Quantitative analysis of SLC34A2 expression in different types of ovarian tumors
Aim: The main purpose of this study was to estimate the SLC34A2 gene expression in normal ovary and different types of ovarian tumors. Methods: We have investigated SLC34A2 gene expression level in papillary serous, endometrioid, unspecified adenocarcinomas, benign tumors, and normal ovarian tissues...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Цитувати: | Quantitative analysis of SLC34A2 expression in different types of ovarian tumors / M. Shyian, V. Gryshkova, O. Kostianets, V. Gorshkov, Yu. Goloev, I. Goncharuk, S. Nespryadko, L. Vorobjova, V. Filonenko, R. Kiyamova // Experimental Oncology. — 2011. — Т. 33, № 2. — С. 94-98. — Бібліогр.: 32 назв. — англ. |
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irk-123456789-1386322018-06-20T03:08:26Z Quantitative analysis of SLC34A2 expression in different types of ovarian tumors Shyian, M. Gryshkova, V. Kostianets, O. Gorshkov, V. Goloev, Yu. Goncharuk, I. Nespryadko, S. Vorobjova, L. Filonenko, V. Kiyamova, R. Original contributions Aim: The main purpose of this study was to estimate the SLC34A2 gene expression in normal ovary and different types of ovarian tumors. Methods: We have investigated SLC34A2 gene expression level in papillary serous, endometrioid, unspecified adenocarcinomas, benign tumors, and normal ovarian tissues using real-time PCR analysis. Differences in gene expression were calculated as fold changes in gene expression in ovarian carcinomas and benign tumors compared to normal ovary. Results: We have found that SLC34A2 gene was highly expressed in well-differentiated endometrioid and papillary serous ovarian carcinomas compared to low-differentiated endometrioid carcinomas, benign serous cystoadenomas and normal ovary. Analysis of SLC34A2 gene expression according to tumor differentiation level (poor- and well-differentiated) showed that SLC34A2 is up-regulated in well differentiated tumors. Conclusion: Upregulation of SLC34A2 gene expression in well-differentiated tumors may reflect cell differentiation processes during ovarian cancerogenesis and could serve as potential marker for ovarian cancer diagnosis and prognosis. 2011 Article Quantitative analysis of SLC34A2 expression in different types of ovarian tumors / M. Shyian, V. Gryshkova, O. Kostianets, V. Gorshkov, Yu. Goloev, I. Goncharuk, S. Nespryadko, L. Vorobjova, V. Filonenko, R. Kiyamova // Experimental Oncology. — 2011. — Т. 33, № 2. — С. 94-98. — Бібліогр.: 32 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138632 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Shyian, M. Gryshkova, V. Kostianets, O. Gorshkov, V. Goloev, Yu. Goncharuk, I. Nespryadko, S. Vorobjova, L. Filonenko, V. Kiyamova, R. Quantitative analysis of SLC34A2 expression in different types of ovarian tumors Experimental Oncology |
| description |
Aim: The main purpose of this study was to estimate the SLC34A2 gene expression in normal ovary and different types of ovarian tumors. Methods: We have investigated SLC34A2 gene expression level in papillary serous, endometrioid, unspecified adenocarcinomas, benign tumors, and normal ovarian tissues using real-time PCR analysis. Differences in gene expression were calculated as fold changes in gene expression in ovarian carcinomas and benign tumors compared to normal ovary. Results: We have found that SLC34A2 gene was highly expressed in well-differentiated endometrioid and papillary serous ovarian carcinomas compared to low-differentiated endometrioid carcinomas, benign serous cystoadenomas and normal ovary. Analysis of SLC34A2 gene expression according to tumor differentiation level (poor- and well-differentiated) showed that SLC34A2 is up-regulated in well differentiated tumors. Conclusion: Upregulation of SLC34A2 gene expression in well-differentiated tumors may reflect cell differentiation processes during ovarian cancerogenesis and could serve as potential marker for ovarian cancer diagnosis and prognosis. |
