Evaluating miRNAs in Circulation
Dominik M. Duelli, Ph.D., assistant professor at Rosalind Franklin University of Medicine and Science, has also been interested in circulating miRNAs, and his lab has evaluated the most accurate and reproducible methods for miRNA detection and quantification from plasma via qRT-PCR.
Dr. Duelli explains that the idea behind testing these various parameters of miRNA profiling stemmed from inconsistencies in detecting certain miRNAs in plasma. “When we first started our work, the original idea for a biomarker measured in blood plasma was that it should reflect what’s inside the malignancy, so an miRNA that’s high inside the cell should also be high outside the cell,” Dr. Duelli explains.
“We profiled miRNAs in an array of cell-line cultures and saw that 60–70% of the miRNAs had the same profile outside and inside cells. But some miRNAs appeared to be exclusively released 90% or more out of the cell. Another category was miRNAs that were retained and were not released at all.” Dr. Duelli wanted to know if this was in fact a biological phenomenon, or were the miRNAs simply not detectable based on the experimental methodology?
His lab began by measuring miR-16 (highly abundant in plasma) and miR-223 (low abundance in plasma) in fresh plasma from 16 individuals to determine the optimal parameters for miRNA profiling. They first noticed that anticoagulants like citrate or KOx/NaF are a much better option than heparin for miRNA quantitation. Heparin can inhibit RT and polymerases, and miRNA detection was possible only if they diluted the heparin or treated raw plasma with heparinase.
Next, Dr. Duelli and his lab evaluated different methods for RNA isolation and noticed that some reagents can selectively precipitate certain miRNAs, so that some miRNAs may not be detected at all in a sample. Instead, using a silica membrane or beads for RNA extraction prevents polymerase inhibitors from co-purifying with miRNAs and leads to better purity and yield.
Additionally, Dr. Duelli notes that using a Taq polymerase such as Hemo KlenTaq™ (New England Biolabs) can solve this problem because some major inhibitors cannot bind to this truncated polymerase. Dr. Duelli’s lab observed a 30-fold increase in miR-16 and miR-223 expression simply by including Hemo KlenTaq in the reaction.
Dr. Duelli’s lab has also used fluorescent SmartFlare™ RNA Detection Probes (EMD Millipore), which utilize a nonenzymatic approach for rapid direct miRNA quantification in live samples. They were able to measure miR-16 expression in plasma within minutes to one hour after venipuncture, he says.
Dr. Duelli expects this technology to be highly useful in the clinical setting. “You can measure miRNA levels right after chemotherapy and correlate it with tumor progression; if the tumor is shrinking, you can measure if these miRNAs go away. It comes at a cost to sensitivity because there is no amplification, but it can be used in plasma or other samples.”
Viral-Mediated miRNA Therapeutics
Along with investigating miRNAs as biomarkers and diagnostic tools, there have also been a number of advances in the field of miRNA therapeutics. Benjamin tenOever, Ph.D., Fishberg professor of medicine at Mount Sinai School of Medicine, has been investigating how miRNAs can be utilized in engineered RNA-based vectors.
Dr. tenOever’s lab has developed a method for exploiting endogenous miRNAs to regulate tissue tropism of viral vectors. “Because viruses lack a mechanism for antagonizing miRNA function, we can exploit miRNA expression to control viruses,” he says. “We have shown that if you incorporate targets for a cell-specific miRNA into an RNA virus genome, you can create a virus that looks identical at a protein level but would be selectively blocked from infecting these particular cells.”
The other side of Dr. tenOever’s research focuses on engineering viral vectors to produce functional miRNAs, which can be used as a therapeutic platform to deliver miRNAs (or other small RNAs) to any tissue in the body. Dr. tenOever states that, while it was known that RNA viruses do not produce miRNAs, it was unclear whether viruses lack the capacity to do so, or whether this activity was perhaps detrimental to the viral life cycle.
His lab addressed this question by incorporating primary miRNAs (pri-miRNA) into various RNA viral vectors that localize to the nucleus or the cytoplasm in different tissues. In all cases, mature miRNAs were properly processed and loaded into the RISC complex. In fact, with cytoplasmic viruses, Drosha, an RNase, was exported out of the nucleus to process the artificial pri-miRNA.
It appears that most RNA viruses are capable of producing functional miRNAs, but do not do so naturally, presumably to prevent some degree of self-attenuation. “What we can capitalize on now is to convert an miRNA that behaves like a tailor-made, sequence-specific siRNA. We can then adapt any RNA virus, regardless of tropism, and use it to generate siRNAs to silence a desired host gene,” Dr. tenOever reported.
The RNA viruses only produce miRNAs for 7–10 days, but transient cytoplasmic RNA viruses have become very useful in Dr. tenOever’s lab for a variety of applications including the reprogramming of fibroblast cells into iPSCs by introducing a few miRNAs.
“The advantage of our system is that we are not going into the nucleus with our vectors and we’re not integrating into the genome; we’re only there for 7–10 days and then our vectors disappear, but by then we have reprogrammed our cells to become pluripotent stem cells.”