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1653│ https://www.e-crt.org │ Copyright ⓒ 2019 by the Korean Cancer Association
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/)
which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Cancer Res Treat. 2019;51(4):1653-1665
pISSN 1598-2998, eISSN 2005-9256
https://doi.org/10.4143/crt.2018.544
Open Access
Paip1 Indicated Poor Prognosis in Cervical Cancer and Promoted Cervical Carcinogenesis
Original Article
PurposeThis study was aimed to investigate the role of poly(A)-binding protein-interacting protein 1(Paip1) in cervical carcinogenesis.
Materials and MethodsThe expression of Paip1 in normal cervical epithelial tissues and cervical cancer (CC) tissueswere detected by immunohistochemistry. In vivo and in vitro assays were performed to val-idate effect of Paip1 on CC progression.
ResultsPaip1 was found to be up-regulated in CC, which was linked with shorter survival. Knock-down of Paip1 inhibited cell growth, induced apoptosis and cell cycle arrest in CC cells,whereas its overexpression reversed these effects. The in vivo tumor model confirmed thepro-tumor role of Paip1 in CC growth.
ConclusionAltogether, the investigation demonstrated the clinical significance of Paip1 expression,which prompted that the up-regulated of Paip1 can presumably be a potential prognosticand progression marker for CC.
Key wordsPaip1, Uterine cervical neoplasms, Proliferation, Cell cycle, Apoptosis
Nan Li, MD1,2
Junjie Piao, PhD1,2
Xinyue Wang, MD1,2
Ki-Yeol Kim, PhD3
Jung Yoon Bae, PhD4
Xiangshan Ren, PhD1,2
Zhenhua Lin, PhD1,2
1Department of Pathology & Cancer Research
Center, Yanbian University Medical College,
Yanji, 2Key Laboratory of the Science and
Technology, Department of Jilin Province,
Yanji, China, 3Brain Korea 21 Project, Yonsei
University College of Dentistry, Seoul, 4Department of Oral Pathology, Oral Cancer
Research Institute, Yonsei University College
of Dentistry, Seoul, Korea
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + +
Correspondence: Zhenhua Lin, PhDKey Laboratory of the Science and TechnologyDepartment of Jilin Province, Yanji 133002, ChinaTel: 86-433-2435056Fax: 86-433-2435104E-mail: [email protected]
Received November 15, 2018Accepted April 17, 2019Published Online April 19, 2019
Introduction
Cervical cancer (CC) is the fourth most common cause ofcancer in women worldwide [1-3]. With the increasing uptake of Pap smear screening and hepatitis B virus vaccinesin recent years, the incidence of CC has been decreased inmany countries [4,5]. However, there are still approximately527,600 new cases and 265,700 deaths worldwide in 2012. Especially in developing countries, CC ranks as the second
most commonly diagnosed cancer and the third leadingcause of cancer death [6]. Unfortunately, there are no definitediagnostic and prognostic biomarkers [7].
Poly(A)-binding protein-interacting protein 1 (Paip1) is a479-residue protein which encoded by PAIP1. Paip1 was involved in the initiation of translation in eukaryotes bybinding polyadenylate-binding protein cytoplasmic 1 andseveral eukaryotic initiation factors 4F complexes [8,9]. Paip1stimulated translation rates and therefore implicated in sev-eral pathogenic roles [10,11]. Scotto et al. [12] mentioned that
Paip1 was up-regulated in invasive CCs and played a role inDNA repair and cell cycle regulation. Subsequently, Piao etal. [13] reported that Paip1 was found to be overexpressedin breast cancer, and its expression was closely related to cellcycle and poor prognosis. These results indicated that target-ing Paip1 might be an effective anti-tumor therapy strategy.However, the biological role and related mechanisms ofPaip1 in CC progression have not been studied.
Therefore, our data examined the relationship betweenPaip1 overexpression and clinicopathological features of CCand assessed its prognostic value. We also observed thatPaip1 promoted CC cell proliferation, arrested cell cycle pro-gression and inhibited cell apoptosis. These studies have pro-moted our understanding of the role of Paip1 in CC deve-lopment and its related molecular mechanisms.
Materials and Methods
1. Reagents
Antibodies against Paip1, cyclin B1, CDK1, P21, Bax, Bcl-2, and actin were purchased from Proteintech Technology(Wuhan, China). Antibody against Ki-67 was purchasedfrom BOSTER (Wuhan, China).
2. Clinical samples
Routinely diagnosed primary CC tissues (130 cases: squa-mous cervical cancers, 121; adenocarcinomas, 9), cervical intraepithelial neoplasia (CIN) tissues (CIN-1, 22; CIN-2, 34;CIN-3, 38) and normal cervical epithelial tissues (47 cases)with clinical features were collected in the present study. Alltissue specimens were taken from patients who underwentCC surgery and who did not receive any preoperative tumortherapy. Diagnosis and staging were based on the StagingManual of the American Joint Committee on Cancer, 7th edi-tion. Clinical information on the samples is summarized inTables 1 and 2.
