Sulfatide epigenetically regulates miR-223 and promotes the migration of human hepatocellular carcinoma cells

Research Article

Sulfatide epigenetically regulates miR-223 and promotes the migration of human hepatocellular carcinoma cells Yi Wei Dong1, Rong Wang1, Qian Qian Cai1, Bing Qi1, Wei Wu1, Yong Hu Zhang2, Xing Zhong Wu1,⇑ 1

Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Key Lab of Glycoconjugate Research, Ministry of Public Health, Shanghai, China; 2People’s Hospital of Beilun District, Ningbo, Zhejiang, China

Background & Aims: The biological relevance and regulation mechanism of aberrant miR-223 expression in human hepatocellular carcinoma (HCC) remain unknown. Our aim was to investigate miR-223 regulation in HCC. Methods: miR-223 and integrin aV dysregulation were verified in 57 HCC specimens. Immunohistochemical analysis of integrin aV and sulfatide levels was performed on another cohort of 103 HCC samples. Epigenetic analysis was used to explore the effect of sulfatide on miR-223 transcription. Orthotopic growth, and intrahepatic and pulmonary metastasis of tumors derived from SMMC-7721 cells expressing miR-223 or cerebroside sulfotransferase were monitored in mice. Results: miR-223 was reduced in HCC specimens and highly metastatic cell lines. Enhanced miR-223 expression had a negative effect on integrin aV-mediated cell migration. In vivo assays of metastasis in an orthotopically implanted model demonstrated that miR-223 effectively inhibited HCC metastasis. Further analysis demonstrated that integrin aV is negatively regulated by miR-223. Moreover, the integrin aV subunit was significantly positively correlated with highly expressed sulfatide in 103 HCC specimens. Intriguingly, miR-223 expression was suppressed by sulfatide in HCC in association with reduced recruitment of acetylated histone H3 and C/EBPa to the pre-miR-223 gene promoter, where monocytic leukemia zinc finger (MOZ) protein, a MYST-type histone acetyltransferase, lost its attachment. The expression of histone deacetylases, HDAC9 and HDAC10, were greatly stimulated by sulfatide and their recruitment to miR223 gene promoter was enhanced. Conclusions: Downregulation of miR-223 in HCC is associated with the epigenetic regulation by highly expressed sulfatide and involved in tumor metastasis. Ó 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Keywords: Liver cancer; microRNA; Sulfated cerebroside; Metastasis. Received 19 September 2013; received in revised form 22 November 2013; accepted 2 December 2013; available online 11 December 2013 ⇑ Corresponding author. Address: Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, China. Tel.: +86 21 5423 7697. E-mail address: [email protected] (X.Z. Wu). Abbreviations: miRNA, microRNA; Gal-Cer, galactocerebroside; CST, cerebroside sulfotransferase; MOZ, monocytic leukemia zinc finger protein.

Introduction Hepatocellular carcinoma (HCC) is the most common liver malignancy and a common cause of cancer-related deaths worldwide. Considering limited conventional treatment options for HCC, identification of new treatment targets is required. Recent studies emphasized causative links between alteration of microRNA (miR) expression profiles and HCC development [1]. Apart from genetic and epigenetic abnormalities, such as modified oncogenes and tumor suppressor genes, dysregulation of miRNAs may be attributable to aberrant signaling pathways, especially in HCC. Evidence is mounting that regulatory causes of abnormal miRNAs are pervasive and play important roles in hepatocarcinogenesis. We reported that miR-223 suppresses HCC cell proliferation [2], and identified transcription factor FOXO1 as a miR-223 target in cell proliferation regulation [3]. Nevertheless, the pathogenesis of miR-223 deregulation and roles of miR-223 in HCC metastasis remain largely undefined. Integrins, serving as cell-cell and cell-substratum adhesion molecules control tumor initiation, progression and metastasis [4], and perform crucial roles in HCC development and malignancy [5]. The adhesion receptor aVb3 is generally highly expressed on the invasive front cells during tumor angiogenesis and is associated with metastatic potential [6]. Several lines of evidence suggest that an epigenetic mechanism is involved in the miRNA dysregulation in cancer [7]. Histone modification and methylation play critical roles in chromatin remodeling and gene regulation, particularly in cancer. Fazi et al. [8] reported that heterochromatic silencing of miR-223 is induced by recruitment of chromatin-remodeling enzymes, including histone deacetylase (HDAC) and DNA methyltransferases, to the pre-miR-223 promoter, and that demethylation contributes to miR-223 expression. MYST-type histone acetyltransferases including TIP60 (KAT5), HBO1 (KAT7/MYST2), MOF (KAT8/MYST1), MOZ (monocytic leukemia zinc finger, MYST3/KAT6A), and MORF (MOZ-related factor/KAT6B), acetylate lysine residues on histones H2A, H3, and H4. They share a highly conserved MYST domain composed of an acetyl-CoA binding motif and a zinc finger, and function in multisubunit protein complexes. Methylation levels of miRNA genes may also affect the malignant phenotype of cancer cells by altering regulatory

Journal of Hepatology 2014 vol. 60 j 792–801

1000

B

miR-223

800

HCC non-tumor tissues

p <0.01

400 200

T: tumor tissues NT: adjacent non-tumor tissues N: normal tissues

21

T
45

79

Relative migration ability

0.0

0

48

72

Control miR-223 miR-223 + integrin αV

-

-

100 80 60 40 20 0

0

48

72 h

Time (h)

10x

72 Scramble miR-223 inhibitor miR-223 inhibitor + si-integrin αV

Relative closure of wound (%)

-

+

48

72 h

Integrin αV

2.5 2.0 1.5 1.0 0.5 0.0

1.0 0.9 0.8 0.7 0.6 0.5

GAPDH **

**

0.7

1.0

0.6

0.5

Control miR-223

0.0

0.5

T1#

T2#

10x Control

miR-223 Control

**

15 10 5 0

Pulmonary metastases

20

Journal of Hepatology 2014 vol. 60 j 792–801

miR-223

No metastase Metastase 120 100 80 60 40 20 0

Control miR-223

F

No. of intrahepatic metastases (%)

miR-223

0

1. Control 2. miR-223

T2# 1 2

No. of mice with pulmonary metastases (%)

10x

n.s.

