Unknown,Transcriptomics,Genomics,Proteomics

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Inhibitors of Differentiation-1 Promotes Transformation of Human Papillomavirus Type 16-immortalized Cervical Epithelial Cells

ABSTRACT: Our previous study implied a correlation of inhibitors of differentiation-1 (Id-1) to cervical cancer development. However, how Id-1 contributes to cervical carcinogenesis is unknown. In the present study, we investigated the role of Id-1 in transforming cervical cells with an in vitro transformation model. The human papillomavirus (HPV) immortalized cervical epithelial cells (H8) were successfully transformed by exposure to the carcinogen N-nitrosopyrrolidine (NPYR). The results showed that both Id-1 RNA and protein expression were significantly increased in transformed H8 cells, suggesting a possible role of Id-1 in cervical cell transformation. Ectopic expression of Id-1 in H8 cells potentiated NPYR-induced cell transformation. In contrast, silencing of Id-1 suppressed NPYR-induced H8 cell transformation. A cDNA microarray assay was performed, which identified suggested potential cell signaling pathways for NPYR-induced H8 cell transformation. The results suggest that Id-1 plays an oncogenic role in the cervix, which sheds light on cervical cancer development and implies potential target for cervical cancer prevention and therapy. Id-1-shRNA H8 and the control pGIPZ-H8 cells, with or without NPYR treatment, were used for cDNA microarray assay. Total RNA was extracted by using TRIZOL Reagent (Life Technologies, Carlsbad, CA, US) following the manufacturer's instructions. Total RNA was further purified by RNeasy micro kit (QIAGEN, GmBH, Germany) and RNase-free DNase Set (QIAGEN, GmBH, Germany). The RNAs were amplified, labeled and purified by using GeneChip 3' IVT Express Kit (Affymetrix, Santa Clara, CA, US) following the manufacturer's instructions. Array hybridization and wash were performed using GeneChipÂ® Hybridization, Wash and Stain Kit (Affymetrix, Santa Clara, CA, US) in a Hybridization Oven 645 (Affymetrix) and Fluidics Station 450 (Affymetrix) following the manufacturer's instructions. The slides were scanned with GeneChipÂ® Scanner 3000 (Affymetrix) using the Command Console Software 3.1 (Affymetrix) with default settings. Raw data were normalized by using the MAS 5.0 algorithm of GeneSpring Software 11.0 (Agilent Technologies, Santa Clara, CA, US).

ORGANISM(S): Homo sapiens

SUBMITTER: Lingxia Xie

PROVIDER: E-GEOD-46842 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Gastric cancer (GC) is one of the most common cancer worldwide. Specific and reliable molecular markers are limited; it is critical to identify new biomarkers for GC to aid in early diagnosis, treatment strategy, and prognosis evaluation. Microarray technology makes it possible to measure the expression levels of thousands of genes, and identifying meaningful and useful molecular targets from these large data. Total RNA was extracted from 10 pairs of GC tissue and adjacent non-tumor mucosa using TRIzolÂ® reagent (Invitrogen, Carlsbad, CA, US) following the manufacturer's instructions. The RNA integrity number (RIN), detected by an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, US), was used to determine the RNA integrity. Total RNA meeting the specified quality criteria (RIN â¥ 7.0 and 28S/18S â¥ 0.7) were further purified using an RNase-Free DNase Set (Qiagen, Hilden, Germany) and an RNeasyÂ® Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Purified total RNA was then used for obtaining biotin-labeled cRNA by applying the GeneChipÂ® 3'IVT Express Kit (Affymetrix, Santa Clara, CA, US), according to the manufacturer's instructions. Then, the GeneChipÂ® Hybridization, Wash, and Stain Kit (Affymetrix, Santa Clara, CA, US) was used for performing the array hybridization and wash, with the use of a GeneChipÂ® Hybridization Oven 645 (Affymetrix, Santa Clara, CA, US) and a GeneChipÂ® Fluidics Station 450 (Affymetrix, Santa Clara, CA, US). All arrays were scanned using a GeneChipÂ® Scanner 3000 (Affymetrix, Santa Clara, CA, US) and Command Console Software 3.1 (Affymetrix, Santa Clara, CA, US) with the default settings. Raw data were normalized by the MAS 5.0 algorithm in GeneSpring Software 11.0 (Agilent Technologies, Santa Clara, CA, US). The significance analysis of microarray (SAM) was used to identify genes that were differentially expressed between the GC and normal tissues. Genes were considered to be differentially expressed when the Tumor versus Normal signal log ratio values were â¥2 or â¤0.5.

Project description:During colonization in the host gastrointestinal tract, the enteric bacteria Campylobacter jejuni is exposed to a variety of signaling molecules including the catecholamine hormones epinephrine (Epi) and norepinephrine (NE). NE has been determined to stimulate the growth of C. jejuni as well as increase its pathogenicity. To investigate the mechanisms of NE or Epi on the biology of C. jejuni, the global gene expression profiles of C. jejuni NCTC 11168 cultured in iron-restricted medium were analyzed in response to NE or Epi. Totally, 183 and 156 genes were differentially expressed by NE and Epi respectively, with 102 differentially expressed genes common between the two treatments. These genes are involved in diverse cellular functions including iron uptake systems, motility, virulence, oxidative stress response, nitrosative stress tolerance, enzyme metabolism, DNA repair and metabolism and ribosomal protein biosynthesis. Adherence to and invasion of Caco-2 cells by C. jejuni were enhanced upon exposure to NE or Epi. These results indicated that NE and Epi have similar effects on the gene expression of C. jejuni and that the effects on gene expression may contribute to elucidate the mechanisms on interaction between host and C. jejuni. Transcriptional profiles were analyzed using microarray to compared the epinephrine (Epi) or norepinephrine (NE) treated and untreated Campylobacter jejuni NCTC 11168. NE or Epi treated and untreated cultures of C. jejuni NCTC 11168 were collected at the mid-log phase (?36 h cultures). Three (for NE or Epi treated culture) or four (for untreated control culture) independent biological replicates were performed. Total RNA was extracted using RiboPure™-Bacteria Kit (Ambion, Life Technologies) according to the manufacturer’s instructions. The quality and quantity of total RNA were determined by an Agilent Bioanalyzer 2100. Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color, following the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (QIAGEN, GmBH, Germany). Each Slide was hybridized with 600ng Cy3-labeled cRNA using Gene Expression Hybridization Kit (Agilent technologies, Santa Clara, CA, US) in Hybridization Oven, according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes with Gene Expression Wash Buffer Kit (Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions. Slides were scanned by Agilent Microarray Scanner with default settings, Dye channel: Green, Scan resolution=5?m, PMT 100%, 10%, 16bit. Data were extracted with Feature Extraction software 10.7. Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0. The genes with fold change ? 1.5 and P < 0.05 were selected as differentially expressed.

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Inhibitors of Differentiation-1 Promotes Transformation of Human Papillomavirus Type 16-immortalized Cervical Epithelial Cells

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