Tiny RNA molecules in food eaten can circulate in the bloodstream and turn genes off in the body; what are the implications of eating genetically modified food? Dr. Mae-Wan Ho
The age-old adage that you are what you eat has taken a literal turn in the rush of startling findings since the human genome sequence was announced 10 years ago, which overturn every tenet of the genetic determinist ideology that had made the Human Genome Project seem a compelling undertaking (see [1, 2] Living with the Fluid Genome, ISIS publications; Death of the Central Dogma and other articles in the series, SiS 24).
In 2009, we reported on nucleic acids circulating in the bloodstream that offer golden opportunities for disease diagnosis, and may also play a role in communication between cells within an organism  (Intercommunication via Circulating Nucleic Acids, SiS 42).
But would circulating nucleic acids cross species barriers? Yes, according to Liu Yongshen at the Henan Institute of Science and Technology in Xinxiang, China . Furthermore, Charles Darwin was really the first person to have proposed a mechanism for it in his theory of pangenesis.
Darwin’s theory of pangenesis suggested that all cells of an organism shed minute particles, gemmules, which circulate throughout the body and are passed on to the next generation through the germ cells. In that way, the characteristics of the parents are passed on to their offspring. And if the cells of the parents undergo changes during their life time, those changes would also be transmitted to the offspring. Liu described Darwin’s theory of pangenesis in some detail and reviewed both historical and more recent evidence in support of it, including fascinating findings on transmission of characteristics through blood transfusion that have been expurgated from the mainstream account. He concluded that : “a considerable revision of views on Darwin’s Pangenesis must occur before a new comprehensive genetic theory can be achieved.”
In my article on the subject  Darwin’s Pangenesis, the Hidden History of Genetics, & the Dangers of GMOs (SiS 42), I raised the potential dangers of genetically modified (GM) nucleic acids in GM food being taken up by cells in our body.
Now, a team of researchers in China have documented just this possibility. Plant nucleic acids are found to survive digestion in the gut, escape into the bloodstream, and taken up into the liver cells to target a very specific gene for silencing .
Zhang Chen-Yu and colleagues at Nanjing University, National University of Defence Technology, Changsha, and Tianjin Medical University, have been researching stable microRNAs, which they found circulating in the bloodstream of mammals that are actively secreted from the tissues and cells in the body. In a paper published in 2008 , the team presented results suggesting that the miRNAs could serve as a novel class of biomarkers for disease, and later showed that they could act as signaling molecules in intercellular communication .
MicroRNAs (miRNAa) are a class of 19-24 nucleotide long non-coding RNAs that silence an estimated 30 percent of protein-coding genes in mammals after the genes are transcribed. They do so by pairing, usually with complementary sequences in the 3’ untranslated regions (UTRs) of the targeted gene transcripts. The targeted genes are involved in a range of vital functions including cell differentiation, apoptosis (programmed cell death), cell proliferation, the immune response, and the maintenance of cell and tissue identity. Dysregulation of miRNAs is linked to cancer and other diseases; and specific miRNA profiles in the blood are potential biomarkers for diagnosis.
Zhang and colleagues had characterized the possible carriers of circulating miRNAs as microvesicles (MVs) shed from almost every cell type under both normal and pathological conditions (rather like Darwin’s gemmules). The MVs carry surface receptors and ligands of the original cells and have the potential to selectively interact with specific target cells to transport lipids, mRNA, proteins, or other signalling molecules between cells. Many MVs also contain miRNAs that could be selectively packaged and delivered into recipient cells where they regulate the expression of target genes and recipient cell function. In other words, miRNAs can serve as a novel class of signalling molecules between cells in the same organism.
To their surprise, Zhang and colleagues found plant miRNAs in the serum and plasma of humans and other mammals . More than half of the plant miRNAs detected are present in MVs. In an extensive series of experiments, they showed that a particularly abundant plant miRNA, MIR168a, can pass through the mouse gut and enter the bloodstream, ending up in various organs especially the liver, where it regulates a specific protein, LDLRAP1, involved in low density lipoprotein uptake.
