Upregulated H19 contributes to bladder cancer cellproliferation by regulating ID2 expressionMing Luo1, Zuowei Li2, Wei Wang2, Yigang Zeng2, Zhihong Liu2 and Jianxin Qiu2

1 Department of Urology, Affiliated Tenth People’s Hospital, Tongji University, Shanghai, China

2 Department of Urology, Affiliated First People’s Hospital, Shanghai Jiaotong University, Shanghai, China

Keywords

bladder cancer; inhibitor of DNA binding/

differentiation 2 (ID2); long noncoding RNA

(lncRNA); long noncoding RNA 19 (H19);

proliferation

Correspondence

J. Qiu, Department of Urology, Affiliated

First People’s Hospital, Shanghai Jiaotong

University, 100 Hai Ning Road, Shanghai,

200080, China

Fax: +86 21 63240825

Tel: +86 21 63240090

E-mail: jasonqiu108@yahoo.com.cn

(Received 18 October 2012, revised 20

January 2013, accepted 25 January 2013)

doi:10.1111/febs.12185

Long noncoding RNAs have been shown to have important regulatory

roles in cancer biology, and long noncoding RNA 19 (H19) is essential for

human tumor growth. However, little is known about how abnormal

expression of H19 contributes to bladder cancer cell proliferation. In this

study, we first evaluated the expression of H19 in bladder cancer tissues by

real-time PCR, and defined the biological functions. We found that H19

expression levels were remarkably increased in bladder cancer tissues as

compared with adjacent normal control tissue, and forced expression of

H19 promoted bladder cancer cell proliferation in vitro. Inhibitor of DNA

binding/differentiation 2 (ID2) expression levels were upregulated in blad-

der cancer tissues and in bladder cancer cells. A significant positive correla-

tion was observed between H19 levels and ID2 levels in vivo. We further

demonstrated that overexpression of H19 resulted in a significant increase

in the expression of ID2, whereas H19 knockdown decreased ID2 expres-

sion in vitro. Gain-of-function and loss-of-function studies demonstrated

that upregulated H19 increased bladder cancer cell proliferation by increas-

ing ID2 expression. In conclusion, upregulated H19 increases bladder can-

cer growth by regulating ID2 expression, and thus may be helpful in the

development of effective treatment strategies for bladder cancer.

Structured digital abstract

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� SPPL2A and TfR1-NTF colocalize by fluorescence microscopy (View interaction)

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Introduction

Human bladder cancer is one of the most common

malignancies worldwide [1]. Bladder cancer comprises

at least two major groups. The most common histolog-

ical type of bladder cancer is urothelial carcinoma,

which usually recurs but rarely progresses [2,3].

Invasive bladder cancer is more aggressive, and

one-half of patients with invasive bladder cancer

develop distant metastases, even after radical surgery

of the primary tumors [4]. Therefore, determining the

molecular mechanism of bladder cancer growth is

Abbreviations

H19, long noncoding RNA 19; ID2, inhibitor of DNA binding/differentiation 2; lncRNA, long noncoding RNA; MEG3, maternally expressed

gene 3; SD, standard deviation; siRNA, small interfering RNA; WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-isulfophenyl)-

2H-tetrazolium, monosodium salt.

FEBS Journal 280 (2013) 1709–1716 ª 2013 The Authors Journal compilation ª 2013 FEBS 1709

indispensable for the development of effective treat-

ment strategies for bladder cancer.

Although only a minority of the transcripts repre-

sent protein-coding genes, the majority of the mamma-

lian genome is transcribed [5]. Long noncoding RNAs

(lncRNAs) have recently been proposed to be key

regulators in carcinogenesis or cancer progression:

examples are maternally expressed gene 3 (MEG3),

HOTAIR, and lncRNA 19 (H19) [6–9]. Zhang et al.

demonstrated that MEG3 is associated with meningi-

oma pathogenesis and progression. MEG3 expression

has been shown to be lost in an expanding list of pri-

mary human tumors and tumor cell lines by gene dele-

tion and promoter hypermethylation [9]. Re-expression

of MEG3 inhibits tumor cell proliferation and colony

formation in soft agar. This growth inhibition is partly

the result of apoptosis induced by MEG3 [9]. MEG3

also suppressed DNA synthesis in meningioma cells by

inducing accumulation of p53 (TP53) protein, and

selectively regulates p53 target gene expression [9–11].HOTAIR expression levels were increased in cancerous

tissues, and upregulation of HOTAIR correlated clo-

sely with the presence of cancer metastasis [12,13].

