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Comparative Study
. 2009 Nov 1;69(21):8472-81.
doi: 10.1158/0008-5472.CAN-09-0744. Epub 2009 Oct 20.

Distinct microRNA alterations characterize high- and low-grade bladder cancer

James W F Catto  1 , Saiful Miah, Helen C Owen, Helen Bryant, Katie Myers, Ewa Dudziec, Stéphane Larré, Marta Milo, Ishtiaq Rehman, Derek J Rosario, Erica Di Martino, Margaret A Knowles, Mark Meuth, Adrian L Harris, Freddie C Hamdy
Affiliations
Comparative Study

Distinct microRNA alterations characterize high- and low-grade bladder cancer

James W F Catto et al. Cancer Res. .

Abstract

Urothelial carcinoma of the bladder (UCC) is a common disease that arises by at least two different molecular pathways. The biology of UCC is incompletely understood, making the management of this disease difficult. Recent evidence implicates a regulatory role for microRNA in cancer. We hypothesized that altered microRNA expression contributes to UCC carcinogenesis. To test this hypothesis, we examined the expression of 322 microRNAs and their processing machinery in 78 normal and malignant urothelial samples using real-time rtPCR. Genes targeted by differentially expressed microRNA were investigated using real-time quantification and microRNA knockdown. We also examined the role of aberrant DNA hypermethylation in microRNA downregulation. We found that altered microRNA expression is common in UCC and occurs early in tumorogenesis. In normal urothelium from patients with UCC, 11% of microRNAs had altered expression when compared with disease-free controls. This was associated with upregulation of Dicer, Drosha, and Exportin 5. In UCC, microRNA alterations occur in a tumor phenotype-specific manner and can predict disease progression. High-grade UCC were characterized by microRNA upregulation, including microRNA-21 that suppresses p53 function. In low-grade UCC, there was downregulation of many microRNA molecules. In particular, loss of microRNAs-99a/100 leads to upregulation of FGFR3 before its mutation. Promoter hypermethylation is partly responsible for microRNA downregulation. In conclusion, distinct microRNA alterations characterize UCC and target genes in a pathway-specific manner. These data reveal new insights into the disease biology and have implications regarding tumor diagnosis, prognosis and therapy.

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Figures

Figure 1
Figure 1
MicroRNA expression in normal and malignant urothelium. Centroid linkage hierarchical clustering was performed after selecting miRs with >90% expression frequency and mean centering data using city block similarity in Cluster 3.0. First dendrogram row indicates subsequent progression (Black box = Progression, White box = No progression (also in brackets after sample ID (0=no, 1=yes)). Boxes outline miRs with similar expression profiles.
Figure 2
Figure 2
MicroRNA expression in low and high-grade bladder cancer. In (a) changes in expression (log scale) and statistical significance (SAM P values plotted as -log 10) of low-grade NMI tumors when compared to normal urothelium from non-UCC cases, reveals both up and down-regulation of numerous miRs. In (b) for high-grade NMI and invasive tumors there is a general upregulation in microRNA expression. Whilst changes in some miRs are shared between the two tumor phenotypes (e.g. miR-133b/204) others appear specific (miR-21 or miRs-100/99a). Expression is relative to the median of 3 RNA molecules and linear median centered.
Figure 3
Figure 3
MicroRNA expression and tumor phenotype. (a). Differentially expressed miRs specific to each phenotype. (b) Signature panels of microRNA for each tissue (c). Progression analysis in all 52 UCC revealed that dichotomous aberrant expression of miRs-21/100/99a was associated with progression to more advanced UCC (Kaplan-Meier method and tested using Logrank analysis).
Figure 4
Figure 4
Analyses of microRNA 99a/100 in UCC. (a) Expression of miRs 100/99a and FGFR3 are inversely correlated (DCT values shown). (b) We found that a 70-80% miR-99a/100 knock-down induced by anti-miRs in NHU cells transcribed with Luciferase and the 3’UTR of FGFR3 was associated with increased Luciferase expression when compared to NHU cells transfected with a scrambled UTR sequence. (c). Upregulation of FGFR3 protein expression was found in NHU cells transfected with anti-miRs to 99a/100 when compared to negative controls (scrambled anti-miR sequence) (Western blot images (FGFR3 is upper band (splice variant seen below)) and photometric relative quantification standardized for B-Actin and measured using ImageJ). (d) The presence of the activating S249C FGFR3 mutation does not alter miR-99a/100 expression (relative miR and mRNA quantification). (e) Reversal of DNA hypermethylation leads to re-expression of 5 down regulated miRs in the low-grade cell line (RT4). Relatively few events were seen in the cell lines representative of high-grade and invasive bladder cancer (RT112 and EJ/T24). Four miRs (mir-127/124a/373/517c) reported to be silenced by DNA hypermethylation are also shown (28, 29).
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