NANOGP8 is a retrogene expressed in cancers
Jingyu Zhang
1,2,
*, Xia Wang
1,2,
*, Meixiang Li
1
, Jin Han
1
, Bing Chen
1
, Bin Wang
1
and Jianwu Dai
1
1 Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology
,
Chinese Academy of Sciences,
Beijing, China
2 The Graduate School, Chinese Academy of Sciences, Beijing, China
Nanog is a recently identified transcription factor that
plays key roles in self-renewal and maintenance of plu-
ripotency in inner cell mass and embryonic stem (ES)
cells [1–3]. It is generally believed that the Nanog gene
is specifically expressed in human ES cells and germ
lineage cells and its expression may be controlled by
an interaction between OCT4 and other proteins
through an adjacent pair of highly conserved Octamer-
and Sox-binding sites of 5¢-flanking region of Nanog
[4,5]. Nanog expression is rapidly down-regulated dur-
ing ES cell differentiation, and constitutive expression

Developmental Biology, Institute of
Genetics and Developmental Biology,
Chinese Academy of Sciences, Beijing,
China.
Tel ⁄ Fax: +86 010 82614426
E-mail:
*The authors contributed equally to this
work.
(Received 11 November 2005, revised 26
January 2006, accepted 20 February 2006)
doi:10.1111/j.1742-4658.2006.05186.x
Nanog is a transcription factor that plays key roles in the self-renewal and
maintenance of pluripotency in human embryonic stem (ES) cells. Among
Nanog’s 11 pseudogenes, NANOGP8 theoretically could be a retrogene,
but was considered unlikely as it has not been identified in any expressed
sequence tags (ESTs). In this study, we found that NANOGP8 was
expressed in several cancer cell lines and in all cancer tissues tested. The
complete coding sequence was cloned and the sequence is highly homolog-
ous to that of Nanog. We were also able to detect its protein expression
using anti-Nanog antibody in recombinant Escherichia coli and some can-
cer cell lines tested. In addition, expression of NANOGP8 in NIH3T3 cells
can promote cell proliferation. The expression of NANOGP8 in cancer cell
lines and cancer tissues suggests that NANOGP8 may play important roles
in tumorigenesis. This work not only has potential significance in stem cell
and cancer research, but it also raises the possibility that some of the
human pseudogenes may have regulatory functions.
Abbreviations
ES, embryonic stem; ESTs, expressed sequence tags; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.
FEBS Journal 273 (2006) 1723–1730 ª 2006 The Authors Journal compilation ª 2006 FEBS 1723
tissues tested. No expression was detected in primarily

embryonic carcinoma NTERA-2, and sequencing
the 444 basepairs PCR product confirmed the findings
(Table 1). Furthermore, when the PCR products were
cloned and sequenced, the results indicated that
NANOGP8 was expressed in human osteosarcoma cell
line OS732, human hepatoma cell line HepG2 and
human breast adenocarcinoma cell line MCF-7
(Table 1). At the same time, other pseudogenes were
found in OS732, HepG2 and MCF-7, while Nanog
gene was also expressed in HepG2 and MCF-7 but not
in OS732 (Table 1 and Fig. 1). In this study, we found
that NANOGP8 was transcribed in all of the human
cancer tissues tested (Table 1 and Fig. 2) as well as
in several cancer cell lines. In contrast, Nanog and
NANOGP8 were not expressed in normal primarily
cultured fibroblast cell line or fetal liver epithelium
cells (data not shown). It is interesting that only
NANOGP8 was expressed in OS732 cell line, uterine
cervix and breast tumor tissues, while Nanog gene was
undetectable (Table 1). Thus, it is true that NANOGP8
was transcribed in cancer cell lines and tumor tissues.
Fig. 1. Detection of Nanog, NANOGP8 and other pseudogenes in
tumor cell lines. RT-PCR analysis the expression of Nanog,
NANOGP8 and other pseudogenes in human fibroblasts and tumor
cell lines OS732, HepG2, MCF-7, THP-1, HeLa, PA-1 and NTERA-2.
Sequencing analysis of PCR products (Table 1) confirmed that
NANOGP8 was expressed in cell lines OS732, HepG2, and MCF-7
while Nanog was expressed in HepG2, MCF-7, PA-1 and NTERA-2.
No-RT data were also shown.
Table 1. The sequencing results of RT-PCR product (403 basepairs)

