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J Mol Hist (2015) 46:291302

DOI 10.1007/s10735-015-9622-7

ORIGINAL PAPER

The role of Cyclin G1 in cellular proliferation and apoptosis


of human epithelial ovarian cancer
Lifei Jiang1,2 Rong Liu3 Yingying Wang2 Chunmiao Li4 Qinghua Xi1,2
Jianxin Zhong1,2 Jian Liu1,2 Shuyun Yang5 Juan Wang1,2 Menghui Huang1,2
Chunhui Tang1,2 Zheng Fang2

Received: 11 March 2015 / Accepted: 11 May 2015 / Published online: 17 May 2015
Springer Science+Business Media Dordrecht 2015

Abstract Cyclin G1 plays an essential role in the development of human carcinoma. Here, we characterized the
clinical significance of Cyclin G1 and investigated its role
in cellular proliferation and apoptosis of epithelial ovarian
cancer (EOC). Western blot was used to evaluate the expression of Cyclin G1 in nine fresh EOC tissues and three
fresh normal ovarian tissues. Immunohistochemistry analysis was performed on formalin-fixed paraffin-embedded
section of 119 cases of EOCs. Using cell counting kit
(CCK)-8 and colony formation assays, we analyzed the
effect of Cyclin G1 in cellular proliferation of EOC. Besides, the immunofluorescence and flow cytometry analysis
was performed to study the role of Cyclin G1 in cellular
apoptosis of EOC. We found Cyclin G1 was up-regulated
in EOC tissues compared with the normal ovary tissues.

Cyclin G1 expression in EOC was closely correlated with


differentiation grade (P = 0.009) and malignant tumor
cells in ascites (P = 0.009). The KaplanMeier curve
showed that higher expression of Cyclin G1 was associated
with significantly shorter survival in EOC patients. Multivariate analysis suggested Cyclin G1 expression was an
independent prognostic factor for overall survival. CCK-8
and colony formation assays revealed that depletion of
Cyclin G1 inhibited the proliferation and clone formation.
Combined immunofluorescence and flow cytometry analysis showed that silencing of Cyclin G1 with shRNA could
promote apoptosis of ovarian cancer cells. Additionally,
the result of immunoprecipitation test showed Cyclin G1
interacted with CDK2 in EOC cells. In summary, our
findings suggest that Cyclin G1 may be involved in the
prognosis of EOC patients and be a useful therapeutic
target for EOC.

Lifei Jiang and Rong Liu have contributed equally to this work.
& Chunhui Tang
ntfytch@163.com
& Zheng Fang
fznt@163.com

Keywords Epithelial ovarian cancer (EOC)  Cyclin G1 


Proliferation  Apoptosis  Prognosis

Department of Obstetrics and Gynecology, Affiliated


Hospital of Nantong University, Nantong, China

Introduction

Jiangsu Province Key Laboratory for Information and


Molecular Drug Target, Nantong University,
Nantong 226001, Jiangsu Province, China

Department of Gynecologic Oncology, Nantong University


Cancer Hospital, Nantong University,
Nantong 226001, Jiangsu Province, China

Clinical Laboratory, Qilu Childrens Hospital of Shandong


University, Jinan 250022, Shandong, China

Department of Pathology, Nantong University Cancer


Hospital, Nantong University,
Nantong 226001, Jiangsu Province, China

The most common ovarian malignant neoplasms are epithelial ovarian cancers (EOC) which may develop from the
epithelium of the fimbrial portion of the fallopian tube and/or
the ovarian surface epithelium (Koshiyama et al. 2014).
Every year 220,000 women develop epithelial ovarian cancer worldwide. Most EOC patients are clinically characterized by an asymptomatic course in early stage disease,
followed by diagnosis at an advanced stage (Aust and Pils
2014). During the last decades, mortality statistics is barely
improved (Vargas-Hernandez et al. 2014). With advance in

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research of the molecular biology of EOC, some studies


