Did you ever hear of “dietary microRNA”? That the tiny miRNA molecules miraculously survive in cooked rice or boiled milk inside your stomach and then genetically modify you from the inside? It sounds silly, but this is peer-reviewed science, dear reader. Didn’t you know that certain miRNAs in plant decoctions can serve as mysterious healing agents, long known to the Traditional Chinese Medicine, to cure all diseases, from cancer to COVID-19? Yes, this all was published in Nature and its family journals.

Now follow Smut Clyde as he bumbles through the “controversial” science of dietary miRNAs!

To send you on the right track, a TCM recipe in Cell Press by Professor Huang-Ge Zhang on how to treat colitis:

“Grapes were washed three times with water in a plastic bucket. After the final washing, grapes with the skin removed manually were pressed in a cold room, the juice collected and diluted with cold PBS. The collected juice was differentially centrifuged and then centrifuged on a sucrose gradient to isolate and purify the exosome-like nanoparticles…”

“The ultimate goal is to learn the language in which our food speaks to us — and to discover whether miRNAs might serve as a Rosetta Stone.” Nature, 2020

As Smut Clyde awoke one morning from uneasy dreams he found himself transformed in his bed into a gigantic Blutwurst, and was swiftly taken into police custody

By Smut Clyde

Three beginnings for a narrative were good enough for Flann O’Brien and they’re good enough for me.

1. Shame on Nature for accepting a five-page compendium of lies from the Saisei Mirai chain of cancer-fraud clinics in exchange for their sponsorship and control over a Special Issue. Saisei Mirai are vile exploiters of patients, the Japanese equivalent of Tijuana medscam clinics, pimping every fraud imaginable. They also cure COVID-19 with their same worthless garbage, because of course they do.

2. “Honeysuckle decoction” is also a cure for COVID-19. ORLY? There is probably a liqueur based on honeysuckle, for no pissant European country is considered to qualify as a real nation-state until it has its own heinously pungent aperitif, but that’s not quite what Zhou et al (2020) had in mind.

Opening 2 was sponsored by the suppliers of Vana Tallinn, Riga Balsam, Gammel Dansk and Becherovka.

3. Harken to the warnings from Heinemann et al 2013. Jack Heinemann’s objections to genetically-modified wheat grew from his concerns that the elevated levels of siRNA within the grain would not only block a plant enzyme and result in slower-digesting starch, but would also disrupt the consumers’ own metabolism when the siRNA molecules are digested and absorbed to become part of the cellular machinery – ultimately transforming the consumers into social pariahs.

Exocannibalism

The unifying theme for all three beginnings is the burgeoning research topic of ‘dietary miRNA’ (which is the explanation for the culinary custom of ‘ceremonial exocannibalism‘, for when you eat the heart of a vanquished enemy you absorb their courageous miRNA, while eating the tongue of a demagogue provides you with the miRNA of oratory). Let me explain.

The first few decades of this century saw the gradual uncovering of an intra-cellular ecosystem of non-coding RNAs (with inter-cellular aspects as well). These are transcribed from chromosomal DNA in the usual way, but not to become part of the central molecular-biological pathway (DNA » RNA » protein) as codon-by-codon instructions for assembling proteins. That pathway – already complicated by the discovery of RNA viruses and reverse transcriptases – turns out be be immersed in an alphabet soup of miRNA, lncRNA, shRNA, siRNA, dsRNA, circular RNA… busily binding to and interfering with genes, ribosomes, mRNA and each other. Their interactions are a feedback loop, or enough loops for a feedback crocheted cardigan, steadying the machinery as multicellular organisms try to grow and replenish their fabric without falling off the tightrope into cancer. Who knew that the nucleus was such a flurry of unproductive activity?

In the interests of full disclosure, I will credit the RNA chapter in ‘The Life Game’ (Calder, 1973) as the source of most of my knowledge of RNA as an active participant in the cellular economy. More recently I’ve learned a little more, including the point that just like mRNA, non-coding RNAs undergo editing after being transcribed from DNA to make them functional.

So this ecosystem is the Dark Matter of molecular biology. In the final analysis it might amount to some fine-tuning of that central pathway; or it might turn out to be a disruptive revolution. Then the Nobel Prizes will rain down like manna from heaven carbonaceous chondrites, and a golden age of research employment will dawn while we develop new software tools and new non-reductive ways of thinking about its mind-stretching complexity. It’s hard to tell, as the field became a spawning ground for fabrication. By staking out territory and stressing the oncological importance of various members of the ribonucleic zoo, researchers sparked off a goldrush. Papermills descended, filling journals with cookie-cutter papers assembled according to templates from Mad-Libs titles and scrapbooked illustrations.

One early application (Frizzi & Huang 2010, by Monsanto) was GE wheat, tweaked to express more of a particular plant-specific dsRNA that hinders the expression of the Starch Branching Enzyme and ultimately makes for higher-amylose starch, slower to digest.

