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Northwestern's Lavker and Ryan on the Role Of miRNAs in Corneal Epithelial Stem Cells

Name: Robert Lavker
Title: professor, dermatology, Northwestern University
Background: Professor, dermatology, University of Pennsylvania — 1994-2002
Research professor, dermatology, University of Pennsylvania — 1989-1994
Associate research professor, dermatology, University of Pennsylvania — 1983-1989
Assistant research professor, dermatology/anatomy, Boston University School of Medicine — 1970-1976
Postdoc, biology, Boston University — 1969
PhD, nutrition/biochemistry, Clemson University — 1968
MS, nutrition, Clemson University — 1966
BS, agriculture, University of Delaware — 1963

Name: David Ryan
Title: Research assistant professor, dermatology, Northwestern University
Background: Postdoc, stem cell genetics, University of Pennsylvania — 1999-2002
Postdoc, molecular enzymology, University of Alberta — 1995-1996
Postdoc, developmental biology, Hospital for Sick Children, Toronto — 1995-1996
PhD, biochemistry, University of Alberta — 1994
MSc, genetics, University College of Dublin — 1985
BSc, industrial microbiology, University College of Dublin — 1983
BSc, chemistry/pharmacology/microbiology, University College of Dublin — 1981

At Northwestern, Robert Lavker and his colleague David Ryan investigate how keratinizing tissues proliferate and differentiate, and how aging affects the epidermis and dermis.
With the growth of the microRNA field, the researchers have begun exploring the role these small, non-coding RNAs may play in epithelial stem cells. Last year, Lavker was awarded a National Institutes of Health R21 grant to characterize miRNAs in corneal epithelial stem cells, as well as to determine how miRNAs may regulate stem cell proliferation and differentiation.
This week, RNAi News spoke with Lavker and Ryan about the work.
Let’s start with an overview of the lab.
RL: Our laboratory has classically been interested in the biology of epithelial stem cells. Even though we’re in the dermatology department, we don’t limit ourselves to skin or the epidermis — we have experience looking at stem cells in the eye and the hair follicle. We’ve also looked at other tissues, so we’re pretty eclectic.
Because microRNAs have been proposed to be involved with cell fate and developmental biology, as well as some stem cell decisions, it was a natural that we started to look at microRNAs and the way they might impact stem cell biology.
When did microRNAs really start coming into the research. Is [the NIH grant project] the first effort involving these non-coding RNAs?
RL: I think we started about a year and a half ago in July. David was aware of microRNAs, as I was, in sort of general terms when we started to do a literature search to see what was done on microRNAs with respect to corneal epithelium, which is one of the tissues that we study. We found there was nothing done [in this area, and] when we looked at what was done on the epidermis at that time, [we found that] nothing was done. In fact, very little was done on any stratified squamous epithelial tissues when we started looking in the summer of 2005.
So we decided, because this was so relatively new to the epithelial biology field, that we would try to get some sense about at least the distribution of microRNAs in some of those tissues and apply for an NIH R21 grant, which is a kind of a kite-flying, exploratory grant.
Can you give a little background on how you did that initial research?
DR: The first approach we took was to use whatever available information was actually out there on the microRNAs that were in the registry and those that had been published — in particular, one or two papers … regarding the cloning of microRNAs from the [Tom] Tuschl lab [at Rockefeller University].
In one paper, he had conducted an experiment where he looked at a variety of different tissues and it appeared that some of the microRNAs [in those tissues] were, at least from the cloning point of view, exclusively expressed in ocular tissue. So we selected those as good candidates for study to do better characterization of the particular ocular tissues they were expressed in.
Another good paper used when working out our strategy was a paper that came out of a group [headed by Ronald Plasterk at the Hubrecht Laboratory] in Holland that is still credited with developing the in situ hybridization technology for looking at microRNAs and where they are expressed in the tissue. … In his set of experiments, he identified ocularly expressed microRNAs. We selected those for study [as well as a few others].
