June 1, 2007 (Vol. 27, No. 11)

New Pathways for Scientific Inquiries Are Being Revealed by a Multitude of Labs

Since they were first discovered in 1993, miRNAs have created a great deal of excitement. These small pieces of RNA, only 21–22 nucleotides long, have emerged as potentially important regulators of cellular developmental pathways, proliferation, apoptosis, and differentiation. They represent the latest addendum to the central dogma of molecular biology—a model already complicated by other types of ’misbehavior’ of RNA such as the retrovirus, the ribozyme, and siRNA.

The discovery of miRNAs has opened new pathways for scientific inquiry, particularly in the field of cancer. Cambridge Healthtech Institute recently sponsored a gathering of the miRNA research community to explore miRNA mechanisms, their role in disease, and their applications, including screening, diagnosis, and therapy.

miRNA in Oncology

Understanding functional relationships between miRNAs and their targets can lead to improved understanding of disease. This idea has generated particular interest in the field of cancer, where it is hoped that miRNAs will be key to controlling the disordered processes of cellular proliferation. Giovanni Stefani, M.D., Ph.D., represented the laboratory of Yale’s Frank Slack in collaboration with Asuragen (www.asuragen.com) in his presentation on the role of the miRNAs let-7 as a tumor suppressor.

Let-7 was one of the first two miRNAs discovered. Loss-of-function mutants of let-7 in C. elegans died in the larval stage, and gain-of-function mutants experienced early expression of adult traits—yielding the first clues that let-7 played a roll in development. A computational search for regulatory targets of let-7 turned up let-60, the C. elegan’s orthologue of Ras. Inactivation of let-7 in humans resulted in increased levels of Ras.

This led to the association of let-7 with lung cancer. Tissue from lung tumors showed decreased levels of let-7 and elevated Ras. A search for other genes regulated by let-7 revealed approximately 500, including cyclins, cdks, cell cycle phosphatases, oncogenes, DNA replication genes, argonates, and Dicer1, which has shown to be associated with poor prognosis in lung cancer when its expression is reduced.

Stefani concluded that let-7 is expressed at high levels in lung tissue, it regulates RAS and other cell cycle genes through the 3´ untranslated region, it is a regulator of cellular proliferation, and it is a likely tumor suppressor in the lung.

The body of work presented depicts let-7 as a crucially important regulator whose effects are just beginning to be understood. The next step is to demonstrate the role of let-7 as a tumor suppressor using in vivo models of lung cancer.

miRNA Meets Microarray

The Luminex (www.luminexcorp.com) platform has provided versatile tool for the discovery and characterization of miRNAs and their targets. Keld Sorensen, Ph.D., director of R&D for Luminex Bioscience Group discussed miRNA microarray experiments on the Luminex bead-based XMap platform using LNA-based (locked nucleic acid) probes from Exiqon (www.exiqon.com). LNA is a bicyclic, high-affinity modified RNA nucleotide with the sugar ring locked in a 3´ endo conformation. When used in oligonucleotide synthesis, it increases the sensitivity and specificity of hybridization and increases the thermal stability of the oligonucleotide.

The Luminex FlexmiR capture probes are a mixture of LNA and DNA, which bind to biotinylated miRNA. The beads are detected by flow cytometer, and the assay produces 9,600 data points in four hours. Competing technologies include planar arrays and qRT- PCR, and each method has its advantages and disadvantages. “The technologies all have a space,” Sorensen noted. “Each method has benefits and drawbacks. You wouldn’t transport sheet rock in a convertible sports car, would you?”

The ability to assay for miRNA in a highly multiplexed manner makes it possible to probe a cell for abnormal patterns. When the Luminex system was used to compare normal tissue vs. carcinoma, a pattern of differentially expressed miRNAs appeared. “MicroRNA is different because it is telling you what is going on metabolically inside the cell. Our prediction is that with many cancers and other diseases, this will tell a patient whether this is something extremely aggressive or not. That’s where microRNA is going in the near future.”

Exiqon’s LNA technology has been described as a “solution looking for a problem.” miRNAs represent an ideal application of the technology, with 20–25 bases being a chemical sweet spot for the molecule. Exiqon’s vp and CSO, Soeren Moeller, Ph.D., gave a talk about using LNA technology to detect miRNAs in breast cancer to use as biomarkers for selecting patients for chemotherapy. Traditionally the decision about whether to use chemotherapy for treatment has been made based on classical pathological characteristics such as tumor size and grade.

