compartment specific micro rna expression profiles (poster) poster
TRANSCRIPT
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Compartment-specific MicroRNA Expression Profiles
in Colorectal Cancer
Supervisors: Dr. TL Chan, Prof. SY LeungDepartment of Pathology,
The University of Hong Kong
Jackie Lau
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Epithelial stem cells within human colon crypts
Confocal imaging of human colon cryptsPutative intestinal stem cell marker Musashi-1 (green) expression
Brittan & Wright (2004) Gut
Epithelial stem cell-pericryptal microenvironment (stem cell niche) interaction is crucial for homeostatic balance between epithelial cell self-renewal, cell division, cell differentiation and lineage determination
Blanpain et al. (2007) Cell Spradling et al. (2001) Nature
Modified from Spradling et al. (2001) Nature
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Gene expression analysis of human colonic epithelial cell differentiation
Kosinski & Li et al. (2007) PNAS; Li et al. (2009) Gastroenterology Identified > 900 cDNA clones differentially expressed between top and basal crypts
in normal human colons Many involved in molecular pathways common to intestinal epithelial cell
development and neoplastic transformation
Modified from Kosinski & Li et al. (2007) PNAS
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Resemblance between stem cell and cancer Immortality Self-renewal, clonal and heterogeneous properties Signals from surrounding microenvironment Common signaling pathways for regulation:
Wnt, Bone morphogenetic protein (BMP), Notch, Hedgehog
Colorectal Cancer (CRC) Tumor cells demonstrate “crypt-progenitor phenotype”, i.e.
phenotypic resemblance to basal crypt Deregulation of multiple signaling pathways mediating normal
intestinal epithelial development
Li, V.S. Profiling of gene expression changes in human colon crypt maturation and study on their dysregulation in tumorigenesis. PhD Thesis, The University of Hong Kong: Hong Kong, January 2008.
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Project Aim
Based on the hypothesis that perturbation of normal colon maturation during tumorigenesis is cell type-dependent, the present study aims to investigate compartment-specific microRNA (miRNA) expression in normal human colons with tumor counterparts.
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Experimental methodology I
Real-time quantitative reverse transcription (qRT)-PCRTaqMan® Human MicroRNA Array Set v2.0 Low Density Array (LDA)
Global expressions of 677 human miRNAs (miRBase v10) Duplicate quantification for each sample
Frozen colonic tissues Tumors Normal counterparts: microdissection-partitioned into
enrichments in whole mucosa, epithelium, stroma, top crypt and basal crypt
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Crypt isolation from Stromal fractionProtocol modified by Dr. Helen Yan from our lab based on Strater et al. (1996) Gastroenterology; Whitehead et al. (1999) Gastroenterology; Hofmann et al. (2007) Gastroenterology
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Microdissection into Samples enriched in: whole mucosa*, top crypt, basal crypt* * Mucularis mucosae and muscles removedKosinski & Li et al. (2007) PNAS; Li et al. (2009) Gastroenterology
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Top Crypt harvested Basal Crypt harvested * Muscularis mucosa and muscles excluded
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Experimental methodology II
Individual qRT-PCR validation of miRNAs differentially expressed across different compartments
TaqMan® MicroRNA Assay, Human Triplicate quantification for each sample
Formalin-fixed, paraffin-embedded (FFPE) tissues Tumors Normal counterparts: microdissection-partitioned into enrichments in whole mucosa, top crypt and basal crypt
Note: Concordance between qRT-PCR data from frozen tissues vs parallel FFPE samples in published reports and in-house data
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Results
qRT-PCR detection for Purity of isolated Crypts and Stroma in Normal Colons
Relative quantification by Comparative Ct method Livak & Schmittgen (2001) Methods
Obtained “near-mesenchyme-free Crypts” &
“near-epithelium-free Stroma” fractions
Perreault & Beaulieu (1998) Exp. Cell Res.
