低氧诱导因子-1α对KLF5表达调控的研究
Study on the regulation of？ KLF5 expression by hypoxia-inducible factor-1α

目的 探索低氧诱导因子-1α（HIF-1α）对转录因子KLF5的表达调控及其在细胞增殖中的作用。方法 用低氧（1%O2或低氧模拟物CoCl2）处理293T细胞，通过qPCR和Western blotting检测HIF-1α、KLF5的mRNA和蛋白水平。用shRNA抑制HIF-1α的表达，再用低氧处理293T细胞后检测KLF5的表达水平。在293T细胞中过表达HIF-1α-P2A质粒，然后检测其对KLF5基因启动子驱动的luciferase及KLF5表达水平的影响。在HIF-1α-P2A过表达的293T细胞转染shKLF5，通过CCK8检测细胞的增殖情况。共同转染KLF5和HRE-luciferase质粒，通过荧光素酶报告系统检测KLF5表达对HIF-1α转录活性的影响。结果 qPCR和Western blotting显示低氧可以稳定HIF-1α蛋白，同时在转录水平增强KLF5的表达；shRNA抑制HIF-1α表达可以抑制低氧诱导的KLF5表达上调，并且过表达外源性HIF-1α可以直接结合KLF5基因的启动子区并上调KLF5的表达；尽管单独过表达HIF-1α-P2A或者抑制KLF5的表达并不影响细胞增殖，但两者同时存在时可以明显促进细胞增殖；KLF5过表达可以明显抑制HIF-1α的转录活性。结论 KLF5是HIF-1α的直接靶基因，HIF-1α在转录水平调控KLF5的表达，而KLF5可能通过抑制HIF-1α的转录活性从而抑制HIF-1α的促增殖作用。
： Objective To study the effects of hypoxia-inducible factor 1α (HIF-1α) on the regulation of transcription factor KLF5 expression and on the cell proliferation. Methods 293T cells were treated with hypoxia (1% O2 or hypoxia mimic CoCl2) and the mRNA and protein levels of HIF-1α and KLF5 were detected with the use of real-time qPCR and Western blotting. The HIF-1α expression was inhibited with shRNA and the KLF5 expression was detected after 293T cells were treated with hypoxia. The HIF-1α-P2A plasmid in 293T cells was over-expressed and the effects of which on expressions of KLF5 promoter-driven luciferase and KLF5 were detected. HIF-1α-P2A-overexpressed 293T cells were transfected with shKLF5 and the cell proliferation was detected with CCK8. 293T cells were co-transfected with KLF5 plasmid and HRE-luciferase plasmid, and the effects of KLF5 expression on HIF-1α transcriptional activity was evaluated with the luciferase reporter system. Results qPCR and Western blotting showed that hypoxia treatment could stabilize HIF-1α protein and transcriptionally upregulate the KLF5 expression. Inhibition of HIF-1α expression with shRNA could suppress hypoxia-induced up-regulation of KLF5 expression. Over-expressed exogenous HIF-1α-P2A could directly bind to the promoter region of KLF5 and up-regulate KLF5 expression. Although over-expression of HIF-1α-P2A or inhibition of shKLF5 alone was unable to affect cell proliferation, their combination could significantly promote cell proliferation. Overexpressed KLF5 could significantly inhibit the transcriptional activity of HIF-1α. Conclusion KLF5 is the direct target gene of HIF-1α and HIF-1α transcriptionally regulates the KLF5 expression. KLF5 may inhibit the HIF-1α-promoted cell proliferation via suppressing the transcriptional activity of HIF-1α