3. Cell culture and stable cell line establishment
The human CC cell lines (SiHa, HeLa, C33A, and Caski)and normal non-malignant breast cells (MCF-10A) (Ameri-can Type Culture Collection, Manassas, VA) were culturedin Dulbecco's modified Eagle medium containing 10% fetalbovine serum and streptomycin-penicillin (100 U/mL). Cellswere incubated at 37°C in an atmosphere of 5% CO2.
Stable knockdown of Paip1 was generated by transducingthe sh-Paip1 lentiviral expression construct in cells (Gene-
chem, Shanghai, China). The shRNA sequence used was thefollowing: 5!-GCTTCTATGCC TACAACTT-3!. Stable lenti-viral transfected SiHa and HeLa were selected using 2 µg/mLpuromycin for 2 weeks. Transfection efficiency was con-firmed by western blot. Human Paip1 cDNA was purchasedform (Genechem) and cloned into the GV144 plasmid. ThePaip1 plasmid and corresponding empty vector were trans-fected into CC cell (C33A) using Lipofectamine 3000 reagent(Invitrogen, Carlsbad, CA) following the manufacturer’s pro-tocol.
4. Western blot
Western blot and protein detection was performed as pre-viously described [14]. The harvested cells were preparedwith RIPA cell lysis buffer containing a protease inhibitormixture, and then run on sodium dodecyl sulfate polyacry-lamide gel electrophoresis and transferred to polyvinylidenedifluoride membranes (Immobilon P, Millipore, Bedford,MA), probed with primary antibodies and second antibodies.At last, the results were analyzed quantitatively using chemi-luminescent and fluorescent imaging system.
5. MTT assay
Cells were plated into 96-well plates and cultured with indicated compounds for 0, 24, 48, and 72 hours, followed bythe addition of 100 µL of MTT solution to each well and fur-ther incubation for 4 hours. Removed the medium from thewells and added 100 µL dimethyl sulfoxide into each well.The relative number of surviving cells was assessed by meas-uring the absorbance at 570 nm.
6. Colony-forming assay
Cells were plated on 6-well plates at a concentration of5,000 cells for each well and the medium was replaced every3 days. Cells were fixed and stained with 1% crystal violetafter incubating for 2 weeks.
7. Flow cytometry assay
Cell cycle status was determined by measuring cellularDNA content after staining with propidium iodide (PI; BDBioscience, San Jose, CA). Briefly, cells in the log growthphase were seeded at a density of 2!105 cells per well in 6-well plates. After treatment, cells were harvested and fixedin 70% cold ethanol at –20°C overnight. The next day, cellswere treated with 100 µg/mL of RNase and incubated at37°C for 30 minutes. Then, cells stained with 100 µg/mL ofPI in the dark at room temperature, for another 30 minutes.Samples were subsequently analyzed with the FACScalibur
Cancer Res Treat. 2019;51(4):1653-1665
1654 CANCER RESEARCH AND TREATMENT
flow cytometer (BD Bioscience), and data were analyzedwith ModFit LT software (Verity Software House, Topsham,ME).
Apoptosis was evaluated in CC cells with Annexin V-FITCand PI staining (20 minutes; BD Biosciences). Cells were analyzed using a flow cytometer (BD Biosciences) accordingto the manufacturer’s instructions [15].
8. In vivo tumorigenesis assays
BALB/c nude mice were housed in specific pathogen-freeconditions following the guidelines of the Institutional Ani-mal Care. To assess the effect of Paip1 on tumorigenicity invivo, CC cells were subcutaneously injected into the left orright flank of the mice. Animals were sacrificed after 6 weeksof treatment, and then the mean tumor weight was meas-
ured. Tumor sizes were measured every 7 days.
9. Immunohistochemical staining
For Immunohistochemical (IHC) studies with a DAKOLSAB kit (DAKO A/S, Glostrup, Denmark), tissue sectionswere deparaffinized, rehydrated and incubated with 3%H2O2 in methanol for 15 minutes at room temperature (RT)to eliminate endogenous peroxidase activity. Antigen retri-eval was performed by placing the slides in 0.01 M sodiumcitrate buffer (pH 6.0) at 95°C for 20 minutes. The slides werethen incubated with the primary antibody at 4°C overnight.After incubation with the secondary antibody at RT for 1hour, immunostaining was developed using DAB, and theslides were counterstained with hematoxylin [16].
Nan Li, Paip1 Expression in Cervical Carcinoma
Diagnosis No. of cases
Paip1 protein expression Positive Strongly
– + ++ +++ rate (%) positive rate (%)
Normal cervix 47 40 5 2 0 14.9 4.3CIN-1 22 11 5 5 1 50.0* 27.3*CIN-2 34 14 9 7 4 58.8** 32.4*CIN-3 38 13 10 10 5 65.8** 39.5**CC 130 27 31 30 42 79.2** 55.4**
Paip1, poly(A)-binding protein-interacting protein 1; CIN, cervical intraepithelial neoplasia. *p < 0.05 and **p < 0.01, com-pared with normal cervical epithelial tissues.
