MicroRNA-194 protects against chronic hepatitis B-related liver damage by promoting hepatocyte growth via ACVR2B - PubMed (original) (raw)

. 2018 Sep;22(9):4534-4544.

doi: 10.1111/jcmm.13714. Epub 2018 Jul 25.

Affiliations

Xue Gao et al. J Cell Mol Med. 2018 Sep.

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.

Keywords: ACVR2B; chronic hepatitis B; liver regeneration; miR-194; microRNAs.

© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

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Figures

Figure 1

Figure 1

Involvement of miR‐194 in liver injury and regeneration in chronic hepatitis B (

CHB

) patients. A and B, The expression of miR‐194 in the plasma and liver tissues of

CHB

patients was detected by

qRT

‐

PCR

. Expression was shown in terms of ΔCt on a log2 scale with lower numbers indicating increasing expression. C, Histological liver damage was detected by H&E staining

Figure 2

Figure 2

miR‐194 was up‐regulated in a mouse model of partial hepatectomy (

PH

). A, Recovery of functional liver mass after

PH

(normalized liver to body weight ratio). Data represent the mean ±

SEM

with n = 6 per group. B, The expression of miR‐194 in mouse liver tissues was detected by

qRT

‐

PCR

. C and D, The

mRNA

and protein expression levels of

ACVR

2B were determined by

qRT

‐

PCR

and Western blotting, respectively. *P < .05, **P < .01. (E) Liver immunohistochemical staining for Ki‐67,

PCNA

, cyclin D1, and

ACVR

2B. Original magnification, 200 ×

Figure 3

Figure 3

miR‐194 binds to and directly targets

ACVR

2B. A,

qRT

‐

PCR

analysis of miR‐194 expression in the human normal liver cell line

HL

7702 transfected with miR‐194, anti‐miR‐194, or negative control. B and C, The

mRNA

and protein expression levels of

ACVR

2B were determined by

qRT

‐

PCR

and Western blotting, respectively. D, Wild‐type or mutant

ACVR

2B 3′‐

UTR

‐binding sites for miR‐194. E, Relative luciferase activity of

HL

7702 cells after cotransfection with wild‐type (

WT

) or mutant (

MUT

)

ACVR

2B 3′‐

UTR

reporter constructs along with miR‐194, anti‐miR‐194, or negative control. N = 3. **P < .01. F, Correlation analysis of the

mRNA

expression of miR‐194 and

ACVR

2B in mouse liver tissues

Figure 4

Figure 4

miR‐194 promotes cell proliferation and inhibits cell apoptosis in hepatic cells. A, Effect of miR‐194 on

HL

7702 cell proliferation. The human normal liver cell line

HL

7702 was transfected with miR‐194, anti‐miR‐194, or negative control and cell proliferation was assessed using the

MTT

assay. B and C, Flow cytometry analysis of the effects of miR‐194 on cell cycle distribution and cell apoptosis. n = 3. *P < .05, **P < .01

Figure 5

Figure 5

ACVR

2B reversed the effect of miR‐194 on hepatic cell growth and apoptosis. A and B, The mRNA and protein expression levels of ACVR2B were determined by qRT‐PCR and western blotting, respectively. C, Cell proliferation was assessed using the

MTT

assay in

HL

7702 cells transduced with

ACVR

2B, miR‐194, miR‐194 +

ACVR

2B, or negative control. D and E, Cell cycle distribution and cell apoptosis were analysed by flow cytometry. F, Expression levels of phosphorylated Smad2/3 were determined by western blotting. n = 3. *P < .05, **P < .01 vs control; #P < .05, ##P < .01 vs miR‐194

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