provide that clinical and molecular biological factors alone
are insufficiently useful as prognostic parameters (Aust and
Pils 2014). Thus, understanding the molecular mechanism of
EOC and focusing on newly characterized specific biologic
targets become more and more important.
Cyclin G1 was firstly found in 1996, located to chromosome 5q-32-q34 with six exons and the cDNA length of
3.17 kb (Ye et al. 2012). It is a subtype of Cyclin G of the
cyclin family that has a positive and negative regulator of
cell growth for different kinds of cells. Although the precise function of Cyclin G1 remains unclear, accumulating
evidence has showed that Cyclin G1 abnormally expressed
in many types of malignant cancers such as cervical carcinoma (Liang et al. 2006), hepatocellular carcinoma (Wen
et al. 2013), lung carcinoma (Zhao et al. 2015) and breast
cancer (Reimer et al. 1999). Unlike other cyclins, Cyclin
G1 has neither a destruction box nor PEST sequences that
are responsible for cyclin degradation (Tamura et al. 1993).
Cyclin G1 levels remain relatively constant throughout the
cell cycle both in vitro and in vivo. Thus, Cyclin G1 may
inhibit proliferation and induce apoptosis in cancer cell
lines and tumor xenografts, while in others it can increase
cell proliferation and even tumorigenesis.
Previous studies mainly focused on the role of Cyclin G1
via several complicated mechanisms. The major findings
are: (1) Some studies found evidence that as a transcriptional
target of p53, Cyclin G1 expression upon DNA damage was
regulated by p53, initiating a feedback regulation of p53
through a mechanism that involves MDM2. It was demonstrated that the interaction between Cyclin G1 and enzymatically active PP2A led to the dephosphorylation of
MDM2 followed by p53 degradation (Okamoto et al. 2002;
Yuan et al. 2014). In addition, a study showed that Cyclin G1
could interact with hepatocyte-specific miR-122a, which
might abrogate p53-mediated inhibition of HBV replication
and therefore contribute to carcinogenesis (Wang et al.
2012). (2) Cyclin G1 was found in complex with a number of
proteins involved in cell cycle checkpoint regulation. For
example, Cyclin G1 could interact with cyclin-dependent
kinase 5 (CDK5) and GAK (cyclin G-associated kinase),
although the physiological significance of these interactions
remained unclear (Kanaoka et al. 1997). (3) Cyclin G1 was
suggested to act as an oncogenic protein, because of its
overexpression in human tumor cells (Kang et al. 2013). (4)
Cyclin G was also involved in G2/M arrest in response to
DNA damage (Piscopo and Hinds 2008) or in the facilitation
of TNF-induced apoptosis (Okamoto and Prives 1999), implicating tumor suppressive function. Despite these findings,
understanding of the biochemical function of Cyclin G1 in
complex with associated partners and the molecular function
of association partners in cellular physiology has been
limiting.

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J Mol Hist (2015) 46:291302

Although Cyclin G1 has been extensively examined in a


wide range of cancers, the issue of whether Cyclin G1 is
regulated and its role in proliferation and apoptosis of
human epithelial ovarian cancer cells remains unclear.
Russell et al. suggested Cyclin G1 amplification was associated with significantly shorter post-surgical survival in
patients with ovarian cancer (Russell et al. 2012). In this
study, we aimed to investigate Cyclin G1 expression in
EOC and clarify its possible molecular mechanisms. According to our results, it was reasonable to consider that
Cyclin G1 could be a novel therapeutic target for EOC.

Materials and methods


Ovarian cancer samples collection
We collected 119 formalin-fixed, paraffin-embedded EOC
tissue samples from the archival files of the Department
of Pathology, affiliated Hospital of Nantong University.
All clinical samples were provided using protocols approved by the Ethics Committee of Affiliated Hospital of
Nantong University. The clinical and pathologic data relating to 119 EOC patients were presented in Table 1,
including age, Figo stage, histologic subtype, lymph node
status and malignant tumor cells in peritoneal fluid, and
so on. All specimens were available for review between
2004 and 2009. Median age of the corresponding EOC
patients at cancer diagnosis was 55 years (range 2480).
These samples were used for IHC to analyze the relationship between Cyclin G1 expression and clinicopathological parameters. Nine fresh EOC specimens and
three fresh normal ovarian epithelial tissues were immediately snap-frozen in liquid nitrogen after surgical removal and stored at -80 C until using for western blot.
And the normal fresh specimens were obtained from patients with normal ovaries who underwent the hysterectomy to treat other diseases such as cervical cancer,
Adenomysis, and so on.
Western blot analysis
The frozen normal and tumor tissues were rapidly lysed with
a homogenization buffer containing protease inhibitors (1 %
NP-40, 50 mmol/l Tris, pH 7.5, 5 mmol/l EDTA, 1 % SDS,
1 % sodium deoxycholate, 1 % Triton X-100, 1 mmol/l
PMSF, 10 mg/ml aprotinin, and 1 mg/ml leupeptin) on ice.
Ovarian cancer cells were disrupted on ice with RIPA buffer
(150 mM NaCl, 1 % Nonidet P-40, 0.5 % deoxycholic acid,
0.1 % sodium dodecyl sulfate, 50 mM TrisHCl, pH 8.0).
After centrifugation at 12,000 rpm for 15 min at 4 C, the
supernatant fraction was harvested. 50 lg of total protein
was resolved by using 10 % SDS-PAGE and transferred onto

