Group Title: Reproductive Biology and Endocrinology 2007, 5:34
Title: Genomic and proteomic profiling I: Leiomyomas in African Americans and Caucasians
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Title: Genomic and proteomic profiling I: Leiomyomas in African Americans and Caucasians
Series Title: Reproductive Biology and Endocrinology 2007, 5:34
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Creator: Pan Q
Luo X
Chegini N
Publication Date: 39317
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Genomic and proteomic profiling I: Leiomyomas in African
Americans and Caucasians
Qun Pan, Xiaoping Luo and Nasser Chegini*


Address: Department of Obstetrics and Gynecology, University of Florida, Gainesville, Florida 32610, USA
Email: Qun Pan panq@obgyn.ufl.edu; Xiaoping Luo xiaoping@obgyn.ufl.edu; Nasser Chegini* cheginin@obgyn.ufl.edu
* Corresponding author


Published: 23 August 2007
Reproductive Biology and Endocrinology 2007, 5:34 doi:10.1 186/1477-7827-5-34


Received: 14 May 2007
Accepted: 23 August 2007


This article is available from: http://www.rbej.com/content/5/1/34
2007 Pan et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Clinical observations indicate that leiomyomas occur more frequently in African
Americans compared to other ethnic groups with unknown etiology. To identify the molecular
basis for the difference we compared leiomyomas form A. Americans with Caucasians using
genomic and proteomic strategies.
Methods: Microarray, realtime PCR, 2D-PAGE, mass spectrometry, Western blotting and
immunohistochemistry.
Results: Using Affymetrix U I33A array and analysis based on P ranking (P <0.01) 1470 genes were
identified as differentially expressed in leiomyomas compared to myometrium regardless of
ethnicity. Of these, 268 genes were either over-expressed (177 genes) or under-expressed (91
genes) based on P < 0.01 followed by 2-fold cutoff selection in leiomyomas of A. Americans as
compared to Caucasians. Among them, the expression E2F I, RUNX3, EGR3, TBPIP, ECM2, ESM I,
THBSI, GASI, ADAMI7, CST6, CST7, FBLN5, ICAM2, EDNI and COL18 was validated using
realtime PCR low-density arrays. 2D PAGE coupled with image analysis identified 332 protein spots
of which the density/volume of 31 varied by greater than or equal to 1.5 fold in leiomyomas as
compared to myometrium. The density/volume of 34 protein-spots varied by greater than or equal
to 1.5 fold (26 increased and 8 decreased) in leiomyomas of A. Americans as compared to
Caucasians. Tandem mass spectrometric analysis of 15 protein spots identified several proteins
whose transcripts were also identified by microarray, including 14-3-3 beta and mimecan, whose
expression was confirmed using western blotting and immunohistochemistry.
Conclusion: These findings imply that the level rather than the ethnic-specific expression of a
number of genes and proteins may account for the difference between leiomyomas and possibly
myometrium, in A. Americans and Caucasians. Further study using larger sample size is required to
confirm these findings.


Background
It has been estimated that 70% of women have a life-long
risk of developing leiomyomas, with symptomatic tumors
accounting for a 1/3 of all the hysterectomies preformed


annually in the United States alone. Such estimates are
even higher among African Americans with leiomyomas
that develop earlier, become larger and more sympto-
matic as compared to other ethnic groups [1-3]. Since lei-


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omyomas develop during the reproductive years and
regress with menopause, ovarian steroids are critical to
their growth, however the molecular environment to
explain these ethnic differences is unknown.

Various conventional and recent large-scale gene expres-
sion-profiling studies have provided valuable information
regarding the molecular environment of leiomyomas [4-
11]. Most of these studies were based on comparing leio-
myomas with myometrium obtained from different eth-
nic groups without examining the influence of ethnicity
on overall gene expression [9,10,12]. Using tissue micro-
array a recent immunohistochemistry study has reported
the expression of a number of proteins in leiomyomas
with some differences in their expression intensities in lei-
omyomas from African Americans as compared to other
ethnic groups [13]. In addition, an increased risk of devel-
oping leiomyomas, including among African Americans,
has been examined in a number of genetic studies,
although in a majority of the cases the evidence of
genomic instability is inconsistent [14-17].

Considering the above information and availability of
limited data differentiating leiomyomas microenviron-
ment in African Americans in the present study we com-
bined genomic and proteomic strategies comparing the
molecular environment of paired leiomyoma and myo-
metrium from African Americans with Caucasians. Fur-
thermore, from microarray data sets we selected 15
differentially expressed genes from different functional
categories rather than levels of their expression and vali-
dated their expression using realtime PCR in a microflu-
idic card format. Additionally, 15 protein spots identified
by proteomic were isolated and subjected to tandem mass
spectrometry (MS/MS) for identification of their content
and verified two of these proteins by Western blotting and
immunohistochemistry.

Methods
Portions of leiomyoma and matched myometrium were
collected from six women, ranging from 29 to 38 years old
who were scheduled to undergo hysterectomy for indica-
tions related to symptomatic leiomyomas. Three of the
patients were Caucasians and three African Americans.
These women were not taking any medication including
hormonal therapy for the pervious 3 months prior to sur-
gery and based on their last menstrual period and
endometrial histology they were from early-mid secretary
phase of the menstrual cycle. The myometrium used in
this study was collected from regions distal from leiomy-
omas. All the leiomyomas were subserosal/intramural
and 3 to 4 cm in diameter. These tissues were collected at
the University of Florida affiliated Shands Hospital with
prior approval from the Institutional Review Board with-
out requiring to obtain informed consent. The tissues


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were snapped frozen and stored in liquid nitrogen until
used.