Uterine cervix 5 0 1 1(NANOGP2)
2(NANOGP4)
1(NANOGP7)
Breast 13 0 8 1(NANOGP4)
1(NANOGP5)
3(NANOGP7)
Urinary bladder 8 1 4 1(NANOGP2)
2(NANOGP7)
NANOGP8 is a retrogene expressed in cancers J. Zhang et al.
1724 FEBS Journal 273 (2006) 1723–1730 ª 2006 The Authors Journal compilation ª 2006 FEBS
NANOGP8 complete coding sequence was
obtained from urinary bladder cancer
Using RT-PCR, the complete coding sequence of
NANOGP8 was cloned from urinary bladder cancer
tissues and its sequence was found to be highly homol-
ogous to Nanog. There are six alternations over 918
sites (Fig. 3 and Supplementary material Fig. 1) and
only one change in the inferred amino acid sequence
from 253 Gln in Nanog to His in NANOGP8. Thus, it
is likely that NANOGP8 and Nanog genes have sim-
ilar functions.
NANOGP8 and ⁄ or Nanog protein was detected
in cell lines
The expression levels of NANOGP8 appear relatively
low in the cancer cell lines and neoplastic tissues
compared to Nanog in EC cells (Figs 1 and 2). Given
the very high degree of homology between Nanog and
NANOGP8 on the basis of nucleotide sequences, it is
likely that the commercially available Nanog antibod-
ies could recognize NANOGP8-translated protein. To

B
Fig. 4. Western blot detection of NANOG and ⁄ or NANOGP8 pro-
tein. Detection of NANOGP8 protein in (B) E. coli (1) and E. coli
with NANOGP8 (2) and NANOGP8 in (A) OS732 and Nanog or
NANOGP8 in MCF-7 and HepG2 using anti-Nanog antibody. The
lack of expression of both Nanog and NANOGP8 in human fibro-
blasts is also shown. Actin (43 kDa) was used as loading control.
J. Zhang et al. NANOGP8 is a retrogene expressed in cancers
FEBS Journal 273 (2006) 1723–1730 ª 2006 The Authors Journal compilation ª 2006 FEBS 1725
showed that NANOGP8 was translated and it sugges-
ted NANOGP8 is a retrogene but not a pseudogene.
NANOGP8 was localized in the nuclei
of transfected cells
Human Nanog is a transcriptional regulator and is
localized in the nucleus [2,3]. Due to the high homol-
ogy between Nanog and NANOGP8, NANOGP8 is
likely a nuclear protein as well. To confirm this, we
constructed a NANOGP8 and GFP fusion protein. As
shown in Fig. 5, the fusion protein was localized in the
nuclei of transfected NIH3T3 (Fig. 5 A and 5B), while
GFP in the control group was present diffused in the
cytoplasm (Fig. 5C,D). Therefore, NANOGP8 is also
a nuclear protein.
NANOGP8 promotes cells to enter into S phase
Cell cycle analysis was performed in NANOGP8 trans-
fected NIH3T3 (pQCXIN-NANOGP8 vector) and
mock control (pQCXIN vector) by flow cytometry
(Fig. 6A). The percentage of S phase in NANOP8-
transfected cells was 53.3%, which was higher in
comparison with the mock control (46.5%). The

vention of pseudogenes [16].
Nanog is a recently identified transcriptional factor
that plays important role in regulating pluripotency
and self-renewal of ES cells [2,3]. Recently, Nanog’s 11
pseudogens were identified, including 10 processed
pseudogenes and one tandem duplicate [12]. They
share sequence homology to the Nanog coding region,
but lack the potential to produce a functional protein
except the NANOGP8 because of critical mutations
[12]. NANOGP8, bearing close similarity to Nanog,
theoretically could be a retrogene, but this was consid-
ered unlikely as it has not been identified in any ESTs.
The EST database provides tremendous gene expres-
sion information from many types of tissues or cells.
Theoretically any gene transcriptions could be found.
We carried out an EST search, where we found it is
difficult to distinguish Nanog from NANOGP8 in
some ESTs (Supplementary material Fig. 2). This
maybe because of the limitation of each clone’s
sequence information in EST database and the high
similarity between NANOGP8 and Nanog, with only
six nucleotides alternations and one amino acid muta-
tion being found between them. In addition,
NANOGP8 expression is extremely low compared with
that of Nanog and this might contribute at least parti-
ally to why NANOGP8 ESTs have not been detected
from human cancers and cancer cell lines.
To investigate whether NANOGP8 could be tran-
scribed, we designed human Nanog-specific primers
and also designed a pair of primers that would amplify

Our preliminary results showed that forced expression
of Nanog gene in 3T3 fibroblasts could greatly increase
cell proliferation rate [18]. When NANOGP8 was sta-
bly transfected into NIH3T3 cells, the cells were pro-
moted to enter into the S stage and, at the same time,
MTT growth assay showed increased cell proliferation.
Our findings that NANOGP8 is expressed in cancer
cell lines and cancer tissues suggest that NANOGP8
60
50
40
30
20
10
0
Mock
Mock
NANOGP8
NANOGP8
1.8
A
B
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2

