Open AccessIdentification of hypoxia‐responsive messengers expressed in human microvascular endothelial cells using differential display RT‐PCR
Author(s) -
Roland Isabelle,
Minet Emmanuel,
Ernest Isabelle,
Pascal Thierry,
Michel Gaëtan,
Remacle José,
Michiels Carine
Publication year - 2000
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2000.01385.x
Subject(s) - differential display , northern blot , biology , microbiology and biotechnology , western blot , complementary dna , cdna library , messenger rna , hypoxia (environmental) , gene , blot , gene expression , chemistry , biochemistry , organic chemistry , oxygen
In order to identify new genes overexpressed in endothelial cells exposed to hypoxia, differential display RT‐PCR was performed on total RNA extracted from human microvascular endothelial cells incubated under hypoxia or under normoxic conditions. Northern blot and reverse Northern blot analyses were used to confirm the results. Sequences corresponding to tissue inhibitor of metalloproteinase‐1, prostate tumor inducing factor‐1, enolase‐α and prothymosin‐α were evidenced as overexpressed in hypoxia. These results were confirmed by Western blot and immunofluorescence experiments. Moreover, several elements homologous to partial sequences of cDNA (expressed sequence tag) were also identified, as well as unknown cDNA sequences. The present study suggests that hypoxia can change the expression of numerous genes in endothelial cells, and that mRNA differential display is useful for cloning known and unknown hypoxia‐responsive genes.