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. 2012 Feb;2(2):279-86.
doi: 10.1534/g3.111.001115. Epub 2012 Feb 1.

Correlation of Global MicroRNA Expression With Basal Cell Carcinoma Subtype

Christopher Heffelfinger, Zhengqing Ouyang, Anna Engberg, David J Leffell, Allison M Hanlon, Patricia B Gordon, Wei Zheng, Hongyu Zhao, Michael P Snyder, Allen E Bale

Correlation of Global MicroRNA Expression With Basal Cell Carcinoma Subtype

Christopher Heffelfinger et al. G3 (Bethesda). 2012 Feb.

Abstract

Basal cell carcinomas (BCCs) are the most common cancers in the United States. The histologic appearance distinguishes several subtypes, each of which can have a different biologic behavior. In this study, global miRNA expression was quantified by high-throughput sequencing in nodular BCCs, a subtype that is slow growing, and infiltrative BCCs, aggressive tumors that extend through the dermis and invade structures such as cutaneous nerves. Principal components analysis correctly classified seven of eight infiltrative tumors on the basis of miRNA expression. The remaining tumor, on pathology review, contained a mixture of nodular and infiltrative elements. Nodular tumors did not cluster tightly, likely reflecting broader histopathologic diversity in this class, but trended toward forming a group separate from infiltrative BCCs. Quantitative polymerase chain reaction assays were developed for six of the miRNAs that showed significant differences between the BCC subtypes, and five of these six were validated in a replication set of four infiltrative and three nodular tumors. The expression level of miR-183, a miRNA that inhibits invasion and metastasis in several types of malignancies, was consistently lower in infiltrative than nodular tumors and could be one element underlying the difference in invasiveness. These results represent the first miRNA profiling study in BCCs and demonstrate that miRNA gene expression may be involved in tumor pathogenesis and particularly in determining the aggressiveness of these malignancies.

Keywords: expression profiling; histopathology; miR-150; miR-183; skin cancer.

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Figures

Figure 1
Figure 1
Twenty miRNAs had statistically significant differences in expression (false discovery rate <0.01) between nodular and infiltrative BCCs. (A) A sample representing a range of expression, indicated by an asterisk, was chosen for validation by qPCR. (B) A heatmap of differentially expressed miRNAs shows that although miRNA expression patterns tend to be consistent within a given class, many tumors show anomalous expression for a handful of miRNAs.
Figure 2
Figure 2
Principal components analysis of 16 tumors on the basis of total miRNA expression. Infiltrative tumors clustered tightly with the exception of Inf 2, which was the smallest of the infiltrative tumors and which had a significant nodular component. Nodular tumors had a much broader distribution, reflecting the increased heterogeneity of the subtype. Nod1 and Nod8, which clustered near the infiltrative tumors, were the largest of the nodular class. The axes, principal components one and two, are produced by determining the variability of miRNA expression within the total dataset and collapsing correlating variance into a reduced set of values. The first two components account for 89.7% of the total variance within the dataset; none of the remaining components account for more than 3.0%.
Figure 3
Figure 3
Characterization of variance within BCC classes. Standard error vs. mean expression was graphed for each miRNA. Nodular tumors (A) have a standard error relative to the mean of 0.1923, approximately twice that of infiltrative specimens (B) at 0.0857. These data suggest that the wide distribution of nodular tumors in principal component analysis is attributable to an overall pattern of higher variance in expression of miRNAs rather than a few highly variable miRNAs.
Figure 4
Figure 4
Validation of differentially expressed miRNAs by qPCR. Six miRNAs were assessed by Taqman assays. Assays for miR-141 and miR-582-5p failed because of low expression levels (see Materials and Methods). A) Among nine tumors from the discovery set in which adequate RNA was available, qPCR mirrored RNAseq in the direction and approximate magnitude of differences between the two tumor types except for Let-7g. (B) A replication set of seven tumors showed concordance in direction with qPCR results in the discovery set. (C) Upon combining the analytical and biological validation sets, three of six miRNAs, miR-150, 183, and 886-5p, were significantly different (P < 0.05) and two others, miR-31 and 146, trended in the same direction as shown by RNA sequencing but failed to reach significance. (D) Expression of miR-183 showed no overlap in nodular tumors compared with infiltrative tumors in both the discovery and replication set. Y-axis values are provided in log(2) such that a value of one indicates a twofold difference between samples.
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