Table 1. Paip1 protein expression in cervical cancers
Table 2. Correlation between Paip1 expression and the clinicopathological features of cervical cancers
Paip1, poly(A)-binding protein-interacting protein 1; SCC, squamous cervical cancer; AC, adenocacinoma; LN, lymph node.
Variable No. of cases Paip1 strongly positive cases, n (%) !2 p-value
Age (yr)
< 50 78 42 (53.8) 0.187 0.666! 50 52 30 (57.7)
Histological types
SCC 121 66 (54.5) 0.498 0.480AC 9 6 (66.7)
Stage
0-II 82 39 (47.6) 5.501 0.019III-IV 48 33 (68.8)
Differentiation
Well 44 17 (38.6) 8.562 0.014Moderately 38 22 (57.9)Poorly 48 33 (68.8)
LN metastasis
Negative 58 25 (43.1) 6.392 0.011Positive 72 47 (65.3)
VOLUME 51 NUMBER 4 OCTOBER 2019 1655
Cancer Res Treat. 2019;51(4):1653-1665
Fig. 1. Poly(A)-binding protein-interacting protein 1 (Paip1) expression was significantly up-regulated in cervical cancer(CC). (A, B) Analysis of Paip1 expression in Oncomine data set. (C) Paip1 protein staining was negative in cervical epithelialtissues. (D, E) Paip1 protein staining was positive in dysplastic cells of cervical intraepithelial neoplasia. (F) Paip1 proteinstaining was positive in adenocarcinoma. (G, H) Paip1 protein staining was positive in squamous cervical cancers. (I) Rela-tionship between Paip1 expression and clinicopathological significance of CCs. LN, lymph node.
C D E
F G H
Paip1-high(n=72)
Paip1-low(n=58)
Clinical stage
1.00
0.75
0
0.50
0.25
I
0-II III-IV
Paip1-high(n=72)
Paip1-low(n=58)
LN metastasis
1.00
0.75
0
0.50
0.25
Negative Positive
Paip1-high(n=72)
Paip1-low(n=58)
Differentiation
1.00
0.75
0
0.50
0.25
Moderately Poorly Well
A4.0
–1.5–1.0–0.5
0.5
1.5
2.5
3.5
1.0
0
2.0
3.0
Log2
med
ian-
cent
ered
inte
nsity
0
Paip1 expression in scotto cervix 2
2 310. Cervix squamous epithelium (n=24)1. Cervical adenocarcinoma (n=2)2. Cervical cancer (n=3)3. Cervical squamous cell carcinoma (n=37)
p < 0.01
B
4.04.55.0
00.5
1.5
2.5
3.5
1.0
2.0
3.0
Log2
med
ian-
cent
ered
inte
nsity
0
Paip1 expression in pyeon multi-cancer
2 3 4 5 6 7 8 9 1010. Normal tissue (n=22)1. Cervical cancer (n=20)2. Floor of the mouth carcinoma (n=5)3. Head and neck cancer (n=1)4. Hypopharyngeal carcinoma (n=2)5. Laryngeal carcinoma (n=2)
6. Oral cavity carcinoma (n=4) 7. Oropharyngeal carcinoma (n=6) 8. Pharyngeal carcinoma (n=1) 9. Tongue carcinoma (n=15)10. Tonsillar carcinoma (n=6)
p < 0.01
1656 CANCER RESEARCH AND TREATMENT
Nan Li, Paip1 Expression in Cervical Carcinoma
Fig.
2. P
oly(
A)-b
indi
ng p
rote
in-in
tera
ctin
g pr
otei
n 1
(Pai
p1) e
xpre
ssio
n w
as a
ssoc
iate
d w
ith p
oor o
utco
me i
n ce
rvica
l can
cer (
CC).
(A) T
he im
plica
tion
of P
aip1
inth
e su
rviv
al o
f pat
ient
s with
CC
was
det
erm
ined
in th
e H
uman
Pro
tein
Atla
s’s c
ohor
t. (B
, C) K
apla
n-M
eier
surv
ival
ana
lyse
s wer
e co
nduc
ted
to e
valu
ate
the
influ
ence
of P
aip1
on
patie
nt d
iseas
e-fre
e sur
viva
l (D
FS) (
B) an
d ov
eral
l sur
viva
l (O
S) (C
). (D
-G) K
apla
n-M
eier
surv
ival
curv
es o
f CC
patie
nts i
n ea
rly an
d la
te st
age.