20 0

T1# 1 2 miR-223 Integrin αV ** *

80 40

10x

D

100 60

10x

Relative protein levels (fold change)

48

Control miR-223

0

-

Control

0.1

+

Intrahepatic metastases

Implanted tumor Intrahepatic metastases

1.0 0.8 0.6 0.4 0.2 0.0

** 0.2

Time (h)

Relative RNA levels (fold change)

si-integrin αV Scramble miR-223 inhibitor

miR-223

10x

E

Control miR-223 **

SMMC-7721 Control

SMMC-7721

12

*** *

C

Percentage

T >NT

SW480 SW620

**

0

Relative closure of wound (%)

0

miR-223 Frequency

Integrin αV

20

Control

40

0.0

miR-223

**

MHCC97L MHCC97H

***

** 60

SMMC-7721

Relative miR-223 levels (fold change)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57

0

LO2 BEL-7404 SMMC-7721 ** ** 1.0 4 0.8 3 0.6 2 0.4 1 0.2

***

600

Relative miR-223 levels (fold change)

A

Relative metastasis ability

JOURNAL OF HEPATOLOGY

793

Research Article networks triggered in cancer. Identification of endogenous modulators that directly or indirectly regulate miR-223 gene expression by epigenetic mechanisms is important in understanding the development and metastasis of HCC. Sulfatide regulation of miRNA has not yet been known according to current documents. Sulfatide, a sulfoglycolipid, is usually elevated in HCC and can protect hepatocytes from ischemia/ reperfusion injury [9], but the relationship between elevated sulfatide and the development of HCC remains unknown [10]. We previously showed that sulfatide enhances integrin aV expression, leading to HCC metastasis [11]. Based on those findings, we investigated the effect of sulfatide on miR-223 transcription and demonstrated the regulatory importance of sulfatide, the endogenous modulator of miR-223, in migration and metastasis of HCC cells.

Immunohistochemistry Immunohistochemical staining of integrin aV and sulfatide was performed. The staining protocol and the scoring process using a semi-quantitative system are described in the Supplementary material. Cell migration and wound healing assays In vitro migration was assayed as previously described [2]. Wound healing was assayed in subconfluent monolayers wounded by scraping off. DNA methylation analysis DNA methylation was determined by methylation-specific PCR [16]. In vivo metastasis assay All experiments involving animals for assessment of metastasis were performed as previously reported [17].

Materials and methods Human samples

Statistical analysis

HCC patients (103 + 57) were pathologically confirmed [12,13]. Histopathological grading was according to criteria of Edmondson and Steiner [14]. Supplementary Table 1 shows the clinicopathological data.

Statistical analysis was performed using SPSS software. Results are presented as mean ± SEM of at least three independent replicates.

Cell lines and reagents

Results

SMMC-7721 HCC cells were infected with lentivirus containing miR-223 (SMMC7721miR-223) or control virus (SMMC-7721control). Purified bovine brain-derived sulfatide and galactocerebroside were from Sigma.

miR-223 is downregulated in HCC

Plasmid construct The sequence encoding pre-miR-223 was cloned into the HpaI/Xhol sites of the lentiviral vector pLL3.7 [2]. Quantitative polymerase chain reaction (PCR) PCR was conducted as previously described [15]. Primer sequences are shown in Supplementary Table 2. Western blotting Western blot analysis was performed as described previously [2]. Chromatin immunoprecipitation (ChIP) assay Cells (5  106) were harvested for ChIP using the EZ-ChIP™ kit (Millipore). Immunoprecipitation was performed following incubation with antibodies against RNA Pol II, AcH3K9/14, C/EBPa, C/EBPb, Sp1, a-50 -methylcytosine, or normal rabbit IgG. The promoter of the miR-223 gene was detected by qPCR.

miR-223 was quantified by qRT-PCR in a cohort of 57 patients with HCC. We compared miR-223 expression in HCC tumor tissue with matching adjacent non-tumor liver tissues or normal liver tissues and found that the level of miR-223 was significantly downregulated by 46.1% and 65.3%, respectively (Fig. 1A). miR223 expression was also downregulated in SMMC-7721 and BEL-7404 HCC cells, compared with non-tumor LO2 hepatocytes, and in cell lines with high metastatic potential compared with those with low metastatic potential (Fig. 1B). We characterized the effect of miR-223 overexpression on migration in SMMC7721 cells infected with lentivirus carrying miR-223 or control virus (Supplementary Fig. 1A–C). The number of migrating SMMC-7721miR-223 cells was significantly lower than that of SMMC-7721control cells (Fig. 1C). The in vitro wound healing assay showed that control cells migrated significantly faster than SMMC-7721miR-223 cells on day 3, and integrin aV overexpression stimulated closure of wounded SMMC-7721 monolayers. Loss-offunction analysis suggested that inhibitor oligonucleotideinduced miR-223 silencing hastened wound closure, while silencing of integrin aV subunit expression diminished its effect on

Fig. 1. Differential expression of miR-223 in HCC. (A) Relative miR-223 expression level in patients P1–P57 was analyzed by qRT-PCR. Differences in miR-223 expression were analyzed between HCC tumor (T) tissue and adjacent non-tumor (NT) tissue or normal (N) tissue adjacent to cavernous hemangioma of three patients. (B) miR-223 expression was measured in LO2, BEL-7404, SMMC-7721 cells (left) and two pairs of cells with high (MHCC97H and SW620) and low (MHCC97L and SW480) metastatic potential (middle and right). (C) Representative micrographs of miR-223-overexpressing (miR233) and control cells in transwell migration assay and quantitative analysis of migration. Bar = 25 lm. miR-223-mediated inhibition of migration in wound healing assays. Relative wound closure is quantified on the right. Magnification: 10. (D) miR-223 and integrin aV mRNA expression in isolated tumor tissues of control and miR-223 groups analyzed by qRT-PCR (left). Integrin aV protein measured by Western blotting and quantified by densitometry (right). T1#: tumor 1, T2#: tumor 2. (E) Representative images of orthotopically implanted tumors in livers and H&E-stained sections of tumors in control and miR-223 groups. Arrows indicate tumors. Boxed fields are shown at higher magnification in corners. (F) Representative images of livers or lungs and H&E stained sections of intrahepatic or pulmonary metastases and quantification of metastatic foci. Arrows indicate visible metastatic foci. Data are representative of at least three independent experiments. ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001, n.s., not significant.

