Inhibitor Found for Family of MicroRNAs

The study of small, non-coding RNA molecules—microRNAs—has ex-ploded in recent years. Beginning with the discovery of the first miRNA in 1993, the finding that these small stretches of genetic material have the ability to turn genes on and off has resulted in an “miRNA frenzy” among cell biologists.

Progress in elucidating the biogenesis of miRNA has been rapid, and it is now known that synthesis of these genetic strands is a multistep process, involving sequential cleaving of precursor forms by specialized protein complexes. The result is an approximately 22-nucleotide molecule with the ability to silence genes, blocking their RNA transcripts and preventing protein synthesis.

Recently, there has been even greater excitement, due to findings that suggest manipulating the miRNA pathway could enable reprogramming of cells into any cell type and, perhaps, reversing cancer.

An interesting observation noted by several researchers is that although miRNA is active in most cells, there appears to be a block in the miRNA-processing pathway in embryonic stem cells and certain cancers. This was a well-recognized phenomenon in the lab of George Daley, HMS associate professor of biological chemistry and molecular pharmacology at Children’s Hospital Boston.

“We’ve been interested in the role of miRNAs in development—as embryonic stem cells differentiate into various lineages,” explained graduate student Srinivas Viswanathan. “But then we became interested in the fact that in embryonic cells you have a buildup of miRNA precursors, which are not processed to mature form. Something seemed to be inhibiting them that was going away as the cells differentiated.”

This blockage appeared to be most significant for a family of miRNAs known as let-7—a 12-member group endogenous to most developed cell types. Interestingly, however, let-7 was found to be entirely absent from embryonic stem cells as well as relatively scant in certain cancers. Until recently, it was not known what was keeping let-7 levels in these cells so low.

In a study published online Feb. 21 in Science, Daley’s team, comprising Viswanathan and Richard Gregory, HMS assistant professor of biological chemistry and molecular pharmacology at Children’s, explored this puzzling observation.

“We knew that let-7 was blocked, but there were many possible ways in which it might have been blocked,” said Gregory.

Using a variety of molecular techniques, the team set about investigating what might be inhibiting let-7 in these developmentally primitive cells. After reproducing in vitro the phenomenon that let-7 levels only increase as the cells become differentiated, the researchers showed that when developed cells were then exposed to extracts from undifferentiated cells, levels of mature let-7 dropped. This was not the case if the cells were exposed to differentiated cell extracts. It looked like something in the undifferentiated extract was specifically binding to let-7 and turning off its processing. “So then we knew we were looking for a specific factor,” said Gregory, “because prior to that, it could have been any other mechanism that was causing the block.”

Continuing the search, the team took the approach of coupling synthetic let-7 precursors to microscopic beads before incubating them with embryonic cells. The samples were then purified, eliminating everything that was not bound to the miRNA–bead complexes, and run through a mass spectrometer, which identified anything that had bound to the complexes. A list of 20 to 30 proteins emerged. “One of the interesting proteins that caught our eye immediately was one called Lin-28 because it’s been known to be highly expressed in stem cells. Many of the other RNA-binding proteins are expressed in all kinds of cells, but Lin-28 is specific to stem cells,” said Gregory. “In fact, during one of our discussions, I said, ‘If it’s not Lin-28, then I don’t know what it is.’ Biologically, it makes sense. So Lin-28 became our number one candidate.”

An interesting twist came when Lin-28 was independently identified by another lab in Wisconsin last December as a factor for reprogramming human somatic cells back into embryonic stem cells. Yet even though this lab had identified the factor, the scientists did not know how it worked. “So what our study did was bring together these different areas of research,” said Daley. “Ours is very interested in fundamental miRNA biogenesis, and the other is interested in the reprogramming phenomenon. Our study provides a mechanism for one of the factors.”

Daley’s team then went on to demonstrate that not only did Lin-28 possess the ability to block processing of all 12 members of the let-7 family in vitro, but that when Lin-28 was added to cells that do not normally express it, it can create a block of let-7 processing. What is more, when Lin-28 levels were knocked down, let-7 levels accumulated.

“We found the first miRNA inhibitor,” said Gregory, “and a selective inhibitor—it only represses the let-7 family of miRNAs.”

Aside from the reprogramming angle, there may also be a cancer angle. For years there has been speculation that in cancer there is the phenomenon of de-differentiation, in which mature cells revert to an earlier state, a kind of reprogramming. So now with increasing studies linking let-7 levels with certain cancers, Lin-28 looks like it could also be an attractive drug target for cancer therapy.

“What makes Lin-28 really exciting to the scientific community is that it’s at the crossroads between two phenomena,” Daley explained. “If your goal is to make embryonic stem cells, you’d want to find a molecule to mimic or activate Lin-28 function. On the flip side, if your concern is abrogating growth and proliferation of cancer, then you’d want to antagonize Lin-28 because then that will lead to upregulation of let-7, which will slow down cancer cell growth.

“So drugs that mimic Lin-28 would be useful for stem cell research, and drugs that antagonize Lin-28 might be anticancer agents in certain contexts,” Daley said.