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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 investigate whether hypoxia-inducible factor 1α (HIF-1α) binding site is a true hypoxia-inducible factor (HIF-1α) by observing the effect of hypoxia on KCC1 promoter and KCC1 gene expression Binding site, the role of this site in hypoxia and the relationship between KCC and hypoxia. Methods Human embryonic kidney epithelial (HEK) 293 cells were transfected with KCC1 wild-type promoter and KCC1 promoter with HIF-1α mutation. The transfected cells were cultured under hypoxia and normal culture conditions respectively. The effect of hypoxia on the luciferase activity of KCC1 promoter was observed by luciferase functional assay: KCC1 mRNA was compared between the two culture conditions by RT-PCR Differences in expression levels. Results The luciferase activity of wild-type KCC1 promoter under hypoxic conditions was 1.8 times that of KCC1 promoter under normal culture conditions, while the KCC1 promoter luciferase activity at the mutated HIF-1α locus was in two No significant difference under the conditions. The expression level of KCC1 mRNA in HEK293 cells under hypoxic conditions was 2.65 times higher than that under normal oxygen conditions, the difference was statistically significant. Conclusion This study found that there is HIF-1α binding site in KCC1 promoter. Hypoxia can enhance the activity of KCC1 promoter. The site of variation determines whether the promoter activity is affected by hypoxia. This result confirms the relationship between KCC and hypoxia and provides an experimental basis for establishing the link between hypoxia-KCC1-tubulointerstitial damage.