Genetic and epigenetic changes of NKIRAS1 gene in human renal cell carcinomas
Renal cell carcinoma (RCC) is the most common malignant tumor of kidney associated with the worst clinical outcome. No molecular markers for RCC diagnostics and prognosis that could be applied in clinics were described yet. Large-scale screening of 3p human chromosome genes/loci in RCC and histologi...
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| Cite this: | Genetic and epigenetic changes of NKIRAS1 gene in human renal cell carcinomas / G.V. Gerashchenko, O.O. Bogatyrova, E.E. Rudenko, A.G. Kondratov, V.V. Gordiyuk, Y.M. Zgonnyk, O.F. Vozianov, T.V. Pavlova, E.R. Zabarovsky, A.V. Rynditch, V.I. Kashuba // Experimental Oncology. — 2010. — Т. 32, № 2. — С. 71-75. — Бібліогр.: 32 назв. — англ. |
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nasplib_isofts_kiev_ua-123456789-1386072025-02-10T00:40:53Z Genetic and epigenetic changes of NKIRAS1 gene in human renal cell carcinomas Gerashchenko, G.V. Bogatyrova, O.O. Rudenko, E.E. Kondratov, A.G. Gordiyuk, V.V. Zgonnyk, Y.M. Vozianov, O.F. Pavlova, T.V. Zabarovsky, E.R. Rynditch, A.V. Kashuba, V.I. Original contributions Renal cell carcinoma (RCC) is the most common malignant tumor of kidney associated with the worst clinical outcome. No molecular markers for RCC diagnostics and prognosis that could be applied in clinics were described yet. Large-scale screening of 3p human chromosome genes/loci in RCC and histologically normal tissues surrounding the tumors using NotI-microarray approach demonstrated that NKIRAS1 gene contained the largest percent of genetic/epigenetic changes in RCC tumor cells. Aim: To validate the results of NotI microarray analysis and study genetic, epigenetic changes, and the expression level of NKIRAS1 gene in human RCC samples. Methods: DNA and RNA were isolated from freshly-frozen renal tumors’ samples (n = 12) and from normal tissues surrounding the tumors. Epigenetic changes (methylation status) of NKIRAS1 were detected by bisulfite sequencing. Genetic changes and expression level were analyzed by Quantitative real-time PCR (qPCR) with SYBR Green. For relative quantification 2-ΔΔCP method was used. Nonparametric tests (Wilcoxon, Kruskal — Wallis and Mann — Whitney) were applied for statistical data analysis using the BioStat software. Results: NKIRAS1 expression was downregulated in 75% of RCC samples (9 of 12) compared with surrounding normal tissue. High grade tumors (3 and 4) showed lower expression of NKIRAS1 at the mRNA level than tumors of low grade (1 and 2). No significant association was found between gene expression level and gender or age. Analysis of NKIRAS1 gene copy number was performed in 19 tumor samples. Changes in the copy number of NKIRAS1 gene were observed in 64% (9 of 14) of cRCC samples. 9 samples displayed ratio ( 0.85) and were considered as normal copy number. Changes in NKIRAS1 gene copy number were detected in all 3 benign oncocytomas, 1 papillary cancer and 1 sarcoma, where hemizygous deletion was observed. No changes in methylation status of NKIRAS1 were found in RCC. Conclusions: We have validated the results of NotI microarray analysis of NKIRAS1 gene in RCC. It was shown the decreased expression level of NKIRAS1 in this type of tumor. This work was supported by National Academy of Sciences of Ukraine, research grants from the Swedish Cancer Society, the Swedish Research Council. 2010 Article Genetic and epigenetic changes of NKIRAS1 gene in human renal cell carcinomas / G.V. Gerashchenko, O.O. Bogatyrova, E.E. Rudenko, A.G. Kondratov, V.V. Gordiyuk, Y.M. Zgonnyk, O.F. Vozianov, T.V. Pavlova, E.R. Zabarovsky, A.V. Rynditch, V.I. Kashuba // Experimental Oncology. — 2010. — Т. 32, № 2. — С. 71-75. — Бібліогр.: 32 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/138607 en Experimental Oncology application/pdf Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions Gerashchenko, G.V. Bogatyrova, O.O. Rudenko, E.E. Kondratov, A.G. Gordiyuk, V.V. Zgonnyk, Y.M. Vozianov, O.F. Pavlova, T.V. Zabarovsky, E.R. Rynditch, A.V. Kashuba, V.I. Genetic and epigenetic changes of NKIRAS1 gene in human renal cell carcinomas Experimental Oncology |
