ORIGINAL ARTICLE

Expression and prognostic value of Ars2 in hepatocellularcarcinoma

Qian He • Yongde Huang • Lei Cai •

Shaobo Zhang • Chenghua Zhang

Received: 12 May 2013 / Accepted: 6 November 2013

� Japan Society of Clinical Oncology 2013

Abstract

Background Hepatocellular carcinoma (HCC) is one of

the most common malignant tumors in China. Arsenic

resistance protein 2 (Asr2) was reported to be important for

microRNA (miR) biogenesis, and its depletion could

reduce the levels of several miRs, including miR-21, which

is over-expressed in HCC. We hypothesized that Ars2 is

also overexpressed in HCC and may be involved in the

biological properties of HCC.

Methods Ars2 immunolabeling was evaluated in 132

HCCs. Ars2 immunolabeling, Ars2 qRT-PCR and miR-21

were evaluated in 20 HCCs and in paired normal tissues.

Ars2 shRNA was transfected into SMCC-7721 and HepG2

HCC cells. The cell proliferation and expression of Ars2

and miR-21 were subsequently evaluated.

Results Ars2 was expressed primarily in the nucleus of

HCC cells. The expression of Ars2 was statistically cor-

related with the loss of HCC differentiation and patho-

logical stage. The survival rates of patients with low Ars2

expression in HCC were statistically higher than patients

with overexpressed Ars2 in HCC. Ars2 and miR-21 were

more highly expressed in HCC specimens than normal

tissues, and they were also correlated. The knockdown of

Ars2 in HCC cells inhibited miR-21 expression and cell

proliferation.

Conclusions Ars2 is overexpressed in HCC and may have

prognostic value; it might play an important role in HCC

proliferation and miR-21 expression.

Keywords Ars2 � Prognostic value � Hepatocellular

carcinoma (HCC)

Introduction

Hepatocellular carcinoma (HCC) is one of the most com-

mon malignant tumors and is the third most lethal tumor in

the world [1]. HCC is the second leading cause of death in

Chinese cancer patients [2]. The treatment of HCC is pri-

marily surgical resection, after which the use of chemo-

therapy drugs is limited. Therefore, research regarding the

impact of proto-oncogenes on the proliferation and inva-

sion of HCC is highly significant in developing new

treatments [3].

Arsenic resistance protein 2 (Ars2/Asr2) was first

identified by screening cDNAs that conferred sodium

arsenite resistance in a hamster cell line; however, this

protein has remained poorly characterized [4, 5]. Several

lines of evidence support the role of Ars2 in RNAi function

during microRNA (miR) biogenesis and cell proliferation.

In addition, it was recently determined that Ars2 depletion

decreased the levels of several miRs, including miR-21,

let-7 and miR-155, and its suppression led to a profound

defect in cell proliferation [6]. MiR-21 was confirmed to be

overexpressed in HCC as an oncomiR, and most of the

targets of miR-21 are tumor suppressors [7]. Therefore, we

Q. He and Y. Huang contributed equally to this work.

Q. He � Y. Huang � L. Cai � S. Zhang � C. Zhang (&)

General Surgery Department, The 180th Hospital of People’s

Liberation Army, Quanzhou 362000, People’s Republic of China

e-mail: modest180@163.com

Y. Huang

Gastroenterology Department, The 180th Hospital of People’s

Liberation Army, Quanzhou 362000, People’s Republic of China

L. Cai

Hepatobiliary Surgery Institute, Southwest Hospital,

Third Military Medical University, Chongqing 400038,

People’s Republic of China

123

Int J Clin Oncol

DOI 10.1007/s10147-013-0642-6

hypothesize that Ars2 also exists in HCC and affects pro-

liferation. In the current study, we determined whether

Ars2 is overexpressed in HCC specimens and whether its

depletion affects the proliferation of HCC cells.

Methods

Patients and HCC specimens

Paraffin-embedded sections were obtained from 132

patients (95 men and 37 women; mean age 57.2 ± 12.3

years). Twenty frozen tumor tissues and their correspond-

ing normal tissues were obtained for real-time PCR and

immunohistochemistry from patients with primary HCC.

