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Endometriosis-Specific Genes Identified by Real-Time Reverse Transcription-Polymerase Chain Reaction Expression Profiling of Endometriosis Versus Autologous Uterine Endometrium

Departments of Obstetrics and Gynecology (W.-P.H., S.K.T) and Clinical Research (Y.Z.), Singapore General Hospital, Republic of Singapore 169608

Address all correspondence and requests for reprints to: A/Professor Sun Kuie Tay, Department of Obstetrics and Gynecology, Singapore General Hospital, Outram Road, Singapore 169608. E-mail: gogtsk{at}sgh.com.sg.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Context: The etiology and molecular pathogenesis of endometriosis, a prevalent estrogen-dependent gynecologic disease, are poorly understood.

Objective: The objective of the study was to identify the differentially expressed genes between autologous ectopic and eutopic endometrium.

Design: Subtractive hybridization was used for a genome-wide search for differentially expressed genes between autologous ectopic and eutopic endometrium. Real-time RT-PCR was used for gene expression profiling in the paired tissue samples taken from multiple subjects.

Patients: The paired pelvic endometriosis and uterine endometrium tissue biopsies were procured from 15 patients undergoing laparoscopy or hysterectomy for endometriosis.

Results: Seventy-eight candidate genes were identified from the subtractive cDNA libraries. Seventy-six of these genes were investigated in approximately 8000 real-time PCR for their differential expression in 30 paired tissue biopsies from 15 patients affected by endometriosis. Cluster analysis on gene expression revealed highly consistent profiles in two groups of genes, despite the clinical heterogeneity of the 15 cases. Thirty-four genes specific to early disease point to their potential roles in establishment and evolution of endometriosis. Most interestingly, 14 genes were consistently dysregulated in the paired samples from the majority of the patients. Of these, there were two uncharacterized transcripts and two novel genes, and 10 were matched to known genes: IGFBP5, PIM2, RPL41, PSAP, FBLN1, SIPL, DLX5, HSD11B2, SET, and RHOE.

Conclusions: Dysregulation of 14 genes was found to be overtly associated with endometriosis. Some of these genes, known to participate in estrogen activities and antiapoptosis, may play a role in the pathogenesis of endometriosis and may represent potential diagnostic markers or therapeutic targets for endometriosis.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
PELVIC ENDOMETRIOSIS IS an estrogen-dependent disease with a detection rate of 2–10% of women at reproductive age (1). It has a propensity to run a chronic and recurrent course after treatment, leading to debilitating chronic pelvic pain and infertility (1). The etiology and pathogenesis of endometriosis are controversial. A long-held belief postulates that endometrial cells from retrograde menstruation are the origin of the disease (2). The possibility of a genetic basis of endometriosis is demonstrated in studies of familial endometriosis, in which the incidence of endometriosis is higher (4.3–6.9%) for first-degree relatives of probands, compared with controls (0.6–2.0%) (3). Aberrant expression of growth factors, cytokines, adhesion molecules, matrix metalloproteinases, and enzymes for estrogen synthesis and metabolism have been implicated in the pathogenesis of endometriosis (4, 5, 6, 7, 8). The plethora of molecular and cellular derangements highly suggests that endometriosis is a polygenic disorder.

In the present study, the molecular mechanisms underlying the development and progression of endometriosis are investigated by a direct comparison of gene expression between autologous ectopic and eutopic endometrium. This investigational approach removes some variables attributable to heterogeneous genetic background between individual subjects and the effects of estrogenic stimulation during different phases of menstrual cycles. The results of this subtractive hybridization and real-time RT-PCR study lead to construction of a minidatabase on 76 differentially regulated genes with chromosomal mapping data and 14 genes that may be playing important roles in the pathogenesis of endometriosis.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
This study was approved by the institutional ethics committee for research involving human tissues, and the tissue samples were obtained after the patients’ informed consent.

