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New Data Suggest Atypical microRNA Can Pass through the Gut, Stir Controversy

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This story has been updated to include comments on the validity of the published research.

NEW YORK (GenomeWeb) – Newly published research out of Nanjing University in China indicates that an atypical microRNA derived from the honeysuckle plant, miR-2911, can be used to combat influenza A viruses including H1N1.

But the study is not without controversy. Among its findings is evidence that miR-2911, which is a degradation fragment of ribosomal RNA, is able to significantly reduce H1N1 infection-associated symptoms and death in mice when delivered orally. This marks the second time the Nanjing team has published data suggesting that miRNAs can pass through the gastrointestinal tract, enter the bloodstream, and regulate target gene expression

The first report focused on miR-168a, which is highly abundant in rice and is capable of inhibiting low-density lipoprotein receptor adapter protein 1 in the liver, thereby decreasing low-density lipoprotein clearance. In their study, the Chinese researchers found the miRNA in the blood of humans and mice and surmised that it was entering their bodies from the rice they consumed.

That report, which came out in 2012, was criticized by many in the miRNA field, and a number of groups, including ones from Monsanto, Miragen Therapeutics, Johns Hopkins School of Medicine, and Brigham and Women's Hospital, published their own contradictory data.

According to Jennifer Broderick, a postdoc in the lab of Phillip Zamore at the RNA Therapeutics Institute at the University of Massachusetts Medical School, a key problem in the latest paper from the Nanjing team is its use of an outdated version of miRBase 14 to identify plant miRNAs.

"Reads from rRNAs in deep-sequencing libraries are usually discarded from further analysis when the reads are mapped to the genome and known microRNAs," she explained in an email to Gene Silencing News. The analysis by the Nanjing researchers, however, was mapped to miRNAs in miRBase 14 rather than the current miRBase 21, in which miR-2911 has been removed.

William Marshall, president and CEO of Miragen, agreed. "Whether [miR-2911] is a microRNA at all is a big question," he told Gene Silencing News.

Chen-Yu Zhang, who led the Nanjing team, noted in an email that his paper does note that miR-2911 is "atypical" as a fragment of rRNA, adding that it might better be described as a plant rRNA fragment that "directly targets flu viruses, functioning as [a] mammalian microRNA in virus-infected cells."

But to Marshall, the likelihood that miR-2911 is functioning through an miRNA mechanism is "essentially nil," based on a number of its characteristics including GC richness, which aren't found in bona fide miRNAs.

The data

As part of traditional Chinese medicine, honeysuckle has been used for thousands of years to treat the flu, with the herb typically boiled in water and the resulting decoction ingested. Yet the mechanism by which this approach is claimed to work has not been elucidated.

In light of their findings on miR-168a, Zhang and his team speculated that plant miRNAs might be responsible for honeysuckle's putative antiviral activity. Following the methods of traditional Chinese medicine, they boiled the plant in double-distilled water for 30 minutes, then analyzed it for RNA fragments.

While most miRNAs were degraded by the boiling process, one — miR-2911 — was unaffected. Noting that it is derived from rRNA and does not arising through classical miRNA biogenesis, the team thoughts that the miRNA's unique sequence — 5'-GGCCGGGGGACGGACUGG-GA-3' — may be responsible for its stability. To test this, they researchers created two mutations that swapped the 5'-GG for a 5'-AA or the 3'-GGA to 3'-AAA. Both mutants were degraded by RNase treatment, while miR-2911 remained largely intact, they wrote in a paper appearing this month in Cell Research.

The investigators then administered the honeysuckle decoction to mice via a single gavage and observed an increase in miR-2911 levels in the animals' lungs, reaching a peak at six hours after administration and returning to baseline at 12 hours.

"Most of the miR-2911 in mouse peripheral blood was detected in the fraction containing cell-derived microvesicles (MVs), implying that the [miRNA] … might initially be taken up from the GI tract, repackaged into MVs by intestinal epithelial cells, and finally secreted into the circulation," they wrote in their paper.

The Nanjing team also examined the effects of long-term administration of the honeysuckle decoction, with mice being provided either the solution or sterile water continually over three days. In treated mice, the levels of miR-2911 increased three-fold in plasma and more than eight-fold in lung tissue, whereas no changes were found in the control animals.

Higher levels of miR-2911 were also observed in the livers of treated mice, but not in the intestines or kidneys.

The researchers then examined whether they could achieve similar results with synthetic, fluorescently labeled miR-2911, which was fed to mice. Indeed, treated animals experienced an increase in levels of the miRNA in both plasma and lungs, with fluorescent dots observed in the lung tissue of the mice. Control animals experienced no changes.

Turning to the potential antiviral activity of miR-2911, the scientists bioinformatically identified several targets of the miRNA in some types of influenza A viruses, and showed in vitro that the miRNA could block the expression of two key proteins — PB2 and NS1 — involved in viral replication. In additional experimentation, they found that H1N1 viral titers were significantly decreased when infected cells were treated with synthetic miR-2911, according to the Cell Research report.

A similar inhibitory effect on H1N1 replication was achieved by transfecting total RNA extracted from honeysuckle decoction into virus-infected cells, they noted in the paper. Treatment of these cells with a miR-2911 antagonist abolished this inhibitory effect.

Aiming to test the therapeutic effects of miR-2911, the scientists administered the miRNA to mice via gavage or through their drinking water one day before infecting the animals with H1N1. The mice were then continually treated with either synthetic miR-2911 or a honeysuckle decoction for seven days after inoculation.

Influenza-infected mice experienced rapid weight loss of around 20 percent by day seven, while those receiving miR-2911 and the honeysuckle decoction were mostly protected, losing less than 10 percent of their bodyweight. This protective effect was completely abolished in mice given miR-2911 antagonists.

Meanwhile, H1N1 viral titers were high in the lungs of infected but untreated mice at days three and five, but significantly reduced in animals treated with either synthetic miR-2911 or the honeysuckle decoction. Treatment with a control non-coding RNA had no effect on viral titers, and the administration of an miR-2911 antagonist reversed the impact of the honeysuckle decoction on viral replication.

To examine the specificity of miR-2911 in vivo, the researchers created a mutant H1N1 virus, in which only the miRNA-binding sites in PB2 and NS1 were mutated, and used it to infect mice. Infection led to the same weight loss and high viral titers as wild-type H1N1, but neither synthetic miR-2911 nor the honeysuckle decoction had any effect, the Nanjing scientists reported.

The researchers lastly demonstrated that treatment with synthetic miR-2911 plus the honeysuckle decoction could significantly reduce H5N1-induced mortality in mice, as well as cut viral titers in the animals' lungs. As in the previous experiments, administration of a miR-2911 antagonist completely eliminated the effects of treatment, while treatment with a control ncRNA had no effect.

Similar results were achieved in vitro and in vivo with the recently reported H7N9 influenza virus.

According to the researchers, these findings are further evidence of the ability of certain miRNAs to pass through the GI tract of mammals and into circulation while retaining their biological activity.

"We suggest that as the first natural product to directly target [influenza A viruses], miR-2911 is the 'virological penicillin' that serves as a novel therapeutic and preventive agent against not only influenza A but potentially also other types of viruses," they concluded.

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