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Table 1 Cyclin G1 and Ki-67 expression and clinicopathologic parameters of 119 EOC patients
Patient and tumor characteristics

Total

Cyclin G1

High n (%)

Low n (%)

Ki-67

High n (%)

Low n (%)

16 (45.7)

19 (54.3)

49 (58.3)

35 (41.7)

37 (60.7)

24 (39.3)

Age
B 50

35

12 (34.3)

23 (65.7)

[ 50

84

40 (47.6)

44 (52.4)

61

30 (49.2)

31 (50.8)

0.182

0.208

Histologic subtype
Serous
Mucinous

0.201

0 (0)

5 (100)

0 (0)

5 (100)

Endometrioid
Clear cell

10
10

3 (30)
4 (40)

7 (70)
6 (60)

8 (80)
5 (50)

2 (20)
5 (50)

Undifferentiated

33

14

19

15 (45.5)

18 (54.5)

0.030*

Differentiation grade
G1

3 (42.9)

4 (57.1)

G2

36

12 (33.3)

0 (0)

24 (66.7)

7 (100)

0.009*

14 (38.9)

22 (61.1)

G3

76

40 (52.6)

36 (47.4)

48 (63.2)

28 (36.8)

Stage I

52

17 (32.7)

35 (67.3)

23 (44.2)

29 (55.7)

Stage II

13

8 (61.5)

5 (38.5)

11 (84.6)

2 (18.4)

Stage III

49

23 (46.9)

26 (53.1)

27 (55.1)

22 (44.9)

Stage IV

4 (80)

1 (20)

4 (80)

1 (20)

Present

28

19 (67.9)

9 (32.1)

Absent

91

33 (36.3)

58 (63.7)

23

12 (52.2)

11 (47.8)

96

40 (41.7)

56 (58.3)

Present

47

28 (59.6)

19 (40.4)

Absent

72

24 (33.3)

48 (66.7)

0.045*

FIGO stage
0.068

0.040*

Malignant tumor cells

Lymph node status


Positive
Negative

0.009*

0.362

19 (67.9)

9 (32.1)

44 (48.4)

47 (51.6)

12 (52.2)

11 (47.8)

51 (53.1)

45 (46.9)

27 (57.4)

20 (42.6)

36 (50)

36 (50)

0.041*

0.256

Ascites

Total

0.005

0.21

119

Statistical analyses were performed by the Pearsons v2 test


* P \ 0.05 was considered significant

polyvinylidene difluoride (PVDF) membranes (Millipore,


Bedford, MA). Membranes were first blocked with 5 % fatfree dried milk for 2 h and incubated with primary antibodies
overnight at 4 C. After washed with TBST (20 mM Tris,
150 mM NaCl, 0.05 % Tween-20) three times for 5 min
each, the membranes were blotted with the peroxidaseconjugated secondary antibody (1:10,000 dilution) for 2 h.
Immunoreactive bands were detected by enhanced chemiluminescence (ECL) detection kit (Pierce, Rockford, IL,
USA).

dilution), rabbit anti-human Ki-67 antibody (1:500 dilution), rabbit anti-human CDK2 antibody (1:100, dilution),
mouse anti-human PCNA antibody (1:500 dilution), mouse
anti-human Cleaved Caspase-9 antibody (1:100 dilution),
rabbit anti-human GAPDH antibody (1:1000 dilution) and
the peroxidase-conjugated secondary antibody (1:10,000
dilution). All the antibodies were purchased from Santa
Cruz Biotechnology, USA.
Immunohistochemistry and tissue microarray
analysis

Antibodies
The monoclonal antibodies (mAb) were used in this study
as follows: rabbit anti-human Cyclin G1 antibody (1:1000

The 119 EOC tissues were sliced in 4 lm thick, dewaxed


in xylene for 15 min twice and rehydrated through graded
ethanols. The sections were performed by heating to