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Reproducibility between LDA Duplicates
15 20 25 30 35 40 4515
20
25
30
35
40
45
R² = 0.970221386323091
Replicate 1 Ct
Re
pli
ca
te 2
Ct
Ct values of all miRNAs on TaqMan® Human MicroRNA Array Set v2.0
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Selection of Endogenous Control for Normalization
15
20
25
30
35 RNU6B
RNU48
RNU44
RNU43
RNU24
MammU6
Ct values of Endogenous Controls
Aver
age
Ct
Samples
6 TaqMan® MicroRNA Assay endogenous controls on each LDA set
RNU48 Lowest Ct variation across samples Ct values within reasonable range (21 – 25)
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Statistical Analysis of LDA data
TM4: MeV (MultiExperiment Viewer) v4.4 Saeed et al. (2003) Biotechniques
Multiple statistical and computational algorithms identified distinct miRNA
expression patterns across compartments
Citical p-value = 0.01
75% amongst 29 miRNAs described to be associated with CRC show agreement in T:N expression ratio to published data
Sensitivity and reliability of high-throughput LDA detection
miRNA expression profilesdocumented in CRC
Lu et al. (2005) Nature
Bandres et al. (2006) Mol. Cancer
Schetter et al. (2009) JAMA
Aslam et al. (2009) Br. J. Surg.
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In silico Target Prediction and Functional Enrichment Analysis
28 miRNAs D.E. between Top vs Basal crypts
Pathways involved in intestinal epithelial development along colon crypt axis
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Candidate miRNAs for Individual qRT-PCR validation
Results collectively suggest D.E. miRNA listHigh concordance with published dataHigh involvement in intestinal epithelial development and CRC tumorigenesis
Selected 5 candidate miRNAs with robustness across various statistical and functional analyses
Formalin-fixed, paraffin-embedded (FFPE) tissues 42 Normal vs CRC Tumor pairs 16 Top vs Basal Crypt pairs (Normal)
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Individual qRT-PCR validation of 5 candidate miRNAs
42 Normal vs Tumor pairs of FFPE Colonic tissues
miR-A miR-B miR-C miR-D miR-E
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Individual qRT-PCR validation of 5 candidate miRNAs
16 Top vs Basal Crypt pairs (Normal)
* Log2 Fold Expression calibrated to Whole mucosa counterparts
miR-A miR-B miR-C miR-D miR-E
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Conclusion
Protocols developed for isolation of compartments and high-throughput detection effective, as evident in data reproducibility and concordance with literature
Fundamental support for a hierarchy of miRNA expression along the colonic proliferation-differentiation axis
Foundation to identify candidate marker miRNAs that define colonic epithelial stem/progenitor cell niche
Based on resemblance between stem cell and cancer, such miRNA markers may constitute targets for mutation and malignant transformation, thus provide novel way to elucidate clonal origin of CRC
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Current Work
Characterization of candidate miRNAs in CRC cell lines and a larger number of clinical samples
Functional Studies in CRC lines
Knockdown/Overexpression of candidate miRNAs
To elucidate biological roles of miRNAs
Monitoring phenotypic changes (cell proliferation, cell differentiation,
apoptosis) in transfected cells
Validate bioinformatically predicted mRNA targets
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Acknowledgement
Department of Pathology, HKU
Dr. Chris TL Chan
Prof. SY Leung
Dr. Helen HN YanMiss Annie SY ChanMiss Grace ChengDr. Vivian Li
Department of Pathology,
St. Paul’s Hospital
Prof. ST Yuen
Department of Surgery, HKUProf. WL Law
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Large intestine: Mucosa and Musculature in Humans
Adopted from Encyclopedia Britannica (http://www.britannica.com/eb/art-68639)
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Why miRNA?
Gene expression profile – done Kosinski & Li et al. (2007) Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors. PNAS, 104(39):15418-23.Li et al. (2009) Frequent inactivation of axon guidance molecule RGMA in human colon cancer through genetic and epigenetic mechanisms. Gastroenterology, 137:176-187.
Diagnostic and prognostic potential of miRNAs implicated in pathogenesis of CRC, either as oncogenes or tumor suppressors.
Potential to regulate multiple target genes or potentially an entire pathway; networks of several different miRNAs may act cooperatively in the translational regulation of individual mRNAs to elicit a biological phenotype
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TaqMan® Human MicroRNA LDA
Fresh Tumors Flash freeze Cryosectioning
Fresh Normal counterparts Crypt isolation from Stromal fraction
Flash freeze Cryosectioning Microdissection into
Samples enriched in: whole mucosa*, top crypt, basal crypt* * Mucularis mucosae and muscles removed
Experimental methodology – Sample preparation