Microarray and gene expression profiling
Small portions of the above tissues were used to isolate
total RNA using Trizol (Invitrogen, Carlsbad, CA). RNA
quality and yield was analyzed with Agilent 2100 Bioana-
lyzer (Agilent Technologies, Foster City, CA). RNA ampli-
fication was carried out as previously described [7], and
following second-strand cDNA synthesis 5 itg of purified
cDNA was reverse transcribed using Enzo BioArray high
yield RNA transcript labeling kit (Affymetrix, Santa Clara,
CA). After purifying the product, 20 itg of cRNA (0.5 itg/
il) was fragmented. The fragment then mixed with 300 pl
of hybridization mixture and 200 il of the mixture was
hybridized to human U133A Affymetrix GeneChip which
consists of 22,277 oligonucleotide probe sets representing
18,400 transcripts and variants, including 14,500 known
genes. The chips were processed after meeting recom-
mended criteria for use of the expression arrays as previ-
ously described [7] and according to manufactures
protocol.

Microarray data analysis
The chips were scanned using Affymetrix Genepix 5000A
scanner and the resulting images were assessed using
Genepix software with a manual supervision to detect any
inaccuracies. The net hybridization values were deter-
mined and subjected to global normalization and trans-
formed expression values were subjected to Affymetrix
Analysis Suite V 5.0 to remove any probe sets that were
flagged as absent on all arrays using default settings. The
expression value of the remaining probe sets was then
subjected to unsupervised and supervised learning, dis-
crimination analysis, and cross validation as previously
described [7]. After variation filtering, the coefficient of
variation was calculated for each probe set across all chips,
ranked and the expression values of the selected genes
were then subjected to statistical analysis in "R" program-
ming as previously described [7]. The gene expression val-
ues having a statistical significance of p _< 0.01 (ANOVA,
Turkey test) were selected and subjected to 2-fold cutoff.
Hierarchical clustering and TreeView analysis was carried
out and the selected genes were subjected to functional
annotation and visualization using Database for Annota-
tion, Visualization, and Integrated Discovery [18] soft-
ware. The integrated GoCharts assigns genes to specific
ontology functional categories based on selected classifi-
cations.

Low-density microfluidic card and realtime PCR
Total RNA isolated from the same paired leiomyomas and
myometrium from African Americans and Caucasians
used for microarray analysis was also subjected to realtime
PCR using microfluidic cards TaqMan Low Density Arrays


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(LDAs) assessing the level of expression of 23 selected
genes, and the house-keeping gene, G3PDH. These genes
were selected based on their functional categories rather
than the level of their expression from the list of differen-
tially expressed genes identified in the present and our
previous [6,7,19] microarray studies. The products of
these 23 genes have been implicated in several biological
processes involved in pathogenesis of leiomyoma and
other fibrotic disorders. Pre-designed TaqMan probe and
primer sets for these genes were selected from the on-line
catalogue and were factory-loaded into customized 384-
well LDA plates (Applied Biosystem, Foster City, CA). The
LDA format was arranged on-line with each array contain-
ing two replicates for each 23-target gene and GAPDH.
Since these LDAs were used to determine the expression of
these genes in several different tissues for establishing the
least variable housekeeping gene as standard prior to
these experiments we utilized a factory-loaded LDA (384-
well plate) specific for 12 housekeeping genes. The results
indicated that GAPDH expression displayed the least var-
iation among the reference genes in these tissues as well as
the tissues used in the accompanied manuscript [20].
Based on these results we selected GAPDH as a house-
keeping gene during the development of the LDA and for
normalization of the expression of other target genes on
LDA. The exact locations and the sequences of the oligo-
nucleotides used in all assays can be downloaded from
the Applied Biosystems website [21] by selecting the
Assays IDs. All genes span exonintron boundaries and
cover the major transcript forms.

The LDAs were analyzed using the 7900HT system with a
TaqMan LDA Upgrade (Applied Biosystems). In brief, 5 jil
of single-stranded cDNA at final concentration of 2 jig
starting RNA was combined with 45 jil water and 50 Wil
TaqMan Universal PCR Master Mix. The mixture was
injected into selected sample ports and the cards were cen-
trifuged twice for 1 min at 1200 rpm and sealed to prevent
well-to-well contamination. Thermal cycling conditions
were as follows: 50C for 2 min, 95 C for 10 min, 97C
for 30s and 60C for 1 min for 40 cycles. In all the exper-
iments total RNA isolated from each sample were assayed
in duplicate and one sample was used in all cards to assess
inter- and intra-assay variability. Since each LDA card has
a unique barcode the Sequence Detection System plate
documents stores the information on the plate type,
detector, sample/target gene configurations, thermal
cycling conditions, data collection, and raw fluorescence
data at each cycle. The data obtained from each card was
analyzed using the comparative method and following
normalization of expression values to GAPDH expression
using relative quantity (RQ) and Sequence Detection Soft-
ware 2.2.1 for automated data analysis according to the
manufacturer's guidelines [21]. For calculating the RQ of
selected mRNA in leiomyomas compared with myo-


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metrium, or tissues from African Americans compared to
Caucasians, myometrial RNA used in each card was desig-
nated as calibrator. To analyze the RQ amounts of selected
genes as an effect of ethnicity, Caucasian myometrium
was used as calibrators. The Ct value >35 was selected as
cutoff for the absence of gene expression. The expression
of 15 of these genes is reported here.

