Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
MicroRNAs (miRNAs) are a class of short noncoding RNA found in both plants and animals. By recognizing complementary sequences on cognate mRNA, miRNAs can sequester the message or mark it for degradation, thereby post-transcriptionally fine-tuning regulation of gene expression.
MicroRNAs have an important role in all biological processes, and aberrant miRNA expression is associated with many diseases including cancer. Here we look at ways in which the miRNA expression signature of cancer cells and tissues is being investigated as a potential biomarker for early cancer detection and monitoring, and the more recent the use of miRNAs as therapeutic agents.
Promiscuity
These ~22 base-pair single-stranded molecules can act very promiscuously in that a single miRNA can affect the expression of multiple target genes, and one gene can be affected by many miRNAs, points out Johanne McGregor, director of strategic marketing in Affymetrix’s expression business unit. When “delivering a specific miRNA as a therapeutic, you have to be really careful about off-target effects,” she says.
The promiscuity of miRNAs means that it’s often difficult to pinpoint any single miRNA that correlates with a given disease state; therefore, when doing biomarker discovery, researchers are apt to search for miRNA signatures—groups of miRNAs that may collectively indicate an individual who is at risk for cancer, as well as someone who is likely to be helped by a specific drug regimen or who may even be in remission.
“Each stage of tumor progression has a very distinct subset of miRNAs that define each stage along the way,” explains Nicholas Farina, a post-doctoral associate in the University of Vermont Department of Biochemistry and member of the Vermont Cancer Center. As long as that signature is a reliable indicator, it may not even be necessary to understand the origin of its components. The signature found in serum, for example, may reflect “either the early tumor development in the tissue, or it could reflect the body’s cells responding to the cancer cells by secreting miRNAs to try to inhibit tumor progression, or it could be from the circulating tumor cells.”
Profiling miRNA
Researchers have a variety of tools at their disposal to profile miRNA from patient samples. “If someone wanted to do a really wide-scale, hypothesis-free discovery experiment, they would typically go with next-gen sequencing [NGS]. NGS enables you to look at everything—all of the small RNAs that might be in the sample,” says Sara Brown, product manager for TaqManTM miRNA qPCR assays at Thermo Fisher Scientific. Yet considerations related to sample, budget, time, throughput and informatics may limit the appeal of NGS. “It really depends on what the researcher is trying to do.”
Hybridization-based microarrays are an alternative to NGS . Affymetrix, for example, offers arrays that probe for all the known human, mouse and rat miRNA sequences contained in miRBase version 20. “That doesn’t just contain mature miRNA sequences, but also precursor sequences for miRNA and other short noncoding RNA sequences,” McGregor notes. “People have found that the processing pathway from pre-miRNA to mature miRNA is becoming extremely important to study—in fact, some cancers are thought to be effected by something going wrong in that regulatory pathway. So we actually include the pre-miRNA on our arrays to enable those kinds of discoveries.”
The other small RNAs on the array—pre-miRNA as well as snoRNA and scaRNA—may be useful for establishing a cancer signature, as well.
For researchers trying to identify which of the miRNAs already implicated in some disease state have altered expression in their samples, qPCR arrays may fit the bill. Brown’s customers often initially examine a small number of samples with NGS, “but then move to a TaqMan qPCR array card and do a more focused profiling—they might have a few more samples and fewer miRNA targets in that study.” In addition to several pre-designed arrays, custom arrays are available. After the number of targets has been narrowed—down to 10 or 20—customers typically move to single-tube (individual) qPCR assays with a greater numbers of samples for validation studies.
“What’s unique about our microRNA assays is that, in addition to the TaqMan probe-based chemistry, we have miRNA-specific RT primers,” Brown explains. “That offers very good specificity for the targets, because you’re only going to be making cDNA from your specific miRNA of interest.”
There are also a host of SYBR-based qPCR miRNA assays available from a variety of vendors, as single assays or in arrays. Qiagen’s miScript miRNA PCR Arrays, for example, according to the website are available as “full miRNome, high content, and biologically or disease relevant panels.”
Don’t just look—take action
Many cancer researchers integrate miRNA-expression data with other datasets, such as mRNA expression and copy number variation, says McGregor. “People can start to prioritize actionable biomarkers more readily, if they can start to combine all these different analytes.”
Alexander Stegh, assistant professor of neurology and medicine at the Northwestern University Feinberg School of Medicine, was interested in finding miRNAs that targeted the glioma oncoprotein Bcl2L12, an inhibitor of the p53 tumor suppressor. “So we did bioinformatics studies, we looked at large-scale gene datasets and looked at negative correlations between miRNA and Bcl2L12, and miR-182 popped out as the top candidate that should be able to target Bcl2L12.”
Introducing miR-182 into cell lines derived from glioma patients enabled Stegh and his colleagues to demonstrate that the miRNA does indeed target Bcl2L12, and in so doing they found that the latter was a critical target in sensitizing the cells to chemotherapy and targeted therapies. “In all cases we saw that when we bring in miR-182 into glioma cells, we see that these cells die better.” The researchers have since systemically injected spherical nucleic acids (SNAs)—gold nanoparticles functionalized with a corona of, in this case, miR-182—into mouse models of glioma and “found very dramatic reduction in tumor burden just using miR-182 as monotherapy,” he says. The next steps are to combine the miR-182 SNA with chemotherapeutic drugs and to combine it with other miRNA-bearing SNAs targeting other pathways.
In the ongoing search to uncover and profile cancer biomarkers, mRNAs offer an exceptional opportunity. In the fight against this disease, miRNAs have been finding their way beyond the role of surrogate marker to become a key defense tool. Profiling and bioinformatic tools assist researchers to identify key miRNAs. Once found, miRNA inhibitors and mimics—chemically synthesized, often modified, RNAs that after introduction into the cells will block or duplicate the action of a miRNA, respectively — help decipher the pathways in which these miRNAs function. When Farina identifies the mechanisms for miRNA’s role in prostate cancer’s metastasis to bone, he hopes to turn such mimics and inhibitors into novel therapeutics.
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