Tissue specimens

Paired pelvic endometriosis and uterine endometrium tissue biopsies were procured from 15 patients undergoing laparoscopy or hysterectomy for endometriosis (Table 1). The patients either had never received any hormonal treatment or had ceased any hormonal medication for at least 6 months before surgery. The endometriosis tissues studied were sampled from lesions with fresh disease, avoiding burnt-out pigmented hemosiderin deposits. One portion of the tissue specimens was used for histopathological diagnosis, whereas the remaining was carefully dissected to select the endometriosis tissues and stored in liquid nitrogen immediately for molecular study. The severity of endometriosis was classified into stage I-IV according to the revised American Fertility Society classification system (9). The timing of menstrual phases (proliferative or secretory) at the time of tissue procurement was recorded.

Total RNA and protein were simultaneously prepared from 10–50 mg of fresh-frozen tissue samples using TRIzol Reagent (Invitrogen, Carlsbad, CA).

cDNA synthesis

mRNA was purified from total RNA using Dynabeads oligo (dT)₂₅ (Dynal Biotech, Brown Deer, WI). First-strand cDNA was synthesized with SuperScript II reverse transcriptase. Unprimed oligo (dT)₂₅ on the Dynabeads was removed by T₄ DNA polymerase. first-strand cDNA on Dynabeads was tailed with poly(dA)_n by terminal deoxynucleotidyl transferase. Second-strand cDNA was then synthesized with Taq DNA polymerase and HT₂₆V (AAGCTTTTTTTTTTTTTTTTTTTTTTTTTTV, V: A, G, or C) primers. The second-strand cDNA was then eluted from the Dynabeads into 30 µl of Tris/EDTA buffer containing 0.6 µg carrier tRNA. All the reactions involving Dynabeads were performed on a roller mixer.

Subtractive cDNA library construction

The protocol for subtractive hybridization based on the solid-phase cDNA technique described by Lönneborg and coworkers (10) was adopted in this study with two modifications (Fig. 1), including: second-strand cDNA was used as tester instead of mRNA; the subtractive process was monitored by PCR amplification of the ß-actin gene in tester after each round of subtraction. Briefly, subtractive hybridization was performed between first-strand cDNA Dynabeads of driver and second-strand cDNA of tester in 30 µl of hybridization solution containing 5x saline sodium citrate, 1 mM EDTA with carrier tRNA (0.01 µg/µl) in a 0.2 ml thin-well PCR tube at 65–68 C for 18–24 h under constant rolling. The tester solution was then collected and the subtractor Dynabeads were regenerated by incubation in 50 µl 0.15 N NaOH at room temperature for 5 min. One microliter of samples was taken from the tester and the eluted solution, respectively, for PCR monitoring of ß-actin level. The above hybridization procedures were repeated twice.

View larger version (32K): [in this window] [in a new window]   FIG. 1. Schematic protocol for subtractive cDNA library construction. Briefly, the tester (left) and driver (right) mRNAs were purified with Dynabeads oligo (dT)25, on which first-strand cDNA was synthesized by reverse transcriptase. To prepare second-strand cDNA tester, the unprimed oligo (dT)25 on the Dynabeads was removed by T4 DNA polymerase, and the first-strand cDNA on the Dynabeads was tailed with poly(dA)n by terminal deoxynucleotidyl transferase. Second-strand cDNA was synthesized with Taq DNA polymerase using HT26V primers and was eluted from Dynabeads. Subtractive hybridization (three rounds) was then performed between first-strand cDNA Dynabeads of driver and second-strand cDNA of tester to enrich the differential sequences. The remaining tester cDNA was isolated with Dynabeads oligo (dT)25 and globally amplified by PCR using HT26V universal primer. The PCR product was then cloned into a TA vector and transformed to generate a subtractive cDNA library.

Screening of subtractive cDNA libraries by PCR and differential hybridization

For dot blot hybridization, cDNA inserts were amplified by colony PCR using the vector-specific primers (T7: 5'-TAATACGACTCACTATAGGG-3'; M13 reverse: 5'-GGAAACAGCTATGACCATG-3'). The PCR product was mixed with equal volume of 0.6 N NaOH, and transferred to Hybond-N+ nylon membrane (Amersham, Little Chalfont, UK). The blotted membranes were neutralized with 0.5 M Tris-HCl (pH 7.5) and cross-linked using Stratalinker (Stratagene, La Jolla, CA). The cDNA probes prepared from the paired endometriosis and uterine endometrium samples, respectively, were labeled with digoxigenin-11-deoxyuridine 5-triphosphate. Colony and dot blot hybridization and chemiluminescent detection were performed according to the manufacturer’s protocol (Roche Diagnostics Asia Pacific, Singapore).