Proteomic: protein isolation and CyDye labeling
Total protein was isolated from 100 to 300 mg of paired
frozen leiomyoma and myometrium from African Ameri-
cans and Caucasians. The tissues were grinded in liquid
nitrogen until fine powder, 5 ml of Trizol reagent and 5 to
15 pl of protease inhibitor cocktail (Sigma-Aldrich, St.
Louis, MO) was added and centrifuged at 10,000 x g for
20 min at 4 C. Following phase extraction with chloro-
form the mixture was centrifuged at 5000 RPM at 40C for
20 min, the inter- and bottom phases were extracted with
100% ethanol and clarified by centrifugation. The protein
was precipitated with isopropanol overnight at -20 C and
the pellets were washed twice with ice cold 80% ethanol,
air dried and dissolved in 300 jil of CyDye labeling buffer
(8 M urea, 2 M thiourea, 4% CHAPS, 20 mM Tris pH 8.5)
and centrifuged at 40,000 x g for 30 min. The superna-
tants were transferred to a clean tube and adjusted to pH
of 8.5 and protein concentration was determined using
EZQ (Invitrogen). Fifty jig of myometrial and leiomyo-
mas protein mixtures was labeled with 400 pM of Cy3 and
Cy5 respectively, for 30 min on ice in dark and the reac-
tion was stopped and quenched with 1 jil of 10 mM Lysin.

Two-dimensional electrophoresis
For the first dimension, the Cy3- and Cy5-labeled proteins
were combined with 450 jig each of unlabeled protein
and 100 mM of DTT ampholyte buffer pH 3 to 11, to
0.5% and adjusted the final volume to 400 il. An 18 cm
of pH 3 to 11 immobilized pH gradient (IPG) strip was
passively rehydrated with above sample solution over-
night in the dark. Isoelectrofocusing (IEF) was carried in
IPGphor (Amersham, GE Healthcare Bio-Sciences Corp.
Piscataway, NJ) and the proteins co-migrated together was
focused at 8000 V at 200C for 60 kV hr. For the second
dimension SDS-PAGE, an 8 to 16% precast Tris Glycine
gel (BioRad, CA) was used. Prior to mounting, the IPG
strips were reduced and equilibrated in 15 ml of 50 mM
Tris-HCl pH 6.8, 6 M Urea, 30% (v/v) glycerol 2% (w/v)
SDS 100 mM DTT for 15 min, then was alkylated with 15
ml of 50 mM Tris-HCl pH 6.8 6 M Urea 30% (v/v) glycerol
2% SDS 2.5% idoacetamide for 15 min. 2nd dimension
was carried in the dark at 20 C for 4 to 5 hrs at 24 mA/gel.
MW markers (New England Biolabs, Beverly, MA) were
added to the 2D-PAGE.






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Image acquisition and spot quantification
The gels were scanned with Typhoon 9400 Variable Mode
Imager (Amersham) and following image acquisition the
protein spots were identified, their volumes were deter-
mined and analyzed using computer-assisted gel analysis
and Progenesis PG220 2D software (Nonlinear Dynam-
ics, Durham, NC). The analysis allows automatic detec-
tion and quantification of protein spots as well as resizing,
alignment, and matching between different 2D gel
images. A database of all protein spots obtained from dig-
ital images was created, and integrated intensity of each
spot was calculated based on spot area and optical density
and expressed as percent volume, namely, spot volume/
volumes of all spots resolved in the gel. The comparative
program was used to geometrically correct spatial differ-
ences and allow comparison between the images. The
information from comparison of the image databases was
then used to establish an average gel. Each protein spot
volume was normalized against the total protein spots
volume present in the gel and the fold difference of each
protein was calculated for the paired myometrium and
leiomyoma sample. The list of protein spots with assigned
identification number was prepared and arranged accord-
ing to the fold difference and the values were statistically
analyzed using ANOVA, Turkey test.

Protein identification by mass spectrometric analysis
The gels were Coomassie blue stained and the spots of
interest were identified and manually excised with a dis-
posable gel puncher and stored in 25% methanol until
tyrptic digestion according to a protocol used by Proteom-
ics Core Facility of the Inter-disciplinary Cores for Bio-
medical Research (ICBR) at the University of Florida.
Peptides derived from tryptic digestion were analyzed by
reversed-phase HPLC-tandem mass spectrometry on a
hybrid quadrupole time-of-flight instrument (QSTAR XL,
Applied Biosystems) equipped with a nano-electrospray
source. Solvent delivery at 200 nl/min was provided by an
integrated capillary HPLC system (Ultimate, LC Packings,
Sunnyvale, CA) in which a 30 min gradient from 3% to
50% acetonitrile in 0.1% acetic acid was employed. Each
information dependent acquisition (IDA) cycle consisted
of a survey scan from m/z 400 to 1500 and three MS/MS
scans obtained by collision-induced dissociation of ions
that demonstrated the largest signal intensity at a given
chromatographic time point. Survey and MS/MS scans
were accumulated for 1 and 2 sec, respectively.

Tandem mass spectrometric data were searched against
the IPI human protein database using the Mascot search
algorithm. Carbamidomethylation of cysteine was
included as a fixed modification whereas oxidation of
methionine, deamidation of asparagine and glutamine,
and pyro-glu formation from N-terminal glutamine or
glutamic acid were included as variable modifications.


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Probability-based MOWSE scores that exceeded the value
corresponding to p < 0.05 were considered for protein
identification. After inputting the pi and MW of proteins
Swiss-Prot Database was searched by TagIdent software
(Swiss Institute of Bioinformatics, Basel, Switzerland) for
matching proteins.

Western blot analysis
Briefly, small portions of paired leiomyoma and myome-
trial tissues were homogenized in homogenizing buffer
containing protease inhibitor cocktail [22]. The laysets
were centrifuged, supernatants protein content was deter-
mined and an equal amount of proteins was subjected to
SDS-PAGE, transfer by electroblotting and the blots were
exposed to polyclonal antibodies generated against 14-3-
3P and mimecan (Santa Cruz Biotechnology, Santa Cruz,
CA), respectively and monoclonal antibody to B-actin
(Sigma) to normalize for protein loading. The blots were
exposed to corresponding HRP-conjugated IgG and visu-
alized using enhanced chemiluminesence reagents
(Amersham). The band intensity was determined using
Kodak gel analysis software (Eastman Kodak, Rochester,
New York).

