Toward the promise of microRNAs - Enhancing reproducibility and rigor in microRNA research

Abstract

The fields of applied and translational microRNA research have exploded in recent years as microRNAs have been implicated across a spectrum of diseases. MicroRNA biomarkers, microRNA therapeutics, microRNA regulation of cellular physiology and even xenomiRs have stimulated great interest, which have brought many researchers into the field. Despite many successes in determining general mechanisms of microRNA generation and function, the application of microRNAs in translational areas has not had as much success. It has been a challenge to localize microRNAs to a given cell type within tissues and assay them reliably. At supraphysiologic levels, microRNAs may regulate hosts of genes that are not the physiologic biochemical targets. Thus the applied and translational microRNA literature is filled with pitfalls and claims that are neither scientifically rigorous nor reproducible. This review is focused on increasing awareness of the challenges of working with microRNAs in translational research and recommends better practices in this area of discovery.

Keywords: Biomarker; miRNA; microRNA; small RNA-seq; transfection; translational research; xenomir.

Figure 1.

microRNA expression is at the cellular level. This schematic representation of an epithelial organ shows shared and unique expression of microRNAs. For ubiquitous microRNAs such as the blue labeled miR-21, the tissue-level signal is a result of expression in almost all cell types. For cell-type restricted microRNAs such as the red labeled miR-150, miR-451a etc. the tissue-level signal is a result of the presence of very specific cell types. Key: epithelial (miR-200b), mesenchymal (miR-143), lymphocyte (miR-150), endothelial (miR-126), red blood cell (miR-451a), macrophage and neutrophil (miR-223).

Figure 2.

Cellular composition is an important driver in changing microRNA signals. Each circle represents a unique cell type making up 1% of a total tissue signal. In the control tissue, 7 different cell types make up 100% of the tissue, with the most abundant cell type (blue) being 40% of the tissue. In disease, the cellular composition can change dramatically and a rare cell type (green) can increase 3-fold. Any cell-specific microRNAs will increase 3-fold, without necessarily altering their expression in the tissue.

Figure 3.

microRNA-seq normalization (RPM) is a dependent normalization. In this example, a single common microRNA (200,000 RPM by microRNA-seq), identified as green *is experimentally doubled (to 400,000 RPM). No other microRNAs (all other colors) are expected to be altered. However, because all are normalized by RPM, which is dependent of the sum of all reads, every other microRNA will decrease proportionately to account for the increase in the single modulated microRNA.

Figure 4.

A comparison of exosomal transfer and culture transfection experiments. (A) In in vivo exosomal transfer, donor cells generate exosomes which then enter the circulation or traverse the local environment of extracellular matrix. These exosomes then bind and fuse to recipient cells. The kinetics and specificity needed to target a particular microRNA from a donor cell to be uptaken by a specific recipient cell to sufficiently modulate levels of that microRNA in the second cell type are challenging. (B) In an ex vivo cell transfection experiment, saturating levels of microRNAs in lipid-based systems (or similar) are used to markedly increase the level of the microRNA in a cell, often to supraphysiologic levels.