into the tongues of SCID/nude mice. The metastatic cells were collected at the LN, separated and cultured, forming an immortal USC-HN1-GFP-G1 cell line. These cells were reinjected into the tongues of mice and the USC-HN1-GFP-G2 cell line was formed by the same LN collection method. The gene expression in these three cell lines were compared using Illumina microarray chips.
Project description:RNA was extracted from cells using Aurum Total RNA kit from Bio-Rad Laboratories, Inc., Hercules, CA following the manufacturerM-bM-^@M-^Ys recommendations. Gene expression profiling was performed using the BeadChip HumanHT-12 v4 Expression kit from IlluminaM-BM-., which contains 47,231 gene-probes (IlluminaM-BM-. Inc., San Diego, CA). The raw signal intensities were imported and analyzed using the GenomeStudioM-BM-. data software. After background subtraction and normalization, the signal intensity values were exported to the PartekM-BM-. genomics expression analysis suite using M-bM-^@M-^\Partek's Report Plug-inM-bM-^@M-^] option in the GenomeStudioM-BM-. software. Differentially expressed genes in the dox- versus vehicle-treated samples were identified using the M-bM-^@M-^\gene expressionM-bM-^@M-^] workflow in the PartekM-BM-. software. T47D cells hoaboring a doxycycline inducible shPIP construct were treated for 24 or 48 hours with 250 ng/mL of doxycycline and their mRNAs analyzed in biological triplicates (a total of 12 samples) using IlluminaM-bM-^@M-^Xs HumanHT-12 v4 BeadChips. The samples for each time point comprised of three samples each for doxycycline or equal volume of water as vehicle control.
Project description:Analysis of the genes involved in the metastasis of head and neck squamous cell carcinoma (HNSCC). We hypothesized that there would be overexpression of certain genes in the highly metastatic USC-HN1-GFP-G1 and USC-HN1-GFP-G2 cell lines compared to the parental USC-HN1-GFP cell line.
Project description:The transcriptional activiy of GATA1s was compared to GATA1 through gene expression analysis in a cell line model with both erythroid and megakaryocyte differentiation. G1ME cells were derived from Gata1- mouse ES cells and have both megakaryocyte and erythrocyte differentiation potential upon reconstitution of GATA-1 expression (Stachura 2006). HA-tagged full length or short GATA-1 were expressed in G1ME cells grown in TPO via retroviral transductions. The cells were sorted for GFP positivity 68 hours post-transduction and then were allowed to recover in normal growth medium for 4h. Total RNA was then isolated using RNeasy kit from Qiagen 72 hours post-transduction.
Project description:Osteosarcoma is the most common primary bone tumours of dogs. Canine osteosarcoma contains a sub-population of cancer stem cells. Here we used canine-specific microarrays to compare the global gene expression profiles of osteosarcoma stem cells to adherent cancer cells and canine mesenchymal stem cells. Canine osteosarcoma spheres were isolated by their ability to form tumourspheres. Spheres, adherent cells and mesenchymal stem cells were harvested and used for RNA extraction and hybridisation on Affymetrix microarrays (Canine 2.0). Four biological replicates of each sample were included.
Project description:MicroRNAs (miRNAs) are critical regulators of gene expression, yet much remains unknown regarding miRNA changes resulting from environmental exposures and whether they influence pathway signaling across various tissues and time. To gain knowledge on these novel topics, we set out to investigate in vivo miRNA responses to inhaled formaldehyde, an important air pollutant known to disrupt miRNA expression profiles. Rats were exposed by inhalation to either 0 or 2 ppm formaldehyde (6 hours/day) for 7 days, 28 days, or 28 days followed by a 7 day recovery. Genome-wide miRNA expression profiles and associated signaling pathways were assessed within the nasal respiratory mucosa, circulating mononuclear white blood cells (WBC), and bone marrow (BM). Male Fischer rats received nose-only inhalation exposures of 2 ppm formaldehyde. Three exposure durations were investigated: (1) 2 ppm formaldehyde exposure, 6 hours/day, for 7 days (7-day group), (2) 2 ppm formaldehyde exposure, 6 hours/day, for 28 days (28-day group), and (3) 2 ppm formaldehyde exposure, 6 hours/day, for 28 days, with a 7 day recovery period following the last exposure (28-day plus recovery group). Control (unexposed) rats were placed in nose-only exposure tubes containing room air for the same duration. After the last exposure period (or the last recovery period for the 28-day plus recovery group), animals were euthanized. RNA were assessed from sampes collected from the nasal epithelium, circulating white blood cells, and bone marrow cells. Genome-wide miRNA expression profiles were evaluated using microarrays.