H19 was initially proposed to have tumor-suppressive

properties, on the basis of its ability to inhibit tumori-

genicity [14]. In contrast, some recent research showed

that H19 RNA levels were upregulated in tumors, and

H19 possessed oncogenic properties [15–17]. Berteauxet al. demonstrated that H19 RNA was actively linked

to E2F1 to promote cell cycle progression of breast

cancer cells. H19 levels were upregulated after

exposure to hypoxia, and showed protumorigenic

properties [17]. Ectopic H19 expression enhances the

tumorigenic potential of carcinoma cells in vivo [17].

However, little is known about whether and how H19

regulates bladder cancer cell proliferation.

On the basis of these findings, we investigated the

molecular mechanism of the regulation of bladder can-

cer cell proliferation by H19. We demonstrated that

H19 expression levels were remarkably increased in

patients with bladder cancer. H19 was associated with

the inhibitor of DNA binding/differentiation 2 (ID2)

expression level, and this association contributed to

cancer cell proliferation.

Results

H19 expression is significantly upregulated in

bladder cancer

In order to assess the role of H19 in bladder cancer

development, we first evaluated the expression of H19 in

bladder cancer tissues and adjacent normal control tis-

sues by using quantitative real-time PCR. Figure 1A

shows that H19 was expressed at low levels in adjacent

normal control tissues, whereas H19 levels were mark-

edly increased in bladder cancer tissue. The mean level of

H19 expressed in adjacent normal control tissues was

2.23, but the mean level of H19 expressed in bladder can-

cer tissues was 5.83 (Fig. 1). We then examined the

expression level of H19 in bladder cancer cell lines (RT4,

RT112, DSH1, 253J, TCCSUP, T24, and KU7). As

compared with normal urothelial cells, H19 expression

levels were significantly increased in these bladder cancer

cell lines (Fig. 1B). These data indicate that upregulation

of H19 may be related to bladder cancer development.

A B

Fig. 1. H19 levels are upregulated in bladder cancer. (A) Analysis of H19 expression levels was performed in bladder cancer tissues or

adjacent normal control tissues (n = 24). Total RNA was extracted and subjected to real-time RT-PCR to analyze the CT values of bladder

cancer normalized to b-actin in each sample. The normalized values (DCT) from all tissues were then compared with normal tissue in each

group (DDCT). The results are expressed as log10(2�DDCt ). (B) H19 levels were evaluated by real-time PCR in seven bladder cancer cell lines.

Normal urothelial cells were used as controls. *P < 0.05.

1710 FEBS Journal 280 (2013) 1709–1716 ª 2013 The Authors Journal compilation ª 2013 FEBS

H19 increases cancer growth M. Luo et al.

H19 promotes bladder cell proliferation in vitro

To study the biological role of H19 in the regulation

of cell proliferation, the bladder cancer cell lines trea-

ted with H19, or H19 small interfering RNA (siRNA),

were analyzed. Figure 2A,B shows that overexpression

of H19 promoted cell proliferation in a bladder cancer

cell line (T24). Furthermore, H19-siRNA treatment

significantly decreased the H19 expression level, and

inhibited T24 cell proliferation (Fig. 2C,D). These data

suggest that upregulation of H19 increases bladder

cancer cell growth.

ID2 levels were upregulated in bladder cancer

and were positively correlated with H19 levels

We next investigated the possible mechanisms involved

in the regulation of cancer cell proliferation by H19.

