Department of Infectious Diseases, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
National Center for Cardiovascular Diseases, Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
J Cell Biochem. 2018 Jan;119(1):358-365. doi: 10.1002/jcb.26188. Epub 2017 Jul 24.
This study aims to evaluate the potential involvement and regulatory mechanism of miR-19a in hepatocytes autophagy of acute liver failure (ALF). The in vitro hepatocytes injury model of primary hepatocyte and hepatocytes line HL-7702 was established by D-galactosamine (D-GalN) and lipopolysaccharide (LPS) co-treatment. Relative expression level of miR-19a and NBR2 was determined by qRT-PCR. Protein expression of AMPK/PPARα and autophagy-related gene was determined by Western blot. In hepatic tissue of 20 ALF patients and D-GalN/LPS-stimulated hepatocytes, miR-19a was upregulated and NBR2 was downregulated. D-GalN/LPS stimulation caused the inactivation of AMPK/PPARα signaling and the decrease of autophagy-related LC3-II/LC3-I ratio and beclin-1 expression in hepatocytes. The expression of both AMPK/PPARα and NBR2 were negatively controlled by miR-19a overexpression or knockdown. Moreover, both NBR2 and PPARα were targeted regulated by miR-19a according to luciferase reporter assay. In D-GalN/LPS-stimulated hepatocytes, AMPK activation promoted PPARα expression. AMPK inactivation inhibited the pro-autophagy effect of miR-19a and caused the decrease of LC3-II/LC3-I ratio and beclin-1 expression. PPARα activation abrogated the anti-autophagy effect of miR-19a mimic and caused the increase of LC3-II/LC3-I ratio and beclin-1 expression. NBR2 knockdown reversed the anti-autophagy impact of miR-19a inhibitor and caused the decrease of LC3-II/LC3-I ratio and beclin-1 expression. In summary, our data suggested that miR-19a negatively controlled the autophagy of hepatocytes attenuated in D-GalN/LPS-stimulated hepatocytes via regulating NBR2 and AMPK/PPARα signaling. J. Cell. Biochem. 119: 358-365, 2018. © 2017 Wiley Periodicals, Inc.
本研究旨在评估 miR-19a 在急性肝衰竭（ALF）肝细胞自噬中的潜在作用和调控机制。通过 D-半乳糖胺（D-GalN）和脂多糖（LPS）共同处理建立原代肝细胞和肝细胞系 HL-7702 的体外肝细胞损伤模型。通过 qRT-PCR 测定 miR-19a 和 NBR2 的相对表达水平。通过 Western blot 测定 AMPK/PPARα 和自噬相关基因的蛋白表达。在 20 例 ALF 患者和 D-GalN/LPS 刺激的肝细胞的肝组织中，miR-19a 上调，NBR2 下调。D-GalN/LPS 刺激导致 AMPK/PPARα 信号失活，自噬相关 LC3-II/LC3-I 比值和 beclin-1 表达降低。miR-19a 的过表达或敲低均负调控 AMPK/PPARα 和 NBR2 的表达。此外，根据荧光素酶报告基因测定，NBR2 和 PPARα 均受 miR-19a 的靶向调控。在 D-GalN/LPS 刺激的肝细胞中，AMPK 激活促进 PPARα 表达。AMPK 失活抑制 miR-19a 的促自噬作用，并导致 LC3-II/LC3-I 比值和 beclin-1 表达降低。PPARα 激活消除了 miR-19a 模拟物的抗自噬作用，并导致 LC3-II/LC3-I 比值和 beclin-1 表达增加。NBR2 敲低逆转了 miR-19a 抑制剂的抗自噬作用，并导致 LC3-II/LC3-I 比值和 beclin-1 表达降低。总之，我们的数据表明，miR-19a 通过调节 NBR2 和 AMPK/PPARα 信号负调控 D-GalN/LPS 刺激的肝细胞自噬。J. Cell. Biochem. 119: 358-365, 2018. © 2017 Wiley Periodicals, Inc.
