Quantitative Analysis of Micrornas in Tissue Microarrays by in Situ Hybridization

Abstract

MicroRNAs (miRNAs) have emerged as key regulators in the pathogenesis of cancers where they can act as either oncogenes or tumor suppressors. Most miRNA measurement methods require total RNA extracts which lack critical spatial information and present challenges for standardization. We have developed and validated a method for the quantitative analysis of miRNA expression by in situ hybridization (ISH) allowing for the direct assessment of tumor epithelial expression of miRNAs. This co-localization based approach (called qISH) utilizes DAPI and cytokeratin immunofluorescence to establish subcellular compartments in the tumor epithelia, then multiplexed with the miRNA ISH, allows for quantitative measurement of miRNA expression within these compartments. We use this approach to assess miR-21, miR-92a, miR-34a, and miR-221 expression in 473 breast cancer specimens on tissue microarrays. We found that miR-221 levels are prognostic in breast cancer illustrating the high-throughput method and confirming that miRNAs can be valuable biomarkers in cancer. Furthermore, in applying this method we found that the inverse relationship between miRNAs and proposed target proteins is difficult to discern in large population cohorts. Our method demonstrates an approach for large cohort, tissue microarray-based assessment of miRNA expression.

Recent trends in microRNA research into breast cancer with particular focus on the associations between microRNAs and intrinsic subtypes

“…The expression of miR-221 in breast cancer has been identified as a good prognostic marker and is associated with ER positivity and lymph node negativity. 42 However, the miR-221/222 cluster is associated with tamoxifen resistance in breast cancer cells. 43,44 Miller et al 45 reported that miR-221/222 expressions were upregulated in endocrine therapy-resistant luminaltype breast cancer cells.…”

miRNA Dysregulation in Breast Cancer

“…Notably, miR-221/222 have yet to be probed in antiendocrine-treated clinical cohorts in breast cancer, but one can only assume these studies are ongoing, given the building evidence of the involvement of these miRNAs in endocrine resistance. The one study that has investigated expression of miR-221 in breast cancer patients found that it was, in fact, a good prognostic marker, and expression of the miRNA was associated with ERpositivity (76), which is at odds with the fact that it has been shown to target the protein in vitro (73). Furthermore, miR-30c, has been shown to be an independent predictor of tamoxifen response and was associated with increased progression-free survival (77).…”

MicroRNAs in adrenal tumors: relevance for pathogenesis, diagnosis, and therapy

“…Since microRNA patterns can be associated with differences in hormone secretion [59], such discrepancies in the composition of patient cohorts might be relevant [60]. For the better evaluation of the diagnostic or prognostic role of certain microRNAs in adrenocortical tumors, the direct assessment of microRNAs in adrenal tissues by in situ hybridization would be a novel possibility [61].…”