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Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
Compartment specific micro rna expression profiles (poster) poster
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Compartment specific micro rna expression profiles (poster) poster

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  • Confocal imaging of human colon cryptsEpithelial 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
  • The second commonest cancer in Hong Kong
  • Comprehensive coverage of Sanger miRBase v10
  • Note: Concordance between qRT-PCR data generated using frozen tissues vs parallel FFPE samples demonstrated in published reports and in-house data
  • Isolation of epithelial lining from mesenchyme in Normal colonsRelative quantification by Comparative Ct method Livak & Schmittgen (2001) MethodsEndogenous Control: GAPDH Calibrator sample: whole mucosa counterparts
  • X Y Scatter plotCt values from Replicate 1 (x-axis) against Ct values from Replicate 2 (y-axis)A representative example: Minimal Ct variation for Ct < 30 Replicate 1 vs Replicate 2:
  • 6 TaqMan® MicroRNA Assay endogenous controls on each LDA set6 small RNAs on each LDA set Lowest variation in endogenous expression across all samples – high stability Ct values within reasonable range (21 – 25 for RNU48)
  • Multiple statistical and computational algorithms identified distinct miRNA expression patterns across compartmentsAmong the 29 miRNAs described to be associated with CRC, 75% show directional agreement in T:N expressions between our data and published dataSensitivity and reliability of the high-throughput LDA detection approach
  • In silico Target Prediction and Functional Enrichment Analysisof predicted targets
  • Selection of candidate miRNAs for downstream workResults collectively suggest D.E. miRNA lists generated from LDA dataHigh potential involvement in intestinal epithelial development and CRC tumorigenesis – novel miRNAs worth looking into
  • To elucidate biological roles of miRNAs Monitoring phenotypic changes (cell proliferation, cell differentiation, apoptosis) in transfected cells
  • Department of Pathology, HKUDr. Chris TL ChanProf. SY LeungDr. Helen HN YanMiss Annie SY ChanDepartment of Pathology, St. Paul’s HospitalDr. ST YuenDepartment of Surgery, HKUProf. WL Law
  • ; networks of several different miRNAs may act cooperatively in the translational regulation of individual mRNAs to elicit a biological phenotype
  • Crypt/Epithelial isolation from Stroma/Mesenchymal fractionPurity of isolated crypt and stromal portions determined by tissue-specific markers Perreault & Beaulieu (1998) Exp. Cell Res.Real-time qRT-PCR using TaqMan® Human MicroRNA LDA
  • Transcript

    • 1. Compartment-specific MicroRNA Expression Profiles in Colorectal Cancer Supervisors: Dr. TL Chan, Prof. SY Leung Department of Pathology, The University of Hong Kong Jackie Lau
    • 2. Epithelial stem cells within human colon crypts Confocal imaging of human colon crypts Putative 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
    • 3. 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
    • 4. 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.
    • 5. 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.
    • 6. Experimental methodology I  Real-time quantitative reverse transcription (qRT)-PCR TaqMan® 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
    • 7. Crypt isolation from Stromal fraction Protocol 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
    • 8. Microdissection into Samples enriched in: whole mucosa*, top crypt, basal crypt* * Mucularis mucosae and muscles removed Kosinski & Li et al. (2007) PNAS; Li et al. (2009) Gastroenterology
    • 9. Top Crypt harvested Basal Crypt harvested * Muscularis mucosa and muscles excluded
    • 10. 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
    • 11. 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.
    • 12. Reproducibility between LDA Duplicates R² = 0.970 15 20 25 30 35 40 45 15 20 25 30 35 40 45 Replicate2Ct Replicate 1 Ct Ct values of all miRNAs on TaqMan® Human MicroRNA Array Set v2.0
    • 13. Selection of Endogenous Control for Normalization 15 20 25 30 35 RNU6B RNU48 RNU44 RNU43 RNU24 MammU6 Ct values of Endogenous Controls AverageCt Samples 6 TaqMan® MicroRNA Assay endogenous controls on each LDA set RNU48  Lowest Ct variation across samples  Ct values within reasonable range (21 – 25)
    • 14. 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 profiles documented 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.
    • 15. 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
    • 16. Candidate miRNAs for Individual qRT-PCR validation Results collectively suggest D.E. miRNA list  High concordance with published data  High 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)
    • 17. 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
    • 18. 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
    • 19. 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
    • 20. 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
    • 21. Acknowledgement Department of Pathology, HKU Dr. Chris TL Chan Prof. SY Leung Dr. Helen HN Yan Miss Annie SY Chan Miss Grace Cheng Dr. Vivian Li Department of Pathology, St. Paul’s Hospital Prof. ST Yuen Department of Surgery, HKU Prof. WL Law
    • 22. Large intestine: Mucosa and Musculature in Humans Adopted from Encyclopedia Britannica (http://www.britannica.com/eb/art-68639)
    • 23. 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
    • 24. 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

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