A team led by Emory University researchers this week published new data showing that a specific microRNA plays a key role in the development of atherosclerosis in a mouse model of the disease, and that inhibiting its human homolog may have therapeutic potential.
Notably, this miRNA is produced through a non-canonical mechanism that also exists in human cells, adding a new facet to the overall understanding of how the small, non-coding RNAs are created and function.
Atherosclerosis is characterized by the thickening of artery walls due to the buildup of calcium and fats, and preferentially occurs in areas of disturbed blood flow, namely arterial regions with curves or branching points such as the heart's coronary arteries.
With a focus on the disease, Emory's Hanjoong Jo and colleagues previously developed a mouse model in which atherosclerosis can be induced by partially ligating one of the carotid arteries.
Using this model, the researchers showed in 2009 that disturbed flow leads to upregulation of proatherogenic genes, downregulation of antiatherogenic genes, endothelial dysfunction, and atherosclerosis. Following up on this work, they conducted a genome-wide microarray analysis of endothelial cells isolated from flow-disturbed and undisturbed carotid arteries in their model, validating a number of known mechanosensitive genes, as well as identifying many novel ones.
Given the growing evidence linking miRNAs to a broad array of biological functions, the scientists have now looked to see whether these ncRNAs are also involved in the atherosclerosis process, Jo told Gene Silencing News.
They ran miRNA array analyses on endothelial-enriched RNAs from partially ligated mouse carotids, then integrated the data from this and their previous microarray experiment, according to a paper appearing in Nature Communications. A total of 45 miRNAs were found to be altered by more than 50 percent in cells from the partially ligated carotid artery. Further analysis singled out one — miR-712 — as upregulated in vivo and under blood flow conditions in vitro.
An in silico analysis pointed to tissue inhibitor of metalloproteinase 3 (TIMP3) as a direct target of the miRNA. Further work showed that miR-712 expression cut levels of TIMP3 in endothelial cells, which activated downstream matrix metalloproteinases, as well as a disintegrin and metalloproteases, and stimulated pro-atherogenic responses, endothelial inflammation, and permeability.
To test the therapeutic potential of miR-712, the investigators delivered locked nucleic acids designed to inhibit the miRNA into the arterial endothelium of mice. The treatment suppressed miR-712 expression compared with control LNAs, and reversed TIMP3 downregulation in partially ligated carotid arteries. Most importantly, miR-712 antagonism prevented atherosclerosis from developing in the animal models.
It was still unclear, however, how the miRNA was produced, and a nucleotide sequence search showed that the pre-miR-712 sequence matched a region called internal transcribed spacer region 2 of the murine pre-ribosomal RNA gene called RN45S, which gives rise to three housekeeping genes.
"Normally, when these three genes are made, the intervening [spacer] sequences are all degraded" by the exonuclease XRN1, Jo explained. "But under bad flow conditions, XRN1 enzyme levels drop," leading to the production of miR-712 through a still-unknown process.
Importantly, knockdown of XRN1 in vitro increased expression of miR-712, while inhibition of the canonical miRNA processor DGCR8 had no effect on levels of the miRNA — all of which suggest that it is produced through a "completely different mechanism" than most other miRNAs.
Curious as to whether this mechanism was specific to murine cells, the investigators examined the human RN45S gene for putative miRNAs using a computational prediction program, and found that miR-663 could be derived from the gene.
"Interestingly, like miR-712, miR-663 was previously identified from a microarray study as one of the most shear-sensitive miRNAs in human endothelial cells and was shown to induce endothelial inflammation, a key atherogenic step, raising a possibility that they share a common biogenesis pathway and roles in endothelial function," they wrote in Nature Communications.
Consistent with this notion, they found that XRN1 expression is inhibited by the stress caused by blood flow, and that siRNA silencing of XRN1 "significantly" increased miR-663 expression in human endothelial cells.
But Jo and his team were left "scratching our heads" about the clinical importance of their findings since miR-712 is only found in mice, he said. "This forced us to look for human homologs … based on the [miRNA's] sequence, and from this search we identified miR-205."
"To validate whether miR-205 targets TIMP3, we treated [murine endothelial cells] and human aortic endothelial cells with pre-miR-205 and found that miR-205 downregulates TIMP3 in a concentration-dependent manner," the investigators noted in their paper.
In light of these data, Jo and his team are now exploring the therapeutic potential of miR-205 inhibition, and are conducting experiments in rabbit and pig models of atherosclerosis, he said.