摘要
目的观察缺氧对HepG2细胞中热休克蛋白（HSP）70—2（基因名为HSPA2）表达的影响，探讨缺氧条件下缺氧诱导因子（HIF）-1在转录水平调控HSP702表达的具体机制。方法以物理缺氧法和化学缺氧诱导剂（DFO或COCl2）诱导法分别模拟肿瘤缺氧环境，Western blot检测HIF-1α和HSP70—2蛋白表达的变化，荧光素酶报告基因分析技术检测缺氧对HSPA2基因的激活作用。HIF—1α抑制剂（YC-1）或HIF—1α小干扰RNA（siRNA）处理后检测缺氧HepG2细胞中HSP70—2的表达变化。构建一系列截短和突变的HSPA2基因启动子荧光素酶报告基因质粒，将其与HIF-1α siRNA共转染HepG2细胞，荧光素酶分析技术检测HSPA2启动子活性的变化，寻找HIF-1 在HSPA2启动子上的结合位点。各组间比较用方差分析，两组间比较用t检验。结果缺氧培养6h后，HepG2细胞中HIF-1α和HSP70—2表达增加，其表达量随着缺氧培养时间的延长而逐渐增加（F=77．369，P〈0．01；F=108．854，P〈0．01）。各组分别加终浓度为0、50、100umol／L的化学缺氧诱导剂DFO或终浓度为0、100、200umol／L的CoCl2，培养24h后检测，随着DFO浓度增加，HIF—1α和HSP702蛋白表达逐渐增加（F=443．174，P〈0．01；F=589．238，P〈0．01）；随着CoCl2浓度增加，HIF—1α和HSP70—2蛋白表达也逐渐增加（F=637．724，P〈0.01；F=692．918，P〈0．01）。与常氧培养相比，缺氧培养后HSPA2启动子活性增加7．09倍（t=43．551，P〈0．01）。随着YC—1浓度升高，HIF-1α表达受到明显抑制（F=883．614，P〈0．01），HSP70—2表达水平也相应下降（F=218．112，P〈0．01）。转染HIF-1α siRNA后HIF1α表达受到明显抑制（F=577．130，P〈0．01），HSP702表达水平也相应下降（F=465．598，P〈0．01）。构建系列截短的HSPA2启动子报告基因载体转染HepG2细胞后检测，转染HSPA2（-1114／＋62）LUC和HSPA2（-653／＋62）-LUC细胞的
Objective To investigate role ofhypoxia inducible factor 1 （HIF-1） in the transcriptional activation of heat shock protein 70-2 （HSP70-2） in hepatocellula carcinoma （HCC） cells under hypoxic conditions. Methods HCC cells were exposed to reduced oxygen atmosphere （1% O2）, or treated with YC-1 or HIF-1α siRNA, the expression of HIF-1α and HSP70-2 were detected by Western blot analysis. Serial deletions of the HSPA2 promoter were cloned in the reporter pGL3-Basic plasmid. These reporter plasmids were co-transfected with HIF- 1α siRNA, and the promoter activities were detected with the dual luciferase assay. Results Western blot analysis showed that both HIF-1α and HSP70-2 proteins were strongly increased after HCC cells were exposed to hypoxic conditions （1% O2） for 6 h, and the expression level of HSP70-2 was increased in a time-dependent manner. Treatment of HepG2 cells with YC-1 or HIF-1α siRNA significantly inhibited the expression of HIF-1α and HSP70-2. In silico analysis of the HSP70-2 promoter using the Gene2 Promoter software revealed the presence of two putative hypoxic response element （HRE） consensuses at -446bp （HRE1） and -238bp （HRE2）. Depletion of promoter sequence between -653 and -385 led to a dramatic reduction of promoter activity, whereas further deletion to position -201 did not reduce the activity further. These data suggested that HRE1 plays an important role in hypoxia-induced activation of the HSPA2 promoter. Site-directed mutagenesis further confirmed these results. Mutation of HRE1 but not of HRE2 abrogated the sensitivity of the HSP70-2 promoter to hypoxia. Conclusions HSP70-2 expression is up-regulated in response to hypoxia and a HIF-1 binding site （HRE 1） in the HSP70-2 promoter is involved in this response.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2009年第3期207-212,共6页
Chinese Journal of Hepatology
