MicroRNA‐194 protects against chronic hepatitis B‐related liver damage by promoting hepatocyte growth via ACVR2B

Abstract Persistent infection with the hepatitis B virus leads to liver cirrhosis and hepatocellular carcinoma. MicroRNAs (miRNAs) play an important role in a variety of biological processes; however, the role of miRNAs in chronic hepatitis B (CHB)‐induced liver damage remains poorly understood. Here, we investigated the role of miRNAs in CHB‐related liver damage. Microarray analysis of the expression of miRNAs in 22 CHB patients and 33 healthy individuals identified miR‐194 as one of six differentially expressed miRNAs. miR‐194 was up‐regulated in correlation with increased liver damage in the plasma or liver tissues of CHB patients. In mice subjected to 2/3 partial hepatectomy, miR‐194 was up‐regulated in liver tissues in correlation with hepatocyte growth and in parallel with the down‐regulation of the activin receptor ACVR2B. Overexpression of miR‐194 in human liver HL7702 cells down‐regulated ACVR2B mRNA and protein expression, promoted cell proliferation, acceleratedG1 to S cell cycle transition, and inhibited apoptosis, whereas knockdown of miR‐194 had the opposite effects. Luciferase reporter assays confirmed that ACVR2B is a direct target of miR‐194, and overexpression of ACVR2B significantly repressed cell proliferation and G1 to S phase transition and induced cell apoptosis. ACVR2B overexpression abolished the effect of miR‐194, indicating that miR‐194 promotes hepatocyte proliferation and inhibits apoptosis by down‐regulating ACVR2B. Taken together, these results indicate that miR‐194 plays a crucial role in hepatocyte proliferation and liver regeneration by targeting ACVR2B and may represent a novel therapeutic target for the treatment of CHB‐related liver damage.

mortality of these diseases is serious, with approximately 600 000 HBV-related deaths estimated annually and 73% of liver cancer deaths attributed to hepatitis viruses. 3 Approximately 8%-20% of patients with chronic hepatitis B (CHB) develop liver cirrhosis, and among these, the risk of hepatocellular carcinoma (HCC) is 2%-5%. 4 HBV infection is responsible for 50%-80% of HCC cases. Chronic liver injury leads to liver cell death and regeneration of parenchymal cells. 5 However, persistent liver injury can lead to failure of regeneration and the replacement of hepatocytes by extracellular matrix (ECM) mainly derived from hepatic stellate cells. 6,7 Therefore, a better understanding of the mechanisms underlying HBV-related liver injury and the development of early detection methods to prevent progression to cirrhosis and HCC are essential for the prevention and treatment of liver diseases.
MicroRNAs (miRNAs) are small endogenous non-coding RNAs that modulate gene expression by binding to the 3 0 -untranslated region (3 0 -UTR) of target mRNAs to inhibit translation or induce mRNA degradation. 8 miRNAs are involved in many physiological processes including cell proliferation, differentiation and apoptosis, and aberrant expression of miRNAs is associated with several diseases. 9 Certain miRNAs play a role in the maintenance of hepatocyte function, such as miR-122a, which promotes the replication of the hepatitis C virus in hepatocytes, and miR-150 and miR-194, which inhibit hepatic stellate cell activation and extracellular matrix production associated with liver fibrosis. [10][11][12] miRNAs such as miR-122 and miR-192 are overexpressed in the livers of mice exposed to druginduced liver injury in a pattern mimicking that of serum aminotransferase levels and the histopathology of liver degeneration. The same miRNAs are overexpressed in humans with acetaminophen-induced acute liver injury, suggesting their value as markers of liver damage. 13,14 Activins are secreted polypeptides that belong to the transforming growth factor beta (TGFb) superfamily, which plays a role in the regulation of cell proliferation, apoptosis, inflammation and differentiation in many cell types and organs including the liver. 15 Activins are homo-or heterodimers of different b subunits that either dimerize with another b subunit to form activins or dimerize with a single a subunit to form inhibins. 15,16 Activin A, which functions as an inhibitor of liver growth, signals through two types of transmembrane serine threonine kinase receptors, binding first to type II activin receptors, which recruit and phosphorylate type I receptors. 17,18 There are two type II receptors, ActR-II or ACVR22 and ActR-IIB or ACVR2B. Similar to other TGFb family receptors, activin A receptors recruit Smads 2 and 3 to transduce signals to the nucleus, although the existence of Smad-independent signalling pathways has been suggested. 15,19,20 In a previous study, we identified a plasma miRNA panel for the diagnosis of hepatitis B-related HCC in a cohort of 934 patients with CHB, cirrhosis and HBV-related HCC using microarray technology. 21 In this study, we expanded this work by exploring the role and mechanisms of miR-194, one of six differentially expressed miRNAs in CHB, and its target ACVR2B in liver injury and regeneration associated with CHB.