ID2 is essential for embryogenesis, and has been func-

tionally implicated in cell proliferation and cell survival

[18]. Gray et al. found that ID2 is upregulated in colo-

rectal cancer, and is important in promoting cell sur-

vival [19]. Here, we found that ID2 mRNA levels were

significantly upregulated in most bladder cancer tissues

as compared with adjacent normal control tissues

(Fig. 3A). In vitro, ID2 overexpression increased T24

cell proliferation (Fig. 3B). Importantly, a significant

positive correlation was observed between ID2 mRNA

levels and H19 expression levels in cancer tissues

(r2 = 0.2184, P = 0.0033; Fig. 3C). We then investi-

gated whether H19 directly regulated ID2 expression

in vitro. We found that H19 overexpression signifi-

cantly increased ID2 mRNA levels in T24 and 253J

cells (Fig. 4A), whereas H19-siRNA treatment

decreased ID2 mRNA levels in T24 and 253J cells

(Fig. 4B). Similarly, upregulation of H19 increased ID2

protein levels in T24 and 253J cells, but H19 downre-

gulation reduced ID2 protein levels in T24 and 253J

cells (Fig. 4C,D). These data indicate that H19 may

increase bladder cell proliferation by regulating ID2.

H19 increases bladder cancer cell proliferation by

upregulating ID2 expression

Upregulation of H19 increased bladder cancer cell

proliferation, and a significant positive correlation

was observed between the ID2 levels and the H19

A C

B D

Fig. 2. H19 increases bladder tumor cell proliferation. (A) T24 cells were transfected with pcDNA-H19, and H19 expression levels were

assayed after 48 h by real-time PCR. (B) T24 cells were transfected with pcDNA-H19, and, at the indicated time points, the numbers of

cells per well were measured by the absorbance (450 nm) of reduced WST-8. (C) T24 cells were treated with H19-siRNA, and H19

expression levels were assayed by real-time PCR. (D) T24 cells were transiently treated with H19-siRNA, and the numbers of cells per well

were measured by the absorbance (450 nm) of reduced WST-8. The results show data from at least three independent experiments,

expressed as the mean � SD. *P < 0.05. NC, normal control.

FEBS Journal 280 (2013) 1709–1716 ª 2013 The Authors Journal compilation ª 2013 FEBS 1711

M. Luo et al. H19 increases cancer growth

expression levels. Therefore, we thought that the effect

of H19 in bladder cancer cells growth was mediated by

modulation of ID2 expression. Figure 5A shows that

T24 cell proliferation was partially suppressed by

knockdown of ID2 in H19-overexpressing cell. In

H19-siRNA-treated cells, ID2 overexpression resulted

in restored cell proliferation (Fig. 5B). These data

confirm that H19, at least in part, increases bladder

cancer progression by regulating ID2.

Discussion

The human transcriptome has been found to be more

complex than a collection of protein-coding genes and

their splice variants [20–22]. With the advent of whole

transcriptome and genome sequencing technologies, it

has been demonstrated that ~ 90% of the genome is

transcribed [22]. Although this was initially argued to be

transcriptional noise, recent studies have shown that the

‘transcriptional noise’ of the genome may play impor-

tant biological roles in diverse biological processes, such

as transcriptional regulation, cell growth, and human

disease [23,24]. Recent evidence has shown that lncR-

NAs have developmental and tissue-specific expression

patterns, and aberrant regulation in a variety of diseases

has been found. However, the role of lncRNAs in tumor

pathogenesis and metastasis is less well characterized.

Studies are beginning to unravel their importance in

tumorigenesis. HOTAIR expression levels are

increased in primary breast tumors, and the HOTAIR

level in a primary tumor is a powerful predictor of

eventual metastasis and death [25]. Enforced expres-

sion of HOTAIR in epithelial cancer cells leads to

altered histone H3 lysine 27 methylation, altered gene

expression, and increased cancer invasion and metasta-

sis in a manner dependent on polycomb repressive

complex 2 [12,26]. Recent studies have shown that

H19 is essential for human tumor growth [16,17].