DNA sequencing

Sequencing of cDNA clones was performed with T7 or M13 reverse primers using ABI Prism BigDye terminator sequencing kit and ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA).

Northern analysis

Total RNA samples (4–8 µg) were electrophoresed on 1.2% formaldehyde denaturing agarose gel before overnight capillary transfer to nylon membranes positively charged. The cDNA clones were miniprepared and labeled with digoxigenin-11-deoxyuridine 5-triphosphate by PCR. Hybridization and chemiluminescent detection were performed according to the manufacturer’s protocol (Roche Diagnostics Asia Pacific).

Gene expression profiling by real-time RT-PCR

Four micrograms of total RNA samples were treated with RNase-free DNase I before reverse transcription with a SuperScript first-strand synthesis system (Invitrogen). Real-time PCR was performed with SYBR Green master mix on an ABI Prism 7700 system (Applied Biosystems), and 0.5% of each cDNA sample prepared from 4 µg of total input RNA was used for each 20 µl PCR with a 96-well reaction plate. Melting curves were generated to check the PCR specificity. The threshold cycle (C_T), which is defined as the cycle number at which the amount of amplified target reaches a fixed threshold, was obtained for each gene in each sample. ß-Actin was used as reference gene for normalization. The validity of ß-actin as a reference gene was confirmed experimentally by measuring the slope of the plot of log input RNA amount vs. C_T as suggested by user bulletin 2 (Applied Biosystems). All the target genes passed the validation tests with absolute values of the slopes less than 0.1.

The expression ratio (endometriosis vs. autologous endometrium) of the target gene in each pair of samples was determined by 2^-C_T, where

The C_T value of each gene in each sample was determined in triplicate, and the mean C_T values were used for analysis.

The primers for real-time PCR of target genes were designed based on the cDNA sequences. The sequence information for cDNA clone EA01-EA78 and 76 pairs of primers (for EA01-EA76) was submitted to the GenBank (accession no. BU197985-BU198062). The primer sequences for ß-actin gene include: ß-actin (forward): 5'-CCAGCACAATGAAGATCAAGATCA-3'; and ß-actin (reverse): 5'-GGGCCGGACTCGTCATACT-3'.

Chromosome mapping

The candidate genes were mapped to chromosomes by searching the human genome, LocusLink, and UniGene databases (http://www.ncbi.nlm.nih.gov).

Western blot analysis

Protein samples were separated by SDS-PAGE in 12% gel and transferred to a nitrocellulose membrane. Western blot analysis was performed with polyclonal antibodies against fibulin-1 (sc-8675, Santa Cruz Biotechnology, Santa Cruz, CA) and actin (sc-1616, Santa Cruz Biotechnology) using the SuperSignal West Femto substrate (Pierce, Rockford, IL).

Data analysis

Differential gene expression measured by the expression ratios given by 2^-C_T for each gene in each pair of samples were analyzed and displayed by unsupervised hierarchical clustering (average linkage) after log₂ transformation using the Cluster and TreeView (11). Paired t test was used to compare the C_T values of gene expression between the paired tissue samples. The mean value of C_T was used to calculate the mean fold change of gene expression in each group. A one-sample t test was used to test the ratios of fibulin-1 protein levels between the paired tissue samples after log transformation. All statistical tests were two sided. Analyses with P < 0.05 were considered to be statistically significant.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Efficacy of subtractive hybridization

A magnetic beads-assisted protocol for subtractive hybridization between single-strand cDNAs coupled with PCR was developed (Fig. 1). Efficacy of the protocol was evaluated with a differential cDNA model system by PCR monitoring the cDNA levels of chloramphenicol acetyltransferase (CAT) and/or ß-actin in the tester and the eluate samples after each round of subtraction. The ß-actin cDNA level decreased rapidly in three consecutive cycles of subtraction, whereas the CAT cDNA level remained fairly stable throughout the subtractive cycles in the tester (data not shown). This confirmed that the common sequences (ß-actin cDNA) but not the differential sequences (CAT cDNA) were effectively removed in this subtractive process.