NANOGP8 gene is regulated in various cancers.
We hypothesize that NANOGP8 may function in
tumor cell self-renewal due to the high homology
between Nanog and NANOGP8 genes. This work
not only has potential significance in stem cell and
cancer research, it also raises the possibility that
some of the human pseudogenes may in fact be
retrogenes and may have important functions in gene
regulation.
Experimental procedures
Total RNA extract and RT-PCR
Total RNA was extracted from cell lines and tissues using
Trizol (Invitrogen, Carlsbad, CA, USA) reagent following
the manufacturer’s instuctions. Total RNA was digested
with RNAase-free DNase I (TaKaRa Carlsbad, CA, USA)
at 37°C for 30 min and inactivated at 60°C for 10 min.
With total RNA (2 lg) as the template and oligo(dT) as
the primer, the first cDNA was synthesized in 25 lL reac-
tion system with Moloney murine leukemia virus (MMLV)
reverse transcriptase (Promega, Madison, WI, USA). First-
strand cDNA and RNA without reverse transcription were
amplified with b-actin primers to confirm the success of RT
reaction and no genomic DNA contamination. At the same
time, no-RT control (RT reaction without reverse transcrip-
tase) was carried out to further exclude the DNA contamin-
ation. cDNA template (3 lL) was used in a 25 lL reaction
volume with rTaq DNA polymerase or LA Taq
TM
DNA
polymerase with GC buffer (TaKaRa). Human NANOGP8

10% fetal bovine serum (Hyclone). For primarily cultured
fibroblast and fetal liver epithelium cells, 20% fetal bovine
serum was added. All cancer tissues used were obtained
from the tissue bank at ZhaoYang Hospital (Beijing,
China) and patients gave written consent for the use of
these tissues for research purposes.
Nuclear protein extraction
Nuclear protein extraction was performed as described [25].
In brief, cells were subsequently rinsed with ice-cold
NaCl ⁄ P
i
(Hyclone), NaCl ⁄ P
i
containing 1 mm Na
3
VO
4
and
5mm NaF, and hypotonic buffer (NaCl ⁄ P
i
including
20 mm Hepes, 20 mm NaF, 1 mm Na
3
VO
4
,1mm Na
4
P
2
O

For western blot analysis, equal protein (30 lg) was
examined by 10% (w ⁄ v) SDS ⁄ PAGE. Proteins on the gel
were transferred onto a nitrocellulose membrane in
1.44% glycine, 0.3% Tris (pH ¼ 8.4), 20% methanol at
80 V for 1 h, and the membrane was then blocked with
NaCl ⁄ P
i
, 5% milk, 0.3% Tween-20. The membrane was
probed with polyclonal goat anti-human Nanog (1 : 1000,
AF1997, R&D Systems, Minneapolis, MN, USA) or
monoclonal mouse anti-human Actin (1 : 500, SC-8432,
Santa Cruz, Santa Cruz, CA, USA). Results were detec-
ted using the WesternBreeze
Ò
kit (Invitrogen). X-ray films
were scanned with a GDS8000 Gel Image Analysis Sys-
tem (Ultra-Violet Products, Cambridge, UK).
Expression constructs and cell transfection
The GFP cDNA was cloned from pEGFP-N1 vector and
inserted into pQCXIN between the BamHI and EcoRI
sites. The NANOGP8 was amplified by RT-PCR and inser-
ted into pQCXIN between AgeI and PacI sites. The GFP
and(or) NANOGP8 were(was) ligated into the pQCXIN
vector to produce the pQCXIN-GFP, pQCXIN-
NANOGP8, and pQCXIN-NANOGP8-GFP. NIH3T3 cells
were transfected with the expression vector pQCXIN,
pQCXIN-NANOGP8 and pQCXIN-NANOGP8-GFP
using Lipofectamine
TM
2000 according to the manufac-

0
⁄ G
1
, S and
G
2
⁄ M phase of the cell cycle were analyzed on a FACScali-
bur and by Modifit software.
For the MTT assay, cells were plated at 1 · 10
4
per 24-well
plates and were cultured for 1–4 days. Viable cells were
evaluated by adding 100 lL of 2.5 mgÆmL
)1
MTT cultured
in 37°C for 4 h. After the removal of MTT solution, 100 lL
dimethyl sulfoxide were added to each well and gently shaken
for 10 min. The absorbance was determined at 492 nm with
plate reader (Sunrise, Tecan, Gr
¨
odig, Austria).
Data analysis
Data were analyzed by Student’s t-test. A value of
P < 0.05 was considered statistical significance.
Acknowledgements
This work was supported by grants from Chinese
Academy of Sciences (KSCW2-SW-205; KSCW2-SW-
218), from NSFC (30428017) and from The Chinese
973 Program (2004CB117404; 2005CB522603).
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Sequence alignment of human Nanog and
NANOGP8 (NgP8) genes. The differences in nucleo-
tides (gray shaded with red characters) are shown.
There is only one change in the inferred amino acid
sequence from Gln (CAG 759, Q) in Nanog to His
(CAC 759, H) in NANOGP8.
Fig. S2. Sequence alignment of human Nanog,