Now RNAs are relatively unstable molecules, which is why no-one is trying to retrieve them from dinosaur blood in the stomachs of insects trapped in Jurassic-era amber. All the same, any suggestion that miRNAs within wheat endosperm would survive a passage through human digestion, and then be conveyed intact across the intestinal lining into one’s bloodstream, calls for compelling evidence. The further suggestion that these molecules would make another leap from the bloodstream into the nuclei of a wheat-consumer’s cells, and then bind promiscuously to human chromosomes, demands even stronger evidence.

This did not assuage Heinemann’s concerns: the risk of eating miRNA-enriched wheat was simply too great. His arguments (which drew heavily on Zhang et al, 2012) were not well-regarded by other academics at the Inquiry, who were unimpressed by his reasoning and selective use of evidence: they pointed out that any plant-based diet is already an constant bombardment of random miRNAs, so one particular double-strand molecule (from modified wheat, or from doubling one’s consumption of the ordinary wheat) will be lost in the background.

Eating Information

One could argue, though, that the bombardment need not be random; in a well-ordered life-style, it could be planned. Opinion is divided on the subject. The main only proponents of the view that dietary miRNAs do involve themselves in mammalian metabolism, and are important for well-being, is Chen-Yu Zhang and his research group (at Jiangsu Engineering Research Center for microRNA Biology and Biotechnology, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University).*

As the American Association for the Advancement of Science (AAAS) informed us 10 years ago in a press release:

“…in addition to eating “materials” (in the form of carbohydrates, proteins, etc), you are also eating “information” (as different miRNAs from distinct food sources could well bear different consequences on the regulation of host physiology once taken by the host due to potential regulation of different target genes as determined by the “information” contained within the miRNA sequence), thus providing a whole new dimension to “You are what you eat”.
[…] plant miRNAs maybe the seventh “nutrient” in the food (the six others are: H2O, protein, FFA, carbohydrate, vitamins and real elements)
[…] provides evidence that plant miRNAs may represent essential functional molecules in Chinese traditional herb medicine“

By 2012 the Nanjing group had already reported finding vegetable miRNA circulating in the bloodstream of laboratory animals, traceable to the animals’ chow (or kibble, or whatever you call it). That 2012 paper advanced to the conclusion that MIR168a (from rice) actually took part in metabolic regulation, lowering LDL in mice and reducing their risk of dying from heart attacks. This was a high-profile discovery which made its way to Cell Research, part of the Nature stable and “[p]ublished in partnership with the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences (CAS)“. [This journal has Chinese editors, a ridiculously phony impact factor of TWENTY-SIX, and caters mostly to authors from China, although the editorial board includes many western bigwigs including of course Guido Kroemer, -LS.]

As Kenneth Witwer of Johns Hopkins University wrote in 2020:

“But despite numerous replication and analysis studies, little or no systemic uptake of dietary RNA has been observed. A faithful replication of the initial experiments, but comparing mice given a nutritionally balanced rice-based diet with animals fed just rice, showed that the cholesterol finding was not the result of miRNA transfer, but rather a starvation response to a nutritionally insufficient rice diet.“

Nor did their progress stop there, with later landmarks including miRNAs as biomarkers of schizophrenia (not necessarily in mice), Wei et al 2015 (criticised by PubPeer contributor ‘Peer 1’). And who can forget such classics as “Reply to Lack of detectable oral bioavailability of plant microRNAs after feeding in mice“, in Nature Biotechnology? In 2020, concerned with maximising the public benefit from their discoveries rather than keeping them confined to academia, they seized at the current global pandemic as a chance to formulate a honeysuckle tea (see above) which would naturally eliminate SARS-CoV-2 because TCM [criticised by PubPeer contributor Leucanthemum Gaudinii].

In the last year’s press release by Nanjing University, Chen-Yu Zhang is quoted:

“We wished we could provide really useful information to help stop the pandemic in the darkest hour“. Chen-Yu Zhang said. “The focus of this study is to demonstrate that absorbed plant MIR2911 in honeysuckle decoction inhibits SARS-CoV-2 replication sufficiently.”

All of these studies involve primers: synthetic RNA sequences with the right order of nucleotides to latch onto trace quantity molecules of the putative miRNA and and present them for the magical amplification of PCR. These necessarily target segments of the miRNA that are unique, and accessible, i.e. not already bound to other segments. RNA likes to double back in hairpin / paperclip structures and cross-bind with itself, in the manner of a strip of packing tape, sticking to itself and to everything else except where you want it to go. When papermills specify primers in their contributions to the field, and copy-paste from elsewhere, they turn out to be designed for the wrong miRNA [H/t PubPeer contributor Aphilanthops Foxi].

Which brings us to an attempt to replicate the Nanjing discoveries. Pastrello et al (2016) (in Scientific Reports, also from Nature) found compelling PCR evidence of brassica miRNA entering the blood of human consumers, which is how broccoli prevents cancer.

The trouble was, Pastrello et al had not used appropriate primers for the task. Appraised of that failure, the crestfallen researchers retracted the paper. Before that point, though, the the Sci.Rep. editors allowed them to repeat the experiment with appropriate primers, which found no trace at all. Extensive RetractionWatch coverage here.

The would-be replicators were commended for Doing The Right Thing but skeptical onlookers continue to marvel at the initial, positive results, given that the garbled antisense primers should have delivered no results at all.

