MicroRNAs (miRNAs) finely regulate gene expression and play an important role in various cellular processes, including cell growth, differentiation, proliferation, and apoptosis. To date, more than 5,000 of these endogenous, noncoding single-stranded RNAs have been identified. miRNAs act through binding to complementary mRNA sequences, thereby preventing their translation into protein or accelerating mRNA breakdown. Investigators are working on exploiting these molecules for experimental and potential therapeutic applications.
As presenters will discuss at CHI’s “MicroRNA in Human Disease and Development” meeting later this month, miRNAs’ mechanistic reach extends well beyond suppression of gene expression and encompasses a complex system of post-transcriptional control. Investigators have found that miRNA regulatory processes involve miRNA activation of gene expression by interacting with complementary regions found in the promoter coding region, as well as the 3´ UTR of their mRNA targets.
Extensive regulation of miRNA itself occurs at the levels of miRNA promoter transcription, methylation, miRNA processing, RNA editing, and miRNA-target interactions.
Brian D. Brown, Ph.D., assistant professor of genetics and genomic sciences at the Mt. Sinai School of Medicine, will describe studies using artificial miRNA target sites to exploit or modulate endogenous miRNA regulation at the meeting. He says that several features of miRNA target sites on mRNA influence the results of miRNA binding to the site. These include complete or incomplete complementarity between miRNA and its mRNA target; the number of target sites on a transcript, which is directly proportional to miRNA-mediated suppression; and the sequence nucleotide sequence around an miRNA target site.
For example, in the nucleotide sequence surrounding a given target, secondary structures in the RNA can reduce target-site accessibility and reduce the chance of transcript regulation by the miRNA.
These observations suggest some guidelines, which will be discussed by Dr. Brown in his presentation, for leveraging endogenous miRNA regulation to improve the outcomes of basic functional studies and potential therapeutic applications including gene and cell therapy.
In the gene-therapy arena, the investigators observed that transgene expression from hepatocyte-specific promoters occurred in antigen-presenting cells, as well as liver cells, causing potential problems because gene therapies targeting liver cells can trigger an antitransgene product T-cell response.
Dr. Brown and his colleagues found that they could prevent this response by modifying the 3´ UTR of a vector to contain four tandem copies of a sequence that is perfectly complementary to miR-142, an miRNA highly expressed in APCs but not in hepatocytes. Studies of transgenic mice showed that miR-142-3p suppressed reporter gene expression more than 100-fold in all mouse cells of hematopoietic lineage, including apes.
The potential therapeutic value of designing vectors under the control of miRNA regulatory mechanisms was verified when hemophilia B mice were injected with the same miRNA-regulated vector and encoding factor IX. The mice were cured of factor IX deficiency, as well as immunological tolerance to factor IX, Dr. Brown explains.
When possible, “miRNA target sites for any type of study should be placed in transcript regions of high accessibility, for example the 3´ UTR A-rich regions, to optimize miRNA binding.”
RNAs have the potential to become a new class of biomarkers to detect cancer at its earliest stages, as well as to characterize specific cancers. Dirk Dittmer, Ph.D., and his colleagues at the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, are focused on understanding viral tumorigenesis—specifically, cancers that are caused by Kaposi’s sarcoma-associated herpes virus (KSHV/HHV-8) and the development of high-throughput tools for viral diagnostics.
Using Kaposi’s sarcoma (KS) as an example, Dr. Dittmer discussed the relative merits of pre-miRNAs vs. mature miRNAs as biomarkers. Dr. Dittmer’s laboratory has shown that tumor suppressor miRNAs (miR-222/221, let-7 family) are significantly downregulated in primary effusion lymphoma (PEL), a lymphoma caused by KSHV or human herpesvirus 8 (HHV-8). PEL occurs most commonly in patients with immunodeficiency diseases, including AIDS. The investigators also distinguished among miRNAs present in latently virus- infected nontumorogenic endothelial cells and Kaposi’s sarcoma cells, identifying 15 virally regulated miRNAs in the endothelial cells.
Two types of miRNAs—MiR-143 and -145—were elevated only in KS tumors, not virally infected endothelial cells, therefore, representing tumor-specific rather than virus-specific miRNAs. “Because many tumor-suppressor proteins are wildtype in KS and PEL, downregulation of multiple tumor suppressor miRNAs provides an alternative mechanism of transformation,” Dr. Dittmer says.
Few studies have simultaneously assessed all three levels of miRNA regulation. Dr. Dittmer says that his group was able to determine changes in gene copy number, pre-miRNA, and mature miRNA levels in a large set of PELs, detecting PEL-specific miRNA gene amplifications and concordant changes in pre-miRNA and mature miRNA. They also identified 68 PEL-specific miRNAs that, according to Dr. Dittmer, define the miRNA signature of PEL.
They further showed that transcriptional regulation of pre-miRNA as well as mature miRNA levels contribute nonredundant information that can be used for the classification of human tumors. “Both pre- and mature miRNA profiling have their place in biomarker research. Pre-miRNAs are ideally suited to investigate signaling events that change miRNA expression, for example, in response to drugs. Mature miRNA profiles integrate miRNA expression, nuclear export, and stability and thus, are more useful in longer-term experiments or if there is a suggestion that a particular tumor changes miRNA processing.”