794

Journal of Hepatology 2014 vol. 60 j 792–801

JOURNAL OF HEPATOLOGY

GAPDH **

**

2 1

Integrin αV

T = NT

14 (13.6%)

24 (23.2%) 103

Sulfatide 73 (70.9%)

16 (15.5%)

14 (13.6%) 103

P103 P1

Patients

L-

5

5

0

0

40

H

4

97 C

2 T NT

Journal of Hepatology 2014 vol. 60 j 792–801

as es

as

st et a o

N

o N

6

m

et M

et

Sulfatide (%)

M

20 40 60 80 100

as es

0

es

20

8

0

BE 10

**

60

0

10

74

72 10

Sulfatide scores

**

st

***

Integrin αV scores 15

et a

0 12

15

Relative fluorescence intensity

T
65 (63.1%)

Patients

2

80

DAPI

T >NT Integrin αV

P1

Sulfatide scores

Sulfatide

T NT

4

C

*** 12 10 8 6 4 2 0

6

4 1 2 LO -740 -772 L C BE MM S

R = 0.425 p <0.01

100

8

H

0

10

M

2

Pearson’s correlation

Sulfatide

4

F Integrin αV (%)

6

Integrin αV scores

8

12

Sulfatide scores

T NT

10

T: tumor tissues NT: adjacent non-tumor tissues

***

***

P103

Integrin αV scores

NT

Integrin αV

T

12

0.0

es

Mann-Whitney U test

E

0.1

as

p <0.001***

0.2

as t

16

m

84

9

L

48

T
** *

97

T >NT

45

Integrin αV 0.3

C

9

12

R = -0.4115 p <0.01

β-actin

0

0

0

Integrin αV

2

C

48

miR-223

-7 M SM 4

2

H

Integrin αV

C

LBE

4

T NT N

10

21 04 77 -74 MCL 2 E M S LO B

6

M

Frequency Percentage

Integrin αV ** 6 ** 8

Relative mRNA levels M H (fold change) M CC H 9 C 7 C L 97 SW H S 4 Relative mRNA levels W 80 62 (fold change) 0

Integrin αV

1

04 74

72 -7 C M Relative mRNA levels (fold change)

D

Integrin αV ** 20 **

04

0

0.0

Control miR-223 inhibitor

1.3 1.2 1.1 1.0 0.9 0.8

3

0.5

Scramble miR-223 inhibitor

+

**

4

1.0

miR-223 levels

T
+

+

**

Control miR-223

1.5

Pearson’s correlation T >NT

+

36

Relative protein levels (fold change)

04

1

Relative mRNA levels (fold change)

Integrin αV

SMMC-7721 BEL-7404

1. Control 2. miR-223

2

Integrin αV

74

72

1

121

L-

-7

2

SM

SM

M

BE

C

-7

Lu

72

r Li

C

C

0.40

**

BE

0.0

** 1.0 0.8 0.6 0.4 0.2 0.0

M

0.45

ng

0.5

**

SM

0.50

1.0

** 1.0 0.8 0.6 0.4 0.2 0.0

1

Control miR-223

Relative mRNA levels (fold change)

**

0.55

ve

Relative protein levels (fold change)

**

Integrin β3

Control miR-223

GAPDH

1.5

1

1

Integrin αV

Integrin αV

SMMC-7721 BEL-7404

as t

B

1. Control 2. miR-223

04

Lung 1 2

74

Liver 1 2

L-

A

1.5 1.0

** MHCC97H MHCC97L

0.5 0.0

795

Research Article the cell migration (Fig. 1C and Supplementary Fig. 1D). These findings indicate that miR-223 is downregulated in HCC and negatively affects integrin aV-mediated cell migration. Ectopic miR-223 expression suppresses HCC metastasis in nude mice We injected SMMC-7721 cells expressing miR-223 into nude mice and monitored body weight and tumor formation. Subcutaneous tumor growth was significantly inhibited in the miR-223 group (Supplementary Fig. 1E and F). The miR-223 level in tumor tissue of the miR-223 group was 1.95-fold higher than in the control group, whereas integrin aV mRNA and protein were downregulated compared to control (Fig. 1D). These differences became more apparent after orthotopic implantation of the subcutaneous tumor tissues into the livers of nude mice (Fig. 1E). Visible intrahepatic metastases occurred in all eight mice (100%) in the control group, but in only six of eight mice (75%) in the miR-223 group. The number of liver metastatic foci in the miR-223 group was significantly reduced by 76.5%; foci were confirmed by histopathology. More mice in the control group (4/ 8, 50%) exhibited pulmonary metastases with a few widely scattered hemorrhagic spots than in the miR-223 group (1/8, 12.5%); metastases were confirmed by histopathology (Fig. 1F). Integrin aV protein in the liver and lung metastatic foci of the miR-223 group were significantly lower compared to control (Fig. 2A). Integrin aV subunit is inhibited in miR-223-overexpressed cells Integrins are important adhesive molecules involved in cell migration [18]. In cells stably expressing miR-223, it was noted that integrin aV and b3 subunits, which can form a heterodimer involved in cell migration, were significantly down regulated (Fig. 2A). However, miR-223 inhibitor enhanced integrin aV protein in SMMC-7721 and BEL-7404 cells (Fig. 2B). qRT-PCR analysis showed that, in 57 HCC patients, integrin aV expression was significantly higher in tumor tissue than in adjacent non-tumor tissue (p <0.01), and the level of integrin aV mRNA in tumor tissue was 96.4% greater than in normal tissue. An inverse correlation between miR-223 and integrin aV expression was observed in most (44/57, 77%) HCC specimens (R = 0.4115, p <0.01) (Fig. 2C). Integrin aV was also expressed at a significantly higher level in cell lines with high metastatic potential than in those with low metastatic potential (Fig. 2D), indicating a correlation between integrin aV expression and metastatic potential. Integrin aV expression was significantly higher in SMMC-7721 and BEL7404 cells than in LO2 hepatocytes (Fig. 2D). These findings indicated that miR-223 had a negative correlation with integrin aV.

