Comment[ArrayExpressAccession]	E-MTAB-1802
MAGE-TAB Version	1.1
Investigation Title	IGR_MESSAI_CCRCC_HIF2
Comment[Submitted Name]	IGR_MESSAI_CCRCC_HIF2
Experiment Description	The aim of the present study was to investigate the potential role of HIF-2a in regulating RCC susceptibility to natural killer (NK) cell-mediated killing. We demonstrated that the RCC cell line 786-O with mutated VHL was resistant to NK-mediated lysis as compared to the VHL corrected cell line (WT7). This resistance was independent of immunological synapse formation and NK ligand expression but was found to be reliant on HIF-2a stabilization in 786-0 cells. In order to elucidate the molecular basis of HIF-2a-induced impairment of NK-mediated killing, we performed global gene analysis using DNA microarray. Three candidate genes (ANGPTL4, ADM and ITPR1) were selected on the basis of their fold change and their involvement in cell death and survival. SiRNA targeting of these genes revealed that only ITPR1 silencing resulted in a significant increase of 786-O susceptibility to NK-mediated lysis. Using gene silencing of HIF2-a and chromatin immunoprecipitation assay, we showed for the first time that ITPR1 was a direct novel target of HIF2-a. Notably, a strong and significant correlation was found between ITPR1and HIF2-a staining in RCC patients. Transcriptional profiling of ITPR1silenced RCC cells indicated that several important genes involved in cell survival and apoptosis were differentially regulated. Using ITPR1 silenced Renca cells, we further found that ITPR1 was involved in the control of tumor progression. Moreover ITPR1 targeting combined with NK depletion significantly enhanced tumor growth as compared to ITPR1 knocked down Renca xenografts.  Our data provide insights into the link between HIF-2a, ITPR1-related pathway and natural immunity and suggest a role of the HIF2/ITPR1 axis in regulating RCC cell survival.
Experimental Design	cellular modification design	dye swap design	co-expression_design
Comment[AEExperimentType]	transcription profiling by array
Comment[AEExperimentDisplayName]	Transcription profiling by array of 786-0, PRC3 and WT7 renal carcinoma cell to investigate the potential role of HIF-2a in regulating renal cell carcinoma susceptibility to natural killer (NK) cell-mediated killing
Experimental Factor Name	SIRNA	cell line
Experimental Factor Type	rnai	cell line
Quality Control Type	dye swap replicate
Quality Control Term Source REF	EFO
Public Release Date	2014-01-11
Person Last Name	Meurice
Person First Name	Guillaume
Person Mid Initials	none
Person Email	guillaume.meurice@igr.fr
Person Phone	 +33(0)142114211
Person Address	114 rue Edouard Vaillant, 94805, Villejuif Cedex
Person Affiliation	Institut Gustave Roussy
Person Roles	submitter
PubMed ID
Publication Author List	Yosra MESSAI, Muhammad Zaeem NOMAN, Meriem HASMIM, Bassam JANJI, Marie BOUTET, Véronique BAUD, Katy BILLOT, Arash NANBAKHSH, Thouraya BEN SAFTA, Catherine RICHON, Sophie FERLICOT, Emmanuel DONNADIEU, Sophie COUVE, Florence ORLANDUCCI, Laurence ALBIGES, Daniel OLIVE, Bernard ESCUDIER and  Salem CHOUAIB
Publication Title	Type 1 inositol tri-phosphate receptor, a novel HIF-2a target, impairs clear cell renal cell carcinoma cell susceptibility to Natural Killer-mediated lysis
Publication Status	in preparation
Protocol Name	P-MTAB-33976	P-MTAB-33977	P-MTAB-33978	P-MTAB-33979	P-MTAB-33980	P-MTAB-33981	P-MTAB-33982
Protocol Type	growth protocol	treatment protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning and feature extraction protocol	normalization data transformation protocol
Protocol Description	786-0, PRC3 and WT7 are renal carcinoma cells obtained from Dr. Kaelin WG Jr. 786-0 cells were maintained in DMEM/F12 medium supplemented with 10% heat-inactivated FCS, 1% penicillin-streptomycin, and 1 mM sodium pyruvate (Life Technologies, Cergy Pontoise, France). WT7 and PRC3 cells were derived from 786-O by stable transfection with pRC-HAVHL or the empty vector respectively and maintained in 786-O medium added with 1 mg/ml of neomycin as selecting agent. NK HD cells were purified from healthy donor peripheral blood mononuclear cells (PBMC) using CD56 positive selection (STEMCELL technologies). They were cultured in RPMI supplemented with 10% human serum, 1% penicillin-streptomycin, 1 mM sodium pyruvate, and IL2 (300U/ml, Institut Gustave Roussy). NKL and NK-92 natural killer cell lines were maintained in RPMI supplemented with 10% heat-inactivated FCS, 1% penicillin-streptomycin, and 1mM sodium pyruvate (Life Technologies, Cergy Pontoise, France). RENCA murine renal carcinoma cell line was originally obtained from a tumor that arose spontaneously in the kidney of BALB/c mice (25). It was maintained in RPMI medium supplemented with 10% heat-inactivated FCS, 1% penicillin-streptomycin, and 1 mM sodium pyruvate (Life Technologies, Cergy Pontoise, France).	Gene silencing of HIF-2a and ITPR1 was performed using predesigned sequence-specific small interfering RNA purchased from SIGMA Aldrich. Briefly, 8 X 106 cells were electroporated twice in 48 h in serum-free medium with 20 mM siRNA in an EasyJect Plus electroporation system (EquiBio; 260 V, 450mF). 	Total RNA was extracted using TRIzol solution (Invitrogen). 	Probe synthesis and labeling were performed by using the two-color Agilent labeling kit (Low Input Quick Amp Labeling Kit 5190-2306)	Hybridization were then performed on microarray using 825 ng of each linearly amplified cRNA labelled Cy3 or Cy5 sample, following the manufacturer protocol (Agilent SureHyb Chamber; 1650ng of labeled extract; duration of hybridization of 17 hours; 40 uL per array; Temperature of 65C). 	After washing in acetonitrile, slides were scanned by using an Agilent G2565 C DNA microarray scanner with defaults parameters (100 PMT, 3 um resolution, at 20C in free ozone concentration environment. Microarray images were analysed by using Feature Extraction software version (10.7.3.1) from Agilent technologies. Defaults settings were used.	Raw data files from Feature Extraction were imported into R with LIMMA (Smyth, 2004, Statistical applications in Genetics and molecular biology, vol3, N1, article3), an R package from the Bioconductor project, and processed as follow: gMedianSignal and rMedianSignal data were imported, controls probes were systematically removed, and flagged probes (gIsSaturated, gIsFeatpopnOL, gIsFeatNonUnifOL, rIsSaturated, rIsFeatpopnOL, rIsFeatNonUnifOL) were set to NA. Intra-array normalization was performed by a loess normalization, followed by a quantile normalization of both Cy3 and Cy5 channel.
Protocol Term Source REF	EFO	EFO	EFO	EFO	EFO	EFO	EFO
Term Source Name	EFO
Term Source File	http://www.ebi.ac.uk/efo
SDRF File	E-MTAB-1802.sdrf.txt