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다낭성증후군이 환자 자궁에 미치는 영향에 관한 연구

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다낭성증후군이 환자 자궁에 미치는 영향에 관한 연구

다낭성증후군이 환자 자궁에 미치는 영향에 관한 연구
- 마이크로어레이를 이용한 유전자 발현 분석

· 송행석 박사(차의과학대학)

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    다낭성증후군이 환자 자궁에 미치는 영향에 관한 연구 다낭성증후군이 환자 자궁에 미치는 영향에 관한 연구 Presentation Transcript

    • Transcriptional profiling with a pathway-oriented analysis identifies dysregulated molecular phenotypes in the endometrium of patients with PCOS Haengseok Song, Ph.D. Laboratory of Molecular Developmental Genetics Department of Biomedical Sciences, CHA University
      • PolyCystic Ovary Syndrome ( 다낭성난소증후군 )
      • Pathway-oriented Analysis for Microarrays
      • Aberrant Signaling Pathway in the Endometrium of the Patients with PCOS
      Transcriptional profiling with a pathway-oriented analysis identifies dysregulated molecular phenotypes of endometrium in patients of PCOS
    • Polycystic Ovary Syndrome (PCOS)
    • H P O Axis H ypothalamus: GnRH 뇌하수체 자궁내막 FSH LH 난소 Estrogen Progesterone 황체 난포 P ituitary Gland O vary 난소 Endometrium
    • Follicle Growth and Ovulation Ovary w/ PCOS
    • Steroidogenesis of Follicles in the Ovary Theca Cell Granulosa Cell insulin↑
      • Chronic anovulation, hyperandrogenism, and frequently accompanying insulin resistance and hyperinsulinemia .
      • In ovaries of patients with PCOS, growth of early antral follicles is typically arrested at 5-10 mm stage .
      • The theca cell layers are prominent in these arrested follicles and represent the major source of the increased circulating androgens in women with PCOS.
      Polycystic Ovary Syndrome (PCOS)
      • A common endocrine & metabolic disorder in women of reproductive age (5-10%).
      • Microarray analysis for cultured theca cells from patients with PCOS has demonstrated distinct biochemical and molecular phenotypes different from the cells of regularly cycling women.
      • Another expression profiling for PCOS showed considerable overlap with those of ovaries from long-term androgen-treated female-to-male transsexuals.
      • Androgens play a key role in the pathogenesis of PCOS .
      • Oocytes from PCOS patients have molecular abnormalities even though they appear to be morphologically normal.
      Polycystic Ovary Syndrome (PCOS)
    • Pathophysiological Characteristics of PCOS Obesity Hirsutism, Acne, .… Follicle Arrest
    • The Endometrium of Patients with PCOS
      • Endometrial cells in women with PCOS (PCOSE) can be aberrantly influenced by various factors, such as insulin, androgens and unopposed estrogens.
      • Due to the absence of ovulation, continuous exposure to the stimulatory and mitogenic effects of estrogens in PCOSE could result in endometrial overgrowth , possibly leading to hyperplasia and cancer.
      • Characteristics of PCOS may cause implantation failure, miscarriage, and cancer in the endometrium.
      • However, mechanisms underlying the pathophysiology of PCOSE have not been thoroughly explored.
      The Endometrium of Patients with PCOS
    • The Endometrium of Patients with PCOS
    • Clinical Features of the patients with PCOS
    • Hierachical Clustering & Heatmaps of Up- and Down-Regulated Genes in PCOSE
      • dCHIP: (Biosun1.harvard.edu/complab/dchip/)
      • Gene Pattern : Designed to identify stage-specific signatures in tumorigenesis
      • Gene Set Enrichment Analysis (GSEA) :
      • Stanford University
      Developed Algorithms for Bioinformatics
      • Significant Analysis of Microarrays (SAM): 2001, PNAS
      • Harvard School of Public Health
      • Broad Institute (Harvard & MIT)
      From Individual Genes to Whole Networks of Genetic Interactions
      • Gladstone Institute (Univ of California at San Francisco) : GenMAPP
      • NIAID (NIH) : DAVID
      • Univ of Michigan : Oncomine
      • Developed at Stanford University (2001, PNAS)
      • Uses data permutations to Provides estimate of False Discovery Rate (FDR) for multiple testing
      • Convenient Excel Add-in
      • Available for both DNA and oligo microarrays
      • Adjustable threshold determines number of genes called significant
      • Gene lists in Excel worksheets, easily exportable into various tools
      • Genes are web-linked to Stanford SOURCE database
      SAM (Significance Analysis of Microarrays) Supervised learning software for genomic expression data mining
      • Able to be applied to protein expression data and SNP chip data
      www~stat.stanford.edu/~tibs/SAM/
    • www~stat.stanford.edu/~tibs/SAM/ . . . .
    • www~stat.stanford.edu/~tibs/SAM/
    • www~stat.stanford.edu/~tibs/SAM/
    • www~stat.stanford.edu/~tibs/SAM/
    • www~stat.stanford.edu/~tibs/SAM/
    • Gene A Gene B Gene C Gene D … . RNA Isolation Microarrays qRT-PCR Next Step ??? Sample Biopsy Purpose of Microarray Analysis Discovering the association of gene expression w/ biological and/or clinical sample characteristics
      • SAM
      • dCHIP
      • Gene Pattern
      • ………
      Single Gene Oriented Analysis