Subtractive cDNA library construction and screening

A total of 738 colonies from two separate subtractive cDNA libraries were screened by colony PCR and/or differential hybridization. Ninety-four percent of the colonies were found to contain cDNA inserts, and 108 cDNA clones were finally identified (Fig. 2).

View larger version (21K): [in this window] [in a new window]   FIG. 2. Screening procedures and numbers of cDNA clones screened and identified. *, False-positive or equal-expressed cDNA clones; , equal-expressed or duplicate cDNA clones; , duplicate clones or the cDNA inserts containing only human repeat sequence inserts, such as Alu.

Sequencing analysis on the 108 cDNA clones through the GenBank databases yielded 78 clones containing different inserts. Of these 78 cDNAs, 49 were identical with or highly homologous to known genes, and 24 matched uncharacterized mRNA and hypothetical proteins. The remaining five clones matched human genomic sequences without homology to known transcript, i.e. novel genes. The detailed information on 78 cDNAs are given in Table 2.

Northern hybridization on the same pair of original RNA samples used for subtractive hybridization was performed with 33 cDNA clones. Detectable hybridization signals were identified for 18 genes, each with a differential expression between the endometrium and endometriosis. Of these, eight were overexpressed (Fig. 3A) and 10 were underexpressed (Fig. 3B) in the endometriosis, compared with the autologous uterine endometrium. Notably, there was a marked overexpression in EA26 (EGR1), EA40 (JUN), and EA62 (PIM2) genes and underexpression in EA27 (SET), EA35 (CTBP1), and EA61 (RPL13A) genes. No specific signals for the other 15 genes suggest that more sensitive technique is required.

View larger version (49K): [in this window] [in a new window]   FIG. 3. Northern blot and real-time RT-PCR analysis of the genes selected from the subtractive cDNA libraries, confirming a high agreement between Northern hybridization and real-time PCR technique. A, Overexpressed candidate genes in endometriosis vs. the paired uterine endometrium confirmed by Northern hybridization. Lane N, total RNA samples from normal uterine endometrium; lane E, total RNA samples from endometriosis; ß-actin, control for normalization. B, Underexpressed candidate genes confirmed by Northern hybridization. C, Linear regression analysis of gene expression data determined by real-time RT-PCR and Northern blot analysis using the same paired RNA samples, as shown in A and B.

Differential expression of 76 genes was studied in a larger group of patients (Table 1) with endometriosis at different stages (I-IV) of severity. Quantitative real-time RT-PCR technique was used for gene expression profiling after an evaluation study confirming a high agreement between real-time PCR and Northern hybridization (r² = 0.9794) (Fig. 3C). About 8000 real-time PCRs were performed to analyze the expression profiles of 76 of 78 candidate genes in 15 pairs of tissue samples (Table 1). The normalized gene expression levels (vs. ß-actin) in the paired samples were displayed on a scatter plot (Fig. 4). The gene expression ratios were within the range from 0.5 to 2.0 in 649 (57%) of data points and were outside this range in 491 (43%) points.

View larger version (26K): [in this window] [in a new window]   FIG. 4. Scatter plot of expression data generated by real-time PCR for 76 genes in 30 paired samples from endometriosis and autologous uterine endometrium. Scatter plot was produced for each pair of samples using normalized expression level of each gene (vs. ß-actin) in endometriosis, plotted against the normalized expression level of that gene in the paired uterine endometrium sample on the x-axis after log2 transformation. The normalized gene expression level of each gene in each sample was determined by 2–CT, where CT [gene(x)sample] = CT [gene(x)sample] – CT [(ß-actin)sample]. The outer diagonal lines represent a 2-fold increase or decrease in gene expression in endometriosis vs. autologous uterine endometrium.