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Renal cell carcinoma (RCC) is the most common malignant tumor of kidney associated with the worst clinical outcome. No molecular markers for RCC diagnostics and prognosis that could be applied in clinics were described yet. Large-scale screening of 3p human chromosome genes/loci in RCC and histologically normal tissues surrounding the tumors using NotI-microarray approach demonstrated that NKIRAS1 gene contained the largest percent of genetic/epigenetic changes in RCC tumor cells. Aim: To validate the results of NotI microarray analysis and study genetic, epigenetic changes, and the expression level of NKIRAS1 gene in human RCC samples. Methods: DNA and RNA were isolated from freshly-frozen renal tumors’ samples (n = 12) and from normal tissues surrounding the tumors. Epigenetic changes (methylation status) of NKIRAS1 were detected by bisulfite sequencing. Genetic changes and expression level were analyzed by Quantitative real-time PCR (qPCR) with SYBR Green. For relative quantification 2-ΔΔCP method was used. Nonparametric tests (Wilcoxon, Kruskal — Wallis and Mann — Whitney) were applied for statistical data analysis using the BioStat software. Results: NKIRAS1 expression was downregulated in 75% of RCC samples (9 of 12) compared with surrounding normal tissue. High grade tumors (3 and 4) showed lower expression of NKIRAS1 at the mRNA level than tumors of low grade (1 and 2). No significant association was found between gene expression level and gender or age. Analysis of NKIRAS1 gene copy number was performed in 19 tumor samples. Changes in the copy number of NKIRAS1 gene were observed in 64% (9 of 14) of cRCC samples. 9 samples displayed ratio ( 0.85) and were considered as normal copy number. Changes in NKIRAS1 gene copy number were detected in all 3 benign oncocytomas, 1 papillary cancer and 1 sarcoma, where hemizygous deletion was observed. No changes in methylation status of NKIRAS1 were found in RCC. Conclusions: We have validated the results of NotI microarray analysis of NKIRAS1 gene in RCC. It was shown the decreased expression level of NKIRAS1 in this type of tumor. |
| format |
Article |
| author |
Gerashchenko, G.V. Bogatyrova, O.O. Rudenko, E.E. Kondratov, A.G. Gordiyuk, V.V. Zgonnyk, Y.M. Vozianov, O.F. Pavlova, T.V. Zabarovsky, E.R. Rynditch, A.V. Kashuba, V.I. |
| author_facet |
Gerashchenko, G.V. Bogatyrova, O.O. Rudenko, E.E. Kondratov, A.G. Gordiyuk, V.V. Zgonnyk, Y.M. Vozianov, O.F. Pavlova, T.V. Zabarovsky, E.R. Rynditch, A.V. Kashuba, V.I. |
| author_sort |
Gerashchenko, G.V. |
| title |
Genetic and epigenetic changes of NKIRAS1 gene in human renal cell carcinomas |
| title_short |
Genetic and epigenetic changes of NKIRAS1 gene in human renal cell carcinomas |
| title_full |
Genetic and epigenetic changes of NKIRAS1 gene in human renal cell carcinomas |
| title_fullStr |
Genetic and epigenetic changes of NKIRAS1 gene in human renal cell carcinomas |
| title_full_unstemmed |
Genetic and epigenetic changes of NKIRAS1 gene in human renal cell carcinomas |
| title_sort |
genetic and epigenetic changes of nkiras1 gene in human renal cell carcinomas |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2010 |
| topic_facet |
Original contributions |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/138607 |
| citation_txt |
Genetic and epigenetic changes of NKIRAS1 gene in human renal cell carcinomas / G.V. Gerashchenko, O.O. Bogatyrova, E.E. Rudenko, A.G. Kondratov, V.V. Gordiyuk, Y.M. Zgonnyk, O.F. Vozianov, T.V. Pavlova, E.R. Zabarovsky, A.V. Rynditch, V.I. Kashuba // Experimental Oncology. — 2010. — Т. 32, № 2. — С. 71-75. — Бібліогр.: 32 назв. — англ. |
| series |
Experimental Oncology |
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2025-12-02T06:17:55Z |
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2025-12-02T06:17:55Z |
| _version_ |
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| fulltext |
Experimental Oncology 32, 71–75, 2010 (June) 71
Renal cell carcinoma (RCC) is the most common
malignant kidney tumor in adults. It stands for approxi-
mately 3% of all human malignancies. Among cancers
of the urinary system, RCC is associated with the worst
clinical outcome [1]. The incidence of RCC is increas-
ing and it is estimated that RCC accounts worldwide
for 95,000 cancer-related deaths per year [2].