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121 C for 3 min in 10 mmol/l citrate buffer (pH 6.0). The


endogenous peroxidase activity was blocked by incubation
with 3 % hydrogen peroxide in methanol for 20 min. The
following panel of antibodies was used: diluted anti-Cyclin
G1 antibody (dilution 1:50) or anti-Ki67 antibody (dilution
1:500) at 4 C for 12 h (Turan et al. 2014). All slides were
processed using the peroxidase antiperoxidase method
(Dako, Hamburg, Germany). After washing with PBS, the
peroxidase reaction was visualized by incubating the sections with the liquid mixture DAB (0.1 % phosphate buffer
solution, 0.02 % diaminobenzidine tetrahydrochloride, and
0.03 % H2O2). All slides were processed using the peroxidaseanti-peroxidase method (Dako, Hamburg, Germany). After rinsing in water, the sections were
counterstained with hematoxylin, dehydrated, and cover
slipped. Two professional observers who were blind to the
background of patients evaluated immunoreactivity by intensity and percentage of epithelial stained by a Leica
fluorescence microscope (Germany). Staining intensity in
every tumor section was scored from 0 to 3 [0, negative(-); 1, weak(?); 2, medium(??); 3, strong(???)],
and the proportion of epithelial cell staining positively was
scored from 0 to 4 (0, 05 %; 1, 625 %; 2, 2650 %; 3,
5175 %; 4, 76100 %) (Ramsay et al. 2007). The expression of Cyclin G1 and Ki-67 was interpreted and
graded according to the sum of the intensity and extent
score. Based on the results from immunohistochemistry, all
patients were divided into two groups: low expression
group (score B 3) and high expression group (score [ 3).

indicated assays. The efficacy of Cyclin G1 inhibition was


tested by western blot analysis.

Cell culture

After transfection, cells were seeded in culture dishes (500


cells/plate) and cultured for two weeks. After washed with
PBS, cells were stained with 1 % crystal violet for 30 s after
fixation with 10 % formaldehyde for 2 h. Experiments were
performed in at least three independent replicates.

We purchased three human ovarian cancer cell lines


(OVCA3, HO8910 and SKOV3) from Shanghai Institute of
Cell Biology. Cells were cultured in RPMI 1640 (Gibco
BRL, Grand Island, NY) supplemented with 10 % fetal
bovine serum (FBS). They were maintained at 37 C in a
humidified atmosphere with 5 % CO2.
Transfection
HO8910 cells were transfected with short hairpin RNA
(shRNAs) targeting the nucleotide sequences (50 -AAATGTT
CAGAAGTTGAAA-30 ,50 -ATTGTCTATCATTGCATTA-30 ,
50 -TCAACTGAAGGCATGTCAT-30 , and 50 -GGACAGATT
CCTGTCTAAA-30 respectively) and control-sh RNA, which
were purchased from Genechem (Shanghai, China). The
transfection of sh Cyclin G1 was performed with Lipofectamine
2000 (Invitrogen) according to the manufactures instructions.
After 48 h, transfected cells were harvested and analyzed in the

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Cell proliferation assay


After transfected with sh Cyclin G1 or control-sh RNA
24 h, cells were seeded into 96-well plates at a concentration of 2 9 104 cells per well in volumes of 90 ll and
grew overnight (Chen et al. 2015). 10 ll CCK8 reagents
(Dojindo, Kumamoto, Japan) were then added into every
well at different time points and then incubated for 2 h in
dark environment. The blanks and samples were measured
at a test wavelength of 450 nm and a reference wavelength
of 630 nm using a microplate reader (Bio-Rad). The experiments were repeated at least thrice.
Flow cytometry
HO8910 cells transfected with sh Cyclin G1 and control-sh
RNA were cultured for 48 h and harvested. Then, add
60 ll of Muse TM Annexin V and Dead Cell Reagent (Part
No. 4700-1485, 100 tests/bottle) and 60 ll of cells in
suspension to each tube. After incubated for 20 min in the
dark, the apoptosis assay was performed by Muse TM Cell
Analyser (EMD Millipore corporation). The experiments
were repeated at least thrice.
Colony formation assays

Immunoprecipitation
Cells were washed three times with PBS and lysed in
immunoprecipitation buffer (50 mM HEPES pH 7.6,
150 mM NaCl, 5 mM EDTA, 0.1 % NP-40). After centrifugation (10 min at 15,000 g), to remove cell debris,
Samples were precleared with nonspecific IgG and albumin
controls. The lysates were incubated with antibodies
(1:200) against anti-Cyclin G1, CDK2 or nonspecific IgG
and rotated overnight. The immunocomplexes were added
with 40 ll of a protein A-Sepharose (Sigma) with rotation
for 1 h at 4 C. The last supernatant liquid was removed,
and the protein was collected. A 50-ll quantity of

J Mol Hist (2015) 46:291302

Fig. 1 Expression changes of Cyclin G1 in normal ovary and EOC


tissues. a Expression of Cyclin G1 in normal ovarian tissues (N1, N2, N3)
and EOC tissues (C1-C9) from grade 1 (G1) to grade 3 (G3). b The bar
chart demonstrated the ratio of Cyclin G1 protein to GD for the above by
densitometry. The relative expression levels were showed by density
photometry. The data were mean SEM (*P \ 0.05 compared Cyclin
G1 expression levels between normal ovarian samples and EOC tissues).