All these specimens were gathered from patients who

underwent radical hepatoma resection at the Department of

General Surgery, the 180th Hospital of PLA (Quanzhou,

China). The clinicopathological data of these patients

are shown in Table 1. In all cases, informed consent

was obtained for the use of the resected tumor speci-

mens. Preoperative examination of all patients found no

metastases.

All resected HCC specimens were histologically

examined by hematoxylin–eosin staining according to the

International Union against Cancer TNM Classification.

HCC was histologically classified into the following three

stages based on the predominant features: well, moder-

ately and poorly differentiated. The survival rate was

determined for the patients who met the following criteria:

first, they did not die preoperatively, and second, they did

not die of causes other than HCC within 5 years after the

surgery.

All specimens were fixed in formalin and embedded in

paraffin wax. Then, 4-lm serial sections were examined by

immunohistochemistry. For real-time PCR, the samples

were immediately frozen in liquid nitrogen and stored at

280 �C until use.

Cell lines

SMCC-7721 and HepG2 HCC cells were purchased from

the ATCC [8, 9].

Ars2 immunolabeling

We used an immunohistochemical assay for the detection

of Ars2 protein levels using a polyclonal anti-Ars2 anti-

body (polyclonal, Catalog No. sc-135083, Santa Cruz

Biotechnology, Inc., CA, USA). Unstained 4-lm sections

were cut from each tissue and de-paraffinized using routine

techniques. Antigen retrieval was accomplished by incu-

bating the tissue sections in citrate buffer at 120 �C (Boster

Bio-Technology Co. Ltd., Wu Han, China) for 130 s fol-

lowed by incubation in 0.5 % Triton-X for 30 min at

37 �C. The sections were then incubated with 4 lg/ml Ars2

antibody at 4 �C overnight. Incubation with the labeled

polymer (Envision Plus Detection kit, Gene Tech Co. Ltd.,

Shanghai, China) was then carried out for 150 min at room

temperature. The peroxidase reaction was visualized by

incubating with the Real EnVision Detection System

(Dako Co., Via Real, CA, USA) for approximately 5 min

under a microscope [10].

The appropriate positive and negative controls were

included [4, 10]. Immunostaining was classified as follows:

-: no immunostaining above the cutoff level, ?: low

expression (between 1 and 33 % positive cells), ??:

moderate expression (between 34 and 66 % positive cells),

???: strong expression (over 67 % positive cells). Two

independent pathologists evaluated each case for the

average expression of Ars2 in five different visual fields.

RNA extraction

The total RNA was isolated using the Tripure isolation

reagent (Roche Molecular Biochemicals Co., Indianapolis,

IN, USA).

Table 1 Clinicopathologic data for HCC specimens

Age (years) Gender Stage Differentiation

T1 43 F T2N0M0 M

T2 52 M T2N0M0 M

T3 61 F T2N1M0 W

T4 51 M T1N0M0 M

T5 38 M T1N0M0 M

T6 65 M T3N1M0 P

T7 47 F T1N0M0 W

T8 41 M T4N1M1 P

T9 44 F T1N0M0 M

T10 64 M T2N0M0 P

T11 52 M T3N0M0 M

T12 48 F T2N0M0 M

T13 37 F T2N0M0 M

T14 55 M T3N0M0 W

T15 68 M T1N0M0 M

T16 50 M T3N0M0 W

T17 46 M T4N1M0 P

T18 54 F T2N0M0 M

T19 41 M T1N0M0 M

T20 45 M T3N0M0 P

Age age at surgery, Gender male (M) and female (F), Stage TNM

staging, Differentiation well differentiated (W), moderately differen-

tiated (M), or poorly differentiated (P)

