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