On investigating the global miRNA profile in human serum, Zhang and colleagues consistently found plant miRNAs in healthy Chinese men and women. Sequencing revealed ~30 known plant miRNAs in Chinese healthy subjects, among which MIR156a and MIR168a were most abundant, and most likely originate from rice eaten by all the subjects. Plant miRNAs were also detected in the serum of other animals such as calves. Plant miRNAs are generally present at one-tenth the level of the most abundant endogenous mammalian miRNAs; and their presence has been confirmed by reverse-transcription PCR and northern blotting. Plant miRNAs are 2’-O-methylated on their terminal nucleotide, which makes them resistant to periodate treatment. In contrast mammalian miRNAs with free hydroxyl groups are sensitive to periodate. This is a further method for distinguishing between plant and animal miRNAs. Whereas MIR156a and MIR168a in rice and serum are highly resistant to periodate, the mammalian miR-16 in liver and serum was oxidized and not detectable after periodate treatment.
In mice, MIR168a and MIR156a were detected in various tissues including liver, small intestine and lung, while MIR166a, another plant miRNA could not be detected in the mouse tissues, although it was present in the serum.
The ordinary chow diet of mice had MIR168a, MIR156a and MIR166a at concentrations less than 1 fm/g (fm, femtomole, is 10-15mole), but 3-10 times as high in fresh rice. The same plant miRNAs are also present in Chinese cabbage, wheat and potato, and are not destroyed by cooking.
Mice fed the chow diet did not show elevated levels of MIR168a and MIR156a. In contrast, the plant miRNAs were significantly increased in both the sera and livers of mice fed with fresh rice for at least 6 h afterwards. It appears that only the mature single-stranded MIR168a was taken up into the blood stream, as precursors and double-stranded forms fed to mice could not be detected in the serum afterwards. Plant miRNAs, being 2’-O methylated, are also protected from acid degradation in the stomach.
Given that functional plant miRNA in food can enter the bloodstream of mammals, Zhang and colleagues wanted to know if it can also silence mammalian genes, the same way it silences plant genes. A bioinformatics analysis identified 50 genes that might be the target for MIR168a silencing in mouse, human and rat genomes. The most highly conserved sequence is located in exon 4 of the low-density lipoprotein receptor apoprotein 1 (LDLRAP1) LDLRAP1 is enriched in the liver, where it plays a critical role in the removal of low density lipoprotein (LDL) from circulation.
Transfection of HepG2 cells (a liver carcinoma cell line) with the precursor of MIR168a resulted in 1 000 fold elevation in MIR168a, indicating that the precursor can be processed to mature MIR168a in the liver. Concomitantly, the LDLRAPI protein was significantly reduced, but the mRNA level was not affected, consistent with MIR168a acting post-transcriptionally to silence the mRNA. The binding of MIR168a to the LDLRAPI coding sequence was confirmed by a luciferase reporter assay, in which a mutant or wild-type human LDLRAPI exon 4, and the human LDLRAPI coding sequence were cloned into a luciferase reporter plasmid and transfected into HEPG2 cells together with pre-MIR168a. As expected, luciferase activity for wild type was significantly reduced through MIR168a binding, where the mutant luciferase activity was not affected.
The possibility that the epithelial cells lining the intestine could take up the plant miRNA and package it into microvesicles (MVs) for transport was investigated in human intestinal Caco-2 cells, which were transfected with single-stranded mature MIR168a and the MVs released were collected and added to HepG2 cells.
The level of MIR168a was boosted 200-fold in Caco-2 cells after transfection, and rose 100-fold in HepG2 cells treated with the MVs released from Caco-2 cells. As a result, the LDLRAP1 protein in the HepG2 cells was significantly decreased, without affecting the LDLRAP1 mRNA level. Repression of LDLRAP1 expression in the HepG2 cells was dose dependent, as Caco-2 MVs containing more MIR168a correspondingly led to greater depression of LDLRAPI protein levels.
The protein Argonaute 2 (AGO2) is present in MVs and facilitates miRNA binding to its target gene via the RNA-induced silencing complex (RISC) (see  Subverting the Genetic Text, SiS 24). To see if plant MIR168a is associated with mammalian AGO2 and LDLRAPI mRNA, the researchers used anti-AGO2 antibody to precipitate the protein from HEPG2 cells treated with Caco-2 MVs. They detected both MIR168a and LDLRAP1 mRNA together with the AGO2.
Low density lipoprotein (LDL) is the major cholesterol-carrying lipoprotein in human plasma, and is generally believed to play an essential role in pathogenesis of atherosclerosis (hardening of the arteries). However, the connection between cholesterol and atherosclerosis is tenuous . Instead, substantial evidence has emerged that it is the oxidation of LDL triggering the inflammation response that’s responsible for atherosclerosis . The probability of oxidation increases with time of circulation in the bloodstream, which is why LDLRAPI is important for clearing it LDL from circulation.
