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Cell-Cell Contact Leads to Global Rise in MicroRNA Levels

NEW YORK (GenomeWeb News) – MicroRNA production is ramped up in response to cell-cell contact, even in cells that fail to arrest at confluence, according to a new study scheduled to appear online this week in the Proceedings of the National Academy of Sciences.

Johns Hopkins University researchers used custom microarrays to evaluate miRNA levels in several normal and cancerous cell types under different growth densities. Unexpectedly, almost all cell types exhibited global miRNA up-regulation following cell-cell contact. This did not seem to be caused by increased transcription of miRNA sequences. Instead, the team found evidence that some components of the miRNA pathway become more efficient when cells are grown at a higher density.

That may not only provide new details about the underlying biology of this system, but also gives researchers a relatively straightforward way to manipulate the effectiveness of miRNA systems in cell cultures.

"From a practical standpoint, we obviously think this phenomenon is important for the miRNA field to be aware of," senior author Joshua Mendell, a molecular biology and genetics researcher at Johns Hopkins University's Institute of Genetic Medicine, told GenomeWeb Daily News. Beyond that, though, Mendell said the finding may offer a new way of directly controlling a cell's overall miRNA levels using cell density.

Contact between cells usually leads to a cell cycle arrest that prevents an overgrowth of cells. In contrast, cancer cells often lack this so-called contact inhibition. "The loss of contact inhibition is thought to be a hallmark of cancer cells," Mendell said.

Because Mendell and his colleagues are interested in both the regulation of miRNAs and miRNAs abnormalities associated with diseases such as cancer, they decided to investigate whether miRNAs have a role in this process.

Using CombiMatrix custom microarrays, the researchers profiled the levels of roughly 500 miRNAs in three cell lines grown at high density: non-transformed human skin cells and transformed mouse fibroblasts — which arrest in response to contact between cells — as well as HeLa cells, cancerous cells lacking contact inhibition.

Even though HeLa cells don't undergo contact inhibition, they showed similar miRNA patterns following cell-cell contact as normal cells: the levels of nearly all the miRNAs tested increased following contact, regardless of whether cells arrested. Similarly, when the researchers plated cells at higher and higher densities, they found that miRNA levels went up as cell density increased.

A luciferase reporter assay suggested that these highly expressed miRNAs are active in confluent cells, though the team has not yet looked at whether the miRNA accumulation affects global messenger RNA levels.

Other cell types showed similar patterns. When the researchers looked at five more mammalian cell lines, including human breast, colorectal, and pancreatic cancers, a mouse kidney cancer line, and a human embryonic kidney line, they found that all but one cell line — the human embryonic kidney line — had increased levels of miRNA following cell-cell contact. Drosophila cells also showed this pattern.

"Virtually all cells we looked at showed this effect very robustly," Mendell said.

The team has been trying to determine what signals in cell culture cause this global miRNA up-regulation. So far they have ruled out the possibility that cells secrete something that spurs miRNA accumulation, since growth media from dense cell cultures did not cause this accumulation in cells that weren't in contact with one another.

Instead, Mendell said, it seems as though the physical contact between cells causes the miRNA phenomenon.

Next, the team looked very carefully at each step of the miRNA biogenesis and processing pathway — from transcription to Drosha and Dicer processing to loading on the RNA-induced silencing complex, or RISC, where miRNAs and their target mRNAs are brought together, to miRNA degradation.

While the researchers did not see any changes in primary transcript levels, they found that cell-cell contact increased the efficiency of both Drosha processing and miRNA loading into RISC.

In the future, the team is interested in unraveling the signaling pathways that drive this increased miRNA efficiency. For instance, since some research suggests a global down-regulation in miRNA biogenesis can promote tumor formation, Mendell said, learning how to reverse that process could potentially restore miRNA deficiencies in cancer cells.

Since cell-cell contact appears to easily convert cells from an inefficient to efficient miRNA state, Mendell added, the team plans to exploit this system experimentally.

"These findings reveal a previously unidentified link between cell-cell contact and miRNA expression, establishing a tractable system to study the global regulation of miRNA biogenesis and providing insight into mechanisms that shape miRNA expression patterns during development and in disease," Mendell and his co-authors wrote.

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