热休克抑制人肝癌SMMC-7721细胞糖皮质激素受体mRNA表达及其机制探讨

Mechanism for decrease of glucocorticoid receptor mRNA in human hepatic cancer cell line SMMC-7721 during heat shock

目的:探讨热休克时细胞糖皮质激素受体(glucocorticoid receptor,GR)mRNA减少的机制。方法:利用RT-PCR半定量的方法研究热休克时人肝癌SMMC-7721细胞GR mRNA的总体变化。制备GR基因跨外显子3、4、5的RNA探针和GR基因内含子E的RNA探针,对热休克后不同时间的细胞进行原位杂交,激光扫描共聚焦显微镜观察,计算机图像分析系统进行分析,研究GR mRNA转录、降解及前体剪接的变化。结果:RT-PCR实验证实,热休克反应时SMMC-7721细胞GRmRNA含量降低。原位杂交实验证实,SMMC-7721细胞热休克时,外显子和内含子探针杂交显色所得的积分灰度值(GRmRNA和GR mRNA前体的量)比未热休克处理组低(P<0.05);加放线菌素D抑制转录后进行热休克反应,内含子探针杂交显色所得的积分灰度值(GR mRNA前体的量)比单纯加放线菌素D(未进行热休克组)高,而外显子探针杂交显色所得的积分灰度值(GR mRNA的量)比单纯加放线菌素D组低(P<0.05)。结论:热休克反应时GR mRNA转录过程受抑,GR mRNA前体剪接过程存在障碍,GR mRNA降解加快。

Objective：To investigate the mechanism for decrease of glucocorticoid receptor mRNA （GR mRNA） during heat shock response. Methods： The changes of GR mRNA level in SMMC-7721 cells were examined by semi-quantitative RT-PCR during heat shock response. We designed 2 RNA fragments paired with GR gene intron E and cross exon 3, 4, and 5, which were used as probes for in situ hybridization with the sequences in SMMC-7721 cells at different periods after heat shock. The result was subjected to confocol microscope observation and computer image analysis. The transcription, degradation, and splicing of GR mRNA were investigated. Results： RT-PCR showed that the GR mRNA level was decreased during heat shock response. In situ hybridization revealed that both GR mRNA and GR pre-mRNA levels were lower in the heat shock group than in the non-heat shock group（P〈0.05）. GR pre-mRNA level was higher in cells treated with actinomycine D before heat shock than in cells only treated with actinomycine D, while the GR mRNA level was lower in cells treated with actinomycine D before heat shock （P〈0. 05）. Conclusion： During heat shock response, GR mRNA transcription is suppressed, the splicing of GR premRNA is suporessed, and GR mRNA degradation is accelerated.