STAT1对hsp90∝基因表达的负调控作用

目的 探讨转录因子STAT1对hsp90∝基因表达的调控作用。方法 将STAT1表达质粒转染Jurkat细胞,利用竞争性RT－PCR定量检测其内源性hsp90∝基因mRNA水平。共转染STAT1表达质粒和hsp90∝基因上游调控区不同长度的片段驱动的氯霉素乙酰转移酶（CAT）报告基因质粒,利用定量RT－PCR检测CAT报告基因转录水平,用Western印迹分析方法检测42℃ 1h热激前后Jurkat细胞中STAT1酪氨酸磷酸化状态,用电泳迁移率变更分析（EMSA）检测STAT1与hsp90∝5‘基因上游调控区中含有STAT1特异结合元件的双链寡核苷酸片段特异性结合程度。结果 STAT1既可抑制Jurkat细胞hsp90∝基因的表达,又能抑制hsp90∝基因5‘上游调控区驱动的CAT报告基因活性。热激以前Jurkat细胞中的STAT1 701位酪氨酸已有一定程度的磷酸化,热激以后其磷酸化程度明显升高,而且STAT1与hsp90∝基因5‘上游调控区中的STAT1结合元件呈特异性结合。结论 STAT1对hsp90∝基因的表达具有负调控作用。

Role of STAT1 on the regulation the human hsp90 alpha gene expression

OBJECTIVE: To investigate the role of STAT1 on the regulation of human hsp90 alpha gene expression. METHODS: We first transfected Jurkat cells with the STAT1 expression construct and analyzed the expression of hsp90 alpha gene expression via quantitative RT-PCR system. Then we co-transfected the STAT1 expression construct and the CAT reporter gene driven by different length of 5' flanking sequence of hsp90 alpha gene. Western blot was carried out to detect the level of tyrosine phosphorylation in Jurkat cells with and without heat shock treatment (42 degrees C 1 h). By electrophoretic mobility shift assays (EMSA), we evaluated the DNA binding activity of a STAT1 responsible element located in the regulatory region of hsp90 alpha gene in Jurkat cell nuclear extracts. RESULTS: The mRNA level of hsp90 alpha gene in Jurkat cells was decreased when transfected by STAT1 expression construct, over-expression of STAT1 down-regulates the expression of CAT reporter gene with the present of a distal fragment from -1756 to -1463 within the 5' flanking regulatory sequences of hsp90 alpha gene. The tyrosine phosphorylation of STAT1 was detectable in Jurkat cells and increased when subjected to heat shock. Electrophoretic mobility shift assays (EMSA) results showed that STAT1 could bind to its responsible element in the regulatory region of hsp90 alpha gene. CONCLUSION: STAT1 could negatively regulate the human hsp90 alpha gene expression.