Hepatology. 2017 Feb;65(2):604-615. doi: 10.1002/hep.28882.

Long noncoding RNA MEG3 induces cholestatic liver injury by interaction with PTBP1 to facilitate shp mRNA decay.

Zhang L1, Yang Z1,2, Trottier J3, Barbier O3, Wang L1,2,4,5.

1 Department of Physiology and Neurobiology and Institute for Systems Genomics, University of Connecticut, Storrs, CT.
2 Veterans Affairs Connecticut Healthcare System, West Haven, CT.
3 Laboratory of Molecular Pharmacology, CHU-Québec Research Centre and Faculty of Pharmacy, Laval University, Québec, Canada.
4 Department of Internal Medicine, Section of Digestive Diseases, Yale University, New Haven, CT.
5 School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.

 

Abstract

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.

CONCLUSION: 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).

 

Study Highlight: LncRNAs in Cholestatic Liver Diseases

Long non-coding RNAs (lncRNAs) have emerged as important regulators of liver function and diseases. In this study, we identified a novel role for maternally expressed gene 3 (MEG3) in modulating bile acid homeostasis and cholestatic liver injury (1). We found that MEG3 serves as a guide RNA scaffold to recruit RNA binding protein PTBP1 to destabilize nuclear receptor SHP mRNA. This results in the loss of feedback inhibition of bile acid synthesis, leading to increased BA levels and cholestasis. On the other hand, SHP represses MEG3 expression via a cyclic adenosine monophosphate response element-binding protein (CREB)-mediated mechanism in a feedback regulatory fashion. Moreover, MEG3 and PTBP1 expression is upregulated in human fibrotic and cirrhotic livers, suggesting that their functional roles are intricately linked to an array of chronic liver diseases (Figure 1).

In addition, we found that lncRNA H19RNA is highly induced in anti-apoptotic protein Bcl2-mediated cholestatic liver injury, as a result of loss of SHP inhibition of H19 expression (2). Further mechanistic studies elucidated that H19 promotes cholestasis by inhibiting Zinc finger E-box-binding homeobox 1 (ZEB1) and activating epithelial cell adhesion molecule (EpCAM) (3). At the molecular level, ZEB1 functions as a transcriptional repressor of EpCAM expression. H19 binds to ZEB1 to prevent its recruitment to the EpCAM promoter, leading to EpCAM upregulation.

Overall, our studies highlight the importance of lncRNAs as new regulators of bile acid homeostasis and cholestatic liver diseases.

 

 

Figure 1: Schematic of MEG3/PTBP1/SHP circuit to modulate BA homeostasis. MEG3 interacts with PTBP1 protein to form a RNA-protein complex. This complex can guide more PTBP1 binding to SHP mRNA to cause its degradation. Loss of SHP results in disruption of BA homeostasis and cholestatic liver injury. In contrast, SHP inhibits CREB-mediated activation of MEG3 expression. The feedback inhibition between MEG3RNA/PTBP1 and SHP mRNA forms a fine-tuned regulation of BA metabolism.

 

References:

1) Zhang L, Yang ZH, Trottier J, Barbier O, Wang L. (2017) LncRNA MEG3 induces cholestatic liver injury by interaction with PTBP1 to facilitate Shp mRNA decay. Hepatology 65, 604-615.

2) Zhang YX, Liu C, Barbier O, Smalling R, Tsuchiya H, Lee SM, Delker D, Zou A, Hagedorn CH, Wang L. (2016) Bcl2 is a critical regulator of bile acid homeostasis by dictating Shp and lncRNA H19 function. Scientific Reports 6, 20559.

3) Song Y, Liu C, Liu X, Trottier J, Beaudoin M, Zhang L, Pope C, Peng GY, Barbier O, Zhang XB, Li L, Wang L. (2017) H19 promotes cholestatic liver fibrosis by preventing ZEB1-mediated inhibition of EpCAM. Hepatology  (in press)

 

Acknowledgements:

This study was supported by grants from NIH R01DK104656, R01DK080440, R01ES025909, R21AA022482, R21AA024935, VA Merit Award 1I01BX002634.

 

Contact:

Li Wang, Ph.D.

Professor, Department of Physiology & Neurobiology

The Institute for Systems Genomics, University of Connecticut

Investigator, VA Connecticut Healthcare System

Adjunct Faculty, Section of Digestive Diseases, Department of Internal Medicine

Member of Yale Liver Center, Yale University

75N. Eagleville Rd., U3156, Storrs, CT 06269-3156

Tel: 860-486-0857; Fax: 860-486-3303

E-mail: li.wang@uconn.edu; http://wanglab.pnb.uconn.edu/