An in vivo hypoxia metagene identifies the novel hypoxia inducible factor target gene SLCO1B3.
A hypoxia-associated gene signature (metagene) was previously derived via in vivo data-mining. In this study, we aimed to investigate whether this approach could identify novel hypoxia regulated genes. From an initial list of nine genes, three were selected for further study (BCAR1, IGF2BP2 and SLCO1B3). Ten cell lines were exposed to hypoxia and interrogated for the expression of the three genes. All three genes were hypoxia inducible in at least one of the 10 cell lines with SLCO1B3 induced in seven. SLCO1B3 was studied further using chromatin immunoprecipitation and luciferase assays to investigate hypoxia inducible factor (HIF) dependent transcription. Two functional HIF response elements were identified within intron 1 of the gene. The functional importance of SLCO1B3 was studied by gene knockdown experiments followed by cell growth assays, flow cytometry and Western blotting. SLCO1B3 knockdown reduced cell size and 3-dimensional spheroid volume, which was associated with decreased activation of the mammalian target of rapamycin (mTOR) pathway. Finally, Oncomine analysis revealed that head and neck and colorectal tumours had higher levels of SLCO1B3 compared to normal tissue. Thus, the knowledge based approach for deriving gene signatures can identify novel biologically relevant genes.
Cancer Research UK Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.
Anassuya
A
Ramachandran
Guy
G
Betts
Sara
S
Bhana
Gemma
G
Helme
Christopher
C
Blick
Carla
C
Moller-Levet
Emma
E
Saunders
Helen
H
Valentine
Stuart
S
Pepper
Crispin J
CJ
Miller
Francesca
F
Buffa
Adrian L
AL
Harris
Catharine M L
CM
West
D015153:Blotting, Western; D018938:Caco-2 Cells; D015687:Cell Hypoxia / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D048429:Cell Size; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D050719:Crk-Associated Substrate Protein / Q000235:genetics; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D045325:HCT116 Cells; D006258:Head and Neck Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D007438:Introns / Q000235:genetics; D009369:Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D029362:Organic Anion Transporters, Sodium-Independent / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D016601:RNA-Binding Proteins / Q000235:genetics; D020218:Response Elements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D018874:Spheroids, Cellular / Q000378:metabolism / Q000473:pathology
C493841:BCAR1 protein, human; D050719:Crk-Associated Substrate Protein; C498779:IGF2BP2 protein, human; D029362:Organic Anion Transporters, Sodium-Independent; D016601:RNA-Binding Proteins; C421209:SLCO1B3 protein, human; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3