Assistant professor, Assistant professor, pediatric/molecular biology and genetics, Johns Hopkins University School of Medicine
• Postdoc, Johns Hopkins University School of Medicine — 2003-2004
• MD, Johns Hopkins University School of Medicine, — 2003
• PhD, genetics, Johns Hopkins University School of Medicine — 2001
• BA, biology/genetics/development, Cornell University — 1996
Johns Hopkins researcher Joshua Mendell and colleagues have published new data showing that when a diversity of cell lines are grown to increasing density, microRNA biogenesis is globally activated, driving up levels of mature miRNAs and enhancing the suppression of their targets.
The findings, which are scheduled to appear this week in the online issue of the Proceedings of the National Academy of Sciences, "uncover a critical parameter necessary for accurate analysis of miRNAs in cell culture settings, establish a tractable system for the study of regulated miRNA biogenesis, and may provide insight into mechanisms that influence miRNA expression in physiologic and pathophysiologic states," the investigators wrote.
RNAi News spoke with Mendell this week about his research.
Let's begin with a little background on where you were coming from when you began the work detailed in the PNAS paper.
One of the major questions we've been interested in is how microRNAs regulate cellular processes that are known to be perturbed as cells are transformed and become tumorigenic.
We know from our work and the work of many labs that microRNAs can be potent regulators of the cell cycle and proliferation. So we were interested in knowing whether microRNAs might participate in the potent inhibition of cell proliferation that occurs in primary cells when they make contact [with each other], a phenomenon called contact inhibition.
We designed a very simple experiment to see if the expression of individual microRNAs was altered as cells undergo contact inhibition … [using] primary human fibroblasts and mouse 3T3 cells, which are non-transformed mouse fibroblasts. Both these cell lines undergo contact inhibition, so when you grow them to high density in cell culture, they stop proliferating, and we simply compared microRNA expression in the cell lines when grown to high density and low density. What was surprising to us was that we observed that most microRNAs were up-regulated when cells were grown to high density.
As a control in that experiment, we had also profiled microRNA expression in a transformed cancer cell line that does not exhibit contact inhibition: HeLa cells. What we were even more surprised about was that HeLa cells also show the same very wide spread up-regulation of most microRNAs when the cells were grown to high density.
So this phenomenon didn't seem linked to contact inhibition, but rather … growth of cells in a high-density state.
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Most microRNAs were up-regulated, but there were some that were down-regulated?
Very few were down-regulated; just a handful, while dozens and dozens, if not more, were up-regulated.
What might be going on here?
We were obviously very interested in determining that. There were three major lines of investigation that we initiated from that initial observation. One was a sort of practical consideration, [postulating that] if this is a widespread phenomenon, i.e., that microRNA expression levels globally are intimately linked to the density of cells grown in culture, then it is important for the research community to be aware of this because this is a major confounding issue in experiments that study microRNA function and expression in cell lines.
So any experiment that assays microRNA expression, any experiment that looks at microRNA targets or reporters that have microRNA target sites, can be affected simply by how dense the cells are grown in culture. Most people don't let their cells get incredibly dense when they do these types of experiments, but … there is not a uniform way of doing [these studies] across all labs or even within labs. So this could be a major confounding issue.
Was this a slow increase in up-regulation, or did you hit a critical mass [of cell density] and all of a sudden saw it?
It's a little bit of both, actually. We definitely see a gradual increase as cells get denser in culture, and that's probably because even if cells are growing at a sub-confluent density, there is … a distribution of the local concentration of cells. So even in cell cultures that are not confluent, there are going to be some areas where … some contact is going on. And the denser the cells get, the more frequent that is. So we definitely see a gradient, and as [the cells] reach complete confluency, there seems to be an even bigger boost.
To see how widespread this effect was, to see whether this was just an artifact of the specific cell lines we initially looked at, we [examined] a larger panel of different … human and mouse cell lines derived from different sources … and all showed this effect [except for human embryonic kidney] 293 cells … and we don't understand why that particular cell line seems to be resistant to these effects.
But [the effect] seems to be extremely widespread and we showed that it occurs even in a Drosophila cell line. … That tells us that it is a fundamental property of animal cells and is shared by very divergent lineages.