| Microarray analysis and quantitative reversetranscriptase PCR (qRT-PCR) validation
This study was approved by the local institutional review boards and informed consent was obtained from all study participants. Between August 2008 and June 2012, 231 participants (91 healthy and 140 CHB) who met the eligibility criteria (Table S1) were recruited from Shanghai Zhongshan Hospital, Huashan Hospital and Public Health Clinical Center. The 231 blood samples were allocated to two phases in chronological order ( Figure S1A). There was no sample overlapping among the two phases. Table S2 shows the clinical characteristics of the study subjects. We used human miRNA microarray 2.0 from Agilent Technologies (Santa Clara, CA) to identify candidate miRNAs in 55 plasma samples (33 healthy and 22 CHB) as described in our previous study. 21 The details of microarray hybridization are described in Appendix S1.

| Histological examination of liver biopsy specimens
Sixty-six CHB patients suspected to have HBV-related liver damage underwent liver biopsy. The biopsy specimens were fixed with 10% formalin, routinely embedded in paraffin, and tissue sections were stained with haematoxylin and eosin (H&E). The H&E sections with at least six portal tracts were blindly and independently assessed by three experienced pathologists, and the severity of chronic liver damage was evaluated according to the Scheuer classification system as follows 23 : G0, none; G1, inflammation but no necrosis; G2, focal necrosis or acidophil bodies; G3, severe focal cell damage; and G4, damage including bridging necrosis.  under isoflurane anaesthesia as previously described. 24 Mice were killed and the remnant liver tissues were collected at 1, 3, 5, 7, and 14 days after surgery. One part was frozen in liquid nitrogen and stored at À80°C; the other part was fixed in 4% paraformaldehyde.

| Animals and operative procedure
Liver and body weight were recorded.

| Immunohistochemical staining
The fixed liver tissues were embedded and 5 lm thick sections were prepared. The slides were stained with anti-Ki-67, anti-proliferating cell nuclear antigen (PCNA), anti-cyclin D1 and anti-ACVR2B (Abcam, Cambridge, UK) and visualized with DAB (Sigma, St. Louis, MO, USA) followed by counterstaining with H&E (Sigma).

| Cell culture and treatment
Human HL7702 normal liver cells were maintained in RPMI-1640 medium (Gibco, USA) supplemented with 10% foetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco) at 37°C in 5% CO 2 .  instructions. Cells were harvested at 24 hours after transfection, and luciferase activity was measured using a dual-luciferase reporter assay system (Promega).

| qRT-PCR
Total RNA was isolated from prepared mouse liver samples or transfected cells using the Trizol reagent (Invitrogen, CA, USA). cDNA was synthesized using Taqman RT reagents (Applied Biosystems, CA, USA) following the manufacturer's protocol. qRT-PCR was performed using a standard SYBR green PCR kit (Qiagen, Hilden,Germany), and PCR-specific amplification was performed using the Applied Biosystems (ABI7300) real-time PCR machine. The relative expression of target genes was calculated with the 2 ÀMMCt method. miR-194 expression was normalized to U6 and ACVR2B to b-actin.

