다낭성증후군이 환자 자궁에 미치는 영향에 관한 연구 - 마이크로어레이를 이용한 유전자 발현 분석 · 송행석 박사(차의과학대학)

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. • 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. Polycystic Ovary Syndrome (PCOS)Polycystic Ovary Syndrome (PCOS)

4. HPO
Axis
Hypothalamus: GnRH
뇌하수체
자궁내막
FSH LH
난소
Estrogen Progesterone
황체난포Pituitary Gland
Ovary
난소
Endometrium

5. Follicle Growth and OvulationFollicle Growth and Ovulation
Ovary w/ PCOS

6. Steroidogenesis of Follicles in the OvarySteroidogenesis of Follicles in the Ovary
Theca Cell
Granulosa Cell
insulin↑

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. • 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. Pathophysiological Characteristics of PCOSPathophysiological Characteristics of PCOS
Obesity Hirsutism,
Acne, .…
Follicle
Arrest

10. The Endometrium of Patients with PCOSThe Endometrium of Patients with PCOS

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. The Endometrium of Patients with PCOSThe Endometrium of Patients with PCOS

13. Clinical Features of the patients with PCOSClinical Features of the patients with PCOS

14. Hierachical Clustering & Heatmaps of Up- andHierachical Clustering & Heatmaps of Up- and
Down-Regulated Genes in PCOSEDown-Regulated Genes in PCOSE

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. • 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. www~stat.stanford.edu/~tibs/SAM/
.
.
.
.

18. www~stat.stanford.edu/~tibs/SAM/

19. www~stat.stanford.edu/~tibs/SAM/

20. www~stat.stanford.edu/~tibs/SAM/

21. www~stat.stanford.edu/~tibs/SAM/

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. 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. 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. http://www.genmapp.org/default.h
tml

26. http://david.abcc.ncifcrf.gov/home.j
sp

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. 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. 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. • 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. Cell Cycle Gene SetCell Cycle Gene Set
PCOSCON

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. 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. 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. 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. Laser Capture Microdissection (LCM)Laser Capture Microdissection (LCM)

37. LCM with VeritasLCM with Veritas

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. Glycolysis Enzymes Are Down-Regulated in PCOSEGlycolysis Enzymes Are Down-Regulated in PCOSE
RT-PCRRT-PCR
RealtimeRealtime
RT-PCRRT-PCR **
**
**
**
**; p<0.01

40. Is the PCOSE Insulin-resistant ?Is the PCOSE Insulin-resistant ?

41. (Diabetes, 2006)
(Metabolism, 2007)

42. Leading Edge AnalysisLeading Edge Analysis

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. 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. Integrin-Rho-cytoskeleton network is cooperativelyIntegrin-Rho-cytoskeleton network is cooperatively
down-regulated in PCOSEdown-regulated in PCOSE
A
B

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. • 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