Using their proprietary Mircury LNA array, Exiqon scientists in collaboration with Herlev University Hospital in Copenhagen, Denmark screened patients looking for differential expression of miRNAs in diseased tissue versus normal. Their results confirmed other reports in the literature that members of the let-7 family, miR-125a/b and miR-21 are deregulated in breast cancer. Additionally, they found new miRNAs that are in the process of being validated. These experiments have become possible only as the technology has become available in recent years.

Mimetics and Inhibitors

The study of miRNAs and their function is greatly facilitated by the development of miRNA mimetics and inhibitors. To meet this experimental need, Dharmacon (www.dharmacon.com), part of Thermo Fisher Scientific (www.thermofisher.com), offers the Miridian® miRNA mimics and inhibitor collections. Miridian includes both inhibitors and mimetics of known miRNAs in human, mouse, and rat. They can be used to alter the balance of any particular miRNA and assess its effect on the cell. This was the topic of the presentation of Thermo Fisher’s Scott Baskerville, Ph.D. at the miRNA meeting.

“Unlike other miRNA mimic designs,” said Bill Marshall, Ph.D., vp for technology and business development, “these modifications inactivate the passenger strand of the Miridian mimic and ensure that it performs similarly to the endogenous mature microRNA without nonspecific passenger strand activity.”

After the double-stranded pre-miRNA is processed by the endonuclease dicer, the 20–25 nucleotide guide strand enters the RISC complex, where it pairs with its complementary mRNA and induces mRNA degradation, effectively turning off the gene. In the Miridian miRNA mimetics, a chemical modification insures that the passenger strand is inactivated and the correct strand enters the RISC complex.

In addition to developing a unique set of miRNA mimics, Dharmacon’ R&D team also performed detailed structure/function studies for development of the next generation of miRNA inhibitors. As detailed in a recent article in RNA, inhibitor potency and longevity are greatly affected by the annealing strength of miRNA inhibitors and the presence of structural elements outside the core (reverse complement) region of the inhibitor.

The company’s Miridian inhibitors adopt design strategies from both domains, using chemical modifications to strengthen inhibitor binding to the miRNA in the RISC complex and incorporating flanking structures around the modified complementary sequence to enhance overall functionality. These reagents were utilized in a recent phenotypic screen to successfully identify miRNA biomarkers that induce unique cellular states. Thermo Fisher is launching a miRNA profiling service based on the Miridian technology.

Tim Davison, Ph.D., senior scientist at Asuragen, discussed the company’s work in biofluid screening for miRNA biomarkers. It is not always clear in a tissue sample what is and is not tumor tissue, nor what the appropriately matched control samples should be. miRNAs are present in biofluids such as serum, plasma, saliva, and urine. These biofluids may potentially be used to define the signature or presence of cancerous tissue. To access biomarkers from fluids, the company developed and validated miRNA isolation methods and a microRNA expression analysis to face the unique sample preparation and analytical challenges presented by working with biofluids.

Asuragen is the successor to Ambion (www.ambion.com). In 2005, Ambion founder, Matt Winkler, sold the Ambion research reagent business to focus on miRNA-based diagnostics and therapeutics. The remaining Diagnostic and Services division was rolled into the new company, Asuragen.

Asuragen has used multiple expression analysis platforms, including qRT-PCR, mirVana™ bioarrays, as well as a custom Affymetrix (www.affymetrix.com) array that includes published miRNAs outside the Sanger miRBase content to screen its biofluids. “The intent was that if we look at the commonly recognized Sanger content and see a separation or depth of information in published microRNA content not found in typical microarrays, we will have the opportunity to screen a large number of microRNA for the potential identification of biomarkers,” said Davison, Ph.D.

“What we’ve been working on as well is understanding what the limitations are of the original sample source. Depending on what tissue you’re looking at, there are different amounts of microRNA you can actually isolate from these tissues; specifically in the context of optimizing isolation procedures and understanding the impact of normalization of array and qRT-PCR data.”

New tools and technologies have new frontiers in RNA research. The field of microRNA has exploded, with notable discoveries regarding their role in cancer and other disorders of development. However, some of the most important news is the rewriting of the rules of molecular biology and the breakdown of the central tenets. Scientists are entering uncharted waters, where the great unanswered questions of biology and human health could finally be adressed.

Asuragen’s Tim Davison speculated on some of the implications of miRNAs, including the possibility that SNPs could have a relationship with them. “This is a whole new realm of understanding,” he remarked. “Just when we thought we had it all figured out, along came microRNA.”

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