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New Research Contradicts Reported Uptake of Dietary Plant microRNAs

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Two independent research groups this month published data indicating that plant microRNAs are not taken up into animals when ingested in concentrations sufficient to regulate gene expression, contrary to a highly publicized report from last year.

While additional work in larger animal populations is required to bear out the latest findings, they suggest that some technical artifact or contamination issue may have been behind the original study, according to Kenneth Witwer, a Johns Hopkins School of Medicine researcher and lead author of one of the new papers.

In early 2012, a team from Nanjing University reported in Cell Research that exogenous plant miRNAs were found in the blood of Chinese individuals. Among these was miR-168, which is highly abundant in rice and is capable of inhibiting low-density lipoprotein receptor adapter protein 1 in the liver and decreasing low-density lipoprotein clearance.

The investigators concluded that exogenous rice miRNAs were passing through the gut of the individuals consuming them and regulating target genes, adding another layer to the debate over the safety of genetically modified foods in light of the efforts of a number of companies to develop crops with altered miRNA profiles.

To Witwer and others researching miRNAs as potential therapeutics, however, the data coming out of Nanjing University were intriguing because they pointed to the possibility of an oral route of delivery of drugs containing the small, non-coding RNAs.

Already there had been indications that small RNAs could be transferred through the gut, including a 2009 report from University of Massachusetts researchers who showed that siRNAs could be encapsulated and delivered into macrophages following oral administration.

Still, the Cell Research paper raised some red flags, Witwer told Gene Silencing News. Firstly, there was significant variation between the levels of miRNAs in the pools of serum that were tested.

“These individuals are all said to have rice being the staple component of their diet,” he said. “We would not expect to see such great inter-individual variation when we’re using a group of ten people per pool,” indicating that an issue such as sample contamination could have influenced the data.

Additional doubt about the Cell Research findings arose in light of a 2012 BMC Genomics paper published by a group at Monsanto, who conducted an analysis of plant miRNA sequences in various mammals, chickens, and insects in order to determine how widespread the passage of plant miRNAs through animals’ gastrointestinal tracts is.

They found that while different plant miRNAs were present in the animals, miR-168 was “extremely over-represented. Furthermore, all or nearly all miR-168 sequences were monocot-derived for most datasets, including datasets for two insects reared on dicot plants in their respective experiments.”

In the end, the Monsanto team determined that the plant miRNAs observed in animals may be “artifactual due to sequencing methodology, and that accumulation of plant miRNAs via diet is not a common faculty among animals.”

To Witwer, the fact that plant miR-168 was appearing most frequently in animals while other miRNAs were detected inconsistently “didn’t make a whole lot of sense … if the transfer of plant microRNAs from the diet is a general mechanism.”

“We were still excited about this possibility of the transfer of dietary microRNAs, so we thought we’d do this simple experiment,” he said of his work, which appeared in RNA Biology.

Witwer and his colleagues conducted a feeding experiment wherein blood was drawn from two pigtailed macaques, which were then fed a commercially available, plant miRNA-rich fruit and protein shake. Blood was then drawn 1, 4, and 12 hours after feeding, and plant and endogenous miRNAs were measured in all the blood samples using RT-qPCR.

“What we found was that there was no consistent difference between what we were seeing before the feeding and at any time point after the feeding,” Witwer said. “That seemed to us to be indicative of a spurious amplification” — a conclusion reinforced by the researchers’ failure to see amplification “until very late” in the cycles and “great variability” in some of the PCR wells.

“All of this is consistent with what we would expect for a target that is either at extremely low concentration or is not present at all, and [if] what we’re seeing is, [for instance], background amplification of some endogenous material that is somehow binding to the primers or probe,” he added.

In order to further examine the potential for extremely low copy numbers of plant miRNA in animal plasma, as well as specificity, Witwer and his team employed droplet digital PCR.

Using that technology, “what we would expect to see for a specific amplification would be a fluorescence intensity level that is approximately the same for all of the droplets that contain amplified material,” he explained.

For spurious amplification, on the other hand, different levels of intensity would be observed in different droplets. “And that is, indeed, what we saw,” he noted.

Witwer’s group wrote in their paper that their use of only two primates allow for the possibility that both animals were “coincidentally deficient in a hypothetical pathway necessary for dietary miRNA uptake,” they noted that they observed same specificity problems in larger numbers of plasma samples from mouse models.

Further reinforcing the findings of Witwer et al. were the results of experiments from a research team led by Brigham and Women’s Hospital clinician Stephen Chan, which tested the delivery of diet-derived miRNAs in humans, mice, and honeybees.

Chan and his colleagues identified “substantial” levels of specific plant miRNAs in fruit — miR-156a, miR-159a, and miR-169a — but found that a cohort of healthy athletes did not carry the miRNAs after ingesting the food, according to their paper, which also appeared in RNA Biology.

“Similarly, despite consumption of a diet with animal fat replete in endogenous miR-21, negligible expression of miR-21 in plasma or organ tissue was observed in miR-21−/− recipient mice,” they wrote. “Correspondingly, when fed vegetarian diets containing the above plant microRNAs, wild-type recipient mice expressed insignificant levels of these microRNAs.”

Lastly, despite the oral uptake of pollen containing plant miRNAs, “negligible delivery” of the small RNAs was observed in recipient honeybees, they added. Taken together, these findings indicate that “horizontal delivery of microRNAs via typical dietary ingestion is neither a robust nor a frequent mechanism to maintain steady-state microRNA levels in a variety of model animal organisms.”

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