Abstract
BackgroundRecent studies indicate that microRNAs (miRNAs) are mechanistically involved in the development of various human malignancies, suggesting that they represent a promising new class of cancer biomarkers. However, previously reported methods for measuring miRNA expression consume large amounts of tissue, prohibiting high-throughput miRNA profiling from typically small clinical samples such as excision or core needle biopsies of breast or prostate cancer. Here we describe a novel combination of linear amplification and labeling of miRNA for highly sensitive expression microarray profiling requiring only picogram quantities of purified microRNA.ResultsComparison of microarray and qRT-PCR measured miRNA levels from two different prostate cancer cell lines showed concordance between the two platforms (Pearson correlation R2 = 0.81); and extension of the amplification, labeling and microarray platform was successfully demonstrated using clinical core and excision biopsy samples from breast and prostate cancer patients. Unsupervised clustering analysis of the prostate biopsy microarrays separated advanced and metastatic prostate cancers from pooled normal prostatic samples and from a non-malignant precursor lesion. Unsupervised clustering of the breast cancer microarrays significantly distinguished ErbB2-positive/ER-negative, ErbB2-positive/ER-positive, and ErbB2-negative/ER-positive breast cancer phenotypes (Fisher exact test, p = 0.03); as well, supervised analysis of these microarray profiles identified distinct miRNA subsets distinguishing ErbB2-positive from ErbB2-negative and ER-positive from ER-negative breast cancers, independent of other clinically important parameters (patient age; tumor size, node status and proliferation index).ConclusionIn sum, these findings demonstrate that optimized high-throughput microRNA expression profiling offers novel biomarker identification from typically small clinical samples such as breast and prostate cancer biopsies.
Highlights
Recent studies indicate that microRNAs are mechanistically involved in the development of various human malignancies, suggesting that they represent a promising new class of cancer biomarkers
The mature miRNAs formed by Dicer cleavage are short dsRNA molecules, one strand of which is incorporated into the ribonucleoprotein complex RISC (RNA induced silencing complex) for subsequent targeting to mRNAs [9]
Comparison of normal and malignant breast tissue has revealed that a small subset of deregulated miRNAs can be identified that unequivocally distinguish normal from malignant breast tissue, as well as other differentially expressed miRNAs that appear to correlate with breast cancer histopathologic features such as tumor size, nodal involvement, proliferative capacity and vascular invasiveness [14]
Summary
Recent studies indicate that microRNAs (miRNAs) are mechanistically involved in the development of various human malignancies, suggesting that they represent a promising new class of cancer biomarkers. MicroRNAs (miRNA) are a class of small non-coding RNAs encoded in the genomes of animals and plants [14] that play a role in targeting messages of protein-coding genes for cleavage or translational repression [5,6]. MiRNAs have been shown capable of distinguishing the different tissue developmental lineages and differentiation states of various human malignancies [13], including breast cancer [14]. While specific miRNAs may be postulated to regulate the expression of genes involved in receptor networks known to drive breast cancer progression, miRNA profiling has not yet been shown capable of independently identifying breast cancer phenotypes clinically defined by the overexpression of ErbB2 and/or estrogen receptor (ER) proteins. Progress in more widespread evaluation of miRNAs as potential cancer biomarkers remains limited by current miRNA assay methods and platforms
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