At present, surgical resection is the most effective
treatment for localized RCC tumors, but no satisfac-
tory treatment is available for patients with advanced-
stage tumor. Some therapies for RCCs have achieved
a response rate of 20%, but severe adverse reactions
are frequent and prognosis for patients does not
seem to have improved overall. Although tumor stage
is consi dered to be the most informative prognostic
factor, little is known about the underlying molecular
mechanisms of renal carcinogenesis.
The most common histological subtypes of RCC
include clear cell (80%), papillary (> 10%), and chro-
mophobe (< 5%) carcinomas. These histological sub-
types are genetically and biologically different [3, 4].
Many genes and signaling pathways are known to be
involved in RCC initiation and progression [5]. However,
until today no tumor suppressor gene, which malfunc-
tion could be responsible or at least contributing to the
development of clear cell renal cell carcinoma (ccRCC)
has been identified. Several potential candidates were
proposed, such as VHL (von Hippel Lindau), FHIT (fragi-
le histidine triad), TTRC1 (two-three-renal-cancer-1),
DUTT1 (deleted in U-twenty twenty), locus NCR-1 (non
papillary renal cell carcinoma 1), and RASSF1A (RAS
association family 1) [6–9].
Papillary RCCs are characterized by trisomies
(chromosomes 3q, 7, 12, 16, 17, and 20) and loss of the
Y chromosome [10]. Chromophobe RCCs are charac-
terized by monosomy of multiple chromosomes (1, 2, 6,
10, 13, 17, and 21). Clear-cell RCCs are characteri zed by
loss of genetic material on the chromosomes 3, 8, 9, 10.
Fifty percent of clear-cell RCCs show somatic mutations
of VHL gene, and, moreover, the 10% to 20% of these
tumors show inactivation of the VHL gene [11]. Loss
of heterozygosity on chromosomes 8p or 9p provides
prognostic significance in patients with locally advanced
ccRCC and PTEN/MMAC1 (chromosome 10) inactiva-
tion that may play a role in the ccRCC progression [12].
However, these molecular markers have not yet gained
general use in RCC diagnostics and prognosis.
GENETIC AND EPIGENETIC CHANGES OF NKIRAS1 GENE
IN HUMAN RENAL CELL CARCINOMAS
G.V. Gerashchenko1, $, O.O. Bogatyrova1, $, E.E. Rudenko1, $, A.G. Kondratov1, V.V. Gordiyuk1,
Y.M. Zgonnyk2, O.F. Vozianov2, T.V. Pavlova3, E.R. Zabarovsky3, A.V. Rynditch1, V.I. Kashuba1, *
1Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Zabolotnogo str. 150,
Kiev 03143, Ukraine
2Institute of Urology, National Academy of Medical Sciences of Ukraine, Kotsubinsky str. 9A, Kiev 04053, Ukraine
3Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institute, Nobels vag. 16, Stockholm
17177, Sweden
Renal cell carcinoma (RCC) is the most common malignant tumor of kidney associated with the worst clinical outcome. No mo-
lecular markers for RCC diagnostics and prognosis that could be applied in clinics were described yet. Large-scale screening of 3p
human chromosome genes/loci in RCC and histologically normal tissues surrounding the tumors using NotI-microarray approach
demonstrated that NKIRAS1 gene contained the largest percent of genetic/epigenetic changes in RCC tumor cells. Aim: To
validate the results of NotI microarray analysis and study genetic, epigenetic changes, and the expression level of NKIRAS1 gene
in human RCC samples. Methods: DNA and RNA were isolated from freshly-frozen renal tumors’ samples (n = 12) and from
normal tissues surrounding the tumors. Epigenetic changes (methylation status) of NKIRAS1 were detected by bisulfite sequenc-
ing. Genetic changes and expression level were analyzed by Quantitative real-time PCR (qPCR) with SYBR Green. For relative
quantification 2-ΔΔCP method was used. Nonparametric tests (Wilcoxon, Kruskal — Wallis and Mann — Whitney) were applied for
statistical data analysis using the BioStat software. Results: NKIRAS1 expression was downregulated in 75% of RCC samples (9 of
12) compared with surrounding normal tissue. High grade tumors (3 and 4) showed lower expression of NKIRAS1 at the mRNA
level than tumors of low grade (1 and 2). No significant association was found between gene expression level and gender or age.
