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Q&A: University of Iowa's Paul McCray on Cystic Fibrosis and miR-138


Paul McCray


Professor, allergy/pulmonary division, microbiology, internal medicine, University of Iowa College of Medicine

• Associate professor, allergy/pulmonary division, University of Iowa College of Medicine — 1996-2001
• Assistant professor, allergy/pulmonary division, University of Iowa College of Medicine — 1991-1996
• Associate pulmonologist, Childen's Hospital Oakland Research Institute — 1988-1991
• MD, University of Iowa — 1981
• BA, biology, Saint Olaf College — 1976

Researchers from the University of Iowa this month reported on the identification of a microRNA network that controls the expression and synthesis of a key gene involved in cystic fibrosis.

According to a paper published in the Proceedings of the National Academy of Sciences, this miRNA-regulated network “directs gene expression from the chromosome to the cell membrane, indicating that an individual miRNA can control a cellular process more broadly than recognized previously. This discovery also provides therapeutic avenues for restoring ... function to cells affected by the most common cystic fibrosis mutation.”

This week, Gene Silencing News spoke with Paul McCray, the paper's senior author, about the findings.

Let's start with some background on you and your lab. Have you been working with microRNAs for a while?

Our lab is interested in cystic fibrosis and airway epithelial cell biology. We developed an interest in microRNAs because they haven't been well studied in the respiratory tract, and we thought they might be involved in host responses to infection and … in the regulation of key cellular processes. We're particularly interested in electrolyte transport because that is a mechanism for regulating the volume and composition of airway secretions, and it is in that physiology where the [cystic fibrosis transmembrane conductance regulator, or CFTR] gene plays a role — that's the gene that is defective in cystic fibrosis.

You mentioned microRNAs haven't been well explored in your area of interest, and the paper focuses on one in particular, miR-138. Going into this work, had you any clues that this microRNA would be the focus?

We had decided upfront that we were interested in identifying microRNAs that might directly or indirectly regulate CFTR. The traditional approach would be to look at the 3' UTR of CFTR for predicted candidates that might directly regulate [the gene]. We have some ongoing work in that area, but the graduate student who had been working with me on the project — Shyam Ramachandran, who is the first author on the PNAS paper — posited a possible relationship between miR-138 and CFTR.

[This microRNA] was one of the abundant microRNAs in airway epithelia based on qPCR, and if you look at the predicted targets for miR-138, the highly conserved target across species is ... a transcriptional repressor protein called SIN3A. Shyam remembered that SIN3A can bind to a DNA-binding protein called CTCF. He further recalled that the CFTR promoter has CTCF binding sites, some of which seem to be involved in transcriptional regulation.

No one has ever said SIN3A has anything to do with regulating CFTR gene expression, but [Ramachandran] thought we should look at it. That's how the project started.

With that background, can you give an overview of the experiments you conducted and what you found?

The first thing we did was validate the interaction between miR-138 and the SIN3A gene product. Once we had confirmed that, we focused on normal human respiratory epithelia. We hypothesized that miR-138 influences CFTR expression in normal cells. We found that repression of SIN3A, either using a miR-138 mimic or an siRNA, caused a very profound increase in mRNA expression for CFTR.

That was the expected finding, and it was confirmed. What was interesting beyond that was that there was a correlative increase in protein. In addition, we could measure more anion transport across the cells. So the whole system was enhanced in terms of CFTR mRNA, protein, and functional expression.

In considering the effect on protein expression, we wondered if it was possible that miR-138 was doing more than just increasing the total abundance of the mRNA, which in turn lead to more protein and more function. We next used a cell line that had a tagged CFTR gene product expressed under the control of the CMV promoter. This allowed us to investigate the effects of miR-138 on CFTR protein expression independent of any effects on the transcription of CFTR.

In that setting, we found that when we knocked down SIN3A expression, there was more CFTR protein at the cell surface. This [suggested] that the miR-138/SIN3A interaction was perhaps regulating a network of gene products that affected the post-translational protein processing. CFTR is a transmembrane protein that undergoes extensive post-translational modifications, beginning with core glycosylation and then additional glycosylation to yield a fully functional membrane channel. Our conclusion from that experiment was that this interaction is regulating something that is more complex than just simply relieving a transcriptional repression.

After we saw the increase in protein in the wild-type CFTR setting, we started wondering what would happen if we used an intervention like this in cells that had the most common mutation associated with cystic fibrosis — the delta-F508 mutation.

Similarly to the wild-type cells, we found that in [cystic fibrosis] cells, there was more transcription of the mRNA when we knocked down SIN3A. But a really exciting finding of this work was that we saw partial rescue of the mutant protein, so that some of the delta-F508 protein in treated cells reached its fully glycosylated state and formed a functional channel at the cell surface that we could measure. The real question now in terms of possible therapeutics is trying to understand the mechanism for this result.

SIN3A is a transcriptional repressor, generally, so CFTR is not the only gene product that it regulates. One possibility is that there are other SIN3A regulated genes involved in CFTR biosynthesis, such that when repression is relieved, we are affecting a network of genes influencing in the post-translational maturation of this protein.

Another, speculative, way to think about it is that if the cell needs more CFTR protein, through regulation by miR-138, it not only enhances gene transcription, but it affects downstream biosynthetic nodes that are important for fully processing the protein to its mature state.

We have performed some microarray expression studies in which we inhibited SIN3A expression in airway epithelia, and then looked for differential gene expression. These studies show that there are a number of genes known to be associated with CFTR and involved in its biosynthetic maturation that were differentially regulated when you knock down SIN3A. This result suggests a SIN3A regulated gene network influences CFTR biosynthesis.

In addition, there were a lot of genes changing that are currently not known to have anything to do with CFTR protein maturation, and we may be able to mine these data to learn more.

What about the therapeutic potential of this work?

The first thought would be [to] use an siRNA against SIN3A or a miRNA mimic as a [cystic fibrosis] therapy. There may be some possibilities there, but currently, efficient delivery of siRNA oligos to the respiratory tract is difficult. It would require further advances in delivery technology to achieve efficiency with that approach. At the same time, since the SIN3A protein is involved in the regulation of hundreds of genes, it might not be the best gene target because it's not CFTR-specific.

A subset of genes in this network may represent better targets for new therapies. We'd definitely be interested in working with collaborators or pharmaceutical partners to try to manipulate this gene network therapeutically.