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The same experiment was repeated at least three times. ch Representative


photographs of Cyclin G1 and Ki-67 immunohistochemistry in paraffinembedded EOC tissue samples. c and f low concentrations of Cyclin G1
and Ki-67 in EOC tissue with grade 1. d and g medium concentrations of
Cyclin G1 and Ki-67 in EOC tissue with grade 2. e and h high
concentrations of Cyclin G1 and Ki-67 expression in EOC tissue with
grade 3. GD, GAPDH. ch were imaged in Magnification 9 400

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Table 2 Survival status and clinicopathologic parameters of 119
EOC patients
Patient and tumor
characteristics

Total

Survival
status

P value

Alive

Dead

v2

Age
B 50

35

19

16

[ 50

84

31

53

61

21

40

0.08

3.064

Histologic subtype
Serous
Mucinous
Endometrioid

10

Clear cell

10

Undifferentiated

33

12

21

0.026*

11.02

Differentiation grade

Fig. 2 The relation between Ki-67 and Cyclin G1 expression in


EOC. Scatter plot of Ki-67 versus Cyclin G1 with regression line
showing a correlation using Spearman correlation coefficient

2 9 loading buffer was added, and the samples were


boiled at 95100 C for 15 min to denature the protein.
Finally, the samples were analyzed by SDSpolyacrylamide gel electrophoresis using enhanced chemiluminescence detection (Amersham International).
Immunofluorescence
The cells transiently transfected with sh Cyclin G1 and
control-sh RNA were washed with phosphate-buffered saline, fixed with 4 % paraformaldehyde (4 h), and permeabilized with 0.1 % Triton X-100 (10 min). Then, the cells were
incubated with both primary antibodies for Cyclin G1 (antirabbit, 1:200; Santa Cruz Biotechnology) and Cleaved
Caspas-9 (a marker of apoptosis, anti-mouse, 1:100; Santa
Cruz Biotechnology) overnight at 4 C. After washing three
times with phosphate-buffered saline, samples were incubated with Hoechst 33342 dye (1 lg/ml, 2 h) for DNA
staining at room temperature. The fluorescence was examined with a Leica fluorescence microscope (Solms, Germany). All assays were performed three times in duplicate.

G1

G2

36

19

17

G3

76

25

51

Stage I

52

34

18

Stage II

13

11

Stage III

49

13

36

Stage IV

Absent

91

42

49

Present

28

20

Negative

96

44

52

Positive

23

17

Absent

72

33

39

Present

47

17

30

Low

67

36

31

High

52

14

38

Low

54

28

26

High

65

22

43

Total

119

0.007*

9.793

\0.001*

21.258

0.099

2.717

0.085

2.970

0.296

1.090

0.003*

9.042

0.048*

3.925

FIGO stage

Malignant tumor cells

Lymph node status

Ascites

Cyclin G1 expression

Ki-67 expression

Statistical analyses were performed by the Pearsons v2 test


* P \ 0.05 was considered significant

Statistical analysis

Results

Statistical Analysis in this study was performed with the


SPSS 17.0 software version (Chicago, USA). Chi square
(v2) and Fishers exact test were used to measure the relationship between the Cyclin G1 and Ki-67 expression and
the clinicopathological features. Multivariate analysis was
performed on the Cox proportional hazards regression
model. Students t test was used to determine significant
differences between 2 means. A P value \0.05 was considered significant. Values are represented as mean
SEM. Significant differences are marked in the graphs.

Expression of Cyclin G1 was up-regulated in EOC


tissues

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To investigate the role of Cyclin G1 in EOC development,


we used western blot to detect Cyclin G1 expression in nine
frozen EOC and three normal ovarian tissues. As shown in
Fig. 1a, b, Cyclin G1 expression was dramatically increased
in nine tumor samples compared with the three normal
ovarian samples (P \ 0.05). To explore the clinicopathological significance of Cyclin G1 in EOC progression, we

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Fig. 3 KaplanMeier survival curves EOC patients showed a highly


significant separation between curves. ae Survival rates of EOC
patients in low versus high Cyclin G1 expression, low versus high Ki-

67 expression, serous versus non serous ovarian cancers, Grade 12


and Grade 3 and Figo III and IIIIV separately are exhibited
(P \ 0.05)

investigated the reactivity for Cyclin G1 and Ki-67 by immunohistochemical staining. Representative examples of
reactivity for Cyclin G1 (Fig. 1ce) and the proliferation
index Ki-67 (Fig. 1fh) were shown. Interestingly, elevated
expression of Cyclin G1 was observed in high grade tissues
compared with that in low grade ones, which was further
confirmed by western blot assay (Fig. 1a). These results
indicated the potential role of Cyclin G1 in EOC
development.