Analysis of NKIRAS1 gene copy number was performed in 19 tumor samples. Changes in the copy number of NKIRAS1 gene were
observed in 64% (9 of 14) of cRCC samples. 9 samples displayed ratio (< 0.85 and ≥ 0.35), thus were considered as hemizygous
deletions. 3 samples showed ratio (> 0.85) and were considered as normal copy number. Changes in NKIRAS1 gene copy number
were detected in all 3 benign oncocytomas, 1 papillary cancer and 1 sarcoma, where hemizygous deletion was observed. No changes
in methylation status of NKIRAS1 were found in RCC. Conclusions: We have validated the results of NotI microarray analysis
of NKIRAS1 gene in RCC. It was shown the decreased expression level of NKIRAS1 in this type of tumor.
Key Words: renal cell carcinoma, genetic and epigenetic regulation, chromosome 3, quantitative real time PCR, methylation status.
Received: May 10, 2010.
$These authors contributed equally to this work.
*Correspondence: E-mail: vlakas@ki.se
Abbreviations used: ccRCC — clear cell renal carcinoma; NKIRAS1 —
NF-B inhibitor interacting Ras-like 1; RCC — renal cell carcinoma;
TBP — tata-box binding protein; VHL− von Hippel Lindau gene.
Exp Oncol 2010
32, 2, 71–75
ORIGINAL CONTRIBUTIONS
72 Experimental Oncology 32, 71–75, 2010 (June)
The most predominant type of RCC, clear cell car-
cinoma, was chosen for this study and the large-scale
analysis of gene expression profiles was performed.
Similar studies, reported by other groups earlier, have
already identified some genes that might be useful
for prognostic purposes or for classification of RCCs
[13–15]. Consequently, relatively little is known about
molecular expression profiles associated with tumor
growth and metastatic progression of ccRCC.
The largest percent of genetic aberrations associ-
ated with ccRCC includes 3p loss or changes. DNA
methylation/deletion is a key mechanism to inhibit the
expression of tumor suppressor genes in cancer; hence
DNA methylation/deletion markers have been applied
in cancer risk assessment, early detection, prognosis,
and prediction of response to cancer therapy.
Genetic/epigenetic changes could be investigated,
using microarrays analysis. NotI microarrays that were
recently developed by us, give the new possibilities for
large scale study of deletion/methylation patterns in
normal compared to pathological cells [16].
Using the NotI microarrays, we have found that
a locus associated with the NF-B inhibitor interacting
Ras-like 1 gene (NKIRAS1) had the largest percent of
genetic/epigenetic changes, and exclusively in RCC
[17–19]. The NKIRAS1 gene encodes a Ras-like protein
that acts as a potent regulator of NF-kappa-B activity.
[20–22]. Function of NKIRAS1 in carcinogenesis is
unknown and its characterization can shed some light
on the mechanism of epithelial cancer development.
In the present work we further analyzed the expres-
sion level, deletion and methylation status of the puta-
tive tumor suppressor gene NKIRAS1 in kidney cancers.
MATERIALS AND METHODS
Tissue samples. Frozen surgically removed tu-
mors and surrounding normal tissues were obtained
from Kyiv National Urological Center (Kyiv, Ukraine).