| format |
Article |
| author |
Shyian, M. Gryshkova, V. Kostianets, O. Gorshkov, V. Goloev, Yu. Goncharuk, I. Nespryadko, S. Vorobjova, L. Filonenko, V. Kiyamova, R. |
| author_facet |
Shyian, M. Gryshkova, V. Kostianets, O. Gorshkov, V. Goloev, Yu. Goncharuk, I. Nespryadko, S. Vorobjova, L. Filonenko, V. Kiyamova, R. |
| author_sort |
Shyian, M. |
| title |
Quantitative analysis of SLC34A2 expression in different types of ovarian tumors |
| title_short |
Quantitative analysis of SLC34A2 expression in different types of ovarian tumors |
| title_full |
Quantitative analysis of SLC34A2 expression in different types of ovarian tumors |
| title_fullStr |
Quantitative analysis of SLC34A2 expression in different types of ovarian tumors |
| title_full_unstemmed |
Quantitative analysis of SLC34A2 expression in different types of ovarian tumors |
| title_sort |
quantitative analysis of slc34a2 expression in different types of ovarian tumors |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2011 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/138632 |
| citation_txt |
Quantitative analysis of SLC34A2 expression in different types of ovarian tumors / M. Shyian, V. Gryshkova, O. Kostianets, V. Gorshkov, Yu. Goloev, I. Goncharuk, S. Nespryadko, L. Vorobjova, V. Filonenko, R. Kiyamova // Experimental Oncology. — 2011. — Т. 33, № 2. — С. 94-98. — Бібліогр.: 32 назв. — англ. |
| series |
Experimental Oncology |
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| fulltext |
94 Experimental Oncology 33, 94–98, 2011 (June)
QUANTITATIVE ANALYSIS OF SLC34A2 EXPRESSION IN DIFFERENT
TYPES OF OVARIAN TUMORS
M. Shyian1, 2,*, V. Gryshkova2, O. Kostianets1, 2, V. Gorshkov3, Yu. Gogolev3, I. Goncharuk4,
S. Nespryadko4, L. Vorobjova4, V. Filonenko2, R. Kiyamova2
1Taras Shevchenko National University of Kyiv, Kyiv, 01601, Ukraine
2 Department of Cell Signaling, Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv, 03680, Ukraine
3Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, Kazan, Russia
4National Cancer Institute, Ministry of Health of Ukraine, Kyiv, 03022, Ukraine
Aim: The main purpose of this study was to estimate the SLC34A2 gene expression in normal ovary and different types of ovarian
tumors. Methods: We have investigated SLC34A2 gene expression level in papillary serous, endometrioid, unspecified adenocar-
cinomas, benign tumors, and normal ovarian tissues using real-time PCR analysis. Differences in gene expression were calculated
as fold changes in gene expression in ovarian carcinomas and benign tumors compared to normal ovary. Results: We have found
that SLC34A2 gene was highly expressed in well-differentiated endometrioid and papillary serous ovarian carcinomas compared
to low-differentiated endometrioid carcinomas, benign serous cystoadenomas and normal ovary. Analysis of SLC34A2 gene expres-
sion according to tumor differentiation level (poor- and well-differentiated) showed that SLC34A2 is up-regulated in well differen-
tiated tumors. Conclusion: Upregulation of SLC34A2 gene expression in well-differentiated tumors may reflect cell differentiation
processes during ovarian cancerogenesis and could serve as potential marker for ovarian cancer diagnosis and prognosis.
Key Words: NaPi2b, SLC34A2, gene expression, ovarian cancer, normal ovary.
The human sodium-dependent phosphate trans-
port protein 2b — NaPi2b, (NPTIIb, NAPI-3B,
FLJ90534, NAPI-IIb) or solute carrier family 34 mem-
ber 2 is a transmembrane protein with at least eight
highly hydrophobic helical regions that form channel
structure via homodimeric assembly [1, 2]. Na/Pi-
cotransport via NaPi2b transporter is electrogenic with
a likely stoichiometry of 3Na:1Pi [3].
