MicroRNA Sequence and Expression Analysis in Breast Tumors by Deep Sequencing

Abstract

MicroRNAs (miRNAs) regulate many genes critical for tumorigenesis. We profiled miRNAs from 11 normal breast tissues, 17 non-invasive, 151 invasive breast carcinomas, and 6 cell lines by in-house-developed barcoded Solexa-sequencing. miRNAs were organized in genomic clusters representing promoter-controlled miRNA expression and sequence families representing seed-sequence-dependent miRNA-target regulation. Unsupervised clustering of samples by miRNA sequence families best reflected the clustering based on mRNA expression available for this sample set. Clustering and comparative analysis of miRNA read frequencies showed that normal breast samples were separated from most non-invasive ductal carcinoma in situ and invasive carcinomas by increased miR-21 (the most abundant miRNA in carcinomas) and multiple decreased miRNA families (including miR-98/let-7), with most miRNA changes apparent already in the non-invasive carcinomas. In addition, patients that went on to develop metastasis demonstrated increased expression of mir-423, and triple negative breast carcinomas were most distinct from other tumor subtypes due to up-regulation of the mir-17~92 cluster. However, absolute miRNA levels between normal breast and carcinomas did not reveal any significant differences. We also discovered two polymorphic nucleotide variations among the more abundant miRNAs miR-181a (T19G) and miR-185 (T16G), but we did not identify nucleotide variations expected for classical tumor suppressor function associated with miRNAs. The differentiation of tumor subtypes and prediction of metastasis based on miRNA levels is statistically possible, but is not driven by deregulation of abundant miRNAs, implicating far fewer miRNAs in tumorigenic processes than previously suggested.

Expression of miR-18a and miR-210 in Normal Breast Tissue as Candidate Biomarkers of Breast Cancer Risk

“…In this study, genes with inversely related profiles to miR-210 included BRCA1, FANCD, FANCF, PARP1, E-cadherin, and Rb1, which were all activated in DCIS and downregulated in the invasive carcinoma. In our study, we found that miR-210 and miR-18a were significantly increased in IDC compared with DCIS, which is consistent with the changes described by Volinia and colleagues in the invasive transition (25,26). However, we did not find significant changes in DCIS when compared with normal breast or CUBs.…”

“…Our data complement a recent profiling study using deep sequencing of miRNAs in DCIS revealed that several miRNAs (including miR-210) were decreased in the transition from normal breast to DCIS, but increased in the transition from DCIS to IDC (25,26). In this study, genes with inversely related profiles to miR-210 included BRCA1, FANCD, FANCF, PARP1, E-cadherin, and Rb1, which were all activated in DCIS and downregulated in the invasive carcinoma.…”

Comparative RNA-sequencing analysis of myocardial and circulating small RNAs in human heart failure and their utility as biomarkers

“…The miRNA content was not significantly different between groups (Fig. S2B) and comparable with other tissues (25). We reviewed the sequence alignments of unannotated reads to the genome and found no evidence of novel highly expressed miRNA genes.…”

“…We reviewed the sequence alignments of unannotated reads to the genome and found no evidence of novel highly expressed miRNA genes. We also analyzed miRNA sequence variation as described previously (25)(26)(27) and found no novel SNPs in positions known to affect miRNA biogenesis or their target mRNA regulation (Dataset S1).…”

“…To investigate myocardial miRNA expression changes, we combined the reads corresponding to miRNA genes organized in miRNA cistrons (25,28). The cistrons are all labeled in lowercase followed by the number of the founding member and the number of cistronic miRNAs in parentheses (for details, see Fig.…”