Survival probability
1.0 0
0.2
0.4
0.6
0
The
Hum
an P
rote
in A
tlas
84
1612
0.8
High
Pai
p1 (n
=76)
Low
Pai
p1 (n
=215
)
A
p <
0.01
DFS
1.0 0
0.2
0.4
0.6
0Ti
me
(mo)60
2040
100
80
0.8
B
Log-
rank
=28.4
74p=
0.000
High
Pai
p1 (n
=72)
Low
Pai
p1 (n
=58)
OS
1.0 0
0.2
0.4
0.6
0Ti
me
(mo)60
2040
100
80
0.8
C
Log-
rank
=29.0
92p=
0.000
High
Pai
p1 (n
=72)
Low
Pai
p1 (n
=58)
Early
stag
eEa
rly st
age
DFS DFS
1.0 0
0.2
0.4
0.6
0Ti
me
(mo)60
2040
100
80
0.8
D
Log-
rank
=10.0
00p=
0.002
High
Pai
p1 (n
=39)
Low
Pai
p1 (n
=42)
OS
1.0 0
0.2
0.4
0.6
0Ti
me
(mo)60
2040
100
80
0.8
E
Log-
rank
=10.5
16p=
0.001
High
Pai
p1 (n
=39)Low
Pai
p1 (n
=42)
Late
stag
eLa
te st
age
1.0 0
0.2
0.4
0.6
0Ti
me
(mo)60
2040
100
80
0.8
F
Log-
rank
=15.6
07p=
0.000
High
Pai
p1 (n
=33)
Low
Pai
p1 (n
=16)
OS
1.0 0
0.2
0.4
0.6
0Ti
me
(mo)60
2040
100
80
0.8
G
Log-
rank
=14.8
43p=
0.000
High
Pai
p1 (n
=33)
Low
Pai
p1 (n
=16)
VOLUME 51 NUMBER 4 OCTOBER 2019 1657
10. Evaluation of IHC staining
Statistics of IHC scores were conducted according to theratio and intensity of positive-staining areas. Staining areaswere scored as ‘0’ (negative, no or less than 5% positive cells),‘1’ (6%-25% positive cells), ‘2’ (26%-50% positive cells), ‘3’(50%-75% positive cells), and ‘4’ (more than 75% positivecells). And the staining intensities were scored as ‘0’ (nostaining), ‘1’ (weak staining), ‘2’ (moderate staining), and ‘3’(intense staining). A final score was calculated by multiply-ing these two parameters. All slides were scored independ-ently by two pathologists, who were blind to all clinical data.
11. Statistical analysis
The data analysis was performed using SPSS ver. 17.0 soft-ware (SPSS Inc., Chicago, IL) and GraphPad Prism 6.0 soft-ware (GraphPad Software Inc., San Diego, CA). Groupcomparisons for continuous data were done by t test or one-way ANOVA. Biochemical experiments were performed intriplicate and a minimum of three independent experimentswere evaluated. The value of p < 0.05 was considered statis-tically significant.
12. Ethical statement
This study complied with the principles of the Declarationof Helsinki and was approved by the human ethics and research ethics committees of Yanbian University MedicalCollege in China. The resected specimens from patients whounderwent surgery were potentially used for scientific researches and publication of identifying information/images (when applicable). Their privacy will be maintained.The follow-up survival data were collected retrospectively
through medical-record analyses.All experiments were performed in keeping with the pro-
cedures and protocols of the Animal Ethics Committee ofYanbian University.
Results
1. Paip1 up-regulated in CC
The Oncomine data sets revealed the fact that Paip1 expression was augmented in tumor tissues in comparisonwith the normal cervical tissues (p < 0.01) (Fig. 1A and B). Toverify of the informatic data, Paip1 expression in a groupwhich consisted 130 cases of CC, 94 cases of CIN and 47 nor-mal cervical tissues was figured out using IHC, and it wasvalidated that Paip1 exhibited remarkable expression levelsin CC tissues when compared with normal cervical epithelialtissues (Fig. 1C-H, S1 Fig.). Here, the positive rate of Paip1expression was only 14.9% (7/47) in normal cervical epithe-lial tissues, but significantly higher in CIN lesions (50.00% inCIN-1, 58.8% in CIN-2, and 65.8% in CIN-3) and CC (79.2%,90/174) (p < 0.05, respectively). Similarly, the strongly posi-tive rate of Paip1 expression was observed in 55.4% (72/130)of CCs, which was significantly higher than in normal cervi-cal epithelial tissues (4.3%, 2/47) (p < 0.01) (Table 1).
The correlations between Paip1 expression and clinico-pathologic features were shown in Table 2. The high expres-sion of Paip1 in CCs was significantly correlated with theclinical stage (p=0.019), differentiation (p=0.014), and lymphnode metastasis (p=0.011) (Fig. 1I), but not with other factorsincluded age or histological types. In all, these results indi-
Cancer Res Treat. 2019;51(4):1653-1665
Table 3. Cox regression model analysis of the clinicopathological features in 130 patients with cervical cancer
B, coefficient; SE, standard error; Wald, Wald statistic; HR, hazard ratio; CI, confidence interval; LN, lymph node; Paip1,poly(A)-binding protein-interacting protein 1.