Immunohistochemistry
For immunohistochemical localization of 14-3-33 and
mimecan, fixed and paraffin embedded leiomyomas and
myometrial tissue sections 3 to 5 jim thick were prepared.
Following standard procedures, the sections were incu-
bated with polyclonal antibodies at 5 jig of IgG/ml pre-
pared in phosphate buffered saline, pH 7.4 [22]. The
sections were than exposed to biotinylated second anti-
bodies and avidin horseradish peroxidase (Vector Labora-
tories, Burlingame, CA) and chromogenic reaction was
developed using 3, 3'diaminobenzidine and counter sec-
tions were counter staining with hemotoxalin as indi-
cated. Tissue sections incubated with normal IgG instead
of the primary antibodies, or deletion of the primary anti-
bodies during immunostaining served as controls.

Results
Gene expression profiling of leiomyomas from African
Americans and Caucasians
Gene expression values obtained from microarray analy-
sis of paired leiomyoma and myometrium from African
Americans and Caucasians were subjected to unsuper-
vised and supervised learning. Based on statistical analysis
with p ranking of P or 8% of the genes on the array, as differentially expressed
among these tissues regardless of ethnicity. Further analy-
sis based on 2-fold cutoff resulted in identification of 268
genes with 177 genes over-expressed and 91 genes under-
expressed in leiomyomas of African Americans as com-
pared to Caucasians (Table 1; for the complete list of
genes see Table 4, Additional file 1). Hierarchical cluster-


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Table I: Differentially expressed genes in leiomyomas of African Americans as compared to Caucasians

Gene Bank Gene Symbol Probability Fold Change Biological Function


P43115 PTGER3 0.007 3.05 signal transduction
NM_006549 CAMKK2 0.0004 2.66 signal transduction
Q92844 TANK 0.00002 2.57 signal transduction
NM_000267 NFI 0.0004 2.57 signal transduction
L35253 MAPK 14 0.0011 2.51 signal transduction
Q 16825 PTPN21 0.004 2.51 signal transduction
P04156 PRNP 0.00003 2.33 signal transduction
P27986 PIK3RI 0.004 2.19 signal transduction
NM_002835 PTPN12 0.0001 2.18 signal transduction
P04901 GNBI 0.0002 2.02 signal transduction
NM_005456 MAPK81PI 0.006 0.50 signal transduction
P17612 PRKACA 0.003 0.50 signal transduction
NM_003331 TYK2 0.001 0.50 signal transduction
P17010 ZFX 0.0004 3.73 transcription factor
000712 NFIB 0.00002 2.56 transcription factor
NM_002040 GABPA 0.004 2.38 transcription
U 18671 STAT2 0.001 2.20 transcription
AF040963 MXD4 0.0002 0.50 transcription
Q9Y6K9 IKBKG 0.004 0.50 transcription
L41066 NFATc4 0.001 0.48 transcription
P26196 DDX6 0.008 0.48 RNA processing
095997 PTTG I 0.009 0.37 oncogenes/tumor suppressors
NM_012141 DDX26 0.0009 0.31 tumor suppressor
P10159 EIF5A 0.0004 2.63 translation
NM 001969 EIF5 0.0001 2.15 translation
NM_000141 FGFR2 0.002 12.66 growth factor receptor
NM_005761 PLXNC I 0.002 3.48 cell receptor
P36897 TGFBRI 0.006 4.81 growth factor receptor
D50683 TGFBR2 0.006 3.54 growth factor receptor
060462 NRP2 0.002 2.43 cell receptors
094816 FZD7 0.004 2.04 cell receptors
M26062 IL2RB 0.002 0.46 cell receptors
NM_004883 NRG2 0.007 0.46 cell signaling
M59040 CD44 0.002 2.70 cell surface antigens
P07585 DCN 0.007 2.00 cell surface antigens
P30408 TM4SF I 0.004 2.00 cell surface antigens
M_002456 MUCI 0.008 0.48 cell surface antigens
Q9NVA2 FLJ 10849 0.002 3.55 cell cycle
P54826 GAS I 0.001 2.12 cell cycle
Q14004 CDC2L5 0.0001 2.05 cell cycle
NM_003644 GAS7 0.005 0.50 cell cycle
P29466 CASPI 0.008 2.28 apoptosis
NM_016315 GULPI 0.0007 2.04 apoptosis
NM_006595 AP15 0.0004 2.02 apoptosis
Q01082 S PTBNI 0.00005 4.52 cytoskeleton/motility
NM_005909 MAPIB 0.002 3.01 cytoskeleton/motility
P35749 MYH I1 0.0005 2.88 cytoskeleton/motility
Q13642 FHLI 0.002 2.59 cytoskeleton/motility
Q9NYL9 TMOD3 0.0002 2.39 cytoskeleton/motility
P35222 CTNNBI 0.005 2.04 cell adhesion receptors
NM_006158 NEFL 0.0006 0.10 cytoskeleton/motility
NM_004995 MMP14 0.003 3.96 protein turnover
NM_013381 TRHDE 0.008 2.33 protein turnover
LTBPI 0.003 0.50 protein kinase activity
NM 000849 GSTM3 0.0002 7.99 metabolism
NM 000850 GSTM4 0.005 2.36 metabolism
P06733 ENO I 0.005 2.07 metabolism
P08294 SOD3 0.0005 0.44 metabolism
NM_004177 STX3A 0.0007 0.53 trafficking/targeting proteins
Q9NZ08 ARTS-1 0.008 2.20 stress response proteins
AF I 12465 OGN 0.003 3.05 membrane channels
NM 000725 CACNB3 0.001 0.54 membrane channels
L22548 COL18AI 0.002 0.51 membrane channels
PTGIS 0.009 2.58 lipid biosynthesis


Partial list of genes from several functional categories differentially expressed in leiomyomas of African Americans as compared to Caucasians as illustrated in figure I and
selected based on p < 0.001 and 2-fold cutoff change (F. Change) as described in materials and methods. The complete list is provided as in Additional file I.





