September 15, 2009 (Vol. 29, No. 16)
Potential of Field Is Catalyst for Emerging Diagnostic and Therapeutic Strategies
As key regulators of gene expression and cellular machinery, microRNAs may be the most significant discovery in molecular biology in the last decade. Because of their enormous potential, Frost & Sullivan projects that current U.S. microRNA revenues of about $20 million in 2008 will increase to more than $98 million in 2015.
The buzz in the industry stems in part from the global presence of microRNAs. These 18- to 25-nucleotide noncoding RNAs operate in organisms ranging from roundworms to mosses and even viruses. The roughly 1,000 human microRNAs discovered so far regulate nearly half of all genes. Dysfunctions are associated with disease processes that range from dementia to cancer. New insights are emerging into the biology of how they work, what happens when they don’t, and their potential use in targeted therapies.
Caenorhabditis elegans provides an ideal model system to study microRNA functions in eukaryotes, according to Alison L. Abbott, Ph.D., assistant professor at Marquette University. “C. elegans has many microRNAs that show nearly complete conservation between worms and humans. Earlier studies in C. elegans demonstrated that microRNAs are critical regulators of developmental timing genes.”
To characterize how individual microRNAs control such genes is no easy task. “There is a cooperativity of microRNA families and on some level they will function together,” Dr. Abbott notes. “So, we decided to begin studying individual microRNAs with a subset of 20–30 that show some sequence conservation with other organisms such as mammals, flies, and fish.”
Dr. Abbott developed a model system in C. elegans to lower the genetic background sufficiently to test if deletion or mutation of the specific microRNAs made a difference in the worm’s development. Her team targeted the genes for Argonaute proteins, which are the catalytic subunits of the RNA-induced silencing complex (RISC). The latter protein complex mediates gene silencing or RNA interference.
“Mutations in two Argonaute-encoding genes, alg-1 and alg-2, result in lower microRNA levels and lethality in C. elegans,” Dr. Abbott reports. “However, knockdown of just alg-1 results in lower microRNA levels but sufficient levels for the worms to progress through embryonic and larval development. This creates an optimal sensitized background that allows us to test the effects of losing an additional single microRNA or a single cluster of microRNAs.”
According to Dr. Abbott, not only can microRNAs regulate a common target, but they also may function through different targets while still regulating the same pathway. For the future, Dr. Abbott hopes “that the combination of bioinformatic approaches and genetics will help us identify key biologically relevant targets.”
Validating microRNA Targets
Although microRNAs control many aspects of an organism’s development, the specifics of microRNA-mRNA regulatory interactions is largely unknown. Liang Zhang, Ph.D., who is currently working at The Rockefeller University, also developed an approach using a C. elegans model while in the laboratory of Min Han, Ph.D., at Howard Hughes Medical Institute and the University of Colorado at Boulder. Dr. Zhang creates and expresses a fusion protein in which green fluorescent protein (GFP) is tagged to AIN-2, an essential member of miRISC (microRNA associated RISC).
“This model can be used to generate a more global look at the dynamics of microRNA-mediated regulation of gene expression during the worm’s development,” Dr. Zhang says. “We prepare lysates during five developmental stages and immunoprecipitate the AIN-2:GFP containing miRISC with an antibody to GFP. Then, we analyze the microRNAs and mRNAs in the immunoprecipitated complexes using deep sequencing and microarrays, which allows us to access the profiles of microRNAs, microRNA targets, and interactions. We found that more than 2,000 mRNAs associate with the AIN-2 family during worm development.”
Using that data, Dr. Zhang then identified thousands of microRNA targets related to each developmental stage. “We used this data to predict more than 1,500 microRNA family-mRNA interactions. Our data indicates that microRNAs have a high degree of specificity and preferentially target genes involved in signaling and do not regulate genes involved in housekeeping functions.
“Further, although perfect complementarity between bases 2–8 of the microRNA and its targets is a highly enriched feature of these microRNA family-mRNA interactions, additional matching between bases 9–10 of the miRNA and its targets are depleted, suggesting that perfect target matchings in the center region of the microRNA are avoided by functional interaction in vivo.”
Dr. Zhang reports that his studies indicate that microRNAs target and have preferences for different stages of development. “MicroRNAs are trying to temporally coordinate processes in different stages of development. Our future studies will begin to analyze spatial patterns of microRNA regulations in specific tissues of the worm, since our current studies used lysates of the whole worm.”
Dissecting how microRNAs work, individually as well as with other regulatory molecules in the complex network within the cell, is a significant hurdle. Bioinformatic modeling can help unravel the intricate workings, says Jiang Qian, Ph.D., assistant professor at Wilmer Institute, Johns Hopkins University School of Medicine.
“Many researchers study only one or a few microRNAs to better understand and profile their activity. It is also important, however, to view the global picture of interactions within such regulatory networks. We use a bioinformatics approach to understand network interactions and gene regulation. Our goal has been to examine a broader scope of network motifs that are the basic building blocks of the regulatory networks.”
Dr. Qian’s group studies not only the interactions of transcription factors with microRNAs, but also other types of network motifs where there could be regulatory targets. “In a recent study, we examined 46 network motifs in order to examine the biological roles they play. We found that transcription factors and microRNAs work together and tend to regulate each other as well as coregulate genes. By looking at global signatures, we found a highly represented pattern showing there is a feedback loop in which two transcription factors regulate each other and one microRNA regulates both of the factors. This is important because it helps explain how microRNAs contribute to development.”