NaPi2b is coded by the SLC34A2 gene and plays
an important role in the maintenance of the overall phos-
phate homeostasis that is essential for proper cellular
functions such as DNA synthesis, cell signaling, bone
formation etc [3, 4]. NaPi2b is highly abundant in the
brush-border membrane (BBM) of small intestine where
it is involved in transcellular flux of inorganic phosphates
via apical membrane of epithelial cells [3, 5].
Besides the small intestine, expression of NaPi2b
has been described predominantly on mRNA level
in some organs of mammalians including lung, pan-
creas, kidney, ovary, placenta, uterus, testis, secreting
mammary gland, thyroid gland, salivary gland, bone,
and epididimus [4–13] where its physiological role
remains to be defined.
NaPi2b expression profile in normal tissues could
be supplemented with sites of expression of MX35 an-
tigen because a molecular identity of both proteins
recently was proved by several techniques [14].
MX35 antigen expression was described by immuno-
histochemical analysis with correspondent MX35 mAbs
in several tissues of epithelial origin. These sites, along
with the already described for NaPi2b include cervix,
Fallopian tubes and sweat glands [15]. Impairments
of SLC34A2 regulation and mutations in this gene
might lead to development of different pathologies.
For instance, several mutations of SLC34A2 gene were
reported to be associated with testicular and pulmo-
nary alveolar microlithiasis [16–18] and downregula-
tion of the NaPi2b cotransporter was associated with
hypophosphatemia [19, 20].
There are plenty of data about aberrant SLC34A2 ex-
pression in cancer cells. The downregulation of NaPi2b
transporter was revealed in non-small cell lung carci-
nomas by RT-PCR analysis [21]. Recently, a strong
overexpression of SLC34A2 in breast cancer and papil-
lary thyroid cancer was found using real-time RT-PCR
[22–24]. Rangel et al. using SAGE and Nothern-blot
analysis reported that SLC34A2 expression was clearly
restricted to the ovarian tumors and certain ovarian
cell lines but absent in normal ovarian tissues [25].
Using real-time RT-PCR analysis authors showed that
NaPi2b expression in serous ovarian carcinomas was
statistically associated with tumor grade and more
differentiated tumors tended to express higher levels
of SLC34A2 mRNA [25]. Papillary serous carcinomas
are the most common ovarian tumors and represent
about 50–60% of all epithelial ovarian cancers (EOC),
and the remaining epithelial ovarian carcinomas exhibit
endometrioid (25%), mucinous (4%) and clear cell
(4%) histology [26]. It was considered that EOC may
arise from ovarian surface epithelium (OSE) that has
“uncommitted” phenotype and retains the capacity
to differentiate into different types of cells in response
to environmental signals. During ovarian carcinogen-
esis, the epithelium of ovary could differentiate into
Received: May 17, 2011.
*Correspondence: E-mail: m.a.shyyan@gmail.com
Abbreviations used: ACTB — actin; AGPC — acid guanidinium
thiocyanate-phenol-chlorophorm; Ct — cycle threshold; EOC —
epithelial ovarian cancer; mAbs — monoclonal antibodies; NaPi2b
(MX35, SLC34A2) — sodium-dependent phosphate transport pro-
tein 2b; OSE — ovarian surface epithelium; SAGE — serial analysis
of gene expression.
Exp Oncol 2011
33, 2, 94–98
Experimental Oncology 33, 94–98, 2011 (June) 95
fallopian tube epithelium (papillary serous tumors), en-
domethrial epithelium (endomethrioid tumors), colonic
or endocervical epithelium (mucinous tumors) and
component of endometriosis (clear cell tumors) [27].
Recently we have described NaPi2b expression
at mRNA and protein levels in different types of ovarian
carcinomas (serous, endometrioid and mucinouse)
using immunohistochemistry, Western-blot and
conventional PCR analysis. We showed that NaPi2b
protein expression detected by anti-NaPi2b mAbs [28]
increased in well-differentiated serous and endome-
trioid tumors compared to poor-differentiated tumors
at protein level but almost uniformly expressed in these
ovarian tumors at mRNA level [29].