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B















D






E


Figure I
Hierarchical cluster analysis of differentially expressed genes in matched leiomyoma and myometrium from African Americans
(AALI, AAL2, AAL3, AAM I, AAM2, AAM3) and Caucasians (CLI, CL2, CL3, CM I, CM2, CM3) selected at P < 0.01 followed
by 2-fold cutoff change. Each column represents data from a single cohort with shades of red and green indicating up- or down-
regulated genes according to the color scheme shown below. Genes represented by rows were clustered according to their
similarities in pattern of expression in each tissue. The dendrogram at the top of the image displays similarity in gene expres-
sion among the cohorts, and relatedness of the arrays is denoted by distance to the node linking the arrays. The clustering
divided the genes into five clusters designated as A to E.




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Reproductive Biology and Endocrinology 2007, 5:34



ing separated these genes into several distinctive clusters
(Fig. 1) and functional pathway analysis indicated that
majority of their products serve as transcriptional, transla-
tional and signal transduction mediators, cell cycle regu-
lation, ECM turnover, cell-cell communication and
metabolic activities etc.

Gene expression using TaqMan LDA
We used customized 384-well LDA cards to determine the
expression of 15 genes in leiomyomas and myometrium,
with each card containing two replicates of the target
genes and GAPDH as a housekeeping gene (Fig. 2). Since
these cards were also used to assess the expression of these
genes in tissues reported in the accompanied manuscript
[20] we first established the least variable housekeeping
gene to use as a standard among these tissues. Using fac-
tory-loaded LDA cards representing 12 housekeeping
genes GAPDH expression displayed the least variation
among these tissues (data not shown). Based on these
results we selected GAPDH as a housekeeping gene and
along with 23 target genes for preparation of customized
LDA for our study. The selection of the 23 genes was based
on the result of the present and our previous microarray
studies [6,7,19]. Figure 2 show the level of expression of
E2F1, RUNX3, EGR3, TBPIP, ECM2, ESM1, THBS1, GAS1,
ADAM17, CST6, CST7, FBLN5, ICAM2, EDN1 and
COL18, representing transcription factors, cell cycle and
apoptosis regulators, matrix remodeling and cell adhesion
signaling, respectively in matched leiomyoma and myo-
metrium from African Americans and Caucasians. The
level of expression of these genes varied significantly in
leiomyoma and myometrium as well as tissues from Afri-
can Americans as compared to Caucasians (Fig. 2; P <
0.05), some displaying a similar pattern of expression
observed with microarray.

Proteomic analysis of leiomyomas and myometrium
A representative of total protein isolated from a paired
myometrium and leiomyoma, labeled with Cy3 or Cy5
respectively, and co-separated in 2D PAGE is shown in
Figure 3A. Using image analysis 332 protein spots were


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identified and their density/volume determined with two
spots (#107 and #192) later extracted for identification
shown in graphic presentation (Fig. 3B). The average den-
sity of each protein spot in leiomyomas and myometrium
was normalized against their density in one of the myo-
metrium from a Caucasian patient. Of the 332 protein
spots identified 28 and 31 spots were unique to leiomyo-
mas and myometrium, respectively with the density/vol-
ume of 31 spots varied by at least 1.5 fold (12 were
overexpressed and 19 underexpressed) in leiomyomas as
compared to myometrium regardless of ethnicity. 109
protein spots identified in leiomyomas the average den-
sity/volume of 34 spots differed by at least 1.5 fold, of
which 26 spots were overexpressed and 8 were underex-
pressed in African American as compared to Caucasians
(Table 2). Mass spectrometric analysis of 15 selected spots
resulted in identification of 137 proteins. A database
search against Swiss-Prot resulted in identification of sev-
eral proteins and the results are summarized in Table 3,
with MS and MS/MS scores showing the reliability of
identification. Among the content of these protein spots
included several keratins, Annexin Al and V, transgelin
(SM22), galgizzarin (S100A11) and EF-hand domin-con-
taining protein 2 (S100A12), vimentin, retinoic acid bind-
ing protein II, cofilin, several isoforms of 14-3-3 and
mimecan. Transcripts corresponding to these proteins
were also identified among differentially expressed genes
in leiomyomas and myometrium of both ethnic groups
(Table 3).

Western blot analysis and immunohistochemical
localization
Western blot analysis indicated that leiomyoma and myo-
metrium from both ethnic groups express 14-3-3p with a
considerable variation among these tissues (Figs. 4). We
were unable to detect mimecan immunoreactive protein
in these tissue extracts by western blotting possibly
because of the nature of the antibody for this application.

Immunohistochemically, 14-3-3p (Fig. 5A and 5B) and
mimecan (Fig. 5C and 5D) were localized in leiomyoma


Table 2: Profile of protein spots detected in leiomyomas from Caucasians and African Americans


Increased Intensity

31 (1.7-8.90 fold)
42 (1.5-13.2 fold)
129 (1.5-14.8 fold)
180 (1.5-41.6 fold)
81 (1.5-35.3 fold)
80 (1.5-14.1 fold)


Decreased Intensity

289 (1.5-48.80 fold)
171 (1.5-46.00 fold)
135 (1.5-128.7 fold)
62 (1.5-37.70 fold)
100 (1.5-86.80 fold)
65 (1.5-38.20 fold)


The number of protein spots detected in leiomyomas from Caucasians (315C, 3 16C and 3 17C) and African Americans (334AA, 353AA and 357AA)
as compared to protein spots detected in myometrium of 3 15C used for normalization. Total number of spots detected in myometrium #3 15 was
332. The column shows the number of spots whose intensity increased, decreased, not changed ( 1.5 fold), or not detected as compared to
myometrium #315C. The range of difference in protein spot intensity is shown as fold change as compared to their intensity in myometrium 315C.