    • Major Limitations of Single Gene-oriented Approaches for Microarrays
      • A long list of statistically significant genes w/o any unifying biological theme
      • Very likely to miss important effects on pathways
      • 30% in all genes of a metabolic pathway vs a gene w/ 20 fold
      • Which one is more important to follow up?
      • Distressingly little overlap in the list of statistically significant genes from the two studies on the same biological system
    • Pathway-oriented Approaches ?
      • Identify underlying genetic abnormalities or signal transduction networks driving disease pathologies
      • Interpretation of Microarray Data at the Level of Gene Sets
      • Gene sets are defined based on prior biological knowledge, e.g., published information about biochemical pathways or coexpression in previous experiments
      • Effectively bridge microarray data with biological significance
    • http://www.genmapp.org/default.html
    • http://david.abcc.ncifcrf.gov/home.jsp
    • Gene Set Enrichment Analysis (GSEA)
      • The goal of GSEA is to determine whether the members of S are randomly distributed throughout L or primarily at the top or bottom .
      • It is expected that sets related to the phenotypic distinction will tend to show the latter distribution.
      Leading edge subset
    • GSEA for Expression Profiles of PCOSE
      • 141/164 gene sets : Up
      • 44 gene sets at FDR < 25%
      • 23/164 gene sets : Up
      • 2 gene sets at FDR < 25%
      PCOS NOR
    • Enriched Gene sets in Normal Endometrium
      • Cell Cycle
      • Cancer_Cell Cycle
      • Cell Proliferation
      • Cell Cycle Checkpoint
      • Proliferation_Genes
      • Cell Cycle Regulator
      • Glucose_Down
      • Glycolysis & Gluconeogenesis
      • Insulin_2 Fold Up
      • Glut_Down
      • Leucine_Down
      • Pyrimidine Metabolism
      • Purine Metabolism
      • Leucine_Up
      • Pyruvate Metabolism
      • Gene Sets Associated with Cell Cycle Are Collectively Down-regulated in PCOSE.
      • Some Gene Sets Associated with Glucose Metabolism Are Down-regulated in PCOSE.
      • Metabolic Pathways Are Systemically Down-regulated in PCOSE.
      GSEA, A Pathway-oriented Analysis Method, Provide Information That …….
    • Cell Cycle Gene Set PCOS CON
    • Cell Cycle Genes Are Down-Regulated in PCOSE TGF β 1 RB1 CDKN1A PRKDC BUB3 CCND2 CDK4 CCNE2 CCNA2 CDC6 ORC MCM WEE1 CDC7 CDC2 CCNA2 CDC2 CCNB1 CCNB2 CDC25B CDC25C MAD2L1 BUB1B BUB1 CHEK1 PCNA MCM2 MCM4 MCM6 MCM5 ORC6L SMC1L1 CDC20 PTTG1 ESPL1 ** ** ** **; p<0.01
    • Cell proliferation in stroma but not in epithelial compartments is severely impaired in PCOSE
    • LCM-Realtime RT-PCR Validated Cell-Type Specific Aberration of Gene Expression in PCOSE LCM Realtime RT-PCR
    • Cell(s) of interest Transfer film Tissue section Glass slide PixCell II LCM system Spot size < 7.5  m ~ 15  m ~ 30  m 40 mW 25 mW 20 mW Power Duration 450  s 1.5 ms 5 ms Activated transfer film The cells of interest are positioned in the center of the field Transfer film is applied to the tissue surface A focused laser beam is pulsed to activated the transfer film Laser Capture Microdissection (LCM)
    • Laser Capture Microdissection (LCM)
    • LCM with Veritas
    • Down-regulation of Glycolysis in PCOSE PCOS NOR HK1 PGM1 ACYP1 PKM2 Glycolysis, but not gluconeogenesis, is cooperatively down-regulated in PCOSE. HK1 (Hexokinase 1) PGM1 (Phosphoglucomutase 1) ACYP1 (Acrylphosphatase 1) PKM2 (Pyruvate Kinase) Glycolysis & Gluconeogenesis Glycolysis Gluconeogenesis
    • Glycolysis Enzymes Are Down-Regulated in PCOSE RT-PCR Realtime RT-PCR ** ** ** ** **; p<0.01
    • Is the PCOSE Insulin-resistant ?
    • (Diabetes, 2006) (Metabolism, 2007)
    • Leading Edge Analysis
    • 1 2 3 4 1 2 3 4 1 2 3 4
      • Integrin Pathway
      • 2. ST_Integrin Pathway
      • 3. Rho Pathway
      • 4. Actin_Cytoskeleton_by_Rho_GTPases
      Integrin-Rho-Cytoskeleton Networks
    • VCL Cell Proliferation Integrin-mediated cell adhesion Integrin-mediated cell adhesion MAP2K1 MAP2K2 MAPK1 MAPK3 SOS1 CSK PXN ACTN1 PIP5K1B PFN1 MYLK VCL CFL1 PAK2 PFN1 CDC42 Integrin-mediated cell adhesion Rho Pathway Integrin-Rho-cytoskeleton networks
    • Integrin-Rho-cytoskeleton network is cooperatively down-regulated in PCOSE A B
      • A variety of signaling pathways such as the cell cycle, DNA replication, apoptosis, glycolysis & integrin-actin cytoskeleton-Rho network are dysregulated in PCOSE.
      • Glucose metabolism is impaired in the endometrium as well as the muscle of patients with PCOS.
      • Proliferation of endometrial stromal cells, but not epithelial cells is severely impaired in PCOSE.
      • Pathway oriented analyses are better approaches to translate profiles of genome-wide expression into biological significance.
      CONCLUSIONS
      • Jin Yeong Kim
      • Sung Ran Hong
      • Tae Jin Kim
      • 제일병원 아이소망센터
      Acknowledgements
      • Hyun Joo Kim
      • Jae Eun Lee
      • Ji Young Choi
      • Soo Jin Hwang
      • Chang Se Lee
      제일병원 분자종양연구실