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HBx protein-mediated ATOH1 downregulation suppresses ARID2 expression and promotes hepatocellular carcinoma - PubMed

. 2017 Jul;108(7):1328-1337.

doi: 10.1111/cas.13277. Epub 2017 Jun 14.

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HBx protein-mediated ATOH1 downregulation suppresses ARID2 expression and promotes hepatocellular carcinoma

Qingzhu Gao et al. Cancer Sci. 2017 Jul.

Abstract

Hepatitis B virus X protein plays a crucial role in the pathogenesis of hepatocellular carcinoma. We previously showed that the tumor suppressor ARID2 inhibits hepatoma cell cycle progression and tumor growth. Here, we evaluated whether hepatitis B virus X protein was involved in the modulation of ARID2 expression and hepatocarcinogenesis associated with hepatitis B virus infection. ARID2 expression was downregulated in HBV-replicative hepatoma cells, HBV transgenic mice, and HBV-related clinical HCC tissues. The expression levels of HBx were negatively associated with those of ARID2 in hepatocellular carcinoma tissues. Furthermore, HBx suppressed ARID2 at transcriptional level. Mechanistically, the promoter region of ARID2 gene inhibited by HBx was located at nt-1040/nt-601 and contained potential ATOH1 binding elements. In addition, ectopic expression of ATOH1 or mutation of ATOH1 binding sites within ARID2 promoter partially abolished HBx-triggered ARID2 transcriptional repression. Functionally, ARID2 abrogated HBx-enhanced migration and proliferation of hepatoma cells, whereas depletion of ATOH1 enhanced tumorigenecity of HCC cells. Therefore, our findings suggested that deregulation of ARID2 by HBx through ATOH1 may be involved in HBV-related hepatocellular carcinoma development.

Keywords: ARID2; ATOH1; hepatitis B virus X protein; hepatitis B virus infection; hepatocellular carcinoma.

© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

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Figures

Figure 1
Figure 1
HB

x downregulated

ARID

2 expression in hepatoma cells. (a)

ARID

2 and

HB

x protein was detected by western blot analysis in HepG2.2.15 cells and HepG2 cells infected with adenoviruses expressing

HB

x or

GFP

control (n = 3, **P < 0.01). (b)

ARID

2 and

HB

x protein expression in

HBV

‐transgenic mice and

WT

littermates (

WT

mice; **P < 0.01). (c) HepG2 cells were transfected with

pCH

‐9(

HBV

1.1),

pCDNA

‐

HB

s,

pCDNA

‐

HB

c,

pCDNA

‐

HB

x, or vector plasmid.

ARID

2 and

HB

x protein was detected using western blotting (n = 3, *P < 0.05; **P < 0.01). (d) HepG2 cells were transiently transfected with full‐length

HBV

(

WT

‐

HBV

), stop‐mutant

HB

x (

HB

x‐stop), or stop‐mutant

HB

x plasmids plus adenovirus encoding

HB

x.

ARID

2 and

HB

x expression was determined by western blot analysis (n = 3, * P < 0.05; **P < 0.01). (e,f) Western blot analysis of

ARID

2 and

HB

x expression in Sk‐Hep1/Sk‐Hep1‐

HB

x, Huh7/Huh7‐

HB

x,

LO

2/

LO

2‐

HB

x, and

HB

x‐transduced hepatoma cells. All data were acquired from three independent experiments, and representative results are shown. Integrated density was quantitatively analyzed using ImageJ software. *P < 0.05; **P < 0.01, Student's t‐test.

Figure 2
Figure 2
HB

x was negatively correlated with

ARID

2 expression in

HCC

tissues. (a) Representative immunohistochemical staining of

HB

x and

ARID

2 in serial tissue slices from 24 paired

HBV

‐related

HCC

tissues and adjacent nontumorous tissues. Immunostaining intensity was assessed using Image‐Pro Plus 6.0 software. *P < 0.05; magnification: 400×. (b) Correlation analysis of ARID2 and HBx

mRNA

expression in 24 paired

HCC

tissues (*P < 0.05, r = −0.42, Pearson's correlation).