Tumors were histologically classified according to
the WHO criteria by TNM classification. This series
included 3 oncocytomas (benign), 18 conventional
clear cell tumors (2 with sarcomatosis), 1 papillary
tumor, and 1 sarcoma. The mean age of patients at
diagnosis was 57.4 ± 11.6 (range 36–69 years), with
a female/male ratio of 1.3. The present study was
performed in accordance with the institutional Ethical
Committee permission. All the samples enrolled in this
study were processed anonymously.
Isolation of DNA and RNA. DNA was isolated
using phenol-chloroform extraction. Total RNA was
isolated from all fresh-frozen renal tumors and from
normal tissues surrounding the tumors by homogeni-
zation with an acid guanidinium thiocyanate-phenol-
chloroform mixture as described [23]. Samples used
for the qPCR reactions were of high molecular weight
(unsheared band of undigested DNA visible on a 0.5%
agarose gel) and pure from contaminations (an OD
260/280 ranging from 1.6 to 1.8). From each RNA
sample, 1 μg of total RNA was treated with DNAseI and
reverse transcribed in duplicates, as well as negative
controls without enzyme, using the reverse transcrip-
tase kit (Fermentas).
Methylation status. The methylation status of
NKIRAS1 was determined in three selected samples by
bisulfite sequencing as described earlier [24, 25]. Bisul-
fite treatment was performed using an EZ DNA Methyla-
tion Kit (Zymo Research Corporation, USA). PCR was
carried out for 35 cycles comprising 30 s denaturation
at 94 °C, 30 s annealing at 56 °C, and 1 min extension
at 72 °C. The cycling started by 2 min denaturation at
94 °C. The PCR products were purified using a DNA
Clean and Concentrator Kit (Zymo Research Corpora-
tion) according to the manufacturer’s protocol, and the
PCR products were cloned using a TOPO TA Cloning Kit
for Sequencing (Invitrogen BV, Netherlands). Plasmid
DNA was isolated using a Zyppy Plasmid Miniprep Kit
(Zymo Research Corporation, USA). Sequencing was
performed using a BigDye Terminator Cycle Sequenc-
ingReady Reaction kit v1.1 and ABI Prism 3100 Genetic
Analyzer (Applied Biosystems, USA).
Primer design. Primers for gene NKIRAS1 were
designed using Primer3 (http://frodo.wi.mit.edu/
primer3/) and PrimerQuest (http://eu.idtdna.com/Sci-
tools/Applications/ Primerquest/), and Oligo6.24 pro-
gram. For genomic sequence following primers were
used: forward 5’-cgagaggtgagagagttggc-3’, reverse
5’-tgcgtgaaacaaacctgttct-3’. For mRNA detection
following primers were used: forward 5’-ctttcaaagagt-
ggagcttctg-3’, reverse 5’-tttccgatctgtaacagtcacc-3’.
Primers for mRNA detection were selected to span at
least one big intron (more than 1kb).
Expression level analysis. Reactions were per-
formed using SYBR Green. TBP was used as a reference
gene [26]. Each qPCR reaction comprised 12.5 μl 2x
SYBR Green PCR Master Mix (Fermentas), forward and
reverse primer at optimized concentrations of 400 nM,
10 ng/μl cDNA template and sterile water up to a final
volume of 25 μl. The qPCR reactions were performed
using Bio-Rad iQ5. The reaction profile was: initial step,
72 °C for 2 min, denaturation 95 °C for 10 min, then
35 cycles of denaturing at 95 °C for 20 s, annealing at
60 °C for 20 s and extension at 72 °C for 40 s.
To generate standard curves for the selected pri-
mers and the reference primers a log10 dilution series
of cDNA was prepared at concentrations ranging from
1 ng to 100 ng. Reference gene TBP was previously
found to be stably expressed in renal tumors. Each
cDNA sample was analyzed in triplicate by real-time
PCR, and detected with SYBR Green Master Mix.
Each qPCR experiment contained duplicates of the
notemplate-controls and patient samples. Quantifica-
tion was based on the increased fluorescence, which
was measured and recorded using the Bio-Rad iQ5.