Say Mooo!

Zempleni’s group at University of Nebraska-Lincoln agree that plant miRNAs are not assimilated from one’s diet. Instead they espouse the cause of dairy-food miRNA as the pathway to optimal gene expression.**

‘Unregistered Submission’ pulled no punches in a trenchant critique of “MicroRNAs are absorbed in biologically meaningful amounts from nutritionally relevant doses of cow milk and affect gene expression in peripheral blood mononuclear cells, HEK-293 kidney cell cultures, and mouse livers” (Baier et al, 2014).

Undeterred, Wang et al (2018) [Zempleni’s group again] detected bovine miRNA in the bloodstream of human dairy-food consumers – not just mice! The paper received the accolade of ‘Editor’s Choice‘ from Kendal Hirschi in Journal of Nutrition. Accolade, I think, is what you make if Life gives you accols, but I digress.

Experts pushed back, pointing out that Wang et al had been misled by deficiencies in the “miRBase” database, and in fact the bovine and human forms of miR-21-5p and miR-30a-5p are identical with no way to tell dietary-sourced from endogenous molecules. The Fall 2017 meeting of the NIH Common Fund Extracellular RNA Communication Consortium must have been more acrimonious than usual. For details I refer the reader to “miR-21-5p and miR-30a-5p are identical in human and bovine, have similar isomiR distribution, and cannot be used to identify xenomiR uptake from cow milk” (Fromm et al 2018), which was published at bioRXiv as a preprint, and then as a Letter to Editor in J. Nutrition as a critical commentary on their Editor’s Choice. There was also a “Reply to B Fromm et al.” by Cui & Zempleni (“observations remain unchallenged“).

None of this weakened the conviction of the Nanjing group that “You are what you eat”, and “let food be your medicine”. Nor did it detract from their ability to secure research funding, as long as they provided the TCM-positive conclusions that the Chinese Government wants to hear. It seems from early press-releases that a key goal for the research was to retcon a rationale for the curative claims made on behalf of Traditional Chinese Medicine (to accompany the usual “Melange of secondary metabolites” rationale). This was not a new enthusiasm for Chen-Yu Zhang, who had already discovered in 2006 that genipin (from Gardenia fruit) cures diabetes, because TCM. Or if it doesn’t, maybe it cures Alzheimer’s. The search for a TCM-compatible application for genipin continues.

If you are thinking that TCM advocates should probably demonstrate these curative claims before dreaming up speculative mechanisms to explain them, you have not paid enough attention to the tooth-fairy-science nature of this research tradition.

Nature Outlooking

So we come to the celebrated 17 June 2020 Outlook issue on “Extracellular RNA”. Among others, Kenneth Witwer was invited to contribute a Review paper, and thereby became embroiled in a classic RetractionWatch post. For Prof Witwer had been unaware that when a well-funded research team pays Nature to advertise their disputed results and to disguise the advertisement within the protective camouflage of recruited articles (or when a gang of lethal medscammers like Saisei Mirai buys advertising for fraudulent cancer cures), that journal’s framework of institutional ethics compels them to grant editorial control over those articles to the customer.

The commissioning editors did not advise Witwer that his contribution should support and work with the Nanjing claims (and was subject to revision and re-titling if it criticised them)… so in the end he withdrew it. The RW piece contains no end of salty observations that I won’t try to summarise here.

That 2012 Saisei Mirai advertorial is mentioned here as indicating the piratical attitude of Nature Outlook. It was stealthily removed from the Nature website at some point after 2016, but it remains on the website of the Clinic’s Heart Online Pharmacy subsidiary, and is still cited by Alt-Med morons as proof that ‘GcMAF cures cancer’ (after all, it was a paper in Nature!!)… so their sponsorship was money well-spent.

In contrast to Flann O’Brien, I have not written three endings to this post. In fact I struggle to complete even one. Instead I’ll remind readers of the value that papermills are extracting from the miRNA field.

The End

Please admire this recent paper in Aging (Tang et al 2019), with Western Blots from the “Bowtie” oeuvre, locating it within the prodigious output of the ‘Contractor’ mill. The alleged experiments on humans and animals were supposedly approved by Ethics Panels at Beijing University of Chinese Medicine Third Affiliated Hospital (also the source of subjects), despite the three authors being employed at two hospitals in Wuhan. After publication, a fourth author from a third Wuhan hospital was added in a Corrigendum. According to the Author Contributions declaration, all the work was done by that fourth author along with six other people, while the three original authors did nothing. It’s almost as if the manuscript was originally written for one group of customers, then accidently sold to another group.

* * * * * * * * * * * * * * * * * *

* In the interests of exhaustive thoroughness I should mention Eric Lam at Rutgers Uni, and his opportunistic 2012 patent on medicinal tomatoes “as oral delivery vehicles for rna-based therapeutics“. In fact Lam’s patent covers Solanaceae plants in general, leaving room for suitably-engineered medicinal eggplants, potatoes, tamarillos, capsicums and chili peppers.

** Not to be confused with the Bovine Milk and Meat Factors discovered by Prof. Harald zur Hausen: DNA plasmids in beef and dairy products which cause cancer.

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