Studies On The Mechanism Of Glucocorticoid Receptor MRNA Decreasing During Heat Shock

It has been reported that glucocorticoid receptor (GR) and GR mRNA were decreased in various tissues and in cultured cells during heat shock . GR content in human liver cancer SMMC-7721 cell line is known to be more than 10 times higher than that in normal human cells. It was found in our experiment that GR content in SMMC-7721 cell line is about twice the amount of that in human osteosarcoma HOS-8603 cell line. It seems that SMMC-7721 cell line is an ideal cell line for the study of the mechanism of GR mRNA decreasing under heat shock .The surviving rate of SMMC-7721 cell line was estimated after its being cultured 1 hour at 43℃-52℃ and returned to 37℃ for 1 hour. The cultured temperature at which the surviving rate over 95% was selected and then the cell line was cultured at these temperature for 1 hour before returning to 37℃ for another 36 hours. The surviving rate of which be cultured at 43℃ or 44℃ was higher than 95%. Thus SMMC-7721 cell line cultured at 44℃ for 1 hour was considered as a model for studying the mechanism of GR mRNA decreasing during heat shock . At 44℃ heat shock protein 70 was also expressed and the capability of [3H]dexamethasone binding to GR was decreased together with GR content decreased .The DNA fragments of rat GR exon between 3 and 5 and human GR intron C and E were obtained from rat GR cDNA plasmid and the genome of SMMC-7721 cell line respectively by PCR method then linked to transcription vector, from which the digoxin-marked antisense were transcripted in vitro. In situ hybridization was done with the RNA probe and optical density of cells was analyzed by computer. Results show that the GR mRNA and GR pre-mRNA all were decreased after heat shock, indicating that GR mRNA transcription was suppressed . GR mRNA was decreased greatly in cells be treated with actinomycine D prior to heat shock in comparison with that only be treated with actinomycine D, on the contrary, GR pre-mRNA was decreased greatly in cells only be treated with aetinomycine D indicating that GR pre-mRNA splicing was also suppressed in heat shock response. GR mRNA was increased in cells be treated with cycloheximide prior to heat shock in comparison with that only be heat shocked. GR pre-mRNA remained a large amount in cells be treated with cycloheximide prior to heat shock , suggesting that some expressed proteins in heat shock response may inhibit GR pre-mRNA transcription and splicing.