Though an increasing number of researchers are looking into how to use microRNAs for diagnostic and therapeutic applications, much about the basic biology behind these small, non-coding RNAs remains unknown.
The National Institutes of Health hopes to change that.
Last month the agency issued three grants supporting research aimed at answering fundamental questions about miRNAs, including their role in human papillomavirus-associated cancers and vertebrate development, as well which miRNAs might be at work in the mammalian cardiac conduction system.
The first grant was awarded by the National Institute of Dental and Craniofacial Research to Amy Gardiner, a postdoc at the University of Pittsburgh, to examine the role miRNAs play in HPV type 16, a strain highly associated with cervical and oral cancers.
“The majority of HPV-associated carcinomas harbor integrated virus, with increased expression of the viral oncogenes E6 and E7,” Gardiner stated in the grant’s abstract. “The viral proteins interact with a number of cellular proteins, enhancing cellular proliferation through deregulation of cell cycle control mechanisms.”
In light of increasing evidence linking miRNAs to cancer due to their roles in cellular development, growth, and proliferation, Gardiner aims to use the NIDCR grant to “gain a better understanding of the interaction between HPV-16 … and the cell during carcinogenesis,” as well as to identify miRNA targets that could be used to develop diagnostics and therapeutics.
In her grant abstract, Gardiner wrote that initial microarray studies by her and her colleagues showed differential expression of miRNAs between HPV-16-positive and HPV-16-negative cell lines, while transfection and siRNA-based gene knockdown experiments showed that expression of a specific miRNA, miR-218, was reduced by HPV-16 E6 in cervical and oral carcinoma cells.
Hypothesizing that HPV-16 affects the expression of cellular miRNAs and encodes miRNAs that might target cellular and viral genes, Gardiner aims to conduct additional expression and protein analyses to characterize how HPV-16 helps regulate miRNAs. She also intends to use the grant to identify HPV-16-encoded miRNAs and their targets.
The one-year grant project began on Aug. 15 and is worth $35,930.
The second grant was awarded by the National Institute of General Medical Sciences to Antonio Giraldez, an assistant professor at Yale University, to investigate the role the conserved miRNA miR-430 plays in cell signaling and lineage specification.
In 2006, Giraldez published data showing that miR-430 is “expressed at the onset of zygotic transcription and regulates morphogenesis during early development” in zebrafish, and that it appears to facilitate the deadenylation and clearance of maternal mRNAs during early embryogenesis.
Last week, Giraldez and colleagues published data in Science showing that miR-430 is a key mediator of Nodal agonist and antagonist levels in zebrafish (see related story, this issue).
“During early development two major events take place: the specification of the primordial germ cells and the formation of the germ layers during gastrulation, endoderm, mesoderm, and ectoderm,” Giraldez wrote in his grant abstract.
He and his colleagues plan to use miRNA-deficient zebrafish embryos to determine how miRNAs regulate gene expression in somatic cells as compared with germ cells, testing the hypothesis that miRNAs dampen germ line-specific gene expression in somatic cells.
This effort is expected to help “identify the molecular mechanisms used by these somatic targets to escape miRNA mediated repression in the germ cells,” according to the abstract.
Giraldez is also planning to use the grant to investigate how miRNAs regulate Nodal signaling, which induces endoderm and mesoderm formation during gastrulation.
“These experiments will explore how miRNAs regulate different components in the Nodal signaling pathway and will analyze the genetic interactions with other elements in the Nodal signaling pathway,” the abstract states.
“We have learned that the dosages of particular transcription factors during precise spatial and temporal windows during cardiogenesis are critical for normal development and maintenance of the cardiac electrical system. How these dosages are regulated has not yet been explored.”
Since miR-430 is the zebrafish homolog of the human miRNAs miR-17 and miR-372, Giraldez expects that pinpointing miR-430’s targets and functions in a vertebrate model system will “provide the necessary context for understanding its roles in humans and learning how their dysfunction might cause human birth defects and contribute to disease.”
Giraldez’s grant began on Aug. 1 and runs through May 31, 2012. It is worth $310,278 in its first year.
The third grant was awarded by the National Heart, Lung, and Blood Institute to Vasanth Vedantham, a postdoctoral fellow at the University of California, San Francisco, to uncover whether miRNAs play a role in the cardiac conduction system of mammals.
“We have learned that the dosages of particular transcription factors during precise spatial and temporal windows during cardiogenesis are critical for normal development and maintenance of the cardiac electrical system,” Vedantham wrote in the grant’s abstract. However, “how these dosages are regulated has not yet been explored.”
Recent work suggesting that miRNAs regulate proliferating ventricular cardiomyocytes during cardiogenesis by silencing a key transcription factor “raises the intriguing possibility that such translational control is equally important in conduction system development,” he wrote in the abstract.
To investigate this possibility, Vedantham aims to eliminate miRNAs from the developing conduction systemusing a tissue-specific deletion of Dicer in order to determine if the non-coding RNAs are needed for normal conduction system development.
He will then perform a microarray analysis of isolated conduction system cells and validate promising miRNA candidates in vivo using in situ hybridization.
Finally, Vedantham will computationally generate a list of the downstream targets of conduction system-specific miRNAs, which will themselves be validated in culture and in vivo using transgenic mice over-expressing the miRNAs.
“This work seeks to unveil novel mechanisms of the molecular regulation of growth, patterning, and maintenance of phenotype in the mammalian cardiac conduction system,” the abstract states. “Understanding how this complex and elegant system is controlled at the molecular level may eventually permit targeted therapies for the millions of patients suffering from disorders of the cardiac electrical system.”
The project began on Aug. 1 and is set to run through July 31, 2009. It is worth $54,842 in its first year.