Sulfatide is upregulated in HCC and positively correlates with integrin aV To further investigate the expression of integrin aV and sulfatide in HCC, paraffin sections of liver specimens from another cohort of 103 patients (Supplementary Table 1) were immunohistochemically stained and histomorphometry was performed using optical density measurements of stained sections (Supplementary Fig. 2A). This analysis showed that 63.1% and 70.9% of patients exhibited integrin aV and sulfatide upregulation (Fig. 2E), respectively, in cancerous tissues, compared to non-cancerous tissues. Immunohistochemical staining scores for integrin aV and sulfatide increased by 21.6% and 25.3%, respectively (Fig. 2E middle & right). Pearson’s correlation analysis indicated that integrin aV expression was significantly positively correlated with sulfatide expression (r = 0.425, p <0.01; Fig. 2F). Patients with lymph node metastases expressed significantly higher levels of integrin aV and sulfatide than those without metastases (Fig. 2F). In highly metastatic MHCC97H cells, the fluorescence intensity of sulfatide staining was much greater than in MHCC97L cells, which showed low metastatic potential (Fig. 2F), as described previously. Sulfatide also significantly impacted cell migration, hastening relative wound closure after 72 h of treatment in SMMC-7721 and BEL-7404 cells (Fig. 3A). Consistently, exogenous sulfatide treatment significantly enhanced integrin aV expression in SMMC-7721 and BEL-7404 cells (Supplementary Fig. 2B), indicating that sulfatide activates integrin aV expression. To confirm the regulatory effect of sulfatide, we established cells stably expressing CST, which catalyzes the conversion of galactocerebroside to sulfatide [19]. CST overexpression stimulated integrin aV expression in CST-1 and CST-8 transfectants, whereas RNAi-mediated CST knockdown substantially reduced integrin aV expression (Supplementary Fig. 2C). Sulfatide suppresses miR-223 expression Because integrin aV was upregulated upon sulfatide treatment and an inverse relationship between integrin aV and miR-223, we hypothesized that sulfatide may exert a substantial impact on miR-223 expression. In SMMC-7721 and BEL-7404 cells, miR-223 expression declined sharply after sulfatide, but not galactocerebroside, treatment (Fig. 3B and Supplementary Fig. 3A) and after cerebroside sulfotransferase transfection, which produces endogenous sulfatide even when cotransfected with the miR-223 precursor. Silence of CST by RNA interference resulted in elevation of miR-223 expression. Intriguingly, sulfatide reversed

Fig. 2. miR-223 negatively regulates integrin aV which is correlated with sulfatide expression. (A) Integrin aV expression in metastasis tissues of liver and lung in control and miR-223 groups measured by Western blotting and quantified by densitometry. The integrin aV and b3 mRNA levels were assessed in miR-223 transfectants by qRT-PCR (right). (B) Integrin aV protein was measured by Western analysis and quantified via densitometry. (C) Integrin aV mRNA level in HCC patients P1–P57 was analyzed by qRT-PCR. Expression levels were compared between HCC (T) and adjacent non-tumor (NT) tissue, and normal (N) liver tissue. Pearson’s correlation between miR-223 and integrin aV expression (right bottom). (D) Integrin aV mRNA level was analyzed in cell lines with high (MHCC97H and SW620) and low (MHCC97L and SW480) metastatic potential. Integrin aV expression was determined by Western analysis and qRT-PCR in the indicated cell types. (E) Representative images of immunochemical staining of integrin aV and sulfatide in HCC (T) and corresponding adjacent non-tumor (NT) liver tissues from 103 patients (40). Immunohistochemical scores for integrin aV and sulfatide expression for individual HCC specimens (T) and the adjacent non-tumor (NT) tissue were plotted (right). Scores presented as box plots, with horizontal and vertical lines representing the median and range, respectively. (F) Pearson’s correlation between sulfatide and integrin aV expression (left). Comparison of integrin aV and sulfatide (right) staining scores in patients (n = 103) with and without metastasis. Immunofluorescent staining of sulfatide and DAPI staining of nuclei in MHCC97H and MHCC97L cells (bottom). Data are representative of at least three independent experiments. ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001.

796

Journal of Hepatology 2014 vol. 60 j 792–801

JOURNAL OF HEPATOLOGY

1.5

SMMC-7721 l ntro A Co TS

* ** **

n.s.

AcH3K9 H3

0.0

0.0

BEL-7404

E

Oligo2 f2 r2

F DAPI

Oligo1 f1 r1

Pri-miR-223

Relative miR-223 levels (fold change)

1547

C/EBPα

miR-223

ChIP

miR-223 pmt (oligo1) Input miR-223 pmt (oligo2) Input

SMMC-7721 er de l ntro l-C fati Co Ga Sul

0.6

0.2

**

Ace-C/EBPα

0.0

1.0

C/EBPα

0.5 0.0 1.0 0.8 0.6 0.4 0.2 0.0

**

C/EBPα

0.4

GAPDH IP: antiAcH3K9/14

Sc C r1 ST -2 si

BEL-7404 Cer ide Gal- Sulfat

3’

C/EBPα binding sites

**

10 4 3 2 5 1 6 9 7 8 AC DAC DAC DAC DAC DAC DAC DAC DAC DAC H H H H H H H H H

AML Pre-miR-223

Relative mRNA levels (fold change)

Sulfatide staining Control

Sulfatide

0

Gal-Cer Sulfatide

3 2 1 0

5’

C/EBP

5

H

6

HD

0

Control TSA

10

SMMC-7721

8

-709 -426

-3415

**

Ac

10

Relative mRNA levels (fold change)