To estimate a quantative mRNA expression
of NaPi2b cotransporter in different histological types
of ovarian tumors and normal ovary we have performed
investigation of the SLC34A2 gene expression using
quantitative real-time RT-PCR analysis.
MATERIALS AND METHODS
Tissue samples. Ovarian tissue samples, in par-
ticular, ovarian carcinomas (n = 14), benign serous
cystoadenomas (n = 4) and normal ovarian tissues
(n = 3) were obtained during surgical treatment of cor-
respondent patients at the National Cancer Institute
(Kyiv, Ukraine). The mean age of patients with ovarian
tumors was 47 years (range 22–69 years). Normal
ovarian tissue samples (n = 3), obtained during surgi-
cal treatment of the patients with endometrial cancer
(mean age 46 years, range 19–69 years) were used
as the control. Tissue specimens were frozen immedi-
ately in liquid nitrogen and stored at –80 ̊ C until further
analysis. The histological types of epithelial ovarian
cancer and cell differentiation status were confirmed
by histopathological examination by clinical pathomor-
phologists at the Department of Pathology, National
Cancer Institute (Kyiv, Ukraine) according Christofer
Fletcher’s Third Edition of Diagnoctic Histopathology
of Tumors, 2007. The study protocol was approved
by the Ethics Committees of the Institute of Molecular
Biology and Genetics and National Cancer Institute.
Isolation of RNA. RNA was purified by a standard
procedure using AGPC (acid guanidinium thiocyanate-
phenol-chlorophorm) method [30]. Tissue samples
(100-150 mg) were homogenized in liquid nitrogen and
mixed with 1 ml of denaturation solution (4 M guani-
dinium thiocyanate, 25 mM sodium citrate, pH 7.0,
5% sarcosyl, 0.1 M 2-mercaptoethanol). Sequentially,
0.1 ml of 2 M sodium acetate, pH 4, 1 ml of phenol
(water saturated), and 0.2 ml of chloroform-isoamyl
alcohol mixture (49:1) were added to the homogen-
ate, with thorough mixing after the addition of each
reagent. The final suspension was shaken for 10 s and
cooled on ice for 15 min. Samples were centrifuged
at 10,000g for 20 min at 4 °C. The aqueous phase with
RNA was transferred to a fresh tube and RNA was
precipitated with 1 ml of isopropanol at -20 °C for 1 h.
After sedimentation at 10,000g the resulting RNA pellet
was dissolved in 0.3 ml of the same denaturation solu-
tion and precipitated with 1 volume of isopropanol at
-20 °C for 1 h. After centrifugation for 10 min at 4 °C the
RNA pellet was washed with 75% ethanol, sedimented,
air dried, and dissolved in 50 μl nuclease-free water.
RNA purty and quality was confirmed by measur-
ing spectrophotometrically A260/A280 ratios, and
by 1% agarose-formaldehyde denaturating gel elec-
trophoresis. Total RNA yield was quantitated spectro-
photometrically.
Preparation of cDNA from RNA samples. Total
RNA (3 μg) was converted to cDNA with M-MuLV Re-
verse Transcriptase (Fermentas) at 37 °C for 60 min
using oligo(dT)18 primers in 20 μl reaction volume
according to the standart protocol of manufacturer.
Real-time RT-PCR analysis. Gene-spe-
cific TaqMan probes and PCR primers were de-
signed using NCBI software Primer-BLAST (URL:
http://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Table).