Yang et al. found that ectopic expression of H19

increased gastric cancer cell proliferation. They further

verified that H19 is associated with p53, and that this

association results in partial p53 inactivation. In the

study, we found that H19 expression levels are remark-

ably increased in patients with bladder cancer in vivo,

and that forced expression of H19 promotes bladder

cancer cell proliferation in vitro. ID2 expression levels

are increased in bladder cancer tissues and in bladder

cancer cells. A significant positive correlation is

observed between H19 levels and ID2 levels in vivo.

We further demonstrated that upregulation of H19

results in a significant increase in the expression of

ID2. We finally demonstrated that upregulation of

H19 increases bladder cancer cell proliferation by

increasing ID2 expression.

Matouk et al. demonstrated that hypoxia increases

the level of H19 RNA, and ablation of tumorigenicity

of Hepatocellular carcinoma in vivo is seen with H19

knockdown, which also significantly abrogates anchor-

age-independent growth after hypoxia recovery [17].

They also showed that ID2 expression levels are

A

B

C

Fig. 3. ID2 levels positively correlated with H19 levels. (A) Analysis

of ID2 mRNA levels was performed in bladder cancer tissues or

adjacent normal control tissues (n = 22). Total RNA was extracted

and subjected to real-time RT-PCR to analyze the CT values of

bladder cancer normalized to b-actin in each sample. The

normalized values (DCT) from all tissues were then compared with

normal tissue in each group (DDCT). The results are expressed as

log10(2�DDCt ). (B) T24 cells were overexpressed with ID2, and, at

the indicated time points, the numbers of cells per well were

measured by the absorbance (450 nm) of reduced WST-8. The

results show data from at least three independent experiments,

expressed as the mean � SD. *P < 0.05. (C) Positive correlation

between ID2 mRNA levels and H19 levels in 22 bladder cancer

samples (r2 = 0.2184, P = 0.0033). NC, normal control.

1712 FEBS Journal 280 (2013) 1709–1716 ª 2013 The Authors Journal compilation ª 2013 FEBS

H19 increases cancer growth M. Luo et al.

reduced at least two-fold by H19 knockdown under

conditions of hypoxic stress. ID2 can regulate the pro-

liferation of squamous cell carcinoma in vitro via the

nuclear factor-jB–cyclin D1 pathway [27]. Enforced

expression of ID2 stimulates the proliferation of squa-

mous cell carcinoma cells and upregulates the tran-

scription of nuclear factor-jB and cyclin D1. ID2

functions as a proproliferative gene that antagonizes

p53-mediated cell cycle regulation in neural progenitor

cells, and may contribute to the malignant proliferation

of glioma-derived tumor stem cells [28]. Therefore, it is

very important to demonstrate the role of H19/ID2 in

the regulation of cancer cell proliferation.

Conclusion

H19 upregulation increases bladder cancer growth by

enhancing ID2 expression, and thus may be helpful in

the development of effective treatment strategies

against bladder cancer.

A B

C D

Fig. 4. H19 increases ID2 expression. Analysis of ID2 mRNA level was performed in bladder cancer cells treated with pcDNA-H19 (A) or

H19-siRNA (B). Western blot analysis of ID2 protein levels was performed in bladder cancer cells treated with pcDNA-H19 (C) or H19-siRNA

(D). NC, normal control.

A B

Fig. 5. H19 increases bladder cancer cell proliferation by upregulating ID2 expression. T24 cells were treated with pcDNA-H19 plus ID2-

siRNA (A) or H19-siRNA plus pcDNA-ID2, and, at the indicated time points, the numbers of cells per well were measured by the

absorbance (450 nm) of reduced WST-8. *P < 0.05. NC, normal control.

FEBS Journal 280 (2013) 1709–1716 ª 2013 The Authors Journal compilation ª 2013 FEBS 1713

M. Luo et al. H19 increases cancer growth

Experimental procedures

Tissue samples and cell lines

Bladder cancer tissues were acquired from the Tenth Peo-

ple’s Hospital affiliated to Tongji University. The protocols

used in the study were approved by the Hospital’s Protec-

tion of Human Subjects Committee. Twenty-four patholog-

ically diagnosed biopsy specimens and adjacent normal

tissues were acquired from patients with bladder tumors

(Table 1). Human bladder cancer cells (RT4, RT112,

DSH1, 253J, TCCSUP, T24, and KU7) were obtained and

maintained as recommended by the ATCC (Manassas, VA,

USA).