0.5

0.0

3K

+ + Sulfatide + + TSA

1.0

0.5

9/

+ DMSO

+

1.0

**

AcH3K9/14

C

C

on

tro TS l A

0.0

tro TS l A

C

miR-223

tro l N A

0.5

Sulfatide

0.0

on

1.0

Relative miR-223 levels (fold change)

1.0

on

Relative miR-223 levels (fold change)

**

SMMC-7721

0.5

0.5

1.5

0.0

1.0

1.0

miR-223

0.5

1.5

Control miR-223 Sulfatide + miR-223 Sulfatide

Integrin αV ** ** * * ** 1.5

9

trol A N Con

U6

1.5

Mock CST CST + miR-223 miR-223

** **

3K

D

miR-223

2.0

96 h

0.0

**

ChIP IP: antiC/EBPα miR-223 pmt (oligo2) Input

Control Gal-Cer Sulfatide

Journal of Hepatology 2014 vol. 60 j 792–801

BEL-7404 l er ide ntro l-C lfat Co Ga Su

Relative mRNA levels (folg change)

BEL-7404

l ntro A Co TS

**

48

0.5

H

0

1.0

14

0

2.0

1.5

Ac

20

LBE

C **

40

**

20x

SMMC-7721

C

SM

M

20x

60

Relative miR-223 levels (fold change)

20x

80

Relative miR-223 levels (fold change)

Relative closure of wound (%)

Gal-Cer Sulfatide

BEL-7404

Gal-Cer Sulfatide

100

**

Relative protein levels (fold change)

96 h

Relative mRNA levels (fold change)

48

Relative protein levels (fold change)

0

-7

0

1.0 0.8 0.6 0.4 0.2 0.0

oc C k3 ST -8

20

1.0 0.8 0.6 0.4 0.2 0.0

**

M

**

40

**

Relative miR-223 levels (fold change)

60

**

1

Relative closure of wound (%)

Gal-Cer Sulfatide

80

74

Gal-Cer Sulfatide

100

SMMC-7721

Gal-Cer Sulfatide

B

72

72

48

Relative miR-223 levels (fold change)

0

04

Time (h)

A

n.s.

AceC/EBPα

0.5 ** 0.4 0.3 0.2 0.1 0.0 Gal-Cer Sulfatide

0.4 0.3 0.2 0.1 0.0

**

Control Gal-Cer Sulfatide

797

Research Article the miR-223-induced inhibition and restored integrin aV expression (Fig. 3B). These findings support our hypothesis that sulfatide modulates endogenous miR-223 expression, leading to enhanced integrin aV expression. However, the mechanism underlying sulfatide regulation of miR-223 remains unknown. To further study, we exposed SMMC-7721 cells to class I and II HDAC inhibitor trichostatin A [20] or sirtuin inhibitor nicotinamide (NA). TSA rapidly elevated miR-223 expression (Fig. 3C) and relieved sulfatide-induced suppression (Fig. 3D left), but NA not. Likewise, TSA markedly increased histone H3 acetylation on lysines 9 and 14 (AcH3K9/14; Fig. 3D right), whereas sulfatide had no effect on total or acetylated H3 levels (Supplementary Fig. 3B). Among HDAC1-10 and Sirt1-7 we noted that expression of HDAC9 and HDAC10 were upregulated after sulfatide treatment (Fig. 3D). Interestingly, sulfatide was able to accumulate in the nucleus apart from membrane in the cells treated with sulfatide (Fig. 3E). These data suggest that the regulation by sulfatide involves deacetylation.

with the miR-223 promoter was also observed in ChIP analysis, and the association was strengthened after sulfatide treatment (Fig. 4B). We further analyzed miR-223 expression in cells exposed to DNA methylation inhibitor 5-aza-20 -deoxycytidine (decitabine, DAC) and observed that DAC significantly enhanced miR-223 expression in HCC cells. Intriguingly, sulfatide still suppressed miR-223 expression in SMMC-7721 cells in the presence of DAC. We also assessed the methylation status of the pre-miR223 promoter. ChIP analysis with a-50 -methylcytosine antibody showed that cytosines on the pre-miR-223 promoter were weakly methylated in SMMC-7721 cells, and unmethylated in LO2 hepatocytes. Sulfatide had no effect on amplification of either unmethylated or methylated sequences (Fig. 4B). Although promoter methylation is a major mechanism by which expression of miR-223 and other tumor suppressor genes is downregulated, [22] sulfatide inactivated miR-223 gene expression through reduced recruitment of acetylated histone H3 and C/EBPa to its promoter.

Sulfatide prevents acetylated histone H3 and C/EBPa recruitment to the miR-223 promoter

CST expression suppresses miR-223 and promotes HCC metastasis in nude mice

Local modification of promoter and histones is required to activate gene expression [21,22], so we completed ChIP assays using anti-AcH3K9/14 antibody and PCR amplification of the pre-miR223 promoter using two pairs of primers (Fig. 3F). Overall, AcH3K9/14 was associated with the miR-223 promoter in both control and galactocerebroside groups, whereas greatly decreased association was observed in the sulfatide group (Fig. 3F). The miR-223 promoter contains two C/EBP binding sites [23], which interact with different transcription factors to promote miR-223 expression. Although no change was observed in total C/EBPa in sulfatide-treated cells, C/EBPa acetylation declined significantly (Fig. 3F). We further investigated C/EBPa recruitment to the miR-223 promoter and noted that C/EBPa occupancy decreased significantly after sulfatide treatment (Fig. 3F). Reduced attachment of both acetylated histone H3 and C/EBPa to the promoter suggests hindered transcription at these sites on the gene. Association of MOZ, HBO1, HADAC9, and HADAC 10 were found in the proximal and distal miR-223 promoter. HDAC9 and HADAC 10 recruitment to the miR-223 gene promoter was enhanced, but MOZ almost lost its binding to the miR-223 promoter after sulfatide treatment in proximal region of the miR223 promoter (Fig. 4A). Occupancy of the miR-223 promoter by BRG1, the central catalytic subunit of the Swi/Snf complex, was slightly increased by sulfatide. Interestingly, sulfatide association

Cerebroside sulfotransferase is the key enzyme for the synthesis of sulfatide. Overexpression of CST enhanced cell migration in a wound-healing assay in SMMC-7721 monolayers, while knockdown of CST hindered the cell migration (Fig. 4C). In the tail vein metastasis assay, the mice receiving CST transfectants had significantly more metastatic lesions in the liver and lung than mock (Fig. 4D). Interestingly, in the metastatic tissues the miR-223 expression level was significantly lower, but integrin aV higher, in CST than that in mock (Fig. 4E). These results indicate that the endogenous sulfatide produced by CST inhibited miR-223 transcription and enhanced the metastatic ability of HCC cells.

