Stanford Study Finds miRNA-320a a Broad Regulator of Glycolysis, Potential Drug Target
Reporter: Aviva Lev-Ari, PhD, RN
A study by Stanford researchers has found that microRNA-320a appears to regulate glycolysis in response to oxidative stress in several biological systems, including lung cancer and wasting of disused muscle.
The Stanford team was initially interested in better understanding the wasting of diaphragm muscles due to mechanical ventilation, but expanded its study to look at lung cancer and an experimental in vitro model of oxidative stress, as well as the similarity of pathogenic glycolytic pathways across these biological systems.
The group profiled miRNA and protein expression in samples from human diaphragm muscles under mechanical ventilation to identify miRNAs associated with the glycolytic rate-limiting enzyme phosphofructokinase, or PFKm, without which glycolysis is reduced.
The group initially identified 28 miRNAs that were significantly downregulated and three that were upregulated in the ventilated human diaphragm samples. Using predictive software, the group pinpointed miR-320a as being potentially involved in the regulation PFKm.
To validate miR-320a, the researchers looked at all three experimental systems — samples of diaphragm tissue, lung cancer, and an in vitro cell model under oxidative stress. In all three, miR-320a was down-regulated in the samples versus the control.
The group also confirmed that miR-320a influences PFKm in each system, and further demonstrated that miR-320a knockdown increased lactate levels in vitro; and thathigher miR-320a levels reduced lactate levels in in vivo mouse experiments.
The group wrote that the study shows for the first time that glycolytic activity “is increased in diaphragm tissue that is noncontractile as a result of full mechanical ventilator support.” The results also confirmed that glycolysis up-regulation, or the Warburg effect, is present in lung adenocarcinoma, and that both otherwise divergent disorders are in fact linked by the influence of miR-320a.
The finding has implications for cancer treatment, as well as more effective treatment for dysfunctional diaphragm muscles following breathing support using a ventilator, according to the team, which published the study online in the FASEB Journal earlier this month.
Glycolysis is the process of converting sugar into energy, and is implicated in the growth of some cancers through a process called the Warburg effect. To the Stanford team, the Warburg effect seen in lung adenocarcinoma “appears to closely mimic” that of dysfunctional human diaphragm tissue after mechanical ventilation therapy, a condition called ventilator-induced diaphragm dysfunction, or VIDD.
The Stanford researchers claim that their study shows that these very divergent biological systems share the same glycolysis regulatory apparatus involving miR-320a, which the authors believe they are the first to identify.
Additionally, “miR-320 regulation of glycolysis may represent a general mechanism underlying other clinical diseases that are associated with changes in energy supply,” the researchers wrote, such as cardiac ischemia, to insulin resistance.
In cancer specifically, down-regulation of miR-320a has been previously reported in a number of malignancies, the group reported. Coupled with the fact that the Warburg effect is thought to be important in many cancers, and the results of the group’s study in adenocarcinoma, this suggests that miR-320a “may be directly related” to the development of cancer, and that the associated glycolysis may be a potential drug target.
Oxidative stress-responsive microRNA-320 regulates glycolysis in diverse biological systems.
Tang H, Lee M, Sharpe O, Salamone L, Noonan EJ, Hoang CD, Levine S, Robinson WH, Shrager JB.
Source
*Division of Thoracic Surgery, Department of Cardiothoracic Surgery.
Abstract
Glycolysis is the initial step of glucose catabolism and is up-regulated in cancer cells (the Warburg Effect). Such shifts toward a glycolytic phenotype have not been explored widely in other biological systems, and the molecular mechanisms underlying the shifts remain unknown. With proteomics, we observed increased glycolysis in disused human diaphragm muscle. In disused muscle, lung cancer, and H(2)O(2)-treated myotubes, we show up-regulation of the rate-limiting glycolytic enzyme muscle-type phosphofructokinase (PFKm, >2 fold, P<0.05) and accumulation of lactate (>150%, P<0.05). Using microRNA profiling, we identify miR-320a as a regulator of PFKm expression. Reduced miR-320a levels (to ∼50% of control, P<0.05) are associated with the increased PFKm in each of these diverse systems. Manipulation of miR-320a levels both in vitro and in vivo alters PFKm and lactate levels in the expected directions. Further, miR-320a appears to regulate oxidative stress-induced PFKm expression, and reduced miR-320a allows greater induction of glycolysis in response to H(2)O(2) treatment. We show that this microRNA-mediated regulation occurs through PFKm’s 3′ untranslated region and that Ets proteins are involved in the regulation of PFKm via miR-320a. These findings suggest that oxidative stress-responsive microRNA-320a may regulate glycolysis broadly within nature.-Tang, H., Lee, M., Sharpe, O., Salamone, L., Noonan, E. J., Hoang, C. D., Levine, S., Robinson, W. H., Shrager, J. B. Oxidative stress-responsive microRNA-320 regulates glycolysis in diverse biological systems.