Copy number analysis. Quantitative real-time
PCR (qPCR) was used for the quantification of marker
copy numbers and has been shown to be precise
enough to discriminate between two and one allele
copies. The parameter CP (threshold cycle) is defined
as the cycle number required for dye fluorescence to
become higher than background fluorescence level.
Experimental Oncology 32, 71–75, 2010 (June) 73
Reactions were performed using SYBR Green and de-
tected with SYBR Green Master Mix (Fermentas) with
similar reaction conditions as for expression analy-
sis. The method is based on the inverse exponential
relationship that exists between initial quantity (copy
number) of target sequence copies in the reactions
and corresponding CP determinations — the higher
the starting copy number of DNA target the lesser the
CP value. This method was used to determine target
sequence copy number in tumor DNA sample relative
to the normal DNA from the same patient (calibra-
tor) and relative to an endogenous control sequence
(reference) — TBP. Quantification was based on the
increased fluorescence, which was measured and re-
corded using the Bio-Rad iQ5. According to histology
analysis contamination of tumour samples with normal
stroma and lymphocytes can reach up to 30–40%.
Therefore, alleles were taken as homozygously deleted
if the highest value of calculated range was below
0.5 and hemizygously deleted if this value was below
1.0. An allele was considered as amplified/multiplied
if the lowest value of the range was over 1.0 [27].
Data analysis. Copy numbers in NKIRAS1 gene
and its expression level was estimated by the 2-ΔΔCP
method of relative quantification [28]. The relative ex-
pression ratio (R) of a target gene is calculated based
on E and the CP deviation of an unknown sample versus
a control, and expressed in comparison to a reference
gene. For the ΔΔCP calculation to be valid, two im-
portant parameters must be considered beforehand.
First, the efficiency of a given PCR amplification must
be close to 100%, and second, the relative efficiency
must be optimal. That is the amplification efficiencies of
the target and reference genes must be approximately
equal. PCR efficiencies (E) were calculated from the
given slopes, according to the equation:
E = (10(-1/slope) – 1) × 100,
where E = 100 corresponds to 100% efficiency. Plots of
the log DNA and cDNA dilutions vs ΔCP were made for
each primer pair. Also for every reaction efficiency was
calculated using sigmoidal and exponential models
from qPCR package [29]. Further calculations were
performed using Microsoft Excel. The range given for
the probes was determined as E-ΔΔCP with ΔΔCP + S
and ΔΔCP-S, where s is the standard deviation of the
ΔΔCP value.
Statistical analysis. Nonparametric Wilcoxon test
was used to compare mRNA expression of target and
reference genes for the same sample. Then groups
of samples were compared in respect to average
level of mRNA decrease (LDav) and the frequency
of decrease (FD). The LD was calculated as 1/R and
reflects the n-fold factor by which the mRNA content
decreased in the tumor compared to normal tissue.
Nonparametric Kruskal — Wallis and Mann — Whitney
rank-sum tests were used to test mRNA differences
(both LDav and FD) for each target gene and with and
without metastases. Nonparametric Spearmen’s cri-
terion was used to calculate the coefficient of correla-
tion between the levels of mRNA decrease (LDav) for
each set of pairs of target genes. P-values < 0.05 were
considered statistically significant. All statistical proce-
dures were performed using the BioStat software [30].
RESULTS AND DISCUSSION
NotI microarray approach allows the search for
hemizigous deletion/methylation that can indicate
a reduction or loss of expression of the certain genes.
Previously, we have found changes in 137 of 181 NotI
clones from human chromosomes 3 [17]. Heterozygous
deletion/methylation events were observed most fre-
quently (428 cases); amplifications (24 cases) and ho-
mozygous deletion/methylation events (24 cases) were
observed rarely. NotI microarray data analysis produced
a profile of the changes in chromosome 3 genes/loci in
the epithelial cancer samples. Among them, 27 genes/
loci showed changes in more than 30% of samples.
To validate NotI microarray results we have chosen
the NF-B inhibitor interacting Ras-like 1 (NKIRAS1)
gene that showed the highest percentage of genetic/
epigenetic changes. Gene NKІRAS1 is localized in
3p24.2 locus on the human chromosome 3, which
has the greatest amount of changes in epithelial can-
cers. Thus, 78% of cases demonstrated hemizigous
deletion/methylation in the NotI locus associated with
NKIRAS1 gene [17].
