Department of Pharmacology, The Second Hospital of Anhui Medical University, Hefei, 230601, China.
Department of Cardiothoracic Surgery, The Second Hospital of Anhui Medical University, Hefei, 230601, China.
Inflamm Res. 2017 Apr;66(4):341-352. doi: 10.1007/s00011-016-1020-4. Epub 2016 Dec 26.
miR-200a has been established as a key regulator of HSC activation processes in liver fibrosis. Epigenetic silencing of miR-200a contributing to SIRT1 over-expression has been discussed in breast cancer; however, whether miR-200a controls SIRT1 gene expression in hepatic fibrosis is still unknown.
We analyzed miR-200a regulation of SIRT1 expression in CCl-induced liver fibrosis and TGF-β1-mediated activation of HSC. miR-200a, SIRT1, α-SMA, Col1A1, Notch1 and NICD expression were estimated by Western blotting, qRT-PCR and Immunohistochemistry. HSCs were transfected with miR-200a mimic, miR-200a inhibitor and SIRT1-RNAi. Luciferase reporter assays further confirmed the interaction between miR-200a and the SIRT1 mRNA 3'-UTR. Cell proliferation ability was assessed by MTT and cell cycle.
We found that treatment activated HSC with miR-200a mimics, restored miR-200a expression and reduced SIRT1 levels. Conversely, treatment activated HSC with miR-200a inhibitors, decreased miR-200a expression and up-regulated SIRT1 levels. Restoration of miR-200a or the knockdown of SIRT1 prevented HSC activation and proliferation. We have established the SIRT1 transcript as subject to regulation by miR-200a, through miR-200a targeting of SIRT1 3'-UTR. Finally, HSC transfected with SIRT1-siRNA increased the levels of Notch1 protein and mRNA expression.
Our study demonstrated that miR-200a regulates SIRT1/Notch1 expression during HSC activation and fibrosis.
miR-200a 已被确定为肝纤维化中 HSC 激活过程的关键调节因子。在乳腺癌中已经讨论了 miR-200a 的表观遗传沉默导致 SIRT1 过表达；然而，miR-200a 是否控制肝纤维化中 SIRT1 基因表达仍不清楚。
我们分析了 CCl 诱导的肝纤维化和 TGF-β1 介导的 HSC 激活中 miR-200a 对 SIRT1 表达的调节。通过 Western blot、qRT-PCR 和免疫组化分析 miR-200a、SIRT1、α-SMA、Col1A1、Notch1 和 NICD 的表达。用 miR-200a 模拟物、miR-200a 抑制剂和 SIRT1-RNAi 转染 HSCs。荧光素酶报告基因实验进一步证实了 miR-200a 与 SIRT1 mRNA 3'-UTR 之间的相互作用。MTT 和细胞周期评估细胞增殖能力。
我们发现，用 miR-200a 模拟物处理激活的 HSC，恢复 miR-200a 表达并降低 SIRT1 水平。相反，用 miR-200a 抑制剂处理激活的 HSC，降低 miR-200a 表达并上调 SIRT1 水平。恢复 miR-200a 或敲低 SIRT1 可防止 HSC 激活和增殖。我们已经确定 SIRT1 转录物受 miR-200a 调节，通过 miR-200a 靶向 SIRT1 3'-UTR。最后，用 SIRT1-siRNA 转染的 HSC 增加了 Notch1 蛋白和 mRNA 表达水平。
本研究表明，miR-200a 在 HSC 激活和纤维化过程中调节 SIRT1/Notch1 表达。
