다낭성증후군이 환자 자궁에 미치는 영향에 관한 연구

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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

  1. 1. Transcriptional profiling with a pathway-oriented analysisTranscriptional profiling with a pathway-oriented analysis identifies dysregulated molecular phenotypes in theidentifies dysregulated molecular phenotypes in the endometrium of patients with PCOSendometrium of patients with PCOS Haengseok Song, Ph.D. Laboratory of Molecular Developmental Genetics Department of Biomedical Sciences, CHA University
  2. 2. • PolyCystic Ovary Syndrome (PolyCystic Ovary Syndrome ( 다낭성난소증후다낭성난소증후 군군 )) • Pathway-oriented Analysis for MicroarraysPathway-oriented Analysis for Microarrays • Aberrant Signaling Pathway in the EndometriumAberrant Signaling Pathway in the Endometrium of the Patients with PCOSof the Patients with PCOS Transcriptional profiling with a pathway-oriented analysisTranscriptional profiling with a pathway-oriented analysis identifies dysregulated molecular phenotypes ofidentifies dysregulated molecular phenotypes of endometrium in patients of PCOSendometrium in patients of PCOS
  3. 3. Polycystic Ovary Syndrome (PCOS)Polycystic Ovary Syndrome (PCOS)
  4. 4. HPO Axis Hypothalamus: GnRH 뇌하수체 자궁내막 FSH LH 난소 Estrogen Progesterone 황체난포Pituitary Gland Ovary 난소 Endometrium
  5. 5. Follicle Growth and OvulationFollicle Growth and Ovulation Ovary w/ PCOS
  6. 6. Steroidogenesis of Follicles in the OvarySteroidogenesis of Follicles in the Ovary Theca Cell Granulosa Cell insulin↑
  7. 7. • Chronic anovulation, hyperandrogenism, andChronic anovulation, hyperandrogenism, and frequentlyfrequently accompanying insulin resistance and hyperinsulinemiaaccompanying insulin resistance and hyperinsulinemia.. • In ovaries of patients with PCOS, growth ofIn ovaries of patients with PCOS, growth of early antralearly antral follicles is typically arrested at 5-10 mm stagefollicles is typically arrested at 5-10 mm stage.. • The theca cell layersThe theca cell layers are prominent in these arrested folliclesare prominent in these arrested follicles and represent the major source of theand represent the major source of the increased circulatingincreased circulating androgensandrogens in women with PCOS.in women with PCOS. Polycystic Ovary SyndromePolycystic Ovary Syndrome (PCOS)(PCOS) • A common endocrine & metabolic disorderA common endocrine & metabolic disorder in women of reproductive age (5-10%).in women of reproductive age (5-10%).
  8. 8. • Microarray analysis for cultured theca cells from patients withMicroarray analysis for cultured theca cells from patients with PCOS has demonstrated distinct biochemical and molecularPCOS has demonstrated distinct biochemical and molecular phenotypes different from the cells of regularly cycling women.phenotypes different from the cells of regularly cycling women. • Another expression profiling for PCOS showed considerableAnother expression profiling for PCOS showed considerable overlap with those of ovaries from long-term androgen-treatedoverlap with those of ovaries from long-term androgen-treated female-to-male transsexuals.female-to-male transsexuals. • Androgens play a key role in the pathogenesis of PCOSAndrogens play a key role in the pathogenesis of PCOS.. • Oocytes from PCOSOocytes from PCOS patients havepatients have molecular abnormalitiesmolecular abnormalities even though they appear to be morphologically normal.even though they appear to be morphologically normal. Polycystic Ovary Syndrome (PCOS)Polycystic Ovary Syndrome (PCOS)
  9. 9. Pathophysiological Characteristics of PCOSPathophysiological Characteristics of PCOS Obesity Hirsutism, Acne, .… Follicle Arrest
  10. 10. The Endometrium of Patients with PCOSThe Endometrium of Patients with PCOS
  11. 11. • Endometrial cells in women with PCOS (PCOSE) can beEndometrial cells in women with PCOS (PCOSE) can be aberrantly influenced by various factors, such as insulin,aberrantly influenced by various factors, such as insulin, androgens and unopposed estrogens.androgens and unopposed estrogens. • Due to the absence of ovulation,Due to the absence of ovulation, continuous exposure tocontinuous exposure to the stimulatory and mitogenic effects ofthe stimulatory and mitogenic effects of estrogens inestrogens in PCOSE could result in endometrial overgrowthPCOSE could result in endometrial overgrowth, possibly, possibly leading to hyperplasia and cancer.leading to hyperplasia and cancer. • Characteristics of PCOS may cause implantation failure,Characteristics of PCOS may cause implantation failure, miscarriage, and cancer in the endometrium.miscarriage, and cancer in the endometrium. • However, mechanisms underlying the pathophysiology ofHowever, mechanisms underlying the pathophysiology of PCOSE have not been thoroughly explored.PCOSE have not been thoroughly explored. The Endometrium of Patients with PCOSThe Endometrium of Patients with PCOS
  12. 12. The Endometrium of Patients with PCOSThe Endometrium of Patients with PCOS
  13. 13. Clinical Features of the patients with PCOSClinical Features of the patients with PCOS
  14. 14. Hierachical Clustering & Heatmaps of Up- andHierachical Clustering & Heatmaps of Up- and Down-Regulated Genes in PCOSEDown-Regulated Genes in PCOSE