Project description:Here we demonstrate the highly efficient derivation of human cortical interneurons in a NKX2.1::GFP hESC reporter line. Manipulating the timing of SHH activation yields three distinct GFP+ populations with specific transcriptional profiles, neurotransmitter phenotypes and migratory behaviors. Total RNA was isolated at day 18 of differentiation from FACS sorted NKX2.1::GFP+ cells from three varying differentiation conditions and a “No SHH” condition (no sonic hedgehog treatment) that did not contain any GFP-expressing cells (Trizol, Sigma). Samples for each group in triplicate were processed for Illumina bead arrays (Illumina HT-12) by the MSKCC genomics core facility according to the specifications of the manufacturer.
Project description:Background: Cancers are commonly characterised by hypoxia and also by global reductions in the levels of mature microRNAs. We have examined the hypothesis that hypoxia might mediate this reduction through repressive effects on microRNA biogenesis proteins. Methods: Breast cancer cell lines were exposed to hypoxia and manipulations of hypoxia inducible factor (HIF) and HIF hydroxylase activity. The effects of hypoxia on the mRNA and protein levels of enzymes involved in microRNA biogenesis (Dicer, Drosha, TARPB2, DCGR8, XPO5) was determined by RT PCR and immunoblotting. The effect of hypoxia on microRNAs was determined with microarray studies, RT PCR and reporter assays. Results: In breast cancer lines there was significant reduction of Dicer mRNA and protein levels in cells exposed to hypoxia. This effect was independent of HIF but dependent on the HIF hydroxylase PHD2 and was partly mediated by feedback effects via microRNAs. Furthermore, several other proteins with critical roles in microRNA biogenesis (Drosha, TARBP2 and DCGR8) also showed significant and co-ordinated repression under hypoxic conditions. Despite these substantial alterations no, or modest, changes were observed in mature microRNA production Conclusion: These observations provide further and important interfaces between oxygen availability and gene expression and a potential mechanistic explanation for the reduced levels of microRNAs observed in some cancers. They provide further support for the existence of feedback mechanisms in the regulation of the microRNA biogenesis pathway and the relative stability of microRNAs. MCF7 cells were treated with three different conditions. Treatment-1: MCF7 cells were exposed to hypoxia (0.1% O2) for 48 h and harvested for RNA extraction (n=3). Treatment-2: MCF7 cells were exposed to normoxia for 48 h and harvested for RNA extraction (n=3). Treatment-3: Dicer inhibition in MCF7 cells by transient transfection of siRNAs targeting Dicer. Cells were transfected with 20 nM siRNA duplexes (Shanghai GenePharma Co., Ltd, China), using Lipofectamine 2000 reagent (Invitrogen) following the manufacturerM-bM-^@M-^Ys protocol. A second transfection was carried out after 24 h following the same protocol. Cells were harvested 24 h after the second transfection and used for RNA extraction (n=3). RNA integrity was assessed using the Agilent 2100 Bioanalyzer. Affymetrix miRNA 3.1 Array Strip was used for RNA analysis. This array consisted probe sets unique to human mature and pre-miRNA hairpins. A detailed protocol can be found in the miRNA 3.1 Array Strips technical manual (Affymetrix). In summary, 100-300 ng of total RNA was used to synthesise double stranded cDNA using random hexamers. The cDNA was then amplified to produce antisense cRNA, which was then reverse transcribed in a second cycle of cDNA synthesis. The second cycle incorporates dUTP into the cDNA sequence, which allows it to be fragmented using uracil DNA glycosylase and apurinic/apyrimidic endonuclease I. Following biotinylation, these fragments were hybridised overnight to a Affymetrix miRNA 3.1 array. The arrays were then washed, stained using a fluorescently-labelled antibody, and scanned using a high-resolution scanner. Intensity data were analysed using PartekM-BM-. software (Partek Inc.). Data were normalised by quantile normalisation and log 2 transformed. Differential expression was determined by ANOVA and corrected for false discovery.