Design and Analysis for Studying microRNAs in Human Disease: A Primer on -Omic Technologies

Abstract

microRNAs (miRNAs) are fundamental to cellular biology. Although only approximately 22 bases long, miRNAs regulate complex processes in health and disease, including human cancer. Because miRNAs are highly stable in circulation when compared with several other classes of nucleic acids, they have generated intense interest as clinical biomarkers in diverse epidemiologic studies. As with other molecular biomarker fields, however, miRNA research has become beleaguered by pitfalls related to terminology and classification; procedural, assay, and study cohort heterogeneity; and methodological inconsistencies. Together, these issues have led to both false-positive and potentially false-negative miRNA associations. In this review, we summarize the biological rationale for studying miRNAs in human disease with a specific focus on circulating miRNAs, which highlight some of the most challenging topics in the field to date. Examples from lung cancer are used to illustrate the potential utility and some of the pitfalls in contemporary miRNA research. Although the field is in its infancy, several important lessons have been learned relating to cohort development, sample preparation, and statistical analysis that should be considered for future studies. The goal of this primer is to equip epidemiologists and clinical researchers with sound principles of study design and analysis when using miRNAs.

Keywords: blood; cancer; circulating biomarkers; lung cancer; microRNA; review.

Figure 1.

Immature, double-stranded microRNAs (miRNAs) are processed by Drosha after transcription followed by cytoplasmic processing by Dicer into separate strands. Single, mature miRNA strands are captured by the RNA-induced silencing complex (RISC) to complement messenger RNA for posttranscriptional modification. The remaining strand from the duplex is usually degraded but can be functional in some cases. In keeping with the traditions of gene nomenclature, pre-miRNAs, pri-miRNAs, and mature miRNAs from the same parent transcript have a nuanced terminology with pre/pri strands designated using a lower case r (“mir”) and the mature transcript designated using a capital “R” (“miR”).

Figure 2.

The number of precursor microRNAs (i.e., raw transcripts) for all species reported over time to miRBase, the key public repository, is displayed for all species along with microRNA publications per analyte type in humans only. Numbers are displayed on a log base 10 scale on the y-axis and with the year on the x-axis. We see here that the current logarithmic phase of discovery and publications is slowing, which is typical of biomarker fields in their infancy. Estimates of publications by microRNA publication category were extracted from GoPubMed (http://www.gopubmed.org/web/gopubmed/) using the following search terms, on January 23, 2014:For all microRNA, [Humans[MeSH] AND microRNA]; for tissue microRNA, [Humans[MeSH] AND microRNA AND (tissue OR tumor)]; and for circulating microRNA, [Humans[MeSH] AND circulating microRNA]. Numbers are reported through 2012 and are estimates only based on the search terms above.