Int J Clin Oncol

123

qRT-PCR for Ars2 mRNA expression

SYBR� Premix Ex Taq II (Takara, Dalian, China) was

used for the qRT-PCR. The amplification primers were as

follows: (1) Ars2: forward 50-GGTGACCTTCGACCGC

AGTGTT-30 and reverse 50-TGGGTGATGCCGTTGATG

TTGC-30; and (2) b-actin: forward 50-TGACGTGGACAT

CCGCAAAG-30 and reverse 50-CTGGAAGGTGGACAG

CGAGG-30. Amplification and detection were performed

using a Roche qRT-PCR system. The fluorescence

threshold value was calculated using the Q system soft-

ware. The conditions were as follows: 20 min at 50 �C

followed by 42 cycles of 15 s at 94 �C for denaturation,

30 s at 60 �C for annealing, and 30 s at 72 �C for exten-

sion. A single fluorescence measurement was obtained at

each extension step. Melting curve analyses and agarose

gel electrophoreses were performed to verify the amplified

products. The concentrations of the PCR products were

calculated using a standard curve prepared from serial

dilutions of the most positive specimen for b-actin and for

the target gene in the specimens.

Transduction with shArs2 lentivirus

The shRNA lentivirus for Ars2 interfering mRNA was

purchased from Sigma-Aldrich (St. Louis, MO, USA).

Confluent cells (70–80 %) were transfected with shArs2 at

a multiplicity of infection (MOI) of 20 along with the

negative control using TurboGFP shRNA (Sigma-Aldrich).

The RNA and proteins were harvested 72 h after trans-

fection. The level of p21 was determined to be unaffected;

therefore, shArs2 does not affect the levels of unrelated

proteins (data not shown).

Western blots

Cells were lysed in RIPA sample buffer (Bi Yun-Tian,

Jiang Su, China) supplemented with protease inhibitors

(complete, EDTA-free; Roche, Nutley, NJ, USA) and

PMSF (Bi Yun-Tian). The protein concentration was

measured using a BCA Protein Assay kit (Bi Yun-Tian).

Cell lysates (50 lg) were electrophoresed on 8–10 %

polyacrylamide gels (Bio-Rad) and transferred to nitro-

cellulose membranes (Whatman International Ltd., Dassel,

Germany). The membranes were blocked with 5–10 %

skim milk and then incubated with the primary antibodies.

Anti-Ars2 (polyclonal, Catalog No. ab55822, Abcam Co.,

Cambridge, MA, USA), anti-b-actin (polycolonal, Catalog

No. sc-10731, Santa Cruz Biotechnology, Inc., CA, USA)

and anti-p21 (polyclonal, Catalog No. sc-756, Santa Cruz

Biotechnology) were used according to the manufacturers’

instructions. Horseradish peroxidase-conjugated goat anti-

rabbit or anti-mouse secondary antibody was used (Thermo

Fisher Scientific, Rockford, IL, USA), and the results were

analyzed using an enhanced chemiluminescence-plus

reagent (GE Healthcare, Buckinghamshire, UK).

Cell proliferation assay

SMCC7721 and HepG2 cells were cultured in RPMI 1640

and DMEM-H supplemented with 10 % fetal bovine serum

(FBS), respectively. The cells were harvested in logarith-

mic phase, plated at a density of 100 cells per well in a

96-well plate, and cultured with 10 % FBS RPMI 1640 or

DMEM-H medium. Cell proliferation was measured using

a CellTiter 96 Aqueous One Solution cell proliferation

assay (Promega, Madison, WI, USA). Following each

treatment, 20 ll dye solution was added to each well in the

96-well plate and incubated for 3 h. Subsequently, the

absorbance was recorded at 490 nm using a Bio-Rad model

550 microplate reader [11].