Discussion Tumor metastasis is a complex process involving cell migration, invasion, angiogenesis, spreading, and colonization of secondary organs. Identification of molecules and pathways contributing to metastasis is critical to understanding carcinogenesis and developing therapeutic strategies. Recent evidence suggests that miR-223 is involved in cancer development [24] and low miR223 expression in chronic lymphocytic leukemia can predict shorter treatment-free and overall survival [25]. However, the mechanisms underlying miR-223 downregulation and the regulatory pathway in metastasis are not fully understood. Here,

Fig. 3. Sulfatide suppresses miR-223 expression. (A) Wound healing assays at indicated time points in SMMC-7721 and BEL-7404 cells treated with 2 lM sulfatide or galactocerebroside, and quantitative analysis (right). (B) miR-223 expression level determined by qRT-PCR in SMMC-7721 and BEL-7404 cells treated with sulfatide or galactocerebroside (Gal-Cer). CST: cerebroside sulfotransferase; Mock: control, siCST: small interfering CST; Scr: scramble. Integrin aV mRNA expression measured in cells transfected with miR-223 and/or treated with sulfatide (2 lM, 24 h, bottom right). Control groups were treated with DMSO equal in volume to sulfatide. (C) miR-223 expression in SMMC-7721 and BEL-7404 cells after exposure to Trichostatin A (TSA, 300 nM, 24 h) or nicotinamide (NA, 20 mM, 48 h) was analyzed by RT-PCR and qRT-PCR. (D) TSA attenuated sulfatide-induced suppression of miR-223 expression (left). Acetylated forms of histone H3 were analyzed in TSA-treated SMMC-7721 cells by Western and quantified by densitometry (right). AcH3K9/K14, H3 acetylated at lysines 9 and 14; AcH3K9, H3 acetylated at lysine 9. The mRNA expression of HDACs was quantified by qRT-PCR after sulfatide treatment (lower). (E) Cellular distribution analysis of the sulfatide by a confocal microscopy. (F) Oligo1 (f1, r1) and oligo2 (f2, r2) span the C/ EBPa binding region and distal C/EBP binding site, respectively, in the miR-223 promoter (upper). ChIP (chromatin immunoprecipitation) assay of sulfatide-treated SMMC7721 cells using oligos 1 and 2 (lower) and anti-AcH3K9/14 antibody, and quantification of amplification. Levels of total and acetylated C/EBPa in sulfatide-treated BEL7404 cells analyzed by Western (right). Recruitment of C/EBPa to miR-223 promoter in BEL-7404 cells was analyzed by ChIP and quantified. IP, immunoprecipitation. Data are representative of at least three independent experiments. ⁄p <0.05, ⁄⁄p <0.01, n.s., not significant.

798

Journal of Hepatology 2014 vol. 60 j 792–801

JOURNAL OF HEPATOLOGY

HDAC9 HDAC10

MOZ

100 80 60 40 20 0

HDAC9 HDAC10 ** **

HDAC9

HBO1

MOZ

HDAC10

miR-223 * ** *

2.5 2.0 1.5 1.0 0.5 0.0

etC os. g. P Ne

5M

U

Gal-Cer Sulfatide U M U M

M

miR-223 pmt (oligo1)

+ DMSO

+

C

60

SMMC-7721

LO2

100

n.s.

Mock3

BRG1

Relative closure of wound (%)

**

AC

Z MO

80 60 40 20 0

20

10x

80 60 40 20 0

0

48

72 h

10x

40 Integrin αV staining

Ac

Ac

H3 0

tro C l ST

on

Lung

C Liver

5

CST

Liver

1.0 0.8 0.6 0.4 0.2 0.0

tro C l ST

***

1.0 0.8 0.6 0.4 0.2 0.0

10

Mock

**

on

0

Mock CST

10

**

C

20

miR-223 Relative miR-223 levels (fold change)

E

30

Mock CST

Liver metastasis count

10x

72 h

Scr1 siCST-2

100

Relative closure of wound (%)

SMMC-7721

CST

48

**

IP: HDAC9 IP: HDAC10 IP: MOZ IP: BRG1

Pulmonary metastasis count

0

Oligo2

Oligo1

Scr1

.

HD

10

40

0

eg

s.

t

A

C9

siCST-2

% Input

60

Mock3 CST-8

72

CST-8

BRG1

Po

48

20 HD

Input

Time (h) 0

40 0

Input

pu

ut

+ + DAC + + Sulfatide

SMMC-7721

% Input

BRG1

In

0

miR-223 pmt (oligo1)

***

Pulmonary metastases

20

80

Input

Mock

Liver metastases

40

l r e ntro Ce tid Co Gal- Sulfa

SMMC-7721 Inp

miR-223 pmt (oligo2) MOZ

*

l r de ntro Ce ati Co Gal- Sulf 60

IP: antisulfatide miR-223 pmt (oligo1) Input

miR-223 pmt (oligo1)

20 0

ChIP SMMC-7721

tide

ut os. eg. ulfa P N Inp S

40

HBO1

N

miR-223 pmt (oligo2) (oligo1)

miR-223 pmt

(oligo2)

**

SMMC-7721

**

miR-223 pmt

*

B

Control Gal-Cer Sulfatide

% Input

l r de ntro -Ce fati Co Gal Sul 60

Input

D

miR-223 pmt (oligo1)

% Input

miR-223 pmt (oligo1)

IP: anti-

SMMC-7721

Relative miR-223 levels (fold change)

