University of Toronto, Canada
Title: Anti-microRNA-378a enhances wound healing process by up-regulating integrin beta-3 and vimentin
Dr. Burton Yang received his Ph.D. degree from the University of Manitoba in 1992, followed by a postdoctoral training in Manitoba Institute of Cell Biology. He moved Harvard Medical School for a second postdoctoral training in 1993. Dr. Yang took a position as a scientist in Sunnybrook Health Sciences Centre and an assistant professor at the Department of Laboratory Medicine and Pathobiology, University of Toronto in 1995. He was promoted to associate professor in 2001 and professor in 2007. Dr. Yang is currently a Career Investigator for Heart and Stroke Foundation of Ontario. Dr. Yang’s group has been working on the extracellular matrix, especially in the area of proteoglycan. Dr. Yang’s group undertakes the approaches of RNA interference including siRNAs and microRNAs to study the roles of matrix molecules in angiogenesis. Dr. Yang has developed a system to study the role of microRNA in tumor growth and angiogenesis in vitro and in vivo. Three approaches are taken to study miRNA function in transgenic mice expressing miR-17, versican 3’UTR, which can bind endogenous miRNAs and relieve mRNAs for translation, and anti-miR-378 construct.
One criterion for microRNA identifi cation is based on their conservation across species, and prediction of miRNA targets by empirical approaches using computational analysis relies on the presence of conservative mRNA 3’UTR. Because most miRNA target sites identifi ed are highly conserved across diff erent species, it is not clear whether miRNA targeting is speciesspecifi c. We aligned all 3’UTRs of fi bronectin and observed signifi cant conservation of all 20 species. Twelve miRNAs were predicted to target most fi bronectin 3’UTRs, but rodent fi bronectin showed potential binding sites for fi ve diff erent miRNAs. One of them, the miR-378, contained a complete matching seed-region for all rodent fi bronectin, which could not be found in any other species. We have previously demonstrated that expression of miR-378 promoted tumorigenesis and angiogenesis by targeting human Fus-1 and Sufu. Consistent with this result, we found that ectopic expression of miR-378 inhibited cell diff erentiation and promoted cell invasion. To understand the specifi c targeting of miR-378 on fi bronectin, we expressed miR- 378 in mouse breast cancer cells and found that overexpression of miR-378 enhanced cancer cell proliferation, migration, invasion, and colony formation, resulting in inhibition of tumor growth. Induced expression of fi bronectin produced opposite results, while silencing fi bronectin displayed similar eff ects as miR-378. To understand how miR-378 works, we generated transgenic mice expression miR-Pirate378 can not only arrest the functions of mature miRNAs by binding to them but it can also induce the “mis-processing” of the target miRNA producing a non-functional truncated miRNA. Th is approach involves generating an expression construct that produces a RNA fragment with sixteen repeat sequences. Th e construct is named miR-Pirate or microRNA-interacting RNA producing imperfect RNA and tangling endogenous miRNA. Th e transcript of the construct contained mismatches to the seed region, and thus it would not target the potential targets of the miRNA under study. Th e homology of the construct is suffi ciently high allowing the transcript to block miRNA functions. Th e functions of the construct were validated in cell cultures, in tumor formation assays, and in transgenic mice stably expressing this construct. We showed that miR-Pirate378 transgenic mice display enhanced wound healing. Expression of vimentin and 3 integrin, two important modulators of wound healing, is elevated remarkably in the transgenic mice. To explore the possibility of adopting this approach in gene therapy, we transfected cells with synthetic miR-Pirate and obtained the results we expected. Th e miR-Pirate, expressed by the construct or synthesized chemically, was found to be able to specifi cally pirate and silence a mature miRNA through its dual roles and thus could be clinically applied for miRNA intervention. Migration assays showed a greater mobility in the miR-Pirate378-transfected cells, which was due to up-regulation of vimentin and 3 integrin. Both molecules were confi rmed as targets of miR-378, and thus their expression could be rescued by miR-Pirate378. Overexpression of vimentin also contributed to fi broblast diff erentiation, and up-regulation of 3integrin was responsible for increased angiogenesis. Treatment with miR-Pirate378 conjugated to nanoparticle enhanced wound healing in mice. Th us, we have demonstrated that knockdown of miR-378 could increase the expression of its target proteins, vimentin and 3 integrin, which accelerated fi broblast migration and diff erentiation in vitro and enhanced wound healing in vivo.