Quantitative real-time PCR

Total RNAs were extracted from tissue samples or cells

with Trizol reagent (Invitrogen, Carlsbad, CA, USA), and

the reverse transcription reactions were performed with

random primers and a Moloney murine leukemia virus

reverse transcriptase kit (Invitrogen). Real-time PCR was

carried out with a standard SYBR Green PCR kit

(Toyobo, Osaka, Japan) protocol on an Applied Biosys-

tems 7300 Real Time PCR system (Applied Biosystems,

Foster City, CA, USA) according to the manufacturer’s

instructions. b-Actin was used as an internal reference for

lncRNAs. Each sample was analyzed in triplicate. The

2�DDCt value was used to determine the relative expression

levels. The results were expressed as log10(2�DDCt ).

Western blot analysis and antibodies

Western blot analysis to assess ID2 and b-actin expression

was performed as previously described [29]. The primary

antibody against ID2 and primary antibody against b-actinwere purchased from Santa Cruz Biotechnology (Santa

Cruz, CA, USA). Briefly, cell lysates were prepared with a

RIPA Lysis kit (Beyotime, Shanghai, China) according to

the manufacturer’s protocol, and protein concentrations

were determined with a standard bicinchoninic acid proto-

col (Pierce, Rockford, IL, USA). Cell lysates were subjected

to electrophoresis on 12% SDS/PAGE gels, and electro-

phoretic transfer to nylon-reinforced nitrocellulose mem-

branes (Osmonics, Minnetonka, MN, USA) was then

performed overnight at 30 V. Membranes were blocked for

2 h in 5% nonfat dry milk and 0.1% Tween-20 (Sigma,

St Louis, MO, USA) in 19 NaCl/Tris. Membranes were

then incubated with goat antibody against ID2 or antibody

against b-actin, and then with appropriate horseradish per-

oxidase-conjugated secondary antibody at a dilution of

1 : 2000.

Cell proliferation assay

Cell proliferation assays were carried out with a Cell Count-

ing Kit-8 (Dojindo, Kumamoto, Japan) [30]. Bladder cancer

cells were plated in 24-well plates at approximately

2 9 105 cells per well. Cells then were transfected with

pcDNA-H19 or with H19-siRNA (Genepharm, Shanghai,

China), and the numbers of cells per well were assayed by

the absorbance (450 nm) of reduced WST-8 (2-(2-methoxy-

4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-isulfophenyl)-2H-tet-

razolium, monosodium salt) at the indicated time points.

Plasmid pcDNA-H19 or plasmid pcDNA-ID2 was con-

structed by introducing a BamHI–EcoRI fragment contain-

ing H19 or ID2 into the same sites in pcDNA3.1. H19 RNA

interference was performed as previously described [26]. The

H19-siRNAs were mixtures of three siRNAs, and the

sequences were are follows: H19-siRNA1, 5′-CCAACAU

CAAAGACACCAUdTdT-3′; H19-siRNA2, 5′-GCAGGA

CAUGACAUGGUCCdTdT-3′; and H19-siRNA3, 5′-UAA

GUCAUUUGCACUGGUUdTdT-3′.

Statistical analysis

Data are expressed as mean � standard deviation (SD)

from at least three separate experiments. Statistical analyses

were performed with Student’s t-test. Differences were con-

sidered to be significant at P < 0.05.

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Table 1. The characteristics of patients with bladder cancer.

Gender

Male, no. (%) 18 (75)

Female, no. (%) 6 (25)

Mean age (years) (range) 62 (43–76)

Relative mRNA level

H19 5.83 � 3.84

Male 5.69 � 3.57

Female 6.25 � 4.92

ID2 4.65 � 3.80

Male 4.90 � 3.65

Female 3.98 � 4.48

T stage, no.

Ta 2

T1 6

T2 9

T3 5

T4 2

N stage, no.

N0N1 16

Higher 8

Grade, no.

1–2 4

3 14

4 6

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