Unsupervised clustering on the expression ratios of 76 genes in the paired samples segregated the 15 cases into two major clusters. The cluster on the right-hand side of Fig. 5A consisted of seven cases of advanced stage endometriosis, and the cluster on the left-hand side included six cases of early disease and two cases of advanced disease. The genes with similar expression patterns aggregated and the 76 candidate genes were generally divided into four distinct groups, including a1, a2, a3, and a4 (Fig. 5A). Group a1, including several immediate-early genes (12), showed a unique expression pattern (Fig. 5B). Group a2 included the genes overexpressed in endometriosis and group a4 underexpressed, whereas the genes in group a3 exhibited a transition from overexpression to underexpression across Fig. 5A as the severity of endometriosis progressed from early to late stage.

View larger version (46K): [in this window] [in a new window]   FIG. 5. Cluster analysis of differential expression of 76 candidate genes in 15 cases. A, Unsupervised hierarchical clustering of expression ratios (endometriosis vs. endometrium) of 76 candidate genes (rows) in 15 pairs of clinical samples (columns). Each square represents an expression ratio after log2 transformation. Red, green, and black colors represent over-, under-, and equal expression, respectively, in endometriosis vs. autologous uterine endometrium. B, Analysis of 15 cases by reclustering based on the expression data of three immediate-early genes EGR1, JUN, and JUND. C, A zoom-in picture for the 14 best candidate genes selected by using the combinatory criteria of mean fold-change of 2.0 and P 0.01 in 15 cases.

Clinical correlations

The majority of cases with early disease clustered on the left-hand side of Fig. 5A, whereas the cases with advanced disease aggregated on the right-hand side. It is noteworthy that moving across Fig. 5A from left to right, the expression patterns of the genes in group a3 exhibited a transition from overexpression to underexpression, indicating differential gene regulation at different stages of the disease. Using the cut-off threshold of 2-fold change in each group, 34 were specific to the early disease, four specific for the advanced disease, and the remaining seven genes were differentially expressed in both groups (Table 4).

The chromosome mapping data on the 14 best candidate genes (Table 3) reveal that seven of the 14 best candidates were mapped to the chromosome loci corresponding to several common chromosomal aberrations previously identified in the endometriotic cells by comparative genomic hybridization (CGH) (13, 14), fluorescence in situ hybridization (FISH) (15), and R-banding (16). Two of these genes mapped to chromosomal losses and/or monosomy were underexpressed, and other five mapped to chromosomal gain, trisomy, or tetrasomy were overexpressed in most cases (Table 3). Our findings of differential expression of these genes may be explained by the chromosomal aberrations found on other cytogenetic studies (13, 14, 15, 16).

Protein expression

To confirm the real-time RT-PCR findings on the gene transcriptional profiles, protein product of an overexpressed gene, fibulin-1, was further studied by Western blot analysis in the paired samples. In all 12 sample-pairs analyzed, fibulin-1 protein was found in high levels in all the endometriotic samples (Fig. 6A). Compared with autologous endometrium, fibulin-1 protein was markedly overexpressed in endometriosis by a median of 4.6-fold (P = 0.006) (Fig. 6B).

View larger version (54K): [in this window] [in a new window]   FIG. 6. Comparison of fibulin-1 protein expression between endometriosis and autologous endometrium. A, Western blot analysis of fibulin-1 expression in 12 pairs of tissue samples. Lane N, the protein samples from normal uterine endometrium; lane E, the protein samples from endometriosis. B, Dot plot of the ratios of fibulin-1 protein levels in endometriosis vs. autologous endometrium. The ratios of fibulin-1 were derived after normalized to actin levels. One-sample t test was performed to test the mean of the log-transformed ratios against zero with P = 0.006.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

The subtractive hybridization-based approach also has certain limitations. Not all the common sequences between the mRNA samples can be completely removed, leading to some false-positive cDNA clones in the subtractive cDNA library. We overcame this pitfall by screening the library for better candidate genes with differential hybridization and DNA sequencing. On the other hand, subtractive cloning and screening are based on a single case and pathological specimens from different individuals are variable; thus, the consistency of gene expression needs to be investigated on multiple cases by using real-time RT-PCR in the current study. In fact, some genes confirmed by Northern blotting on one pair of RNA samples (Fig. 3) were not on the final lists based on the data from 15 cases (Tables 3 and 4), including a down-regulated candidate EA19 (RPL41) that was later proven up-regulated in 13 of 15 cases studied. These data demonstrated the importance of careful verification of gene expression data with more samples.