  15. 15. • dCHIP: (Biosun1.harvard.edu/complab/dchip/) • Gene PatternGene Pattern : Designed to identify stage-specific signatures in tumorigenesis • Gene Set Enrichment Analysis (GSEA) :Gene Set Enrichment Analysis (GSEA) : • Stanford University Developed Algorithms for BioinformaticsDeveloped 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 InteractionsFrom Individual Genes to Whole Networks of Genetic Interactions • Gladstone Institute (Univ of California at San Francisco) : GenMAPP • NIAID (NIH) : DAVID • Univ of Michigan : Oncomine
  16. 16. • Developed at Stanford University (2001, PNAS)Developed at Stanford University (2001, PNAS) • Uses data permutations to Provides estimate of False DiscoveryUses data permutations to Provides estimate of False Discovery Rate (FDR) for multiple testingRate (FDR) for multiple testing • Convenient Excel Add-inConvenient Excel Add-in • Available for both DNA and oligo microarraysAvailable for both DNA and oligo microarrays • Adjustable threshold determines number of genes called significantAdjustable threshold determines number of genes called significant • Gene lists in Excel worksheets, easily exportable into various toolsGene lists in Excel worksheets, easily exportable into various tools • Genes are web-linked to StanfordGenes are web-linked to Stanford SOURCESOURCE databasedatabase SAM (Significance Analysis of Microarrays)SAM (Significance Analysis of Microarrays) Supervised learning software for genomic expression data mining • Able to be applied to protein expression data and SNP chip dataAble to be applied to protein expression data and SNP chip data www~stat.stanford.edu/~tibs/SAM/
  17. 17. www~stat.stanford.edu/~tibs/SAM/ . . . .
  18. 18. www~stat.stanford.edu/~tibs/SAM/
  19. 19. www~stat.stanford.edu/~tibs/SAM/
  20. 20. www~stat.stanford.edu/~tibs/SAM/
  21. 21. www~stat.stanford.edu/~tibs/SAM/
  22. 22. Gene A Gene B Gene C Gene D …. RNA Isolation Microarrays qRT-PCR Next Step ??? Sample Biopsy Purpose of Microarray AnalysisPurpose of Microarray Analysis Discovering the association of gene expression w/Discovering the association of gene expression w/ biological and/or clinical sample characteristicsbiological and/or clinical sample characteristics • SAMSAM • dCHIPdCHIP • Gene PatternGene Pattern • ……………… Single Gene Oriented Analysis
  23. 23. Major Limitations of Single Gene-orientedMajor Limitations of Single Gene-oriented Approaches for MicroarraysApproaches for Microarrays • A long list of statistically significant genesA long list of statistically significant genes w/o any unifyingw/o any unifying biological themebiological theme • Very likely to miss important effects on pathwaysVery likely to miss important effects on pathways 30% in all genes of a metabolic pathway vs a gene w/ 20 fold30% in all genes of a metabolic pathway vs a gene w/ 20 fold Which one is more important to follow up?Which one is more important to follow up? • Distressingly little overlapDistressingly little overlap in the list of statistically significantin the list of statistically significant genes from the two studies on the same biological systemgenes from the two studies on the same biological system
  24. 24. Pathway-oriented Approaches ?Pathway-oriented Approaches ? • Identify underlying genetic abnormalities or signal transductionIdentify underlying genetic abnormalities or signal transduction networks driving disease pathologiesnetworks driving disease pathologies • Interpretation of Microarray DataInterpretation of Microarray Data at the Level of Gene Setsat the Level of Gene Sets • Gene sets are defined based on prior biological knowledge,Gene sets are defined based on prior biological knowledge, e.g., published information about biochemical pathways ore.g., published information about biochemical pathways or coexpression in previous experimentscoexpression in previous experiments • Effectively bridge microarray data with biological significanceEffectively bridge microarray data with biological significance
  25. 25. http://www.genmapp.org/default.h tml
  26. 26. http://david.abcc.ncifcrf.gov/home.j sp
  27. 27. Gene Set Enrichment AnalysisGene Set Enrichment Analysis (GSEA)(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
  28. 28. GSEA for Expression Profiles of PCOSEGSEA for Expression Profiles of PCOSE • 141/164 gene sets : Up141/164 gene sets : Up • 44 gene sets at FDR < 25%44 gene sets at FDR < 25% • 23/164 gene sets : Up23/164 gene sets : Up • 2 gene sets at FDR < 25%2 gene sets at FDR < 25% PCOSNOR
  29. 29. Enriched Gene sets in Normal EndometriumEnriched 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
  30. 30. • Gene Sets Associated with Cell Cycle Are CollectivelyGene Sets Associated with Cell Cycle Are Collectively Down-regulated in PCOSE.Down-regulated in PCOSE. • Some Gene Sets Associated with Glucose MetabolismSome Gene Sets Associated with Glucose Metabolism Are Down-regulated in PCOSE.Are Down-regulated in PCOSE. • Metabolic Pathways Are Systemically Down-regulatedMetabolic Pathways Are Systemically Down-regulated in PCOSE.in PCOSE. GSEA, A Pathway-oriented Analysis Method,GSEA, A Pathway-oriented Analysis Method, Provide Information That …….Provide Information That …….