ChIP

% Input

A

Lung

MOZ

H3

Ac C/EBPα

High expression miR-223

Sulfatide C/EBPα

H3

H3 HDAC

Low expression miR-223

Journal of Hepatology 2014 vol. 60 j 792–801

799

Research Article miR-223 downregulation was confirmed in tumor, compared with non-tumor tissue, consistent with the previous report [24]. miR-223 downregulation was also observed in tumor cells with high (vs. low) metastatic potential. We showed unequivocally that ectopic miR-223 expression reduced SMMC-7721 and BEL-7404 cell migration, whereas miR-223 inhibitor reversed this trend, and integrin aV silence diminished the effect of miR-223 inhibitor (Fig. 1). These observations indicate that miR-223 plays a significant role in HCC cell migration. Integrin aV is a subunit of the vitronectin receptor [26], which forms a heterodimer with integrin b3 and contributes to HCC metastasis. We observed that integrin aV was negatively regulated in miR-223-overexpressed cells (Figs. 1 and 2, Supplementary results), and an inverse correlation between miR-223 and integrin aV expression in HCC specimens and cell lines with different metastatic potential; the data suggested that miR-223 deregulation may lead to alterations in integrin aV expression in HCC. We previously observed that sulfatide [11] induces expression of integrin aV. Here, integrin aV and sulfatide were upregulated in HCC tumor tissue compared to non-tumor tissue. Sulfatide significantly increased integrin aV mRNA and protein expression, indicating sulfatide-induced activation of integrin aV transcription and translation. Overexpression of CST, which catalyzes production of sulfatide, also enhanced integrin aV mRNA and protein expression, and sulfatide treatment significantly facilitated relative wound closure, demonstrating that sulfatide activates integrin aV upregulation and impacts cell migration in HCC. Because integrin aV were upregulated by sulfatide, we postulated that sulfatide had an impact on miR-223 expression and activated its downstream targets in HCC. Sulfatide treatment significantly reduced miR-223 expression in SMMC-7721 and BEL7404 cells, and HDAC inhibitor relieved this repression. We then investigated whether sulfatide influenced AcH3K9/14 or C/EBP recruitment to the miR-223 promoter in vivo. ChIP analysis indicated that promoter binding of AcH3K9/14 and C/EBPa declined substantially in sulfatide-treated cells. Reduced attachment of both proteins suggested hindered transcription of the miR-223 gene. CpG methylation [8] is closely associated with gene silencing; however, sulfatide treatment did not change the methylation status of the pre-miR-223 promoter in SMMC-7721 cells, suggesting that sulfatide-induced miR-223 suppression was not mediated by promoter methylation. Hence, sulfatide-induced miR-223 downregulation in HCC more likely occurs through local histone and C/EBPa deacetylation than by DNA methylation, due to the dissociation of MOZ, the histone acetyltransferase, from the promoter. It is also possible that HDAC9 and HDAC10 catalyze the deacetylation since their recruitment to the miR-223 promoter was enhanced by sulfatide (Fig. 4E bottom). Occupancy

of BRG1, the core subunit of ATP-dependent chromatin remodeling complex, was only slightly increased in the promoter, suggesting that ATP-independent remodeling was dominating in sulfatide regulation. The epigenetic regulation of miR-223 expression by sulfatide may be attributed to chromatin remodeling, which might result from the accumulation of sulfatide in the nucleus. This study is the first to present evidence that sulfatide suppresses miR-223 expression and to demonstrate that sulfatide overexpression contributes to low miR-223 expression in HCC. Highly expressed sulfatide and CpG methylation in HCC lead to miR-223 downregulation. Reduced miR-223 expression promotes integrin aV expression which is important in HCC migration and metastasis. Sulfatide may represent not only an adhesion molecule on membrane, but also a novel bioregulator of miR-223 expression in nucleus.

Financial support Supported by grants from the National Key Basic Research Program of China (2012CB822104), The Natural Science Foundation of China (30970641, 31170767), and the Shanghai Leading Academic Discipline Project (B110).

Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Acknowledgments The authors thank Prof Huichuan Sun, Drs Boyi Liao and Jie Hu from Liver Cancer Institute and Zhongshan Hospital, Shanghai, China for their generous help in our experiments. Authors thank Drs Chengyou Jia, Weiwei Guo, Dongwei Jia, Liping Liang and Xuchao Zhu for their expert technical assistance. Authors also thank Drs Eric N. Olson, Ronald M. Evans, Qunying Lei for HDAC plasmids.

Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2013.12. 004.

Fig. 4. Sulfatide reduces acetylated histone H3 and C/EBPa recruitment to miR-223 promoter. (A) Chromatin immunoprecipitation with antibodies against HDAC9, HDAC10, HBO1, MOZ, BRG1, RNA Pol II, and normal rabbit IgG, respectively. The promoter fragment of miR-223 gene was amplified by qPCR. (B) ChIP analysis of sulfatide association with miR-223 promoter. miR-223 expression in SMMC-7721 cells treated with DNA methylation inhibitor 5-aza-20 -deoxycytidine (DAC, 3 lM, 24 h) and sulfatide (2 lM, 24 h), alone or together, was analyzed by qRT-PCR (left). ChIP assay using oligo 1 and a-50 -methylcytosine (5MetC) antibody showed cytosines methylated on pre-miR-223 gene (middle). In vitro methylated DNA served as positive control. Methylation-specific PCR analysis of miR-223 promoter regions in SMMC-7721 cells treated with sulfatide and galactocerebroside and in LO2 hepatocytes (bottom right). U, unmethylated; M, methylated. (C) Migration in wound-healing assays was observed in cells with overexpression (CST-8) and silence (siCST-2) of CST. Relative wound closure is quantified on right. Magnification: 10. (D) Representative images of livers and lungs isolated from nude mice 4 weeks after tail vein injections. H&E-stained sections and quantification (right) of liver and pulmonary metastases in CST and control groups (n = 8). Arrows indicates metastatic foci. Boxed fields are shown at higher magnification in corners. (E) miR-223 PCR measurement and integrin aV immunohistochemistry staining in liver and lung metastatic foci of CST and control groups. Regulation is summarized on right. Data are representative of at least three independent experiments. ⁄p <0.05, ⁄⁄p <0.01.