Mice were fed with chow or fresh rice for 7 days after 12 h fasting . There was no difference in body weight although the rice-fed group ate more. The serum level of MIR168a in the chow-fed group was not significantly altered, while the rice-fed mice had significantly increased levels of MIR168a circulating in serum over the entire period. Levels of MIR168a in the liver similarly, were increased in rice-fed mice over chow-fed controls after 1 day.
Concomitantly, LDLRPI expression decreased dramatically in the rice-fed mice after 1 day, while LDL levels in plasma were significantly elevated on days 3 and 7. Interestingly, the level of liver LDLRAPI was not related to the levels of plasma cholesterol or triglycerides. If anything, plasma cholesterol level was decreased, while ApoA, the protein in LDL, and triglycerides were unchanged.
The findings of Zhang’s team  have profound health implications for food. Food provides not just energy, building blocks, and the usual vitamins and minerals, but also ‘informational’ nucleic acid molecules across kingdoms that influence the expression of our genes. Group leader Zhang is quick to agree with me  that their study says nothing about the health of rice, and definitely not that rice is bad for health. After all, healthy Chinese men and women all have plant miRNA circulating in their bloodstream. What it does reveal is the intimate relationship between our traditional diet and our biology, which have co-evolved and co-adapted for millennia, if not longer. We are, to quite an extent, what we eat. A staple, such as rice, is particularly important, as it forms the major part of the diet, and hence deeply embedded in our physiology.
This raises the key question on the safety of genetically modified (GM) food, which Prof. Zhang has declined to comment on.
The safety of GM food is very topical, as clear evidence has now emerged on the health hazards of GM feed (see  GM Feed Toxic, New Meta-Analysis Confirms, SiS 52).
We have previously warned of the potential dangers of circulating nucleic acids . Most relevant to the new findings of Zhang’s team is an experiment in gene therapy using a precursor of miRNA. It turned out to have so many off-target effects that it killed more than 150 of the experimental mice (see  Gene Therapy Nightmare for Mice, SiS 31).
Genetic modification of plants and animals is notorious for being completely unpredictable and uncontrollable, as well as unstable (see  FAQ on Genetic Engineering, ISIS tutorial). That applies to new and unpredictable complement of miRNAs that could result, to which humans (as well as other animals) are totally unprepared. It could account for some of the illnesses and deaths observed in the field as well as in lab experiments [13, 16] (see GM Science Exposed, ISIS e-book).
The miRNA profiles of all GM food and feed need to be carefully documented now, especially if proponents are still intent on introducing them into our food chain in the face of all the damning evidence on health and environmental impacts.
Article first published 30/11/11
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Prof Frances Burton Comment left 1st December 2011 08:08:52
The field of epigenesis, while dating to Waddington in the 1960s and 70s, really took off at the beginning of this century, and now has burgeoned into a major interest of genetics. The now-known fact that environmental substances affect genetic expression is of paramount importance,and undoubtedly had its effect on our ancestors. In my book, FIRE: THE SPARK THAT IGNITED HUMAN EVOLUTION, I speculated that early hominins were able to associate with fire, and in so doing exposed themselves to the LIGHT emanating. This light affected circadian rhythms through the melatonin pathway, as well as other hormonal systems detailed in my book.
Todd Millions Comment left 4th December 2011 09:09:16
Dr Mae-Wan Ho Thanx for this.Such a continent this shore becomes. On both these rna circulating fragments and the rest of any organisams DNA-RNA -zoo.Are referent materials from before GM modification even available for comparision?Stored suitably,by whom? I would have some concern that samples could be switched,too make it appear that escapes had always being part of a genome.
Sharlene Comment left 4th December 2011 20:08:59
I applaud your work in this area. I know that our food source,especially gm foods are altering our bodies. I have an extreme case of morgellons disease and have been tested positive for agrobacteria tumefaciens. I have also produced actual plants,pods and insects in my body.I have thousands of photos of these abberations. I will be so thankful when the truth comes out about our food supply.I hope it is exposed and stopped before it is too late. Thank you for being brave enough to speak up with the facts.
Mae-Wan Ho Comment left 4th December 2011 20:08:55
Hi Todd, Yes, there is a lab in Europe that is supposed to collect standard materials deposited by companies. Yes, companies have been allowed to change their standards when it became clear that the GMO has mutated from its original state, or turned out not to match what the company had described when checked by other scientists. I have been challenging companies to prove that their GMOs are genetically stable, and as yet received no proof, or answer. Transgenic instability multiplies unknown upon unknown. That is why people like myself want nothing more to do with GMOs.