Real-time PCR assays and Northern blots for mature

miR-21

The expression of mature miR-21 and U6 was assessed by

real-time PCR analysis. Reverse transcription was per-

formed on RNA isolated using the TaqMan� MicroRNA

Reverse Transcription Kit, and the cDNA was amplified

using the specific primers provided in the TaqMan�

Human MicroRNA Assay kit (Applied Biosystems, Foster

City, CA, USA) according to the manufacturer’s instruc-

tions. The target sequence in these assays for miR-21 was

50-UAGCUUAUCA GACUGAUGUU GA-30 and for U6

was 50-CTGCGCAAGG ATGACACGCA AATTCGT-

GAA GCGTTCCATA TTTTT-30. The cycle passing

threshold (Ct) was recorded and normalized to U6

expression. The values of the candidate specimens were

calculated using standard curves derived from serial dilu-

tions of the standard miR-21 and U6. For Northern blots,

the total RNA was fractionated on 15 % urea-PAGE gels,

transferred to Hybond? membranes, and cross-linked by

UV irradiation. Hybridization of 32P-end-labeled DNA

oligonucleotide probes antisense to miR-21 was performed

using ULTRAhyb-Oligo buffer (Ambion) at 37 �C over-

night. The probe sequences for miR-21 was 50-TAGGTA

GTTTCATGTTGTTGGCCTGTCTC-30 and for U6 was

50-ACGAATTTGCGTGTCATCCTT -30 [10].

Statistical analyses

Cumulative survival analysis was performed using the

Kaplan–Meier method and was analyzed using the log-rank

test. The correlation between Ars2 expression and each

clinicopathological factor was evaluated using the chi-

squared test. The paired samples t-test was performed to

Int J Clin Oncol

123

determine the difference in Ars2 mRNA and miR-21

expression between HCCs and corresponding normal tis-

sues, and linear regression analysis was performed to

determine the correlation of Ars2 mRNA and miR-21

expression in all the 40 specimens. Each statistical analysis

was performed with SPSS 13.0 (SPSS, Apache Software

Foundation, USA). Differences with a P value of 0.05 or

less were considered statistically significant.

Results

Immunohistochemical results

The Ars2 antibody produced a primarily nuclear staining

pattern in all HCC cells of each sample. The results of the

immunohistochemical staining are shown in Fig. 1 and

Table 2. Ars2 staining was positive (score ?, ?? and

???) in 115 cases of HCC tissues (87.1 %) (Fig. 1). The

weakly positive (?), moderately positive (??) and

strongly positive staining (???) were 26/132 (19.7 %),

51/132 (38.6 %) and 38/132 (28.8 %), respectively. The

expression of Ars2 was statistically correlated with the loss

of differentiation (P = 0.000), tumor topography (P =

0.015), lymph node metastasis (P = 0.006) and patholog-

ical stage (P = 0.010. However, statistical analysis showed

that there was no significant correlation between the

expression of Ars2 and the age, gender and distant

metastasis of the patient.

Survival analysis

The Kaplan–Meier post-operative survival curve shows the

survival rate of patients with HCC and Ars2 expression

(Fig. 2). The post-operative mean survival time of all

patients with HCC was 37.227 ± 1.409 months. The mean

survival time of patients with strongly positive (???)

Ars2 expression was 29.218 ± 1.270 months, and of

patients with moderately positive (??), weakly positive

(?) and negative Ars2 expression were 34.214 ± 1.395,

43.147 ± 2.934 and 54.511 ± 3.898 months, respectively

(pairwise comparison all log-rank test: P \ 0.05). The

post-operative median survival time of all patients with

Ars2 expression was 35.000 ± 1.166 months. The median

survival time of patients with strongly positive (???)

Ars2 expression was 29.000 ± 1.270 months, and of

Fig. 1 Immunohistochemical detection of Ars2 in sections of HCCs (9200). The Ars2 was primarily stained in the nucleus. -: no

immunostaining, ?: 1 to 33 % positive cells, ??: 34 to 66 % positive cells, ???: over 67 % positive cells

Int J Clin Oncol

123

patients with moderately positive (??), weakly positive

(?) and negative Ars2 expression were 35.000 ± 2.574,

41.000 ± 3.399 and 53.000 ± 2.920 months, respectively.