  31. 31. Cell Cycle Gene SetCell Cycle Gene Set PCOSCON
  32. 32. TGFβ1 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 Cell Cycle Genes Are Down-Regulated in PCOSECell Cycle Genes Are Down-Regulated in PCOSE ** ** ** **; p<0.01
  33. 33. Cell proliferation in stroma but not in epithelialCell proliferation in stroma but not in epithelial compartments is severely impaired in PCOSEcompartments is severely impaired in PCOSE
  34. 34. LCM-Realtime RT-PCR Validated Cell-Type SpecificLCM-Realtime RT-PCR Validated Cell-Type Specific Aberration of Gene Expression in PCOSEAberration of Gene Expression in PCOSE LCM Realtime RT-PCR
  35. 35. Cell(s) of interest Transfer film Tissue section Glass slide PixCell II LCM systemPixCell II LCM system Spot size < 7.5 µm ~ 15 µm ~ 30 µm 40 mW 25 mW 20 mW Power Duration 450 µs 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)
  36. 36. Laser Capture Microdissection (LCM)Laser Capture Microdissection (LCM)
  37. 37. LCM with VeritasLCM with Veritas
  38. 38. Down-regulation ofDown-regulation of Glycolysis in PCOSEGlycolysis in PCOSE PCOSNOR 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
  39. 39. Glycolysis Enzymes Are Down-Regulated in PCOSEGlycolysis Enzymes Are Down-Regulated in PCOSE RT-PCRRT-PCR RealtimeRealtime RT-PCRRT-PCR ** ** ** ** **; p<0.01
  40. 40. Is the PCOSE Insulin-resistant ?Is the PCOSE Insulin-resistant ?
  41. 41. (Diabetes, 2006) (Metabolism, 2007)
  42. 42. Leading Edge AnalysisLeading Edge Analysis
  43. 43. 11 22 33 44 11 22 33 44 11 22 33 44 1.1. Integrin PathwayIntegrin Pathway 2. ST_Integrin Pathway2. ST_Integrin Pathway 3. Rho Pathway3. Rho Pathway 4. Actin_Cytoskeleton_by_Rho_GTPases4. Actin_Cytoskeleton_by_Rho_GTPases Integrin-Rho-Cytoskeleton NetworksIntegrin-Rho-Cytoskeleton Networks
  44. 44. MAP2K1 MAP2K2 MAPK1 MAPK3 SOS1 CSK PXN VCL ACTN1 PIP5K1B PFN1 MYLK VCL CFL1 PAK2 Cell Proliferation PFN1 Integrin-mediated cell adhesion Integrin-mediated cell adhesion CDC42 Integrin-mediated cell adhesion Rho Pathway Integrin-Rho-cytoskeleton networks
  45. 45. Integrin-Rho-cytoskeleton network is cooperativelyIntegrin-Rho-cytoskeleton network is cooperatively down-regulated in PCOSEdown-regulated in PCOSE A B
  46. 46. • A variety of signaling pathways such as the cell cycle, DNAA variety of signaling pathways such as the cell cycle, DNA replication, apoptosis, glycolysis & integrin-actin cytoskeleton-replication, apoptosis, glycolysis & integrin-actin cytoskeleton- Rho network are dysregulated in PCOSE.Rho network are dysregulated in PCOSE. • Glucose metabolism is impaired in the endometrium as wellGlucose metabolism is impaired in the endometrium as well as the muscle of patients with PCOS.as the muscle of patients with PCOS. • Proliferation of endometrial stromal cells, but not epithelialProliferation of endometrial stromal cells, but not epithelial cells is severely impaired in PCOSE.cells is severely impaired in PCOSE. • Pathway oriented analyses are better approaches to translatePathway oriented analyses are better approaches to translate profiles of genome-wide expression into biological significance.profiles of genome-wide expression into biological significance. CONCLUSIONSCONCLUSIONS
  47. 47. • Jin Yeong Kim • Sung Ran Hong • Tae Jin Kim • 제일병원 아이소망센터 • Hyun Joo Kim • Jae Eun Lee • Ji Young Choi • Soo Jin Hwang • Chang Se Lee 제일병원 분자종양연구제일병원 분자종양연구 실실

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