Cyclin G1 expression was associated


with clinicopathological parameters and prognosis
of EOC patients
We evaluated the association of Cyclin G1 and Ki-67
expression with clinicopathological variables in Table 1.
The statistical analysis showed that expression of Cyclin
G1 was significantly associated with differentiation grade
and malignant tumor cells in ascites (P \ 0.05), but there

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Table 3 Contribution of various potential prognostic factors to survival by Cox regression analysis in 119 EOC patients

Histologic subtype

Hazard ratio

95 % CI

0.996

0.8621.150

0.951

Differentiation grade

1.575

0.9432.630

0.083

FIGO stage

1.638

1.2372.169

0.001*

Cyclin G1 expression

1.944

1.1833.194

0.009*

Ki-67 expression

0.940

0.5111.729

0.842

Histologic subtype (non-serous vs serous); Differentiation grade


(Grade 12 vs. Grade 3); FIGO stage (FIGO III vs. FIGO IIIIV);
Cyclin G1 expression (High expression vs Low expression); Ki-67
expression (High expression vs Low expression)
* P \ 0.05 was considered significant

was no relation between Cyclin G1 and other clinicopathological variables such as lymph node status and
histologic subtype. According with Cyclin G1 and Ki-67
expression in the nuclei, we evaluated the proportion of
Cyclin G1 and Ki-67 positive tumor cells. After analysis,
we found that there was a positive correlation between
Cyclin G1 expression and Ki-67-based proliferative activity (P \ 0.001; Fig. 2). When all variables were compared separately with survival status, we found Cyclin G1
and Ki-67 expression, histologic subtype, differentiation
grade, and FIGO stage (P \ 0.05) could significantly influenced survival (Table 2 and Fig. 3). Only 14 (26.9 %)
of 52 patients in the Cyclin G1 high expression group
were alive versus 36 (53.7 %) of 67 in the Cyclin G1 low
expression group. More importantly, the patients in the
Cyclin G1-high group exhibited shorter survival
(P \ 0.05) than those in the Cyclin G1-low group
(Fig. 3a). Multivariate analysis suggested Cyclin G1 expression (P = 0.009) as well as FIGO stage (P = 0.001)
were independent prognostic factors for EOC (Table 3).
Thus, Cyclin G1 expression could serve as a valuable
predicting factor for poor survival of EOC patients.
The depletion of Cyclin G1 inhibited
the proliferation of ovarian cancer cells and Cyclin
G1 interacts with CDK2
In cell level, we first evaluated the different expression
level of Cyclin G1 in three human ovarian cancer cells:
HO8910, SKOV3, and OVCA3. After normalizing to
GAPDH, the endogenous Cyclin G1 was higher in HO8910
and SKOV3 than OVCA3 cells, especially HO8910 cells
(Fig. 4a). To further investigate the potential effects of
Cyclin G1 in EOC cells, a series of short hairpin RNAs (sh
RNAs) were used to inhibit the expression of endogenous
Cyclin G1 in the HO8910 cells. After transfection for 48 h,
western blot analysis showed Cyclin G1 protein level was

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Fig. 4 The depletion of Cyclin G1 inhibited the proliferation of c


ovarian cancer cells and Cyclin G1 interacts with CDK2. a Western
blot analysis showed that Cyclin G1 protein level was higher in
HO8910 and SKOV3 than OVCA3 cells, especially HO8910 cells.
The bar charts were quantifications of the western blots. Experiments
were repeated at least three times. b HO8910 cells were transfected
with control-sh RNA and sh Cyclin G1#1, 2, 3, 4. The protein levels
of Cyclin G1 were detected by western blot 48 h after transfection.
The bar charts were quantifications of the western blots. Experiments
were repeated at least three times. c Western blot analysis showed that
sh Cyclin G1#2 markedly decreased Cyclin G1 and PCNA levels. The
bar charts were quantifications of the western blots. Experiments
were repeated at least three times. d The proliferation of HO8910 and
SKOV3 cells was measured using CCK-8 assay. Data showed sh
Cyclin G1#2 knockdown represses HO8910 (left) and SKOV3 (right)
cells proliferation, especially HO8910. e Cyclin G1 knockdown in
HO8910 cells suppressed clone formation. The bar chart demonstrated the number of colonies of HO8910 cells treated with sh Cyclin
G1#2 markedly decreased. f The immunoprecipitation experiments
showed Cyclin G1 was able to interact with CDK2. HO8910 cells
were harvested for IP. The lysates were incubated with antibodies
(1:200) against anti-Cyclin G1, CDK2 or nonspecific IgG and rotated
overnight. The immune complexes were analyzed by western
blotting. All experiments were repeated at least three times using
independent samples and data were shown as mean SEM
(*P \ 0.05). Asterisks (*) indicated a significant difference, compared with the control group. GD, GAPDH