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Tissue


Fibroid-315C
Fibroid-316C
Fibroid-317C
Fibroid-344AA
Fibroid-353AA
Fibroid-357AA


Not Changed


Not-Detected








Reproductive Biology and Endocrinology 2007, 5:34


2.5
S* *CM OCL
.1 AAM AAL
2 -


w 1.5












*CM
z






S 1 -- -- --- -------
2 *
E2F1 RUNX3 EGR3 TBPIP

2.5
MCM CM
a-





C- CL
S-m AAMM
SDAAAAL
x 1.5







0
ECM2 ESM1 THBS1 GAS1



CL
AAM I *'*
3 0 AAL

X*









ADAM17 CST6 CST7 FBLN5 ICAM2 EDN1 COL18



Figure 2
Bar graphs show the relative level of expression of 15 of the differentially expressed genes in paired myometrium (M) and leio-
myomas (L) from Caucasians (C) and African Americans (AA) determined by realtime PCR low density array. Values on the y-
axis represent an arbitrary unit derived from the mean expression of each gene independently with the mean value of myo-
metrium from Caucasian set at I for each gene. The asterisks are statistically different from ** comparing paired myometrium
and leiomyomas from African Americans and Caucasians with arrows pointing out the difference between the expression of
these genes in leiomyoma and myometrium in each group. A probability level of P < 0.05 was considered significant.





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Reproductive Biology and Endocrinology 2007, 5:34



and myometrial smooth muscle cells, connective tissue
fibroblasts and vascular cells, with a considerable variabil-
ity in their intensity among these cells within the same tis-
sue (Figs. 5). Incubation of the tissue sections with normal
rabbit and/or goat serum instead of the primary antibod-
ies resulted in considerable reduction in staining intensity
(Fig. 5E and 5F; the insert images show a higher magnifi-
cation portion of these figures indicated by the arrows).

Discussion
Using genomic and proteomic strategies the present study
provided further insight into the molecular environments
of leiomyoma and myometrium in Caucasians and Afri-
can Americans. At genomic level 1470 genes or about
10% of the transcripts on the array were identified as dif-
ferentially expressed in leiomyomas as compared to myo-
metrium regardless of ethnicity. Of these genes 268 were
identified as either over-expressed (177 genes) or under-
expressed (91 genes) in leiomyomas of African Americans
as compared to Caucasians. Hierarchical cluster analysis
separated these genes into several clusters reflecting their
tissue as well as ethnic-dependent and -independent asso-
ciation. However, the profile of some of the genes in these
clusters displayed an area of relatedness between myo-
metrium and leiomyomas within each ethnic group. This




_+ I


http://www.rbej.com/content/5/1/34


suggests that some of the differences in leiomyoma's gene
expression might be attributed to differences in myome-
trial gene expression between the ethnic groups, as well as
the differences in leiomyomas vs. myometrium. Func-
tional analysis indicated that the majority of these genes
serve as transcriptional, translational and signal transduc-
tion mediators, cell cycle regulation, ECM turnover, cell-
cell communication and transport/metabolic activities.

At proteomic level we identified several proteins display-
ing both ethnic-dependent and -independent profiles,
with 34, or 10% of total protein spots identified, display-
ing altered intensity in leiomyomas of African Americans
as compared to Caucasians. Tandem mass spectrometry
analysis of 15 selected protein spots revealed an array of
proteins as part of their content (Table 2) of which the
transcripts of some of them, or their related proteins, were
among the genes identified in myometrium and leiomyo-
mas. Although the genomic and proteomic results of our
study in part confirmed the previous reports involving lei-
omyomas and myometrium [8-10,12,13,19,23,24], previ-
ous microarray analysis did not consider the influence of
ethnicity as part of their comparative assessment. We rec-
ognize that low sample size is a limitation of our study,
however validating the expression of a large number of


Spot # 192 (green outline) representing leiomyomas #317 (Caucasian)
and #352 (A. American) with a 3.5 fold average increase in expression.


Spot# 192


9-- --- .



Spot # 107 (green outline) representing leiomyomas #317 (Caucasian) and
#352 (A. American) with a 1.5 fold average increase in expression.


Figure 3
Two-dimensional gel electrophoresis of total protein isolated from leiomyoma and myometrium. Protein spots with differenti-
ate expression are encircled and identified by a number. The spots were identified using MALDI-TOF and peptide matching.
Figures B to E show the three-dimensional images with differential expression of two protein spots (#107 and 192) in leiomyo-
mas of African American (C and E) and Caucasians (B and D)



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2
1 ,









Reproductive Biology and Endocrinology 2007, 5:34


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Table 3: List of protein spots and their contents identified by MS/MS in leiomyomas and myometrium


Protein Spots Description


Accession Theoretical
Number MW


Theoretical pl Score Match Transcripts *


76 Annexin A I
Aflatoxin BI Aldehyde
reductase 2
Splice Isoform I
Heterogeneous nuclear ribonucleoprotein H3
PDZ and LIM domain protein I
PTD012 protein
LIM and SH3 domain protein I
93 CGI-150 protein
Inorganic pyrophosphatase
107 Annexin V
Mimecan
Microtubule-associated protein RP/EBI
EF-hand domain-containing protein 2
Tubulin-specific chaperone B
126 14-3-38
14-3-3T
14-3-3y
14-3-3plu/
Annexin V
Proteasome u5
Vimentin
Acidic leucine-rich nuclear phosphoprotein 32A
183 a crystallin B chain
187 Transgelin (SM22)
192 Myosin regulatory light chain 2, smooth muscle
isoforms
Myosin regulatory light chain 2, nonsarcomeric
204 Splice Isoform CFL2b of
Cofilin, muscle isoforms
Cofilin, non-muscle isoforms
Transgelin (SM22)
206 Transgelin (SM22)
Destrin alpha
209 Actin-related protein 2/3
complex subunit 5
243 Retinoic acid-binding protein II, cellular
14 kDa phosphohistidine phosphatase
303 Calgizzarin (S0OA I I)
Cytochrome c oxidase polypeptide Vib
324 Ubiquitin