Characteristic B SE Wald HR 95% CI p-value
Univariate
Age 0.154 0.183 0.708 1.166 0.815-1.669 0.400Stage 0.551 0.188 8.595 1.735 1.200-2.507 0.003Differentiation 0.178 0.114 2.437 1.194 0.956-1.493 0.118LN metastasis 0.388 0.178 4.745 1.474 1.040-2.091 0.029Paip1 0.932 0.191 23.764 2.540 1.746-3.694 0.000
Multivariate
Stage 0.557 0.215 6.743 1.746 1.146-2.658 0.009LN metastasis 0.317 0.203 2.442 1.373 0.923-2.043 0.118Paip1 1.076 0.203 28.097 2.932 1.970-4.364 0.000
1658 CANCER RESEARCH AND TREATMENT
cated that high-level expression of Paip1 appears to be asso-ciated with CC progression.
2. Elevated Paip1 expression correlated with poor progno-sis in CC patients
Since we have confirmed the correlation between highPaip1 expression and CC progression, we next explored thepotential roles of Paip1 in CC patients’ clinical outcome. By
Nan Li, Paip1 Expression in Cervical Carcinoma
Fig. 3. Poly(A)-binding protein-interacting protein 1 (Paip1) attenuated the ability of proliferation of cervical cancer (CC)cells in vitro. (A) The protein expression of Paip1 in CC cells was examined by western blot analysis. Actin was used as aloading control. (B) The protein expression of Paip1 in the constructed CC cells was confirmed by western blot analysis.Actin was used as a loading control. (C, D) Cell proliferation was examined by MTT (C) and colony formation (D) in theconstructed cells. Data are represented as mean±standard error of mean of at least three independent experiments, **p < 0.01.
A
Paip1
Actin
MCF-10A
SiHaHeLa
C33A Caski
BSiHa
Paip1
Actin
sh-Con sh-Paip1 sh-Con sh-Paip1HeLa C33A
Vector Paip1
C
0 24
SiHa
Time (hr)
OD va
lue
(570
nm
)
2.5
2.0
0
1.5
0.5
1.0
48 72
sh-Consh-Paip1
0 24
HeLa
Time (hr)
OD va
lue
(570
nm
)2.5
2.0
0
1.5
0.5
1.0
48 72
sh-Consh-Paip1
0 24
C33A
Time (hr)
OD va
lue
(570
nm
)
3.02.52.0
0
1.5
0.51.0
48 72
Paip1Vector
D
sh-Con sh-Paip1
SiHa
Cell c
olon
y num
ber
400
300
0
200
100
SiHash-Con sh-Paip1
HeLash-Con sh-Paip1
sh-Con sh-Paip1
HeLa
Cell c
olon
y num
ber
500
400
300
0
200
100
C33AVector Paip1
Vector Paip1
C33A
Cell c
olon
y num
ber
800
400
0
300
200
VOLUME 51 NUMBER 4 OCTOBER 2019 1659
Cancer Res Treat. 2019;51(4):1653-1665
Fig.
4.
Poly
(A)-b
indi
ng p
rote
in-in
tera
ctin
g pr
otei
n 1
(Pai
p1) r
egul
ated
cell
cycle
pro
gres
sion
of ce
rvica
l can
cer (
CC) c
ell l
ines
in v
itro.
(A) C
ell c
ycle
dist
ribut
ion
was
iden
tifie
d by
flow
cyto
met
ry. D
ata
are
repr
esen
ted
as m
ean±
stand
ard
erro
r of m
ean
of a
t lea
st th
ree
inde
pend
ent e
xper
imen
ts. **
p <
0.01.
(B) T
he p
rote
in
expr
essio
n of
mol
ecul
es as
socia
ted
with
the c
ell c
ycle
in th
e con
struc
ted
CC ce
lls w
as co
nfirm
ed b
y w
este
rn b
lot a
naly
sis. A
ctin
was
use
d as
a lo
adin
g co
ntro
l.
