MicroRNAs—as an entirely new class of gene-expression regulators (discovered in humans in 2001)—have elicited widespread interest. These noncoding, single-stranded, 20 to 25 nucleotide RNAs regulate gene expression through sequence-specific mRNA hybridization (usually at the 3´ untranslated region) and have been shown either to block translation or steer the degradation of mRNAs.
But because miRNA function does not require perfect binding complementarity to mRNA targets, a single miRNA may regulate multiple mRNAs. This, coupled with an integral involvement in several biological processes (including immune responses, cell-cycle control, stem cell differentiation, and metabolism), as well as their sheer collective number (as many as 1,100 identified in humans to date), renders miRNA both a rich and confounding source of study.
A broad range of challenges and innovations in miRNA research were presented at CHI’s recent conference on microRNA in Human Disease and Development.
Kai Wang, Ph.D., senior research scientist at the Institute for Systems Biology, discussed extracellular miRNA as a new source of biomarkers. Dr. Wang and colleagues in David Galas’ lab are studying many aspects of miRNA, both functional and disease-correlative. By profiling both miRNA and mRNA expression levels in numerous body fluids, including breast milk, urine, and plasma, they determined various miRNA signatures (profiles) discretely associated with various biological states (e.g., pregnancy, presence of disease, stage of disease).
The identification of stable miRNAs in extracellular environments suggests a biological function that may relate to cell-cell communication. Dr. Wang’s research aims to understand the potential of miRNAs as biomarkers, as well as their mechanisms of function, intra- and extracellularly, through study of human samples and animal models.
The challenges, as he acknowledged in his presentation, are current limitations in miRNA measurement and assessment. “When we evaluate miRNA measurement data, we need to be extremely judicious.” Using different platforms may yield significantly different results, which can complicate data verification and validation.
It is commonly acknowledged, for instance, that fluorescence- or other colorimetric-based microarray data can vary from that obtained with the same samples using qPCR, Dr. Wang noted. The imperative is, then, as much as possible, to use the same platform within a study and to be aware of possible differences between measurement methods, he advised.
Employing multiplexed assays to identify both miRNA and protein markers can strengthen conclusions about microarray data. Lorenzo Sempere, Ph.D., research assistant professor of medicine at Dartmouth Medical School, has adopted a multiplexing strategy combining in situ hybridization and immunohistochemistry for co-detecting, respectively, miRNA and clinically relevant protein markers at single-cell resolution.
Standard microarrays can fail to distinguish “altered miRNA expression within the cancer cell compartment or other elements of the tumor microenvironment; for example, upregulation of a miRNA in cancer cells or immune cells may each have etiological relevance and clinical significance, but obviously indicate vastly different biological processes,” he said.
“In addition, the ratio of cancer cells to stromal cells in different tumors can vary substantially, confounding interpretation of results. A tumor sample with a high percentage of cancer cells expressing a particular miRNA at low levels may yield similar results as a tumor sample with a low percentage of cancer cells expressing that same miRNA at very high levels.”
Dr. Sempere has developed multiplexed in situ fluorescence assays which he described as appropriate for current diagnostic workflows in typical clinical pathology labs. His team is implementing a fully automated pipeline of marker detection and computer-assisted analysis for “morphology-driven tissue slide-based assays,” applying patented Dartmouth miRNA biomarkers and detection methods. His goal is to “accurately and reproducibly” determine the role of miRNA-mediated processes in different cellular compartments.
Dr. Sempere has applied in-house synthesized fluorochromes for detection of other RNA markers to assess RNA quality and integrity, and protein markers to characterize particular cellular components associated with specific cell identities. He has used the multiplexed assays to localize expression of miR-21 and miR-155 (putative oncogenes known to be upregulated in cancer), to cancer cells and reactive stroma, and to immune cells in solid tumors, respectively. Such characterizations will serve to “increase the diagnostic power” of aberrant miRNA expression analyses, he said.