We believe that our approach in this study carries specific advantages. Studies of gene expression in clinical samples are subject to effects of individual variations in genetic backgrounds from each patient. Furthermore, endometriosis, being an estrogen-dependent disorder, may show different gene expression patterns from the changes in the levels of estrogen at different menstrual phases. The effects of genetic diversity and variations in estrogenic stimulation of menstrual phases are minimized in this study in which a direct comparison of gene expression is made on autologous ectopic and eutopic endometrium. This should enhance the precision in identifying the underlying molecular changes in the pathogenesis of endometriosis. To prioritize the significance of these genes, 14 best candidate genes showing highly consistent expression profiles are identified. It seems highly likely that these are candidate genes critical to the development and/or progression of endometriosis and representing potential diagnostic markers or therapeutic targets. These genes may carry an important implication in the pathogenesis of endometriosis via estrogen-mediated pathway, antiapoptosis, and other mechanisms.

Of particular interest are those genes known to be regulated by estrogen or to influence the synthesis of estrogen because endometriosis is an estrogen-dependent disorder, and some molecular aberrations leading to local accumulation of estrogen at the site of endometriosis have been identified in endometriosis (8). Previous studies suggest that gene expression of IGF-binding protein (IGFBP)-5, prosaposin (PSAP), and fibulin-1 (FBLN1) may be influenced by estrogen, whereas 11ß-hydroxysteroid dehydrogenase type 2 (HSD11B2) by inactivating glucocorticoid may affect the estrogen synthesis pathway. The IGFBP-5 gene, encoding the IGFBP-5, is consistently overexpressed in endometriotic tissue by up to 58-fold. In human endometrium, IGFBP-5 mRNA is preferentially expressed in the proliferative phase, suggesting a role of IGFBP-5 in promoting endometrial cell proliferation (18). The stimulatory property of estrogen on IGFBP-5 expression (19) and the interaction of IGFBP-5 with TNF in protecting cells from apoptosis (20) suggest that overexpression of IGFBP-5 may play an important role in promoting ectopic growth and survival of endometrial cells, leading to development of endometriosis.

PSAP and FBLN1 encode the secretory proteins prosaposin and fibulin-1, respectively. Prosaposin, the glycoprotein precursor of saposins, is abundantly expressed in the reproductive system (21). Prosaposin interacts with procathepsin D in human breast and ovarian cancer cells, suggesting an involvement in tumor invasiveness and metastasis (22). FBLN1, a calcium-binding extracellular matrix protein (23), is aberrantly expressed in ovarian and breast cancer cells and has been identified as a breast cancer-restricted antigen (24). Expression of both PSAP and FBLN1 is stimulated by estrogen treatment (25, 26).

HSD11B2 encodes 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), which inactivates glucocorticoid by converting cortisol to cortisone. Glucocorticoid has been shown to stimulate expression of aromatase (27), which converts C19 steroids to estrogens. Aromatase activity is essential for the growth of ectopic uterine tissue (28). Down-regulation of 11ß-HSD2 expression in endometriotic tissue may maintain a high cortisol level at the endometriotic site and, by promoting aromatase activity, play a role in the pathogenesis of endometriosis.

Several of the candidate genes identified in the current study are related to apoptosis. PIM2 oncogene encodes a serine/threonine kinase Pim-2. Pim-2 functions as an apoptotic inhibitor and confers long-term resistance to a variety of apoptotic stimuli (29). Overexpression of PIM2 is found in various human cancer cells (30). Overexpression of RPL41 may stimulate the activity of protein kinase CK2 (CK2) (31), and CK2 has been shown to possess the antiapoptosis properties (32). On the other hand, the SET gene encodes a potent and specific inhibitor (I₂^PP2A) of protein phosphatase 2A (PP2A) (33). PP2A regulates Pim kinases at the posttranslational level, and inhibition of PP2A activity stabilizes the Pim proteins (34).

