Department of Obstetrics and Gynecology, Harbor-UCLA Medical Center, Torrance, CA, United States; LA Biomed Research Institute, Torrance, CA, United States.
Department of Obstetrics and Gynecology, Harbor-UCLA Medical Center, Torrance, CA, United States; LA Biomed Research Institute, Torrance, CA, United States.
Life Sci. 2017 Oct 1;186:87-91. doi: 10.1016/j.lfs.2017.08.007. Epub 2017 Aug 8.
The objective of this study was to determine the underlying mechanism by which glucocorticoids (GCs) induce of miR-29c expression in vascular smooth muscle cells.
QRT-PCR was used for miR-29c detection. Protein levels were determined by western blotting. Knockdown of SP1, DNMT1 and DNMT3A was achieved through transfection with their specific respective siRNAs. The effect of GCs on SP1 activity was determined by luciferase reporter assay and the methylation status in miR-29c promoter was detected by methylation specific PCR. CHIP assay was used to determine the binding ability of SP1 and glucocorticoid receptor (GR) in miR-29c promoter.
Treatment of RASMC with SP1 siRNA or SP1 inhibitor, mithramycin A, as well as DNMT1 and DNMT3A siRNAs and an inhibitor of DNMTs, Decitabine, resulted in increased expression of miR-29c (P<0.05). Treatment RASMC with dexamethasone (DEX, 0.1μM) for 24h reduced the expression of SP1 and phosphorylated SP1 at threonine 453 protein levels, repressed SP1 activity, and inhibited the expression of DNMT1 and DNMT3A proteins (P<0.05). Treatment with mifepristone, a GR antagonist, blocked the inhibitory effect of DEX on DNMT1 and DNMT3A protein expression. DEX also suppressed SP1 binding ability in miR-29c promoter and inhibited methylation of miR-29c promoter (P<0.05). Treatment of RASMC with DEX (0.1μM) significantly (P<0.05) increased the binding of GR to the miR-29c promoter.
The stimulatory effect of GCs on miR-29c expression is mediated by these three mechanisms: transcriptionally regulated by SP1, and epigenetically through a methylation-dependent process and GR.
本研究旨在确定糖皮质激素（GCs）诱导血管平滑肌细胞中 miR-29c 表达的潜在机制。
使用 QRT-PCR 检测 miR-29c。通过转染其特异性 siRNA 来确定蛋白质水平。通过转染其特异性 siRNA 来实现 SP1、DNMT1 和 DNMT3A 的敲低。通过荧光素酶报告基因测定法测定 GCs 对 SP1 活性的影响，并通过甲基化特异性 PCR 检测 miR-29c 启动子中的甲基化状态。使用 CHIP 测定法确定 SP1 和糖皮质激素受体（GR）在 miR-29c 启动子中的结合能力。
用 SP1 siRNA 或 SP1 抑制剂米屈肼 A 以及 DNMT1 和 DNMT3A siRNAs 和 DNMT 抑制剂地西他滨处理 RASMC，导致 miR-29c 的表达增加（P<0.05）。用地塞米松（DEX，0.1μM）处理 RASMC 24 小时可降低 SP1 表达和丝氨酸 453 磷酸化 SP1 蛋白水平，抑制 SP1 活性，并抑制 DNMT1 和 DNMT3A 蛋白的表达（P<0.05）。用 GR 拮抗剂米非司酮处理可阻断 DEX 对 DNMT1 和 DNMT3A 蛋白表达的抑制作用。DEX 还抑制 miR-29c 启动子中 SP1 的结合能力并抑制 miR-29c 启动子的甲基化（P<0.05）。用 DEX（0.1μM）处理 RASMC 可显著增加 GR 与 miR-29c 启动子的结合（P<0.05）。
GCs 对 miR-29c 表达的刺激作用是通过这三种机制介导的：通过 SP1 转录调控，以及通过依赖于甲基化的过程和 GR 进行表观遗传调控。