markedly decreased in HO8910 cells transfected with sh


Cyclin G1#2 compared with others sh RNAs
(Fig. 4b).Therefore, sh Cyclin G1#2 was used for all the
subsequent experiments. In HO8910 cells treated with sh
Cyclin G1#2, PCNA protein level was significantly decreased than the control group (Fig. 4c).
Some studies found Cyclin G1 promoted G2/M cell
cycle arrest in response to DNA damage (Russell et al.
2012; Kimura et al. 2001). In our study, Cyclin G1 expression was significantly associated with differentiation
grade and malignant tumor cells in ascites. Therefore,
Cyclin G1 might affect EOC cells proliferation. Using
cell counting kit (CCK)-8 assays, we further investigated
if Cyclin G1 activity affected cell proliferation. After
transfected with sh Cyclin G1#2, the cells proliferation
rate of SKOV3 and HO8910, especially HO8910, exhibited a significant decrease compared with the other groups
(Fig. 4d). Besides, the colony formation assay showed
inhibition of the expression of Cyclin G1 suppressed
clone formation of HO8910 cells (Fig. 4e). Moreover,
genome-wide two-hybrid screens suggested that Cyclin G
interacted with different CDKs, including CDK2 and
CDK4 (Stanyon et al. 2004). To further study the
mechanism of Cyclin G1 in EOC, we found Cyclin G1
interacted with CDK2 in ovarian cancer cells by immunoprecipitation (Fig. 4f). Taken together, we considered that Cyclin G1 might promote ovarian tumor cell
proliferation by interacting with CDK2.

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Loss of Cyclin G1 expression promoted EOC cells


apoptosis is involved in many of the functions
regulated by p53 such as apoptosis
Recent reports indicated that Cyclin G1 was involved in
diverse cellular processes, including regulation of the
cell cycle, apoptosis, and so on (Kimura and Nojima
2002; Kimura et al. 2001). Thus, we presumed that the
changes in Cyclin G1 expression might involve ovarian
cancer cells apoptosis regulation. To further address this
hypothesis, we used sh RNA to knockdown Cyclin G1
expression in HO8910 cells and detected the expression
of the apoptosis -related protein Cleaved Caspase-9. As
shown in Fig. 5a, western blot analysis showed a significant increase in the expression of Cleaved Caspase-9
after transfected with sh Cyclin G1#2, which was opposite to the protein level of Cyclin G1. Besides, we
investigated whether Cyclin G1 induced apoptosis in
EOC cells by the flow cytometry assay. The results revealed that there were more apoptotic cells in the sh
Cyclin G1#2 group compared with the control group
(Fig. 5b). Considering the pattern of Cyclin G1 localization in the nucleus and the potential functions of
Cyclin G1, we explored the possibility of the association
between Cyclin G1 and Cleaved Caspase-9. Moreover,
Combined immunofluorescence and flow cytometry
analysis showed that silencing of Cyclin G1 with shRNA
could promote apoptosis of ovarian cancer cells
(Fig. 5ck). The immunofluorescent staining and flow
cytometry analysis showed that silencing of Cyclin G1
with shRNA could promote apoptosis of ovarian cancer
cells.

Discussion
Tumor development results from a multistep process.
Despite increasing knowledge in the etiology of EOC,
there has been only a slight change in the mortality of
EOC patients in the past 30 years. Recent studies show
disrupted cell cycle regulations and targeting these cell
cycle proteins may bring beneficial effects for EOC patients (Aust and Pils 2014). Cyclin G1 was described as
both a positive and negative regulator of human cancer
cells growth (Li et al. 2009). Although exact mechanism
of Cyclin G1 remains unclear, Cyclin G1 may be associated with oncogenic potential and involve growth control, DNA repair, and apoptosis (Kimura et al. 2001). Our
study showed Cyclin G1 expression level in EOC was
closely correlated with differentiation grade (P = 0.009)
and malignant tumor cells (P = 0.009). Multivariate
analysis showed Cyclin G1 expression was an independent prognostic factor for EOC patients. Thus, Cyclin G1