We selected several samples with genetic/epige-
netic changes for bisulphite sequencing and found
an absence of DNA methylation in all samples, both in
tumor and normal tissue (data not shown). To explain
the high percent of changes obtained by NotI microar-
ray approach we measured the expression level and
estimated the copy number of NKIRAS1 gene.
Significant (from 2 to 94 fold) decrease of the mRNA
level (LD) of gene NKIRAS1 was observed in 75%
of RCC samples (9 of 12, P < 0.002) in comparison
to surrounding normal tissue (Table 1). TBP expres-
sion level was found to be constant in tumor and nor-
mal renal tissue. While no significant association was
found between gene expression level and gender or
age, it should be pointed out that NKIRAS1 expres-
sion correlated with histological grade. Thus, tumors
of grade 3–4 had lower NKIRAS1 mRNA level than
tumors of grade 1–2 (Figure).
–1.80
–1.60
–1.40
–1.20
–1.00
–0.80
–0.60
–0.40
–0.20
0.00
0.20
0.40
1 2 3 4 5 6 7 8 9 10 11 12
Sample number up to increase of atypia stage
NK
IR
AS
1
ex
pr
es
si
on
le
ve
l,
lg
(R
)
1–2 stage 3–4 stage
Figure. NKIRAS1 expression (n = 12) in renal cell carcinoma
Analysis of NKIRAS1 copy number was performed in
19 samples (3 benign oncocytomas, 1 papillary cancer,
1 sarcoma, and 14 ccRCCs), using qPCR (Table 2).
NKIRAS1 copy number decrease was observed in
74 Experimental Oncology 32, 71–75, 2010 (June)
64% (9 of 14) of cRCC samples: 9 samples displayed
ratio < 0.85 and ≥ 0.35 and were conside red as
hemizigous deletions. 3 samples had ratio > 0.85 and
were considered to have unmodified copy number sta-
tus. Copy number changes were found in all 3 benign
oncocytomas, 1 papillary cancer, and 1 sarcoma, where
hemizigous deletions were observed. All these data
suggest that NKIRAS1 gene could be involved in the
carcinogenesis as a tumor suppressor gene.
The NKIRAS1 gene encodes a Ras-like protein that
acts as a potent regulator of NF-kappa-B activity. Func-
tions of NKIRAS1 in carcinogenesis are still unknown.
Mutations of other members of the Ras gene family
(HRAS, KRAS2 or NRAS) are identified in approximately
of 15% of human neoplasm cases [31]. Here we de-
scribe a novel member of this family which is associated
with tumor growth and metastasis in epithelial cancer.
We have reported earlier that the CpG islands in
the ITGA9 gene/locus were homozygously methy-
lated in one of the tissue samples, heterozygously
methylated in the second sample, and unmethylated
in the third sample from colorectal cancer [32]. These
results correlated with the microarray results. The ratio
of the hybridization signal intensity for tumor/normal
tissues was 0.2, 0.55 and 0.97 for the three samples.
No methylation of CpG islands was revealed for the
corresponding normal tissues as well as no deletions
were found. Our work demonstrated that the NotI
microarray produces valuable data for analysis. The
NotI microarray protocol might be useful as a primary
screening method due to its ability to determine the
global genetic/epigenetic changes in a genome. This
may lead to the subsequent selection of gene candi-
dates that may participate in carcinogenesis.
In summary, we have found that NKIRAS1 is down-
regulated in malignant renal tumors and copy number of
NKIRAS1 gene is decreased in RCC. Due to the results
of NotI microarray analysis NKIRAS1 was selected for
further investigation as a gene with the highest number of
genetic alterations in epithelial tumors. We plan to analyze
the association of NKIRAS1 expression with the age of pa-
tients, and perform further studies to test if NKIRAS1 gene
expression could be regarded as dia gnostic marker for
RCC. The function of this protein in the development of
renal cancer should be elucidated further.
ACKNOWLEDGEMENTS
This work was supported by National Academy
of Sciences of Ukraine, research grants from the Swe-
dish Cancer Society, the Swedish Research Council.
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