Lipopolysaccharide Downregulates Kruppel-Like Factor 2 (KLF2) via Inducing DNMT1-Mediated Hypermethylation in Endothelial Cells
Lipopolysaccharide Downregulates Kruppel-Like Factor 2 (KLF2) via Inducing DNMT1-Mediated Hypermethylation in Endothelial Cells
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脂多糖通过诱导内皮细胞中 DNMT1 介导的高甲基化下调 Kruppel 样因子 2 (KLF2)
DOI：
10.1007/s10753-017-0599-0
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发表时间：
2017-06
期刊：
影响因子：
5.1
通讯作者：
Ou HL
中科院分区：
文献类型：
--
作者：
Yan ZH;Deng Y;Jiao F;Guo J;Ou HL
KLF2在抗炎和内皮功能方面具有保护作用，并可通过启动子甲基化改变进行调节。脂多糖(LPS)是炎症反应的中介物，引起宿主细胞某些基因的表观遗传改变。因此，我们的目的是确定内毒素能否通过诱导启动子区域甲基化来控制KLF2的表达。用亚硫酸氢盐测序聚合酶链式反应检测KLF2启动子区域16个CpG位点的DNA甲基化。结果显示，人脐静脉内皮细胞暴露于脂多糖后，有12个CpG位点的甲基化水平显著增加，平均水平提高了57%。逆转录、实时定量聚合酶链式反应和Western印迹法检测KLF2的表达与未经内毒素模拟的细胞相比明显下调。此外，脂多糖和DNA甲基转移酶(DNMT)1小干扰RNA共同作用于HUVEC后，KLF2的信使RNA和蛋白表达水平均显著高于单纯脂多糖处理组。当细胞与内毒素和5-氮杂-2‘-脱氧胞苷(AZA)共同孵育时，也得到了类似的结果，提示内毒素引起的KLF2表达的降低可以被DNMT1抑制所逆转。AZA的存在改变了脂多糖刺激的HUVECs中依赖KLF2的基因的表达，下调了E-选择素和VCAM的表达，增加了eNOS和血栓调节蛋白的表达。我们的数据表明，脂多糖暴露导致HUVECs中KLF2启动子的高甲基化，从而导致KLF2表达下调。本研究提示，表观遗传学改变参与了脂多糖诱导的炎症反应，为动脉粥样硬化的发生提供了新的视角。
KLF2 plays a protective role in antiinflammation and endothelial function, and can be regulated by promoter methylation alteration. Lipopolysaccharide (LPS) is a mediator of inflammatory responses, which causes epigenetic change of certain genes in host cells. We thus aimed to determine whether LPS could control the KLF2 expression by inducing methylation in promoter region. DNA methylation of 16 CpG sites within KLF2 promoter region was detected by bisulfite sequencing PCR. Results showed that methylation at 12 CpG sites were significantly increased in HUVECs after exposure to LPS among the total 16 sites, and the average level was increased by 57%. The KLF2 expressions assessed by reverse transcription quantitative real-time PCR and Western blot were significantly downregulated compared that without LPS simulation. Moreover, both messenger RNA and protein levels of KLF2 in HUVEC co-treated with LPS and DNA methyltransferase (DNMT) 1 small interfering RNA were dramatically higher than that treated with LPS only. Similar result was obtained when the cells were incubated in combination with LPS and 5-aza-2'-deoxycytidine (AZA), suggesting that the reduction of KLF2 expression induced by LPS can be reversed by DNMT1 inhibition. Finally, the presence of AZA changed the expression of genes that depends on KLF2 in LPS-stimulated HUVECs, which downregulated the E-selectin and VCAM and increased the eNOS and thrombomodulin expression. Our data demonstrated that LPS exposure resulted in hypermethylation in KLF2 promoter in HUVECs, which subsequently led to downregulation of the KLF2 expression. The study suggested that epigenetic alteration is involved in LPS-induced inflammatory response and provided a new insight into atherogenesis.
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