P04083
043488

P31942

000151
Q6F188
Q14847
Q9Y3E8
Q9H2U2
P08758
P20774
Q15691
Q96C19
Q99426
Q04917
P27348
P61981
P31946
P08758
P28066
P08670
P39687
P0251I
Q5UOD2
P24844

P19105
Q9Y281

P23528
Q01995
Q01995

015511

P29373
Q9NRX4
P31949

P62988


6.64 361 10 +
6.70 359 II +

6.37 327 9 +


4.67 224 9 +
7.66 263 10 +
+
8.26 172 6 +
8.88 96 3 +
8.88 329 14 +
309 17 +
5.47 245 14 +


5.43 208
5.65 105
6.56 164
64
6.56 203


The identified proteins and their corresponding MASCOT scores obtained by 2D-PAGE separation combined with MALDI-TOFMS PMF and nano-HPLC-ESI-MS/MS peptide
sequencing. Spot numbers corresponds to data in Fig. 3 with changes in their expression reported in Table 2. The corresponding transcripts (*) of the proteins identified by
microarray in leiomyomas and myometrium at p < 0.05 and 2-fold cutoff are shown as + in the right hand side column. The Accession numbers, theoretical MW and pl are
according to and calculated from Swiss-Prot


transcripts simultaneously in the same samples by quan-
titative realtime PCR (low density array) and identifica-
tion and confirmation of two proteins, mimecan
(osteoglycin) and 14-3-3p in paired leiomyoma and myo-
metrium compensated for this limitation.


Of the genes functionally relevant to processes that con-
tribute to characteristic and pathogenesis of leiomyomas
was the identification of several components ofTGF-P sys-
tem and their elevated expression in leiomyomas of Afri-
can Americans as compared to Caucasians. Based on this
and our previous studies we consider over-expression of


TGF-P and TGF-P receptors in leiomyomas of African
Americans result in further amplification of their signal-
ing, targeted transcription factors and downstream genes
expression, which are already altered in leiomyomas as
compared to myometrium [7,19,22,25-28]. In contrast,
leiomyomas of African Americans expressed lower levels
of LTBP-1, a member of fibrillin super-family that associ-
ates and stores TGF-P into the ECM. LTBPs consist of
LTBP-1 to LTBP-4 [29] with LTBP-1 and -2 expression
reported in leiomyomas [30,31]. Commencing our study
a recent report provided evidence for the expression of
LTBP-1 and FBN-1 in leiomyomas of three different sizes



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Reproductive Biology and Endocrinology 2007, 5:34


Af. Americans


I.T ]/


14-3-3 13

B-actin


Leiomyoma


Caucasians Af. Americans


Figure 4
Western blot analysis of 14-3-3p (30 Kd) and P-actin (control) in paired leiomyoma (L) and myometrium (M) from African
Americans (Af. American) and Caucasians. The bar graph shows the relative expression (band density) of 14-3-33 in leiomyo-
mas and myometrium in ethnic-dependent and independent manners. The asterisks ** are statistically different from (P <
0.05).





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Caucasians


1T ]/% 1. ]/% 1T ]/% 1. ]/% 1T ]/%


10000


8000


6000


4000


2000


0


Myometrium


10000


8000


6000


4000


2000


0


http://www.rbej.com/content/5/1/34


----------- T -----------------







Reproductive Biology and Endocrinology 2007, 5:34


Figure 5
Immunohistochemical localization of 14-3-3p (A and B) and mimecan (C and D) in myometrium (A and C) and leiomyomas (B
and D) associated with leiomyoma and myometrial smooth muscle cells and cellular components of connective tissue and vas-
culature with inserts showing a higher magnification portion of these tissues. Incubation of tissue sections with non-immune
rabbit (E) and goat (F) IgGs instead of primary antibodies during immunostaining, served as controls, reduced the staining inten-
sity. Mag: XI50; inserts = X265


with higher levels of expression in the medium-sized (3-
5 cm) tumors compared with myometrium in the prolif-
erative phase, while FBN-1 mRNA expression was size-
independent [31]. Although the results suggested that
ECM turnover in leiomyomas might be size-dependent,
the larger tumors undergo significant alteration in their
tissue structure, specifically at the center that must be
taken into account during such comparative analysis. Our
results that LTBP-1 is expressed at lower levels in leiomy-
omas of African Americans as compared to Caucasians
suggest that TGF-P upon secretion and activation becomes
readily available for binding rather than being stored into
the ECM, a mechanism that regulates local availability of
many growth factors and cytokines, including TGF-P.

Plasmin, integrins, thrombospondin-1 (TSP-1), mannose
6-phosphate, and decorin are known to activate TGF-P
[32-34] and their differential expression suggests the pres-
ence of regulatory mechanism that control TGF-P action


in leiomyoma where it regulates the expression of several
genes involved in tissue fibrosis [19,25-28,35]. Addition-
ally, we identified TCF8/ZB-1, a member of zinc finger
transcription factors that include Smad interacting protein
1 (SIP1/ZEB-2), as differentially expressed gene in leiomy-
omas of African Americans. TCF8 through interaction
with Smad and recruitment co-activator (p300) and co-
repressor (CtBP), is involved in ER and TGF-P receptors
signaling [35-37]. Leiomyomas express these and several
other transcription factors that interact with TCF8 includ-
ing Runx, CITED, EGR1, EGR3, E2F1, Nurr77, c-Myc,
Max, and Mad, whose expression is validated in this and
our previous study [7,19]. These transcription factors reg-
ulate the expression of group of genes involved in cell
cycle regulation, apoptosis, angiogenesis and inflamma-
tion mediated by the action of TGF-P receptor and ER acti-
vation [38-43]. Because leiomyomas grow more rapidly in
African Americans as compared to other ethnic groups,
altered expression of these genes regulated by TGF-P and


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Reproductive Biology and Endocrinology 2007, 5:34



ovarian steroids could influence the outcome of this proc-
ess.