800

Journal of Hepatology 2014 vol. 60 j 792–801

JOURNAL OF HEPATOLOGY References [1] Ladeiro Y, Couchy G, Balabaud C, Bioulac-Sage P, Pelletier L, Rebouissou S, et al. MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations. Hepatology 2008;47:1955–1963. [2] Jia CY, Li HH, Zhu XC, Dong YW, Fu D, Zhao QL, et al. MiR-223 suppresses cell proliferation by targeting IGF-1R. PLoS One 2011;6:e27008. [3] Wu L, Li H, Jia CY, Cheng W, Yu M, Peng M, et al. MicroRNA-223 regulates FOXO1 expression and cell proliferation. FEBS Lett 2012;586:1038–1043. [4] Ruoslahti E, Pierschbacher MD. New perspectives in cell adhesion: RGD and integrins. Science 1987;238:491–497. [5] Ke AW, Shi GM, Zhou J, Huang XY, Shi YH, Ding ZB, et al. CD151 amplifies signaling by integrin alpha6beta1 to PI3K and induces the epithelialmesenchymal transition in HCC cells. Gastroenterology 2011;140:e1615. [6] Koistinen P, Ahonen M, Kahari VM, Heino J. AlphaV integrin promotes in vitro and in vivo survival of cells in metastatic melanoma. Int J Cancer 2004;112:61–70. [7] Dickstein J, Senyuk V, Premanand K, Laricchia-Robbio L, Xu P, Cattaneo F, et al. Methylation and silencing of miRNA-124 by EVI1 and self-renewal exhaustion of hematopoietic stem cells in murine myelodysplastic syndrome. Proc Natl Acad Sci U S A 2010;107:9783–9788. [8] Fazi F, Racanicchi S, Zardo G, Starnes LM, Mancini M, Travaglini L, et al. Epigenetic silencing of the myelopoiesis regulator microRNA-223 by the AML1/ETO oncoprotein. Cancer Cell 2007;12:457–466. [9] Arrenberg P, Maricic I, Kumar V. Sulfatide-mediated activation of type II natural killer T cells prevents hepatic ischemic reperfusion injury in mice. Gastroenterology 2011;140:646–655. [10] Takahashi T, Suzuki T. Role of sulfatide in normal and pathological cells and tissues. J Lipid Res 2012;53:1437–1450. [11] Wu XZ, Honke K, Zhang YL, Zha XL, Taniguchi N. Lactosylsulfatide expression in hepatocellular carcinoma cells enhances cell adhesion to vitronectin and intrahepatic metastasis in nude mice. Int J Cancer 2004;110:504–510. [12] Qian YB, Zhang JB, Wu WZ, Fang HB, Jia WD, Zhuang PY, et al. P48 is a predictive marker for outcome of postoperative interferon-alpha treatment in patients with hepatitis B virus infection-related hepatocellular carcinoma. Cancer 2006;107:1562–1569. [13] Qiu Y, Xu L, Zhou YH, Shi M, Ma Y, Li M, et al. Involvement of genetic instability in the downregulation of sFRP1 in Chinese patients with hepatocellular carcinoma. Anat Rec (Hoboken) 2010;293:2020–2026.

[14] Edmondson HA, Steiner PE. Primary carcinoma of the liver: a study of 100 cases among 48,900 necropsies. Cancer 1954;7:462–503. [15] Wu W, Dong YW, Shi PC, Yu M, Fu D, Zhang CY, et al. Regulation of integrin alphaV subunit expression by sulfatide in hepatocellular carcinoma cells. J Lipid Res 2013;54:936–952. [16] Wong IH, Lo YM, Zhang J, Liew CT, Ng MH, Wong N, et al. Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients. Cancer Res 1999;59:71–73. [17] Zheng F, Liao YJ, Cai MY, Liu YH, Liu TH, Chen SP, et al. The putative tumour suppressor microRNA-124 modulates hepatocellular carcinoma cell aggressiveness by repressing ROCK2 and EZH2. Gut 2012;61:278–289. [18] Albelda SM, Buck CA. Integrins and other cell adhesion molecules. FASEB J 1990;4:2868–2880. [19] Honke K, Tsuda M, Hirahara Y, Ishii A, Makita A, Wada Y. Molecular cloning and expression of cDNA encoding human 30 -phosphoadenylylsulfate:galactosylceramide 30 -sulfotransferase. J Biol Chem 1997;272: 4864–4868. [20] Karakatsanis A, Papaconstantinou I, Gazouli M, Lyberopoulou A, Polymeneas G, Voros D. Expression of microRNAs, miR-21, miR-31, miR-122, miR-145, miR-146a, miR-200c, miR-221, miR-222, and miR-223 in patients with hepatocellular carcinoma or intrahepatic cholangiocarcinoma and its prognostic significance. Mol Carcinog 2013;52:297–303. [21] Peterson CL, Workman JL. Promoter targeting and chromatin remodeling by the SWI/SNF complex. Curr Opin Genet Dev 2000;10:187–192. [22] Agalioti T, Chen G, Thanos D. Deciphering the transcriptional histone acetylation code for a human gene. Cell 2002;111:381–392. [23] Fazi F, Rosa A, Fatica A, Gelmetti V, De Marchis ML, Nervi C, et al. A minicircuitry comprised of microRNA-223 and transcription factors NFIA and C/EBPalpha regulates human granulopoiesis. Cell 2005;123: 819–831. [24] Wong QW, Lung RW, Law PT, Lai PB, Chan KY, To KF, et al. MicroRNA-223 is commonly repressed in hepatocellular carcinoma and potentiates expression of Stathmin1. Gastroenterology 2008;135:257–269. [25] Stamatopoulos B, Meuleman N, Haibe-Kains B, Saussoy P, Van Den Neste E, Michaux L, et al. MicroRNA-29c and microRNA-223 down-regulation has in vivo significance in chronic lymphocytic leukemia and improves disease risk stratification. Blood 2009;113:5237–5245. [26] Hughes AL. Evolution of the integrin alpha and beta protein families. J Mol Evol 2001;52:63–72.

Journal of Hepatology 2014 vol. 60 j 792–801

801