SiHa HeLa C33A
DNA
cont
ent
Cell numberA
BSi
Ha
Cycl
in B
1
CDK1 p2
1
sh-C
onsh
-Pai
p1
Actin
Cycl
in B
1
CDK1 p2
1
Actin
Cycl
in B
1
CDK1 p2
1
Actin
HeLa
sh-C
onsh
-Pai
p1
C33A
Vect
orPa
ip1
G1G2
/MS
Percentage100 80 060 40 20
sh-C
onsh
-Pai
p1
G1G2
/MS
Percentage100 80 060 40 20
sh-C
onsh
-Pai
p1
G1G2
/MS
Percentage100 80 060 40 20
Paip
1Ve
ctor
sh-C
on
Chan
nels
Number 1,000 0
2,000
3,000
4,000
5,000
0120
150
6090
30
Debr
isAg
greg
ates
Dip
G1Di
p G2
Dip
S
sh-P
aip1
Chan
nels
Number 1,000 0
2,000
3,000
4,000
0120
150
6090
30
Debr
isAg
greg
ates
Dip
G1Di
p G2
Dip
S
sh-C
on
Chan
nels
Number 1,000 0
2,000
3,000
4,000
5,000
6,000
0120
150
6090
30
Debr
isAg
greg
ates
Dip
G1Di
p G2
Dip
S
sh-P
aip1
Chan
nels
Number
0
800
1,600
2,400
3,200
0120
150
6090
30
Debr
isAg
greg
ates
Dip
G1Di
p G2
Dip
S
Vect
or
Chan
nels
Number
0
400
800
1,200
1,600
0120
150
6090
30
Debr
isAg
greg
ates
Dip
G1Di
p G2
Dip
S
Paip
1
Chan
nels
Number 1,000 0
2,000
3,000
4,000
0120
150
6090
30
Debr
isAg
greg
ates
Dip
G1Di
p G2
Dip
S
1660 CANCER RESEARCH AND TREATMENT
Nan Li, Paip1 Expression in Cervical Carcinoma
Fig.
5.
Poly
(A)-b
indi
ng p
rote
in-in
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ctin
g pr
otei
n 1
(Pai
p1) r
egul
ated
cell
apop
tosis
of c
ervi
cal c
ance
r (CC
) cel
l lin
es in
vitr
o. (A
) Cel
l apo
ptos
is of
CC
cells
was
dete
cted
by fl
ow cy
tom
etry
. Dat
a wer
e pre
sent
ed as
the m
ean±
stand
ard
devi
atio
n. **
p <
0.01.
(B) W
este
rn bl
ot an
alys
is of
apop
tosis
-rela
ted
prot
ein in
the c
onstr
ucte
dCC
cells
. Act
in w
as u
sed
as a
load
ing
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rol.
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ropi
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de.
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on
SiHa 102
101
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104
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106 10
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210
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%Q3
4-UL
1.8%
Q34-
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4-LR
0.9%
sh-P
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102
101
107.2
103
104
105
106 10
110
210
610
7.2
10410
510
3
Q42-
UR0.2
%Q4
2-UL
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2-LR
6.4%
sh-C
on
HeLa 102
101
107.2
103
104
105
106 10
110
210
610
7.2
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510
3
Q35-
UR0.0
%Q3
5-UL
0.3%
Q35-
LL97
.1%Q3
5-LR
2.6%
sh-P
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102
101
107.2
103
104
105
106 10
110
210
610
7.2
10410
510
3
Q40-
UR0.5
%Q4
0-UL
1.6%
Q40-
LL90
.3%Q4
0-LR
7.6%
Vect
or
C33A 102
101
107.2
103
104
105
106 10
110
210
610
7.2
10410
510
3
Q41-
UR0.5
%Q4
1-UL
0.7%
Q41-
LL95
.2%Q4
1-LR
3.6%
Paip
1
102
101
107.2
103
104
105
106 10
110
210
610
7.2
10410
510
3
Q32-
UR0.4
%Q3
2-UL
1.7%
Q32-
LL97
.8%Q3
2-LR
0.1%
Anne
xin V
PI
sh-Con
sh-Paip1
Apoptosis (%)10 8 06 4 2
sh-Con
sh-Paip1
Apoptosis (%)10 8 06 4 2
Vector
Paip1
Apoptosis (%)
5 4 03 2 1
AB
SiHa
Bax
Bcl-2
Actin
sh-C
onsh
-Pai
p1
HeLa
Bax
Bcl-2
Actin
sh-C
onsh
-Pai
p1
C33A
Bax
Bcl-2
Actin
Vect
orPa
ip1
VOLUME 51 NUMBER 4 OCTOBER 2019 1661
using The Human Protein Atlas, we showed that CC patientswith high expression of Paip1 displayed a poor overall sur-vival (OS) (Fig. 2A). Then Kaplan-Meier survival curves sug-gested that patients with high Paip1 expression had signi-ficantly shorter disease-free survival (DFS; p < 0.001) (Fig. 2B)and OS (p < 0.001) (Fig. 2C). Additionally, CC patients withoverexpression of Paip1 had decreased DFS and OS com-pared to those with low Paip1 expression in either early-stage or late-stage cases (both p < 0.05) (Fig. 2D-G). More-over, clinical stage, lymph node metastasis, and Paip1 expre-ssion status were correlated with shorter survival time in uni-variate analysis (Table 3). Multivariate Cox regression ana-lysis further substantiated that Paip1 and clinical stage wasan independent risk factor for lower DFS and shortened OSin patients with CC (Table 3). Based on the results above, weconclude that the overexpression of Paip1 can be regardedas a poor prognostic index in CC.
