低氧对钾-氯协同转运子1启动子的调控作用

Regulative function of hypoxia to the promoter of human K-Cl cotransporter

目的通过观察低氧对钾-氯协同转运子1(KCC1)启动子的作用及对KCC1基因表达的影响,探索低氧诱导因子1α(HIF-1α)结合位点是否为真正的低氧诱导因子结合位点、在低氧环境中该位点的作用以及KCC与低氧间的关系。方法用KCC1野生型启动子和HIF-1α位点变异的KCC1启动子转染人胚胎肾上皮(HEK)293细胞。转染细胞分别在低氧条件下和正常培养条件下培养,用荧光素酶功能分析的方法观测低氧对KCC1启动子荧光素酶活性的影响:用RT-PCR方法比较两种培养条件下KCC1mRNA表达水平的差异。结果低氧条件下野生型KCC1启动子荧光素酶活性为正常培养条件下的KCC1启动子荧光素酶活性的1.8倍,而变异的HIF-1α位点的KCC1启动子荧光素酶活性则在两种条件下差异无统计学意义。低氧条件下HEK293细胞的KCC1mRNA表达水平是正常氧条件下表达量的2.65倍,差异有统计学意义。结论本研究发现了KCC1启动子中有HIF-1α结合位点,低氧可增强KCC1启动子的活性。该位点的变异与否决定了启动子活性是否受低氧的影响。该结果证实了KCC与低氧间的关系,为建立低氧-KCC1-肾小管间质损害之间的链接提供了实验依据。

Objective To confirm whether the binding site of hypoxia-inducible factors 1α (HIF-1α) in the promoter of human K-C1 cotransporter is a real and functional transcriptional factor binding site and explore its functional role in hypoxia environment.Methods Plasmid of wild 700 bp KCCl promoter and the mutated 700 bp promoter with point mutation at HIF-1α site were constructed into luciferase vector pGL-2B and transfected into HEK293 cells under the normoxia and hypoxia culture conditions. Luciferase activities of KCCl promoter were compared.RNA from normoxia and hypoxia cultured HEK293 cells was extracted and RT-PCR was conducted to evaluate the expression level of KCCl mRNA.Results The luciferse activity of wild type KCCl promoter transfected and cultured under hypoxia condition was 1.8 times as that under normoxia condition. No significant difference of luciferase activity was observed between normoxia and hypoxia cultured HEK293 cells if plasmid point mutated at HIF-1α binding site was used. RT-PCR showed that KCCl mRNA expression level in hypoxia condition was 2.65 limes as that in normoxia.Conclusions This is the first report about the discovery of a functional HIF-1α binding site in human KCCl promoter. Hypoxia can increase the activity of KCCl promoter. This function was achieved by the HIF-1α binding site because mutated plasmid at this site loses such function.There is a close relationship between hypoxia and KCCl regulation. Experimental data are provided for the link of hypoxia-KCCl-renal tubular and interstilial damage.