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DOI 10.1007/s10735-015-9622-7
ORIGINAL PAPER
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.
Lifei Jiang and Rong Liu have contributed equally to this work.
& Chunhui Tang
ntfytch@163.com
& Zheng Fang
fznt@163.com
Introduction
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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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*
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
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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
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Cell culture
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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
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296
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
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
Ascites
Cyclin G1 expression
Ki-67 expression
Statistical analysis
Results
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297
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.
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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
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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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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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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