3. Paip1 promoted the proliferation of CC cells
To investigate its potential function in CC progression, wefirstly examined the expression of Paip1 in CC cell lines bywestern blot, and then stably knocked down Paip1 in highPaip1-expressing cells (SiHa and HeLa), and overexpressedPaip1 in low Paip1-expressing cell (C33A) (Fig. 3A). The expression levels of Paip1 in transfected cells were verifiedby western blot (Fig. 3B). Then, we assessed cell growth byMTT assays and found that CC cell lines with decreasedPaip1 expression had slower growth rates, while CC cell lineswith increased Paip1 expression had higher growth rates(Fig. 3C, S2A Fig.). Next, as determined by colony formationassays, Paip1 knockdown reduced the number of coloniesformed, while Paip1 overexpression enhanced (Fig. 3D, S2BFig. S3 Fig.). These results indicated that Paip1 promote theproliferation of CC cells in vitro.
4. Paip1 knockdown induced cell cycle arrested and apop-tosis
For the purpose of decided on whether the impact of Paip1on CC cell proliferation was the key observations of thePaip1-mediated alternations in the progression of the cellcycle, we carried out the flow cytometry assay in CC celllines. As suggested by our observations, Paip1 knockdowngave rise to increased G2/M phase together with decreasedG1 phase, as compared with the control cells (Fig. 4A). In addition, Paip1 knockdown altered the expression pattern ofthe key regulators that were indispensable in G2/M cell cycleregulation. Western blotting results indicated that cyclin B1and CDK1 were decreased after Paip1 silencing, while cyclin-dependent kinase inhibitor P21, which was identifiedas tumor suppressor [17], was increased in the sh-Paip1
group (Fig. 4B, S2D-S2E Fig.). Conversely, the opposite effectwas found in Paip1 overexpressed cells (Fig. 4A and B).
Subsequently, in order to study whether Paip1 can affectthe apoptosis of CC cell lines, we carried out the flow cytom-etry assay in CC cell lines. As highlighted by the flow cytom-etry assay, Paip1 knockdown cells manifested higher apop-totic rate in comparison with the control group, whereasPaip1 overexpression displayed the opposite effect in CCcells (Fig. 5A, S2C Fig.). To further investigate which apop-tosis pathway components were involved in CC cells res-ponse to Paip1 knockdown [18], we detected the expressionof apoptosis-associated molecules by western blot. As shownin Fig. 5B, a marked increase in the pro-apoptotic Bax, anddecrease in the anti-apoptotic Bcl-2 was observed after Paip1knockdown. Meanwhile, the overexpression of Paip1 per-formed reserved trend (Fig. 5B). Collectively, Paip1 not onlyinhibited cell apoptosis and regulated cell cycle progressionof CC cell lines, but also promote cell proliferation.
5. Paip1 promoted CC growth in vivo
To further verify the cellular function of Paip1 in vivo, axenograft mouse model was employed to study the role ofPaip1 in CC growth. There was good agreement between invivo and in vitro data. CC cells with stable knockdown oroverexpression Paip1 were injected into nude mice, tumorsderived from Paip1 knockdown group grew slower, and theaverage tumor weight was significantly lower. While tumorvolume was markedly higher, and tumors were also heavierin mice which inoculated with Paip1 overexpressed cells(Fig. 6A and B, S4 Fig.). Next, we performed immunohisto-chemistry for Ki67, Bcl-2, and Bax in the xenografted tissues.As expected, knockdown Paip1 decreased the number Ki67positive cells, reduced the expression of Bcl-2, and up-regu-lated the expression of Bax (Fig. 6C, S5 Fig.). The result wasfurther confirmed in Paip1 overexpressed group (Fig. 6C, S5 Fig.). In conclusion, as an oncogene, Paip1 performs a sub-stantial function in CC proliferation.
Discussion
The regulation of gene expression occurs in the process ofmRNA translation of eukaryotes, particularly at the transla-tion initiation [19,20]. Deregulation at this step of the trans-lation process results in abnormal expression of genes, whichin turn changes cell growth and may lead to the developmentof cancer [21,22]. As a translational enhancer, Paip1 can stim-ulate translation rates in vivo, which have a promoter func-tion in several tumors [11]. Besides, PAIP1 also plays a role
Cancer Res Treat. 2019;51(4):1653-1665
1662 CANCER RESEARCH AND TREATMENT
in the decay of RNA. Grosset et al. [23] demonstrated thatPaip1 was identified as one of the five mCRD-associated pro-teins. These proteins can form a multi-protein complex, sta-
bilized mCRD-containing mRNA by impeding deadeny-lation. Nevertheless, the role of Paip1 in CC is still consideredto be part of a growing sphere, which needs further investi-
Nan Li, Paip1 Expression in Cervical Carcinoma
Fig. 6. Poly(A)-binding protein-interacting protein 1 (Paip1) promoted tumor growth in vivo. (A, B) Indicated cells were injected into the nude mice, and statistical comparison of the differences in tumor weight was shown. (C, D) Expressionlevels of Ki-67, Bcl-2, and Bax in the xenograft tumors tissues were detected by immunohistochemistry.
