Zhang Li, Yang Zhihong, Trottier Jocelyn, Barbier Olivier, Wang Li
Department of Physiology and Neurobiology and Institute for Systems Genomics, University of Connecticut, Storrs, CT.
Veterans Affairs Connecticut Healthcare System, West Haven, CT.
Hepatology. 2017 Feb;65(2):604-615. doi: 10.1002/hep.28882. Epub 2016 Dec 24.
Bile acids (BAs) play critical physiological functions in cholesterol homeostasis, and deregulation of BA metabolism causes cholestatic liver injury. The long noncoding RNA maternally expressed gene 3 (MEG3) was recently shown as a potential tumor suppressor; however, its basic hepatic function remains elusive. Using RNA pull-down with biotin-labeled sense or anti-sense MEG 3RNA followed by mass spectrometry, we identified RNA-binding protein polypyrimidine tract-binding protein 1 (PTBP1) as a MEG3 interacting protein and validated their interaction by RNA immunoprecipitation (RIP). Bioinformatics analysis revealed putative binding sites for PTBP1 within the coding region (CDS) of small heterodimer partner (SHP), a key repressor of BA biosynthesis. Forced expression of MEG3 in hepatocellular carcinoma cells guided and facilitated PTBP1 binding to the Shp CDS, resulting in Shp mRNA decay. Transient overexpression of MEG3 RNA in vivo in mouse liver caused rapid Shp mRNA degradation and cholestatic liver injury, which was accompanied by the disruption of BA homeostasis, elevation of liver enzymes, as well as dysregulation of BA synthetic enzymes and metabolic genes. Interestingly, RNA sequencing coupled with quantitative PCR (qPCR) revealed a drastic induction of MEG3 RNA in Shp liver. SHP inhibited MEG3 gene transcription by repressing cAMP response element-binding protein (CREB) transactivation of the MEG3 promoter. In addition, the expression of MEG3 and PTBP1 was activated in human fibrotic and cirrhotic livers.
MEG3 causes cholestasis by serving as a guide RNA scaffold to recruit PTBP1 to destabilize Shp mRNA. SHP in turn represses CREB-mediated activation of MEG3 expression in a feedback-regulatory fashion. (Hepatology 2017;65:604-615).
胆汁酸（BAs）在胆固醇稳态中发挥关键生理功能，胆汁酸代谢失调会导致胆汁淤积性肝损伤。长链非编码RNA母系表达基因3（MEG3）最近被证明是一种潜在的肿瘤抑制因子；然而，其基本的肝脏功能仍不清楚。我们使用生物素标记的正义或反义MEG3 RNA进行RNA下拉，随后进行质谱分析，鉴定出RNA结合蛋白多嘧啶序列结合蛋白1（PTBP1）为MEG3相互作用蛋白，并通过RNA免疫沉淀（RIP）验证了它们的相互作用。生物信息学分析揭示了小异二聚体伴侣（SHP）编码区（CDS）内PTBP1的假定结合位点，SHP是胆汁酸生物合成的关键抑制因子。在肝癌细胞中强制表达MEG3可引导并促进PTBP1与Shp CDS结合，导致Shp mRNA降解。在小鼠肝脏中体内瞬时过表达MEG3 RNA会导致Shp mRNA快速降解和胆汁淤积性肝损伤，同时伴有胆汁酸稳态破坏、肝酶升高以及胆汁酸合成酶和代谢基因失调。有趣的是，RNA测序结合定量PCR（qPCR）显示Shp肝脏中MEG3 RNA显著诱导。SHP通过抑制MEG3启动子的cAMP反应元件结合蛋白（CREB）反式激活来抑制MEG3基因转录。此外，MEG3和PTBP1的表达在人类纤维化和肝硬化肝脏中被激活。
MEG3通过作为引导RNA支架招募PTBP1来使Shp mRNA不稳定，从而导致胆汁淤积。SHP反过来以反馈调节方式抑制CREB介导的MEG3表达激活。（《肝脏病学》2017年；65：604 - 615）