A balance between cell growth and apoptosis is critical in
progression of tissue fibrosis and the expression of several
genes functionally related to these categories, including
Bcl-XL, Bad, Bax, p27Kipl, p57Kip2, Gasi, Gas7, CST6,
CST7, caspases, etc., were identified in leiomyomas and
myometrium. Differential expression of some of these
genes in leiomyomas of African Americans most likely
contributes toward their rapid growth as compared to
other ethnic group. Local apoptotic and non-apoptotic
cell-death also results in regulation of multiple genes
involved in inflammation, angiogenesis, fibrogenesis and
tissue turn over. Among these genes involved in these
processes are collagens, versican, fibromodulin, syndecan
4, TSP-1, tenascin-C, osteonectin/SPARC and ECM2,
endothelial cell specific molecule-1 (ESM-1), ICAM2,
EDN1, FZD7, 31 catenin, CST6, CST7 as well as several
member of integrins, MMPs, TIMPs and ADAMs [44-49]
that are expressed in leiomyomas [7,26,27] with altered
expression in African Americans. The identification of 14-
3-33 and mimecan as differentially expressed genes in lei-
omyomas of African Americans is also of interest. 14-3-3s
are regulatory proteins that bind to a variety of cellular tar-
gets, including Raf kinase, cell cycle-dependent phos-
phatase Cdc25, pro-apoptotic protein Bad and many
others proteins and are considered to regulate hyper-
trophic scar formation through regulation of MMPs
expression [50-52]. The ability of 14-3-3 proteins to bind
and regulate various oncogenic gene products as well as
various tumor suppressor gene products points to a
potential role in cancer As such 14-3-3 has been shown to
inhibit TSC1/TSC2 complex functions and overexpression
of either TSC1 or TSC2 in Hela cells has been reported to
increase the expression of various 14-3-3 isoforms [53]
suggesting that deregulated expression of 14-3-3 and TSC
can be associated with leiomyoma pathogenesis. Mime-
can is a member of small leucine-rich proteoglycans fam-
ily, which includes lumican, fibromodulin, decorin and
biglycan, and has been implicated in collagen fibrillogen-
esis, cellular growth, and migration [54-56]. Mimecan is
secreted and proteolytically cleaved by a serine protease to
form the 25-30 kDa form found in association with ECM
of connective tissue and considered to play a structural
role by maintaining the tensile strength and hydrated
nature of the tissue [55,56]. Mimecan is expressed in
mouse pituitary gland and in human anterior pituitary
gland and Pit-1 is reported to activate the human mime-
can promoter through Pit-1 response element sites [57].
As such mime can and 14-3-3 as well as other genes prod-
ucts in these categories may have a similar biological
activity in leiomyoma pathogenesis, specifically in African
Americans with more symptomatic tumors.


http://www.rbej.com/content/5/1/34


Because the ovarian steroids are critical to fibroid growth
it is essential to assess their relationship with genomic and
proteomic profiling presented here since the expression of
some of these genes are the target of ovarian steroids reg-
ulatory functions. Since the tissues used in our study come
from the early-mid secretary phase of the menstrual cycle
it is possible to assume that both estrogen and progester-
one-dependent and independent mechanism influence
the expression of these genes. Additionally, we only iden-
tified the content of 15 of the protein spots isolated from
the proteomic protein profiling as compared with a large
number of genes identified by microarry. This limited our
ability to assess any overlapping expression between
genomic and proteomic results which determine whether
different genes or proteins are regulated at different
molecular level since some genes are regulated at tran-
scription level, while others at protein level.

In conclusion, subjecting paired leiomyoma and myo-
metrium from African Americans and Caucasians to
genomic and proteomic analysis we identified a consider-
able similarity between their molecular environments
with differences seen at the level rather than ethnic-spe-
cific expression of a number of genes. The area of related-
ness among some of the gene clusters between
myometrial and leiomyomas within each ethnic group
suggests that some of the differences in leiomyoma's gene
expression might be attributed to differences in myome-
trial gene expression between the ethnic groups, in addi-
tion to differences in leiomyomas vs. myometrium.
Because many of the differentially expressed genes identi-
fied in these cohorts are know to regulate inflammatory
response, angiogenesis, cell cycle, apoptosis and ECM
matrix turnover, their products may account for rapid
growth and associated symptoms in African Americans as
compared to other ethnic groups.

Competing interests
The authors) declare that they have no competing inter-
ests.

Authors' contributions
QP, XL and NC participated in all aspect of the experi-
ments presented here with the final microarray gene chips
and two-d gels were performed at Interdisciplinary Center
for Biotechnology Research at the University of Florida.
Gene expression analysis performed by XL, proteomic
analysis and immunohistochemistry by NC, realtime PCR
and Western blot by XL and QP. All the authors read and
approved the final manuscript.








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Reproductive Biology and Endocrinology 2007, 5:34


Additional material


Acknowledgements
We thank Drs. Mick Popp and Stanley Steven at Interdisciplinary Center for
Biotechnology Research at the University of Florida for their assistance
with microarray and proteomic analysis. The work presented here is sup-
ported by a grant HD37432 from the National Institute of Health. The
work was presented in part at 53rd Annual Meeting of the Society for
Gynecological Investigation, Reno NA, March 2007.

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Table 4 I ii-.. al ,, expressed genes in leiomyoma of African Ameri-
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