Figure 3
Figure 3
HB

x inhibited ARID2

mRNA

expression at the transcriptional level. (a) Luciferase activity of the human ARID2 promoter construct

pGL

3‐

ARID

2 in Huh7 cells. Huh7 cells were transfected with

pGL

3‐

ARID

2 (position ‐1040 to +101) for 24 h and then infected with Ad

GFP

control or Ad

HB

x. At 36 h postinfection, cells were harvested for luciferase assays. Data are presented as the mean (±

SD

) relative luciferase activity compared with the activity of the

pGL

3‐Basic control sample. Three independent experiments were performed. *P < 0.05; **P < 0.01 by Student's t‐test. (b) Luciferase assays of human ARID2 promoter constructs with the wild‐type sequence (‐1040/+101 nt) or the indicated serial deletion mutations in Ad

HB

x‐ or Ad

GFP

‐infected Huh7 cells (n = 3, *P < 0.05). (c) Ch

IP

assays of cell extracts from Huh7 cells infected with Ad

HB

x or Ad

GFP

. Huh7 cells were infected with Ad

HB

x or Ad

GFP

control. At 48 h post‐infection, cell lysates were collected, and Ch

IP

analysis was performed using control IgG or anti‐

HB

x antibodies. Transcriptional factor E2F1 recruited on ARID2 and CCND1 promoter were used as Ch

IP

positive control.

Figure 4
Figure 4
HB

x downregulated

ATOH

1 expression. (a) Illustration of the predicted transcription factor binding sites in the 1‐kb ARID2 promoter region according to the

JASPAR

database analysis. (b) The

mRNA

expression levels of transcription factors were detected by real‐time

PCR

.

SK

‐Hep1 cells were infected with Ad

HB

x virus or Ad

GFP

control. Total

mRNA

was isolated at 36 h after infection. Expression of genes encoding the indicated transcription factors was determined by

RT

‐

qPCR

(n = 3, *P < 0.05 versus

GFP

control). (c) Protein expression of

ATOH

1.

SK

‐Hep1 cells were treated as described above. At 48 h after infection, cell lysates were used for western blot analysis (n = 3, *P < 0.05; **P < 0.01).

Figure 5
Figure 5
HB

x inhibited ARID2 promoter activity via an

ATOH

1‐dependent pathway. (a) Luciferase activity of the human ARID2 promoter reporter

pGL

3‐

ARID

2 in Huh7 cells. Huh7 cells were transfected with

pGL

3‐

ARID

2 and then co‐infected with Ad

GFP

control or Ad

HB

x together with Ad

ATOH

1. Luciferase activities were measured at 24 h after infection (n = 3, *P < 0.05). (b)

ARID

2 protein expression was determined by western blotting in Sk‐Hep1/

SK

‐Hep1‐

HB

x cells infected with Ad

ATOH

1 or Ad

GFP

control (n = 3, **P < 0.01). (c) Ch

IP

assays of cell extracts from Huh7 cells infected with Ad

HB

x or Ad

GFP

using anti‐

ATOH

1 antibodies. IgG served as a negative control. The relative fold enrichment (bound/input) was measured by

qPCR

. Data represent the means ±

SD

s (n = 3, *P < 0.05). (d) and (e) The

mRNA

and protein expression levels of

ARID

2 in

ATOH

1‐depleted

SK

‐Hep1 cells.

SK

‐Hep1 cells were infected with lentiviruses carrying

ATOH

1 sh

RNA

or control sh

RNA

. Cells were performed

qRT

‐

PCR

and Western blot assay (n = 3, *P < 0.05; **P < 0.01). (f) Schematic representation of the ARID2 promoter region with the potential

ATOH

1 binding sites indicated. (g) Luciferase assay of ARID2 promoter constructs with the wild‐type (

WT

) or mutated

ATOH

1 binding site in Ad

GFP

‐ or Ad

HB

x‐infected Huh7 cells. Data are shown as means ±

SD

s (n = 3 independent experiments; *P < 0.05 by Student's t‐test).

Figure 6
Figure 6
ARID

2 partially reversed the enhanced migration and proliferation of hepatoma cells induced by

HB

x. (a) Transwell assays of cell migration in Sk‐Hep1/Sk‐Hep1‐

HB

x and Huh7/Huh7‐

HB

x cells. Cells were infected with Ad

GFP

control or Ad

ARID

2 and then subjected to transwell assays. Data represent the results of three independent experiments (means ±

SD

s; *P < 0.05; **P < 0.01 versus the

GFP

control; magnification: 200×). (b) E‐cadherin protein expression was determined by western blotting in Sk‐Hep1/

SK

‐Hep1‐

HB

x and Huh7/Huh7‐

HB

x cells infected with Ad

ARID

2 or Ad

GFP

control (n = 3, *P < 0.05; **P < 0.01). (c) Cell proliferation was analyzed by EdU incorporation assays. Cells were treated as described above. Data are presented as the means ±

SD

s (n = 3, *P < 0.05 versus the Vector control).

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