At the time, in June, we had constraints on the time we had to put together enough data for our R21. So we essentially … [took] a bunch of microRNAs from various ocular tissues, and epithelial tissues that were related to the ocular tissues, and [did] a Northern characterization to see which ones of those were expressed in corneal epithelium or skin epithelium.
We also, as an aside, decided to carry out in situ hybridization of mouse eyes to see where and at what layer of cells these microRNAs were expressed. One of the other components [of the work] was to do a straightforward microarray that contains all of the microRNAs on a chip so we could get a rough approximation of quantitative amounts of the various microRNAs and which ones in relation to other tissues were more preferentially expressed in corneal epithelium.
What were the key findings of this work?
RL: This [work] has recently been published in Molecular Vision. In that paper, we characterized the distribution of microRNAs in the adult mouse eye, looking at three primary portions of the eye: the anterior segment, which is composed of the corneal epithelium [and] the limbal epithelium … which also happens to be the site of the corneal epithelial stem cells; … the lens epithelium, [which is a] single layer of epithelium that is actually involved in producing the lens; and the retinal tissues.
We found that the various microRNAs had really distinct and overlapping tissue specificity. For example, we found microRNA-184, which is almost exclusively localized to the corneal epithelium and the lens epithelium — we find it in no other tissues in the body, and we surveyed a lot of tissues. So this seems to be a corneal/lens epithelial-specific microRNA, and it’s one of the [microRNAs for which] we’re trying to find targets.
We also found that there were other microRNAs that were more ubiquitous [and located] not only in the corneal epithelium but also the epidermis. Again, we validated the microRNA expression profiles using Northerns and in situ hybridization.
So the idea now is to take these microRNAs and find their targets, find out about what they do?
RL: Absolutely. I think as with most individuals who are in this field, knowing what microRNAs are present is a start, and understanding its distribution is also helpful, and seeing if you can modulate also is a help. But the real name of the game is what the target is. What protein is this microRNA either attenuating or obliterating.
Any thoughts on the long-term impact of this work? Is the idea to find targets for therapeutic intervention?
RL: Taking a pure hypothetical … if we find a microRNA that is very specific to the corneal epithelium, and it turns out that this microRNA somehow regulates or inhibits the production of factors that would allow for angiogenesis … the therapeutic impact is vast. You now have the ability to construct a drug that would inhibit angiogenesis in many other areas where angiogenesis may not be necessary. Conversely, by eliminating this microRNA, you could stimulate angiogenesis. That’s the potential pharmacological impact of all this microRNA work, not just what I’m working on.
But remember, this is a total hypothetical situation. We don’t know if this will ever come to pass.
DR: Many people are very interested in the use of RNAi technology and siRNA technology in order to modify processes that may be apparent in the cancer or other [diseases]. It seems to me that there is a great potential to use the knowledge that will be gained from looking at microRNAs, which are naturally occurring and obviously toning down certain things that would not want to be aberrantly expressed in certain cellular situations.
I can see that the microRNA field could actually be very useful for somebody who would want to take an siRNA approach, but not to have to deal with the various side effects that seem to occur in many of these siRNA experiments.
Although it’s really early days right now in the mammalian microRNA field … microRNAs seem to be very intimately associated with differentiation events [and] microRNA expression seems to be misregulated, whether directly from the genes that lead to the production of microRNAs or from the machinery that produces microRNAs in situations of tumorogenesis, cancer, [and] metastasis. It would seem to be that it would be a very obvious thing that, if we could drive the processes that regulate microRNAs in cells, we may be able to revert back some tumor situations.
Are you guys collaborating with anybody on this work, either academically or from industry?
RL: No, it’s all being done by [our lab]. That’s not to say we’re opposed to collaborations by any means, and each of us has our own long-standing collaborations with other individuals in other aspects of our work. But right now we’re sort of our own Ma-and-Pa operation.

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