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J Mol Hist (2015) 46:291302

expression could serve as a valuable predicting factor for


EOC patients.
Cyclin G1 belongs to cyclin family, which contain a
highly conserved motif called the cyclin box. The abnormal expression of many cyclin proteins has been found in
many human cancer tissues, such as Cyclin A, B, D1, D3,
and E (Ye et al. 2012). Cyclins have been classified into
different groups on the basis of their structural similarity
and functional period in the cell division cycle. Currently
the cyclin family comprises 17 subtypes denoted AM, O,
T and Y in mammalian cells. The Cyclin G subfamily
includes Cyclin G1 and Cyclin G2. Though the structures
of Cyclin G1 and G2 proteins are similar, their functions
are separated. In contrast to Cyclin G2, Cyclin G1 is the
only known cyclin that is activated transcriptionally by the
p53 tumor suppressor gene and apparently constitutive
throughout the cell cycle.
Most cyclins have been shown to associate with and
regulate the cyclin dependent kinases (cdks) to promote
the progression of the cell cycle (Peyressatre et al. 2015).
Previous reports suggested that control of cell growth and
cell cycle by Cyclin G might be achieved via interaction
with the cyclin-dependent kinases CDK2. Piscopo and
Hinds found co-expression of dominant-negative CDK2
with cyclin G1 in U2OS cells resulted in a substantial
increase in the half-life of cyclin G1 (Piscopo and Hinds
2008). Faradji et al. built a Cyclin G interactor network
based on some data, which indicated that top interactors
could include the cyclin-dependent kinases CDK4 and
CDK2 (Faradji et al. 2011). In our study, cell counting kit
(CCK)-8 and colony formation assays showed Cyclin G1
played a critical role in cells proliferation of EOC. The
results of immunoprecipitation showed Cyclin G1 was
able to bind CDK2 in EOC cells, Hence, Cyclin G1 might
promote ovarian tumor cell proliferation by interacting
with CDK2.
In a recently published paper, Cyclin G1 and MEF2D, a
transcriptional factor established as an oncogenic gene for
liver cancers, have been shown to mediate the anti-tumor
activity of oleanolic acid, reinforcing the notion that Cyclin
G1 is an important gene involved with cancer biology
(Zhao et al. 2015). Interestingly, MEF2D can promote or
depress the expression of target genes by directly binding
the recognition site located within their promoter regions.
Furthermore, Ma et al. showed that MEF2D could regulate
the progression of cell cycle progression by modulating the
expression of G2/M transition gene in liver cancers (Ma
et al. 2014), indicating that MEF2D was closely associated
with cell cycle progression. In combination with the coinvolvement of Cyclin G1 and MEF2D in the antitumor
function of oleanolic acids, we hypothesize that MEF2D
may be associated with the expression of Cyclin G1. Thus,
further study can be done to establish the link between

J Mol Hist (2015) 46:291302

301

Fig. 5 Loss of Cyclin G1 expression promoted EOC cells apoptosis.


a The expression of Cleaved Caspase-9 in HO8910 transfected with
sh Cyclin G1#2 had a significant increase. b HO8910 cells were
treated with control-sh RNA and sh Cyclin G1#2, respectively, and
then assayed by flow cytometry. cj Double-immunofluorescence
analysis of Cyclin G1 (red) and the apoptosis-related protein Cleaved
Caspase-9 (green) in ovarian cancer cells. cf HO8910 cells
transfected with control-sh RNA; G-J, HO8910 cells transfected with

sh Cyclin G1#2. Cells were immunostained with anti- Cyclin G1 and


Cleaved Caspase-9 antibodies. Cell nucleus was stained with Hochest.
k Quantitative analysis of Cyclin G1 and Cleaved Caspase-9-positive
cells in HO8910 cells transfected with control-sh RNA or sh Cyclin
G1#2. All results were representative of at least three repeated
experiments and data were shown as mean SEM (*P \ 0.05).
Asterisks (*) indicated a significant difference, compared with the
control group. GD, GAPDH

MEF2D and Cyclin G1, in order to elucidate the


mechanism of Cyclin G1 overexpression in EOC.
In conclusion, these findings provide multiple lines of
evidence to Cyclin G1 implicate as a key determinant of EOC
development. Cyclin G1 expression is associated with
clinicopathological parameters and prognosis of EOC patients. The down-regulation of Cyclin G1 by sh RNA could
inhibit ovarian cancer cells proliferation and promote cell
apoptosis. Thus, Cyclin G1 might be a useful biomarker in
diagnosis and provide a new treatment strategy for EOC
patients.

Conflict of interest All authors declare that they have no conflict of


interests.

Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81302285), and A Project
Funded by the Priority Academic Program Development of Jiangsu
Higher Education Institutions (PAPD).

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