Ars2 immunolabeling, Ars2 mRNA and miR-21 qRT-

PCR assays indicate higher levels of Ars2 and miR-21

in HCC specimens than normal tissues and a correlation

between Ars2 mRNA and miR-21

Immunohistochemistry for Ars2 was performed on 20

paired normal and cancer tissues. Overexpression of Ars2

was observed in the cancers; however, underexpression or

loss was observed in the paired normal tissues, and there

was a significant difference between the two as determined

by chi-squared tests (Pearson chi-squared = 28.923,

P \ 0.01). qRT-PCR assays to measure Ars2 mRNA andFig. 2 Kaplan–Meier post-operative survival curve for groups of

patients with Ars2 expression

Table 2 Correlation between clinicopathological characteristics and Ars2 expression

Parameters Total (n = 132) Ars2

- (n = 17) ? (n = 26) ?? (n = 51) ??? (n = 38) P value

Age (years)

B39 36 4 6 16 100.849

[40 96 13 20 35 28

Sex

Male 89 11 17 23 27

0.582Female 43 6 9 18 11

Differentiation

W 28 7 12 6 3

0.000M 73 8 9 34 22

P 31 2 5 11 13

TNM classification

T

T1 32 6 5 18 3

0.015T2 53 7 12 21 13

T3 29 4 6 8 11

T4 18 0 3 4 11

N

N0 121 17 26 48 300.006

N1 11 0 0 3 8

M

M0 126 17 26 49 340.156

M1 6 0 0 2 4

Stage

I 28 5 5 15 3

0.010II 48 8 12 20 8

III 50 4 9 14 23

IV 6 0 0 2 4

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miR-21 were performed on 20 corresponding normal and

HCC tissues. Normal tissues had uniformly lower expression

of Ars2 mRNA and miR-21, as shown in Fig. 3a. The rela-

tive normalized qRT-PCR values were 15.02 ± 3.35 and

11.07 ± 3.03, respectively. However, the HCCs displayed

more variable expression, with relative normalized values of

32.78 ± 8.40 and 24.84 ± 5.14, respectively. All tumors

displayed higher Ars2 and miR-21 than the normal speci-

mens. The fold-difference between the cancer and normal

groups was 2.18 and 2.24 for Ars2 and miR-21, respectively

(all P \ 0.01, n = 20, paired-samples t-test), as shown in

Fig. 3b. Ars2 is related to the expression of miR-21 and

accurately discriminates between HCC and paired normal

tissues. Linear regression analysis shows that the R2 of the

expression of Ars2 mRNA and miR-21 is 0.469 (P \ 0.001,

n = 40) in HCC and paired normal tissues (Fig. 3c).

Ars2 knockdown suppresses proliferation of HepG2

and SMCC7721 cell lines

HepG2 and SMCC7721 cells were infected with a lentiv-

iral shRNA targeting Ars2 or infected with an empty vector

control (shVec) and cultured for 3 days. Western blot

analysis was performed to detect the expression of Ars2

(Fig. 4a). The HepG2 and SMCC7721 cell lines were

plated at approximately 100 cells per well in 96-well plates

and cultured for 3, 6 or 9 days. Each assay was performed

in triplicate. The data represent the mean ± SD of 490 nm

absorbance (Fig. 4b). The shArs2 hairpin suppressed the

proliferation of both the HepG2 and SMCC7721 cell lines

by approximately 56.6 and 57.9 %, respectively (all

P \ 0.01, n = 3, independent samples t-test) compared

with the shVec-infected control cells.

Fig. 3 Realtime-PCR data in 20 HCC and corresponding normal tissue specimens. a, b Ars2 mRNA and miR-21 paired sample t-test, P \ 0.01.

c Scatter plot analysis of all T/NT specimens of Ars2 and miR-21 with R2 and P-values

Int J Clin Oncol

123

qRT-PCR assays and Northern blotting for miR-21

in HCC cells transduced by empty vector control

and shArs2

As reported, Ars2 acts during the RNAi function of miR

biogenesis and cell proliferation. It was recently observed

that Ars2 depletion decreases the levels of several miRs,

including miR-21, which was reported to be a novel onco-

gene similar to miR in HCC, and this may be the signaling

pathway by which Ars2 affects HCC cell proliferation.