Table. Gene-specific TaqMan probes and PCR primers
Gene Oligonucleotide Oligonucleotide sequence (5’ — 3’) Length
SLC34A2 Forward primer ttg-gag-gaa-aaa-tgg-cag-gac-ag 23
Reverse primer gca-aga-gca-cca-aca-cgg-aca-g 22
Probe (FAM)cc-cga-aca-g(T-BHQ1)g-agc-
aat-gaa-gag-gac-acc
29
ACTB Forward primer ggc-acc-cag-cac-aat-gaa-g 19
Reverse primer gcc-gat-cca-cac-gga-gta-ct 20
Probe (FAM)tc-aag-atc-a(T-BHQ1)t-gct-cct-
cct-gag-cgc
26
The real-time reverse transcription-polymerase
chain reaction (real-time RT-PCR) procedure was
carried out in 25 μl of reaction mix containing diluted
1:10 cDNA mixture (3 μg — 2 μl), forward and reverse
primers (20 pmole each), gene-specific TaqMan probe
(20 pmole), 10 x Taq Buffer (2.5 μl), 10 mM dNTP
mix (5 nmole — 0.5 μl), Taq DNA Polymerase 5 u/μl
(1.25 u — 0.25 μl), 25 mM MgCl2 (50 nmole — 2 μl)
and nuclease-free water. The following thermal con-
ditions were applied: 95 °C for initial denaturation
(30 s) and 40 cycles consisting of 95 °C denaturation
(10s), 55 °C annealing (5 s) and 60 °C extension (60s).
Thermal cycling and fluorescent monitoring were
performed using iCycler iQ5 (Bio-Rad, CA, USA).
Amplified products were separated on a 1% agarose
gel and visualized.
Calculations. The point at which the PCR product
is first detected above a fixed threshold, termed cycle
threshold (Ct), was determined for each sample. When
PCR product was not detected above a fixed threshold
we defined its Ct as 40 (number of final PCR cycle).
To determine the quantity of gene-specific tran-
scripts present in ovarian tumor cDNA relative to nor-
mal ovarian tissue, their respective Ct values were
first normalized by subtracting the Ct value obtained
from the actin (ACTB) endogenous control (ΔCt =
Ct SLC34A2 – Ct ACTB). For 3 normal ovarian cDNA
samples normalized ΔCt was calculated as the mean
value. The relative concentrations of gene-specific
mRNAs in ovarian cancer cDNAs compared to normal
ovarian tissue (ΔΔCt) were calculated by subtracting
the normalized mean ΔCt value obtained for normal
ovarian cDNAs from those obtained for each of 17 ovar-
ian tumor samples (ΔΔCt = ΔCt of tumor — mean ΔCt
96 Experimental Oncology 33, 94–98, 2011 (June)
for 3 normal ovaries), and the relative concentration
was determined as 2^- ΔΔCt [31].
Immunohistochemical analysis. Representative
sections of tumor samples were prepared from paraffin
blocks and stained with hematoxylin-eosin according
as previously described [29]. Endogenous peroxidase
was quenched with H2O2 (3%) in 0.01% PBS. After
blocking of nonspecific binding by avidin–biotin
blocking solution (Vector Laboratories, Burlingame,
CA, USA), tissue sections were incubated overnight
at 4 °C with anti-NaPi2b mAb (10 μg/ml). Then, sec-
tions were incubated with biotinylated secondary
antibodies for 2 h at room temperature (1 : 400, goat
anti-mouse biotinylated IgG, Sigma), followed by incu-
bation with avidin–biotin–peroxidase complex (Vector
Laboratories, Burlingame, CA, USA). The immune
complexes were developed with diaminobenzidine
solution. Hematoxylin was used for counterstaining.
Prepared slides were examined with the use of Zeiss
Universal microscope (Zeiss, Germany); images were
captured using digital Axiocam software.
RESULTS
The expression of the SLC34A2 gene in ovarian car-
cinomas (n = 14), benign serous cystoadenomas (n = 4)
and normal tissues (n = 3) was examined at mRNA level
by real-time RT-PCR. In a panel of 14 ovarian carcino-
mas, there were 7 well-differentiated papillary serous
tumors, 5 endometrioid tumors (2 well-differentiated
and 3 poor-differentiated carcinomas), and 2 unspeci-
fied poor-differentiated adenocarcinomas. Differences
in SLC34A2 gene expression were calculated as fold
changes in gene expression in ovarian carcinomas
and benign serous cystoadenomas compared to three
normal ovaries (27 ov, 30 ov, and 35 ov). Since the PCR
product in these three samples of normal ovary was not
detected above a fixed threshold, which means that
NaPi2b is not expressed in normal ovary or is expressed
at very low levels we have defined it’s Ct as 40 in our
calculations (see Materials and Methods).
