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Q&A: ISB's David Galas Discusses Extracellular microRNAs

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galas.jpgName:
David Galas

Position:
Professor/senior vice president of strategic partnerships, Institute for Systems Biology

Background:
• CSO, life sciences, Battelle Memorial Institute — 2005-2008
• Chancellor/CSO, Keck Graduate Institute of Applied Life Sciences — 2002-2005
• President/CSO, Chiroscience R&D — 1997-1998
• PhD, physics, University of California, Davis-Livermore — 1972
• MS, physics, University of California, Davis-Livermore — 1968

A team of researchers from the Institute for Systems Biology last week reported in Nucleic Acids Research on the discovery that serum deprivation of various human cell lines triggered the export of a "substantial amount" of microRNAs into the culture medium.

The investigators also identified a number of proteins that may be involved in the export system, including one that appears to play a role in protecting the miRNAs from degradation outside of the cell.

The report comes on the heels of a publication from a different group, which showed that cells can selectively package microRNAs in microvesicles, which are actively secreted and trigger target gene inhibition in recipient cells (GSN 7/8/2010).

This week, Gene Silencing News spoke with David Galas, the study's lead author, about the findings.

How about a little background on you and the focus of your research?

I've been at the Institute for Systems Biology for about five years now. I was educated as a physicist, but I have done a lot of [different] things — mostly in academia, but I did spend three and a half years in Washington … [taking] charge of the [Department of Energy's] biological and environmental research, which included the Human Genome Project. I've done a lot of human genetics over the past 20 years, as well.

When did microRNAs come onto your radar, and how did this work come about?

I've been interested for a long time in understanding complex networks, particularly the regulatory and dynamic aspects of these networks and how they relate to phenotypes, and ultimately how to connect it with genetics.

When I began reading about microRNAs, I guess about five years ago, I came to the realization that there is no way we can understand these networks without including microRNAs. At that time, that was extremely difficult to do, and it's still very hard to understand how they fit into regulatory networks. That was my first blush of trying to understand the importance of microRNAs to systems biology.

The second thing was that I started collaborating with … [Ohio State University researcher] Clay Marsh on pulmonary disease. He pointed out that there were microRNAs in microvesicles that you found floating around in the blood. We started looking in plasma and realized that there are all kinds of the microRNAs in plasma. And several other labs were, of course, finding the same thing.

After looking at that for a very short period of time, I realized that there are fundamental biological questions: how did they get there and what are they doing? But just trying to understand how they got there was the motivation for this study.

We began studying cells in culture to see if we could see microRNAs moving outside of the cell under various conditions. The study that was written up here was the result.

Can you provide an overview of the experiments and what you found?

What we were really interested in was whether or not microRNAs [are] actively exported [out of cells] in any way separate from the obvious way in which they could [be released] … by lysing the cells. The question was: would intact, healthy cells export microRNAs? I worked on this closely with Kai Wang, a senior scientist in my lab.

We cultured a number of different cell types. Because fetal calf serum contains a lot of bovine microRNAs that sometimes interfered with our ability to accurately measure microRNA export, we stumbled across the idea of using serum deprivation as a way of shocking the cells and having them change states, thereby [enabling us to] see if we got a specific response, including microRNA export.

The first thing we did was look at cultured cells in the medium to see if there were different profiles inside and outside the cells — and we did. That was more of a qualitative answer to the question. Were the cells lysing? We looked very carefully and used a number of methods to be sure they weren't. Our conclusion was that microRNAs must be exported.

To go about studying this, we moved to the serum-deprivation system. That has a real advantage in that it allows you to study and measure microRNAs in medium that has absolutely no other microRNAs present, and therefore you can get a much more accurate picture. We were able to look at the dynamics after replacing serum-supplemented medium with serum-free medium with these different cell types.

Basically, we saw that after serum deprivation, you got a rapid export of different microRNAs — not the same microRNAs that were inside the cell; it's not just a reflection of the profile inside the cell. Surprisingly, we noticed that when you cultured different cell lines, you saw different microRNAs being preferentially exported.

These are all hints of a much more complex system that people had not studied yet. Since microRNAs are a really natural way of moving information around, the idea that had been floated in the literature in several previous papers … [is that] microRNAs are actually used for cell-cell communication, at least in some cases.

Are there plans to follow up on these findings in your lab?

We'll follow up the in vitro work by trying to characterize the protein complex [involved in the export process]. The evidence is reasonably strong that microRNAs are present outside of the cells in nucleoprotein complexes. What that means is that characterizing that complex may give us some hints about how the export pathway works.

Then, there is just an enormous number of interesting questions: How are microRNAs targeted for export, what does that system look like, and — probably one of the most important ones from the point of view of understanding if there is information transfer between cells — are they taken up by other cells? We know that they can be taken up if they are present inside various types of lipid vesicles … but we have no evidence yet that nucleoprotein complexes can be taken up.

Then, [there are] questions like: what does the uptake system look like [and] what is actually being signaled by microRNAs in a biological situation? All of these questions will require years of interesting work.

But the first thing we're going to focus on is trying to understand the biochemistry of the in vitro system a little better.

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