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

作　　者：王晓博[1] 王豪雨 姚俊吉 曾琬琴 刘海山[1] 赵克温[1] 

机构地区：[1]上海交通大学医学院病理生理学教研室细胞分化与凋亡教育部重点实验室,上海200025

出　　处：《上海交通大学学报（医学版）》2016年第5期619-625,共7页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(81171888)~~

摘　　要：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%O_2或低氧模拟物CoCl_2)处理293T细胞,通过q PCR和Western blotting检测HIF-1α、KLF5的m RNA和蛋白水平。用sh RNA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sh KLF5,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 q PCR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;sh RNA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（ H IF-1α） on the regulation of transcription factor KL F5 expression and on the cell proliferation. Methods 293 T cells were treated with hypoxia（ 1% O_2 or hypoxia m im ic CoCl_2） and the m RN A and protein levels of H IF-1α and KL F5 were detected with the use of real-tim e q PCR and W estern blotting. The H IF-1α expression was inhibited with sh RN A and the KLF5 expression was detected after 293 T cells were treated with hypoxia. The H IF-1α-P2 A plasm id in 293 Tcells was over-expressed and the effects of which on expressions of KLF5 prom oter-driven luciferase and KLF5 were detected. H IF-1α-P2A-overexpressed 293 T cells were transfected with sh KLF5 and the cell proliferation was detected with C C K8. 293 T cells were co-transfected with KLF5 plasm id and H RE-luciferase plasm id, and the effects of KL F5 expression on H IF-1α transcriptional activity was evaluated with the luciferase reporter system. Results qPCR and W estern blotting showed that hypoxia treatm ent could stabilize H IF-1α protein and transcriptionally upregulate the KLF5 expression. Inhibition of H IF-1α expression with sh RN A could suppress hypoxia-induced up-regulation of KLF5 expression. Over-expressed exogenous H IF-1α-P2 A could directly bind to the prom oter region of KLF5 and up-regulate KL F5 expression. Although over-expression of H IF-1α-P2 A or inhibition of sh KL F5 alone was unable to affect cell proliferation, their com bination could significantly prom ote cell proliferation. Overexpressed KL F5 could significantly inhibit the transcriptional activityof H IF-1α. Conclusion KLF5 is the direct target gene of H IF-1α and H IF-1α transcriptionally regulates the KL F5 expression. KLF5 m ay inhibit the H IF-1α-prom oted cell proliferation via suppressing the transcriptional activity of H IF-1α.

关 键 词：低氧诱导因子-1Α KLF5 低氧 细胞增殖 

分 类 号：R363[医药卫生—病理学]