Other mechanisms involved in the pathogenesis of endometriosis may involve the changes in cell adhesiveness and maturation. Expression of RHOE in mammalian cells inhibits the formation of actin stress fibers and integrin-based focal adhesions and induces loss of cell-substrate adhesion (35). Down-regulated RhoE may contribute to the establishment of endometriosis by promoting cell adhesion and attachment to peritoneum. DLX5 expression has been shown to correlate with osteoblastic differentiation (36), and misexpression of DLX5 markedly reduces chondrocyte proliferation and promotes maturation (37). We speculate that highly down-regulated DLX5 may contribute to the persistent proliferative state of the endometrial cells at ectopic locations by failure of entering end-stage differentiation.

Another interesting observation is that, of the 10 down-regulated transcripts shown by Northern blot analysis in Fig. 3B, six encode ribosomal proteins. This may suggest that protein synthesis was slower in endometriotic lesions than paired uterine endometrial tissues. We cannot exclude the possibility that the limited sensitivity of Northern hybridization, which tends to detect transcripts in high abundance such as ribosome proteins, failed to detect other genes at low abundance.

Some early studies on microarray analysis of gene expression profiles associated with endometriosis have identified a number of potential candidate genes (38, 39), but it appears that none of the 14 best candidates is common to the findings in these early studies (38, 39). This may be partly attributed to the different technical approaches and variable pathological specimens from different individuals. Because a majority of these candidate genes are not previously associated with endometriosis and some of these are eventually uncharacterized or novel genes, the data suggest the presence of yet undefined molecular pathways involved in the pathogenesis of endometriosis.

In summary, this study has generated a minidatabase on differential expression profiles of 76 genes in 30 paired tissue samples from patients affected by endometriosis. Of these 76 genes, 47 match known genes, and the other 29 are either uncharacterized or novel genes. Forty-five genes are found to differentially express in the paired samples in either the early disease or the advanced disease or in both groups of patients. Most significantly, 14 best candidate genes showing consistent expression profiles have been identified. The interactions and exact roles of these molecular events in the development, persistence, and progression of endometriosis as well as their potential uses for diagnostic purpose are being further investigated.

	Acknowledgments

 
We thank Dr. Valerie Lin and Ms. Stephanie Fook for helpful discussions.

	Footnotes

 
Current address for W.-P.H.: MP Biomedicals Asia Pacific, Singapore 118259. E-mail: weiping_hu{at}hotmail.com.

The sequence data for cDNA clone EA01-EA78 and 76 pairs of primers (for EA01-EA76) have been submitted to the GenBank databases under accession no. BU197985-BU198062.

This work was supported by National Medical Research Council (NMRC/0311/1998), SingHealth Cluster Research Fund (BF007/2001), and Department of Clinical Research (DCR/P29/2002 and DCR/P22/2003), Singapore General Hospital, Singapore.

The data in this manuscript were presented in part at the VIII World Congress on Endometriosis, San Diego, CA, February 24–27, 2002, and the 18th World Congress on Fertility and Sterility, Montreal, Canada, May 23–28, 2004.

The authors have no conflict of interest.

First Published Online October 25, 2005

Abbreviations: CAT, Chloramphenicol acetyltransferase; CGH, comparative genomic hybridization; CK2, protein kinase CK2; C_T, threshold cycle; FBLN1, fibulin-1 gene; FISH, fluorescence in situ hybridization; HSD11B2, 11ß-hydroxysteroid dehydrogenase type 2 gene; 11ß-HSD2, 11ß-hydroxysteroid dehydrogenase 2; IGFBP, IGF binding protein; PP2A, protein phosphatase 2A; PSAP, prosaposin.

Received August 10, 2004.

Accepted October 19, 2005.

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Top Abstract Introduction Patients and Methods Results Discussion References

 

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