The second line of investigation we've begun exploring — and I don't think we've fully answered this — asks, "What is the trigger for this global up-regulation of microRNA biogenesis?"
In cells that are grown to high density, there are a couple of things that could be happening. Even if the cell line doesn't undergo contact inhibition, like a cancer cell line, the proliferation rate might still slow down as [the cells] get very dense, and microRNA biogenesis might be linked to the proliferation rate, not contact.
We grew [HeLa cells] to full density and far beyond, and we found that there was no measurable slowing of proliferation when they get confluent, but right when they make contact we saw the switch where microRNA levels are globally up-regulated. We also arrested cell proliferation by other mechanisms such as serum starvation … and that did not affect microRNA levels. So [the phenomenon] does not seem to be due to the proliferation rate of cells.
We also assessed whether the cells secrete a signaling molecule of some kind when get very dense. Media was transferred from confluent cultures of cells to sub-confluent cultures. This didn't cause any change in microRNA expression indicating that [the up-regulation] is unlikely to be due to a secreted factor.
So it looks like it is really [due to] the cell-cell contact that is occurring as cells are grown at high density. Consistent with that, when we look at cells growing in suspension that don't form stable cell-cell contacts, those cell lines seem to be resistant to the effect.
The final line of investigation that we addressed was the molecular mechanism of up-regulation of the microRNAs. We assessed whether transcription of microRNAs is affected by cell density by measuring the levels of the primary transcripts, and we looked at later steps in [microRNA] biogenesis.
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We found that microRNA transcription was unchanged, but later steps of their biogenesis were enhanced. Specifically, the Drosha-processing step — the first step in microRNA biogenesis — seemed to be enhanced, and the loading of processed microRNAs into the RISC machinery also seemed to be enhanced when cells make contact. So it's a post-transcriptional effect.
Scott Hammond [at the University of North Carolina at Chapel Hill] has shown that during early mouse development … the biogenesis of many microRNAs is inefficient, and as development proceeds there seems to be a switch that occurs which leads to greater processing efficiency. There is some evidence that in certain tumor cells, there is a reversal of that process leading to less efficient microRNA biogenesis.
One of the challenges in elucidating the mechanisms that underlie these effects has been that there is not a simple, tractable system in which one can study regulation of the activity of the microRNA biogenesis machinery. Now, this phenomenon that we have documented in these cell lines will allow us to study the microRNA biogenesis machinery and its regulation in the same cells just by inducing this switch.
We don't understand why there is a system in place to link cell-cell contact to global microRNA biogenesis. But I think what we are observing is mechanistically related to the similar effects that have been observed during development and in cancer cells. Importantly, it has been shown that global down-regulation of microRNAs in cancer cells can actually enhance tumorigenesis.
I think that if we can understand the mechanisms through which cells globally regulate microRNA biogenesis, we might be able to reverse those effects in tumors for therapeutic benefit.
We have spent a lot of time functionally studying individual microRNAs, and we know from our work and others' that if we restore the expression of even one microRNA in a tumor where it is lost, it can have very potent anti-tumorigenic effects. So we think that if we could restore global microRNA expression in tumors where microRNA expression is globally reduced, that could have an even more powerful [impact] … and in the future could lead to new ways to attack tumor cells therapeutically.
The work in PNAS was done in cell culture, but are there any in vivo situations where this phenomenon might be at work?
There is an interesting observation that has been made in the past that we think could be relevant to this. When one looks at … tissues versus cell lines, there is generally higher microRNA abundance in tissues. For example, if you isolate total RNA from liver, in general there seems to be more microRNAs per unit of total RNA than in a cell line derived from that tissue.
This has been a phenomenon that has been talked about in the field, and we've certainly encountered it in our own research … but the mechanistic basis for this has been mysterious. We think that the phenomenon we have now described in cell culture could be at least one reason why there is this difference.
In tissue, cells elaborate a lot of cell-cell contact, obviously. … So it's likely this pathway is operative in cells in tissue and is leading to more efficient microRNA biogenesis. And we're designing experiments now to test that idea directly.