During this study we observed overexpression
of the SLC34A2 gene in all papillary serous tumors
compared to normal ovarian tissues (30.5-398.1 with
mean 152,9-fold increase) (Fig. 1, A). In addition, we de-
tected a heterogeneous level of the SLC34A2 gene
expression in endometrioid tumors, unspecified poor-
differentiated adenocarcinomas and benign tumors
compared to normal ovarian tissues (Fig. 1, B, C, and
D). It should be noted that the level of SLC34A2 gene
expression in benign ovarian tumors differed slightly
compared to normal ovary, in contrast to the expression
level of SLC34A2 gene in ovarian carcinomas where
it differed significantly compared to normal tissues.
Figure 2 demonstrates SLC34A2 gene expression
in the ovarian carcinoma samples grouped according
to their differentiation level. SLC34A2 gene was highly
expressed in all well-differentiated papillary serous
tumors and well-differentiated endometrioid tumor
samples 14ov and 18ov (19.8-54.1 with mean 36.9-fold
increase) compared to poor-differentiated endometri-
oid tumor samples 5ov, 6ov and 22ov (0.18-0.75 with
mean 0.44-fold decrease) and poor-differentiated
adenocarcinoma tumor sample 16ov (0.29) (Fig. 2).
1000
100
10
1
0,1
0,01
Fo
ld
c
ha
ng
e
Numbers of tumor samples
8o
v
13
ov
15
ov
19
ov
21
ov
24
ov
29
ov 5o
v
6o
v
14
ov
18
ov
22
ov
16
ov
23
ov 9o
v
17
ov
25
ov
28
ov
A B С D
Fig. 1. Real-time RT-PCR results for relative expression level
of SLC34A2 in ovarian tumor cDNA samples grouped according
to histomorphological type. The mean of SLC34A2 expression
level in 3 normal tissues referred as 1 in all cases. A. Papillary
serous carcinomas (8ov, 13ov, 15ov, 19ov, 21ov, 24ov, 29ov);
B. Endometrioid carcinomas (14ov, 18ov — well-differentiated;
5ov, 6ov, 22ov — poor-differentiated); C. Unspecified adenocar-
cinomas (16ov, 23ov — poor-differentiated); D. Benign tumors
(9ov, 17ov; 25ov, 28ov — serous cystoadenomas)
Data concerning mRNA expression of SLC34A2 in se-
rous and endometrioid ovarian tumors accurately cor-
related with NaPi2b protein expression in these types
of ovarian tumors detected by Western-blot and immu-
nohistochemical analyses in our previous investigation
[29]. Summarizing the results we can conclude that
SLC34A2 gene is up-regulated in well-differentiated
ovarian carcinomas compared with poor-differentiated
ovarian carcinomas and benign serous cystoadeno-
mas both at the mRNA and protein levels.
1000
100
10
1
0,1
0,01
Fo
ld
c
ha
ng
e
Numbers of tumor samples
5o
v
6o
v
16
ov
22
ov
23
ov 8o
v
13
ov
14
ov
15
ov
18
ov
19
ov
21
ov
24
ov
29
ov
A B
Fig. 2. Real-time RT-PCR results for relative expression level
of SLC34A2 in ovarian cancer samples grouped according to tu-
mor differentiation level. The mean of SLC34A2 expression level
in 3 normal tissues referred as 1 in all cases. A. Poor-differentiat-
ed ovarian tumors; B. Well-differentiated ovarian tumors
Immunohistochemical analysis with anti-NaPi2b
monoclonal antibodies in our previous investigation
and in this study has shown that NaPi2b is expressed
predominantly at the surface membrane (M) of cancer
cells in well-differentiated serous and endometrioid
ovarian carcinomas (Figures 3 a, b) [29]. In addition
to membrane localization, in some cases we observed
a weak NaPi2b positive staining in the cytoplasm (C)
and the nuclei (N) of cancer cells and cells of certain
benign tumors (Figures 3 a, b, c).