| Western blot analysis
Tissues and cells were lysed in RIPA lysis buffer (Santa Cruz Biotechnology, CA, USA). The lysates were sonicated and centrifuged at 11 750 g at 4°C for 10 minutes. Equal amounts of protein were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. For immunodetection, membranes were incubated with primary antibodies against ACVR2B, p53, p-Smad2, p-Smad3, and Smad2/3 (Abcam).
The immunoblots were developed using horseradish peroxidase (HRP)-coupled secondary antibodies (Abcam) followed by detection with an ECL kit (Pierce Biotechnology, Rockford, USA). b-actin was used as a control.

| Statistical analysis
For microarray analysis, the Mann-Whitney unpaired test with Benjamini-Hochberg correction was used for the comparison between CHB patients and healthy individuals. Hierarchical clustering analysis was performed with GeneSpring GX10 software (Agilent Technologies, Santa Clara, CA). For the data obtained by qRT-PCR, the Mann-Whitney unpaired test was used for pairwise comparisons (CHB vs. healthy and CHB G0-1 vs G2-4).
The data shown in animal and cell experiments are presented as the mean AE standard deviation (SD). All P values were two-sided and a P-value of <.05 was considered statistically significant. The Student's t test was used to compare continuous variables between two groups.

| miR-194 is up-regulated in correlation with increased liver damage in CHB patients
Hierarchical clustering analysis of microarray data showed that 33/  (Table S3).
According to the Scheuer Classification System of histological grades of liver damage, G0 and G1 patients had no necrosis, whereas G2, G3, and G4 patients showed necrosis. Therefore, CHB patients were divided into two groups, G0-1 without significant liver damage and G2-4 with significant liver damage. Analysis of the 118 CHB patients who underwent liver biopsy showed higher expression levels of miR-122, miR-148a, miR-192, miR-194, miR-215, and miR-27b in CHB patients with G2-4 than in CHB patients with G0-1 (fold changes: 3.1, 3.2, 2.6, 2.6, 3.3, and 1.9, respectively; Table 1), whereas there was no statistically significant difference between G0 and G1, or G2 and G3/4. Further analysis of miR-194 expression in plasma and tissues of CHB patients with different histological grades of liver damage also showed higher expression levels in the G2 and G3 groups than in the G0 and G1 groups ( Figure 1A and B).

| miR-194 is induced in response to partial hepatectomy
To determine the impact of miR-194 on liver hepatocyte growth, mice were subjected to 2/3 partial hepatectomy (PH). As shown in  Figure 2E). Taken together, these results implicate miR-194 and its target ACVR2B in the response of hepatocytes to injury.

| miR-194 regulates ACVR2B at the posttranscriptional level
To determine the effect of miR-194 on hepatocyte growth, miR-194 was overexpressed or silenced in the normal human liver cell line  Figure S2B). These results were confirmed using the dual-luciferase reporter assay system, which showed that miR-194 overexpression and silencing significantly inhibited and promoted luciferase reporter activity, respectively, in the wild-type but not the mutant construct ( Figure 3D and E). Correlation analysis showed a significant negative correlation between the mRNA expression of miR-194 and ACVR2B in mouse liver tissues ( Figure 3F).  The miRNA selected for study is shown in bold.

GAO ET AL.
| 4537 inhibition had the opposite effect ( Figure 4A). Flow cytometry showed that miR-194 overexpression accelerated cell cycle G1 to S phase transition ( Figure 4B) and significantly inhibited cell apoptosis ( Figure 4C), whereas miR-194 knockdown had the opposite effects.

| miR-194 promotes hepatocyte proliferation and inhibits apoptosis by targeting ACVR2B
The mechanism underlying the effect of miR-194 on hepatocyte growth was further assessed in HL7702 cells overexpressing  Shanghai Rising-Star Program (16QA1401000).

CONF LICT OF I NTEREST
All the authors have no conflict of interest to declare.

AUTHOR CONTRI BUTIONS
Xue Gao performed the research and wrote the manuscript.