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. 2013 Apr;182(4):1171-9.
doi: 10.1016/j.ajpath.2013.01.006. Epub 2013 Feb 12.

A common MicroRNA signature consisting of miR-133a, miR-139-3p, and miR-142-3p clusters bladder carcinoma in situ with normal umbrella cells

Angela Y Jia  1 , Mireia Castillo-Martin, Josep Domingo-Domenech, Dennis M Bonal, Marta Sánchez-Carbayo, Jose M Silva, Carlos Cordon-Cardo
Affiliations

A common MicroRNA signature consisting of miR-133a, miR-139-3p, and miR-142-3p clusters bladder carcinoma in situ with normal umbrella cells

Angela Y Jia et al. Am J Pathol. 2013 Apr.

Abstract

miRNAs are small noncoding RNAs with critical roles in a large variety of biological processes such as development and tumorigenesis. miRNA expression profiling has been reported to be a powerful tool to classify tissue samples, including cancers, based on their developmental lineage. In this study, we have profiled the expression of miRNAs in bladder carcinoma in situ (CIS) and distinct cell compartments of the normal bladder, namely umbrella and basal-intermediate urothelial cells, as well as the muscularis propria. We identified several miRNAs differentially expressed between umbrella and basal-intermediate cells (miR-133a, miR-139-3p, miR-142-3p, miR-199b-5p, and miR-221). In situ hybridization confirmed the expression of miR-133a and miR-139-3p in umbrella cells, and miR-142-3p in basal-intermediate cells. Strikingly, miRNA expression levels of CIS most closely resembled the miRNA profile of umbrella cells. Finally, we examined well-established umbrella and basal-intermediate cell immunohistochemical biomarkers in an independent series of CIS samples. Again, this analysis revealed the significant expression of umbrella-specific markers in CIS when compared to non-CIS lesions. Overall, our studies represent a comprehensive and accurate description of the different miRNAs expressed in CIS tumors and three distinct histological areas of the urinary bladder. Notably, this study provides evidence of the possible origin relationship between CIS and normal umbrella cells.

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Figures

Figure 1
Figure 1
Experimental design and isolation of UM and B/i cells from normal bladder urothelium. A: Representative immunohistochemical analyses of UPKII and CK5 in normal urothelium; prominent UM cells (arrows). Scale bar = 50 μm. B: Microphotograph of frozen tissue section was taken with PALM Microbeam IV Laser Capture Microscope (LCM; Zeiss). UM, B/i, and CIS samples were microdissected and analyzed with both miRNA and mRNA microarrays. MP samples were macrodissected (MD) and analyzed only in the miRNA microarray. C: Delineation of UM cells, before and after laser capture. Scale bar = 75 μm. D: RT-qPCR on UM and B/i cells for UPKII and CK5. Graph shows B/i log2 fold expression; B/i expression normalized to UM. Horizontal line indicates the sample median. Square indicates the sample mean. *P < 0.05 by Student’s t-test.
Figure 2
Figure 2
Hierarchical clustering of normal urothelium and CIS. A: Samples are in columns and miRNAs are in rows. Individual samples of normal urothelium and CIS were clustered according to the expression signature of 41 differentially expressed miRNAs (P ≤ 0.05). B: Samples are in columns, mRNAs are in rows. Normal urothelium and CIS samples were clustered according to the expression signature of 1113 differentially expressed mRNAs (P ≤ 0.05). C: Venn diagram illustrating 698 mRNAs upregulated in common between CIS compared to normal UM and B/i cells. There were 753 mRNAs upregulated in CIS compared to B/i cells, and 738 mRNAs upregulated in CIS compared to UM cells. Arbitrary assignment of cluster groups is as follows: A, UM cells; B, B/i cells; C, CIS.
Figure 3
Figure 3
Validation of differentially expressed miRNAs from microarray data. A: RT-qPCR on UM and B/i cells for various miRNAs. Graph shows B/i log2 fold expression; B/i expression is normalized to UM. Horizontal line indicates the sample median. Square indicates the sample mean. *P < 0.05 by Student’s t-test. B: Localization of miR-133a, miR-139-3p, and miR-142-3p in human bladder samples by in situ hybridization. Scale bar = 100 μm. Inset: miR-142-3p shows UM cells. Scale bar = 25 μm. No staining was observed in a negative control in situ hybridization using the sense strand of miR-424/503, whereas a strong signal was observed in a positive control in situ hybridization using U6 in representative cases of normal bladder. Scale bar = 100 μm.
Figure 4
Figure 4
Phenotypic association of CIS with UM cells by RT-qPCR and immunohistochemistry. A: RT-qPCR results on B/i and CIS samples for various miRNAs. Graph shows CIS log2 fold expression; CIS expression is normalized to B/i. Horizontal line indicates the sample median. Square indicates the sample mean. *P < 0.05, **P < 0.005 by Student’s t-test. B: Representative immunohistochemical analyses of UPKII, CK20, and high molecular weight cytokeratins (HMWCK) in four different CIS tissue samples. Scale bar = 100 μm. C: Quantification of the percentage of cases with expression of the respective biomarkers.
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