Experimental Oncology 33, 94–98, 2011 (June) 97
Fig. 3. Immunohistochemical analysis of NaPi2b expression
in ovarian tumors: a, serous papillary carcinoma; b, well-dif-
ferentiated endometrioid carcinoma; c, serous cystadenoma.
NaPi2b positive staining at the membrane of cells marked
as M, in cytoplasme and in nuclei as C and N correspondently.
Magnification x 400
DISCUSSION
In our previous study, we have analyzed NaPi2b
expression at protein and mRNA levels in different
types of epithelial ovarian cancer and normal ovarian
tissues using Western blot, immunohistochemical and
qualitative RT-PCR techniques [29]. We have shown
a differential expression profile of NaPi2b phosphate
transporter at protein level in various histological types
of epithelial ovarian cancer and have not identified
any protein expression in normal ovary. Intere stingly,
we didn’t reveal any correlation between NaPi2b
protein expression and SLC34A2 mRNA expression
in ovarian tumor samples detected by conventional
RT-PCR analysis [29]. This contradiction could be ex-
plained in part by putative regulation of NaPi2b expres-
sion at the posttranscriptional level [32] in ovarian
tumors or by limitation of qualitative RT-PCR analysis.
The main purpose of this investigation was to perform
quantitative analysis of the SLC34A2 gene expression
by real-time polymerase chain reaction in samples
of normal ovary and ovarian tumors.
In this study we have analysed the same tissue
samples that were used in our previous investiga-
tion [29] and have shown a heterogeneous level
of SLC34A2 gene expression in ovarian tumor samples
of different histomorphological types using quantita-
tive real-time RT-PCR analysis in contrast to previ-
ous studies where conventional RT-PCR analysis
which showed uniform expression SLC34A2 in these
ovarian tumors has been used. mRNA expression
of SLC34A2 in serous and endometrioid ovarian
carcinomas, benign serous cystoadenomas as well
as in normal ovary accurately correlated with protein
expression level that was detected in our previous
investigation of these tumor samples by Western-blot
and immunohistochemical analyses. These data clear-
ly indicate that the use of quantitative analysis is more
sensitive for studying of SLC34A2 gene expression
profile in different types of ovarian tumor samples and
in normal ovary. In addition, analysis of SLC34A2 gene
expression according to tumor differentiation level
(poor- and well-differentiated) in the endometrioid
and serous histological types of ovarian cancer as well
as in unspecified ovarian adenocarcinomas showed
that SLC34A2 is up-regulated in well-differentiated
tumors. Thus, our results are well consistent with
our previous data about NaPi2b protein expression
profile in different types of ovarian carcinomas and
data reported by Rangel et al. [25] concerning the
various SLC23A3 gene expression profile in serous
ovarian carcinomas according to differentiation level.
In addition to data Rangel et al. [25], we have showed
heterogeneous SLC34A2 expression profile according
to differential level in endometrioid tumors as well.
According to immunohistochemical analysis phos-
phate transporter protein was located predominantly
at the surface membrane of cancer cells, but in a few
cases cytoplasmic and nuclear localization of NaPi2b
in ovarian cancer cells was also observed. Since NaPi2b
function in cancer cells has not been studied so far
we can only suggest that NaPi2b might be involved
in transport of inorganic phosphate from the peritoneal
cavity fluid via surface membrane of cancer cells, while
NaPi2b function in the cytoplasm and nuclei of cancer
cells is completely unclear. NaPi2b function in cancer
cells should be investigated in further studies.
Thus, quantitative real-time PCR and immuno-
hystochemical analyses allowed us to investigate
more detailed features of NaPi2b expression in dif-
ferent types of ovarian cancer. We have shown that
SLC34A2 gene expression detected by real-time
RT-PCR analysis is up-regulated in well-differentiated
ovarian carcinomas that correlates with NaPi2b pro-
tein expression in cancer cells of the same tumor
samples. Taking into account that better differenti-
ated tumors usually have better prognosis, evaluation
of SLC34A2 gene expression could serve as a potential
marker for ovarian cancer diagnosis and prognosis.
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