4-HNE increases intracellular ADMA levels in cultured HUVECs: evidence for miR-21-dependent mechanisms.
4-HNE increases intracellular ADMA levels in cultured HUVECs: evidence for miR-21-dependent mechanisms.
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4-HNE 增加培养的 HUVEC 细胞内 ADMA 水平：miR-21 依赖性机制的证据
DOI：
10.1371/journal.pone.0064148
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Chen XP
中科院分区：
文献类型：
--
作者：
Chen L;Zhou JP;Kuang DB;Tang J;Li YJ;Chen XP
探讨4-羟基壬烯醛（4-HNE）是否通过不依赖于与二甲基精氨酸（ADMA）代谢酶直接形成加合物的途径调节人脐静脉内皮细胞（HUVECs）的不对称二甲基精氨酸（ADMA）代谢以及microRNA（miRNA）miR-21的参与。用4-HNE（浓度分别为1、5和10 µM）或1‰ DMSO（溶剂对照）处理培养的HUVEC 24 h。在4-HNE处理前1小时转染miR-21抑制剂（终浓度为100 nM）。HUVEC也用miR-21（浓度为50 nM和100 nM）转染并分别培养12、24和48 h。半定量真实的时间PCR检测HUVECs中DDAH mRNA和miR-21的表达。Western blot分析DDAH 1和DDAH 2蛋白表达。通过ELISA分析细胞培养基和细胞裂解物中的ADMA。测定了细胞裂解物的ADMA代谢活性。MiR-21显著降低HUVECs中DDAH 1和DDAH 2的表达以及ADMA代谢活性，同时显著增加细胞内ADMA积累。10 μM 4-HNE处理24 h后，细胞裂解液中miR-21的表达增加，细胞内ADMA浓度升高，DDAH 1/2 mRNA和蛋白的表达降低，ADMA代谢活性显著降低。MiR-21抑制剂可完全逆转4-HNE对DDAH 1表达的抑制作用，并部分逆转10 μM 4-HNE引起的ADMA代谢活性和细胞内ADMA积累的变化。4-HNE通过miR-21依赖性机制下调DDAH 1表达并增加HUVEC中细胞内ADMA积累。
To investigate whether 4-hydroxynonenal (4-HNE) regulates asymmetric dimethylarginine (ADMA) metabolism through pathway independent of direct adduct formation with ADMA metabolizing enzyme and the involvement of microRNA (miRNA) miR-21 in human umbilical venous endothelial cells (HUVECs). Cultured HUVECs were treated with 4-HNE (at concentrations of 1, 5, and 10 µM, respectively) or 1‰ DMSO (vehicle control) for 24 h. MiR-21 inhibitor (final concentration of 100 nM) was transfected at 1 h before 4-HNE treatment. HUVECs were also transfected with miR-21 (at concentrations of 50 nM and 100 nM) and cultured for 12, 24, and 48 h, respectively. DDAH mRNA and miR-21 expression in the HUVECs were determined by semi-quantitative real time PCR. DDAH1 and DDAH2 protein expression were analyzed by Western blot. ADMA in the cell medium and cell lysates were analyzed by ELISA. ADMA metabolizing activity of the cell lysates was also determined. MiR-21 decreased DDAH1 and DDAH2 expression and ADMA metabolic activity significantly, while increased intracellular ADMA accumulation significantly in HUVECs. 10 µM 4-HNE treatment for 24 h increased the expression of miR-21 and intracellular ADMA concentration, decreased the expression of DDAH1/2 mRNA and protein, decreased ADMA metabolizing activity of the cell lysates significantly. MiR-21 inhibitor reversed the inhibitory effects of 4-HNE on DDAH1 expression completely, and partially reversed the changes in ADMA metabolizing activity and intracellular ADMA accumulation challenged by 10 µM 4-HNE. 4-HNE down-regulates DDAH1 expression and increases intracellular ADMA accumulation in HUVECs through a miR-21-dependent mechanism.
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