Another finding was that there are two overall classes of microRNAs. “It is clear that specific microRNAs show distinct preference for either functioning in embryonic conditions (class I) or for functioning in adult tissues (class II). We feel that this is a valid finding since these expression patterns are conserved across species including humans, mice, and zebrafish. Also, we used several platforms to come to the same conclusion.”
Dysfunction in Multiple Myeloma
Mature microRNAs play a critical role in the pathogenesis of solid tumors as well as hematologic malignancies such as multiple myeloma. “Very little is known about the characteristics of multiple myeloma at the epigenetic level,” says Aldo M. Roccaro, M.D., Ph.D., department of medical oncology, Dana-Farber Cancer Institute and Harvard Medical School.
Dr. Roccaro’s goal is to better understand how microRNAs regulate multiple myeloma progression within the bone marrow milieu. “We conducted a series of studies evaluating microRNA signatures in multiple myeloma patients,” he reports. “We showed that plasma cells isolated from patients with multiple myeloma are characterized by lower expression of a specific microRNA cluster called microRNA-17-92, which is located on chromosome 13.”
To perform the studies, Dr. Roccaro and his team investigated 318 microRNAs using liquid-phase Luminex microbead microRNA profiling. In this system, total RNA is labeled with biotin and hybridized to beads containing different fluorophores coated with oligonucleotides complementary to each known microRNA. Binding is detected with streptavidin-phycoerythrin. The team validated its results using quantitative RT-PCR.
“We found a multiple-myeloma-specific signature that was characterized by down expression of a specific set of microRNAs as well as overexpression of another set,” he reports. “We next focused our attention on some of the deregulated microRNAs and looked specifically at microRNA-15a and -16 and how they could potentially regulate myeloma cell growth both in vitro and in vivo. Overall our results suggest that both microRNA-15a and -16 play important roles in the biology of this disease. This information may provide a rationale for developing microRNA-targeted therapies in myeloma.”
Unique Disease Signatures
Rapidly entering the microRNA playing field are strategies to identify altered expression of microRNAs associated with hereditary diseases such as retinitis pigmentosa (RP), the most common form of inherited retinal degenerations. The eye disorder is characterized by progressive photoreceptor-cell death.
According to Arpad Palfi, Ph.D., post-doctoral fellow in the laboratory of G. Jane Farrar, Ph.D., at Trinity College, they generated a microRNA-expression profile in the mouse retina using microRNA microarray technology and real-time RT-PCR.
“Initially, we analyzed a transgenic mouse model in which the gene for rhodopsin has a specific mutation resulting in an RP phenotype,” Dr. Palfi explains. Rhodopsin is a retinal pigment responsible for light detection in rod photoreceptors. Following normalization to wild-type mice, we found an altered microRNA-expression profile involving several microRNAs (e.g., miR-1, -96, -133, and -183) in the rhodopsin-mutant transgenic mice. This data suggested that the normal microRNA expression profile is altered in the disease state.”
The group decided to expand on those studies by examining other RP mouse models. “We examined three RP mouse models using the same strategies and found a common signature in which levels of microRNAs-96, -182, and -183 were decreased while expression of microRNA-1, -133, and -142 were upregulated.”
The group will next examine what protein targets and pathways in the retina are affected by these altered microRNA expression patterns. “If we can understand how microRNAs work in normal and degenerating retinas, perhaps we can use microRNAs as a therapeutic tool, possibly in conjunction with other therapies, to beneficially influence outcome of RP.”
Chronic inflammation is a key feature in many human diseases such as asthma, arthritis, and inflammatory bowel disease. Rene Lutter, Ph.D., principal investigator at the Academic Medical Center, University of Amsterdam, is trying to understand these inflammatory mechanisms and how microRNAs may be implicated.
“Inflammatory processes are tightly controlled at multiple levels to ensure a limited and transient response. The production of mediators such as interleukins and chemokines drive the inflammatory response. A common feature of mRNAs encoding these mediators is the presence of AU-rich elements in the 3´-untranslated region that target the mRNA for rapid degradation.”
Currently, Dr. Lutter and post-doctoral fellow Saheli Chowdhury, Ph.D., are exploring the mechanisms for mRNA degradation of the inflammatory mediators. They manipulate candidate regulatory molecules in cultured airway-epithelial cells and analyze cells derived from the airways of asthmatic and healthy subjects before and after infecting them with rhinovirus 16.
“We see that a family of proteins that binds to these AU-rich mRNA sequences facilitates mRNA degradation,” Dr. Lutter says. Furthermore, it appears that AU-binding proteins act in conjunction with specific microRNAs, targeting a number of mRNAs to modulate production of several inflammatory mediators.”
A key extension of the concept is that aberrant regulation by this system may underlie the development of chronic inflammation. “Recent experiments indicate that manipulating the cellular level of certain microRNAs markedly affects the production of inflammatory mediators. This also suggests their therapeutic potential.”
The young field of microRNA continues to grow and mature. Its enticing potential is driving new strategies and technologies for research, diagnostic, and therapeutic applications.