C
Ki-67
Bcl-2
Bax
sh-Con sh-Paip1SiHa
D
Ki-67
Bcl-2
Bax
Vector Paip1C33A
sh-Con
sh-Paip1
A
500
0
200
100
300
400
Tum
or w
eigh
t (m
g)
sh-Paip1sh-Con
sh-Con sh-Paip1
Vector
Paip1
B600
500
0
200
100
300
400Tu
mor
wei
ght (
mg)
Paip1Vector
Vector Paip1
VOLUME 51 NUMBER 4 OCTOBER 2019 1663
gation. Through this study, we identified for the first timethe function of Paip1 in CC progression and the underlyingmechanism.
We firstly studied the clinicopathological significance ofPaip1 in CC, by performing immunohistochemistry. Accord-ing to our results, Paip1 may have a tumor promoter func-tion, and its expression could be a crucial prognostic indi-cator in CC. Our data showed that the expression of Paip1was significantly elevated in CC tissues, while being absentin normal tissues. More importantly, the high Paip1 expres-sion level was significantly associated with some clinico-pathological parameters like clinical stage, differentiation,and lymph node metastasis. However, no differences werefound with age or histological types. In particular, we foundthat CC patients exhibiting high Paip1 expression had poorerOS and DFS than patients with low expression. As a result,we believed that Paip1 plays a crucial role in patient prog-nosis, and the overexpression of Paip1 was an importantcharacteristic in CC occurrence and progression.
Previous studies reported that Paip1 was overexpressed inbreast cancer and can promote tumor-cell growth [13]. Con-sistent with these findings, our in vitro and in vivo studieshave demonstrated a strong role of Paip1 in CC growth.Since cell cycle progression was a key step for tumor growth,and the aberrant cell cycle was closely related to the occur-rence and development of cancers [24]. So, we assessed thechange of cell cycle in CC cells transfect with or withoutPaip1, and found that Paip1 knockdown leads to G2/M cellcycle arrested, while Paip1 overexpression exhibited the opposite results. Moreover, we detected the change of cycle-related proteins cyclin B1 and CDK1, which were responsiblefor the transition of the cell from the G2 to the M phase andtheir down-regulation would lead to G2/M cell cycle arrest[25]. As indicated by our data, Paip1 knockdown led to decreased expression of the cyclin B1 and CDK1, whereasPaip1 overexpression enhanced cyclin B1 and CDK1 expres-sion. These results supported that Paip1 can control the pro-gression of the cell cycle by regulating the expression of cellcycle proteins, thus affected CC cell proliferation.
Apoptosis was also a critical step for tumor development,and there were complex links between cell progression andcell apoptosis in the tumor. Bcl-2 and Bax were respectivelyanti-apoptotic and pro-apoptotic genes, and both of them belonged to Bcl-2 gene family [26]. Increased levels of pro-apoptotic proteins and/or decreased anti-apoptotic proteinscan lead to apoptosis [27]. In the present report, Paip1 knock-down was found to induce apoptosis and followed a decrease of Bcl-2, an increase of Bax expression. Currentthinking suggested that the ratio of Bax to Bcl-2 may play avital role in cell apoptosis [28]. Thus, we conclude that Paip1might be closely bonded with the apoptosis of CC cellsthrough regulating the expression of apoptotic proteins,
thereby influenced the proliferation of CC cell.To summarize, as suggested by our research, Paip1 was
overexpressed in CC samples and associated with the adverse outcomes in patients with CC, Paip1 knockdown induced inhibition of cell growth, G2/M arrest and apoptosisin CC cells (Fig. 7). These findings may provide the potentialuse of Paip1 as a therapeutic target of CC. Before that, a morecomprehensive study was urgently needed to determine themechanisms of Paip1 as an informative biomarker in patientswith CC.
Electronic Supplementary Material
Supplementary materials are available at Cancer Research andTreatment website (https://www.e-crt.org).
Conflicts of Interest
Conflict of interest relevant to this article was not reported.
Acknowledgments
This study was supported by grants from National Natural Sci-ence Funds of China (NO. 31760313), The Funds of Changbai Moun-tain Scholar Project and Key Laboratory of the Science and Tech-nology Department of Jilin Province (NO. 20170622007JC).
Cancer Res Treat. 2019;51(4):1653-1665
Fig. 7. Schematic diagram. Poly(A)-binding protein-inter-acting protein 1 (Paip1) enhanced proliferation and inhib-ited apoptosis in cervical cancer.
Apoptosis
G2/M phasearrest
M
G2
G1
S
Membrane
Cytoplasm
Mitochondria
NucleusCyclin B1CDK1
Bcl-2
Bax
p21
Sh-Paip1
1664 CANCER RESEARCH AND TREATMENT
Nan Li, Paip1 Expression in Cervical Carcinoma
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References
VOLUME 51 NUMBER 4 OCTOBER 2019 1665