Therefore, qRT-PCR assays and Northern blotting for miR-

21 were performed on HCC cells transfected with control and

shArs2 to determine whether changing the expression of

Ars2 affects miR-21 expression. Each experiment was per-

formed in triplicate. The results showed that Ars2 depletion

by shArs2 reduced the level of miR-21 by 50.1 and 64.1 %

(all P \ 0.01, n = 3, independent samples t-test) in SMCC-

7722 and HepG2, respectively, as shown in Fig. 4c.

Discussion

Ars2 was originally identified in a screen of cDNAs that

conferred sodium arsenite resistance to a hamster cell line.

Fig. 4 SMCC-7721 and HepG2 cells were infected with retroviral

shArs2 and empty vector control. a Western blot detection of Ars2 in

SMCC-7721 and HepG2 cells infected with retroviral shArs2 and

empty vector control for 3 days. b Plated for MTS proliferation assay

in triplicate, the data represent mean ± SD of 490 nm absorbance.

c Northern blots and qRT-PCR to detect the levels of mature miR-21

Int J Clin Oncol

123

The gene was located in the 7q21 human chromosome;

however, the protein has remained poorly characterized

[4]. Ars2 was found to play an important role in cell pro-

liferation, viral infection and neural stem cell characteris-

tics in a Drosophila model and in a variety of cell-based

experiments [4, 5, 12]. Furthermore, evidence showed that

Ars2 plays an essential role in miRNA-mediated silencing

by interacting with the microprocessor and stabilizing pri-

miRNAs [4–6]. Gruber et al. [4] recently reported that Ars2

plays a critical role in the proliferation of mammalian cells.

Mammalian cells cannot maintain proliferative expansion

in vitro if Ars2 is knocked down [13]. Consistent with a

critical role in proliferation, Ars2 is selectively expressed

in proliferating cells. Cells with knocked-down expression

of Ars2 undergo cell-cycle slowing at all stages of the cell

cycle, as evidenced by impairment of proliferation without

discernible changes in the cell-cycle profile and under

expression of several miRs, including miR-21, let-7 and

miR-155 [6]. Here, we characterized Ars2 expression in

formalin-fixed, paraffin-embedded esophageal cancer

specimens using immunohistochemical staining methods.

The data indicate that overexpression of Ars2 is associated

with HCC carcinogenesis, possibly through regulating the

expression of mature miR-21. Ars2 expression was also

confirmed to be upregulated in HCC cancer tissues com-

pared to the adjacent normal tissues, as determined by

immunolabeling and real-time PCR. This is also signifi-

cantly correlated with the expression of miR-21 in human

HCC specimens.

MicroRNAs are small non-coding RNA molecules found

in plants and animals, which function in both transcriptional

and post-transcriptional regulation of gene expression [14].

MiRNAs are encoded by eukaryotic nuclear DNA and

function via base-pairing with complementary sequences

within mRNA molecules, usually resulting in gene silencing

via translational repression or target degradation. The human

genome may encode over 1000 miRNAs, which may target

approximately 60 % of mammalian genes and are abundant

in many human cell types [15–17]. miR-21 is one of the first

miRs to be described as an oncomiR. Because the majority of

the targets of miR-21 are tumor suppressors, miR-21 is

associated with a wide variety of cancers, and it is an

important over-expressed miR in HCC. miR-21 can decrease

the levels of programmed cell death 4 (PDCD4), tissue

inhibitor of metalloproteinase 3 (TIMP3) and phosphatase

and tensin homolog deleted on chromosome ten (PTEN)

[18–21]. In our study, we observed growth inhibition and

found that decreasing the Ars2 protein inhibits the expres-

sion of mature miR-21, consistent with previous reports.

They may be the reason that Ars2 is significantly correlated

with a predictor of survival in human HCC specimens.

Our work confirmed that Ars2 is overexpressed in HCC

and might contribute to the signaling pathways that have

important HCC prognostic value; depletion of it decreases

HCC cell proliferation and mature miR-21 expression.

Further studies are necessary to determine the signaling

pathways of Ars2 in HCC and the biochemical mechanisms

through which Ars2 influences HCC tumorigenesis.

Acknowledgments This work was partially supported by the

National Science Foundation of China (NSFC) grants 81201948/

H1617.

Conflict of interest No author has any conflict of interest.

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