This is a seminal discovery in biochemistry, metabolic control, and the Warburg effect. The last step of the glycolytic pathway is the conversion of pyruvate to lactic acid. The generation of lactic acid and accumulation in serum is lactic acidemia. This occurs with diabetic crisis, and with shock from withdrawal of peripheral circulation to preserve the viscera, and it requires the pyridine linked enzyme LDH and the coenzyme NADH. The assay for lactic acid converts NAD to NADH followed at 340 nm, as I published in Methods in Enzymology some 35 years ago. The physiological importance of FPK is that it is a reverse one-way reaction. If the PFK activity is down-regulated, that the reaction pyruvate to lactate is favored. This miR320a story has more to be found.
The work in Nathan O. Kaplan’s lab at Brandeis and in La Jolla established that the LDH is a tetramer composed of H- and M-type subunits – HHHH, HHHM, HHMM, MMMH, MMMM. The enzyme, oxidized coenzyme and pyruvate form a ternary complex in heart, but not in skeletal muscle. A method was worked out to assay the reaction by following it on an Aminco stop-flow instrument. So the question then arises as to a linkage between the PFK event, and the predominant species of LDH, the heart being regulatory and the liver not. In addition, Kaplan presented the pyridine nucleotide coenzymes, NADH and NADPH in an equilibrium by way of the transhydrogenase in his Harvey Lecture, I think in 1971. His idea was that synthetic organs – liver, endocrine – are NADPH organs, and the the heart and skeletal muscle are energy transformers tied to NADH.
At the same time, I studied the citric acid cycle linked isoenzyme mitochondrial malate dehydrogenase that my predecessor found had no tryptophan residue at the active site, which I confirmed, discrediting work that made that claim. This was not the interesting part of the work. The mitochondrial malate dehydrogenase forms a ternary complex with oxaloacetate and NAD as the reaction procedes, at a much faster rate that the LD H-type reaction. The cytoplasmic isoenzyme also is regulatory, unlike the M-type LD. There also were multiple forms of the isoenzyme that were identified by Barry Kitto, but whose importance we disregarded. I didn’t look at or consider the Warburg Effect until I was at my first appointment after the Vietnam War, when I designed an assay to identify the mitochondrial content in a variety of animals and sea creatures as well as in different tissues that was consistent with column chromatography and with centrifugation. Having this work done (I filed a patent and a subpatent), I used Herschel Sidranski’s minimal deviation hepatomas. The mitochondrial enzyme was as expected, but the cytoplasmic MDH was unsaturable for oxaloacetate.
This genomic and metabolomic work is really important. The question might be raised as to whether the same mechanism also has an effect on the entry into the Krebs cycle. This is also a feature of diabetic ketoacidosis.
Dr. Larry,
Thank you for the comprehensive reply.
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On the question you raised as to whether the same mechanism also has an effect on the entry into the Krebs cycle — I would assume that it has, positive effect on.
Thank you. If the PFK (one-way) reverse pathway is down-regulated, then this would draw substrate from entry into the Krebs cycle. The Krebs cycle pathway generates something like 16 ATPs, while the entire travel through glycolysis generates 2 ATPs. It was Fritz Lippman who won the Nobel Prize for his discovery of coenzyme A with the S-adenosyl moiety, which brings into play another matter unrelated to this discussion. NO Kaplan and S Colowick were coworkers at Johns Hopkins at the time. But this is where we encounter the high energy phosphate bond.
Warburg pioneered in mitochondrial “respiration”. The Warburg hypothesis was that there is a “defect” in respiration in malignant transformation leading to a bacteria like cellular metabolism. Our knowledge was unable to grasp the events at the time, and there was serious criticism and disbelief.
If the down-regulation moved the substrates through to lactic acid, the a step would be bypassed in the entry into the Krebs cycle. What does this mean for hydrogen transfer in the mitochondrion?
The conenzymes – NADH and NAD do not cross the mitochondrion. So H transfer is done through the transport of reduced/oxidized substrates – malate/oxaloacetate. Of course malate is critical for entry into the Krebs cycle. It would appear that the flow of hydrogen ions would be impaired by OAA sitting in the cytoplasm and malate not accumulating in the mitochondrion. So we are actually looking at the redox potential of a cancer cell. This would be further exaggerated if (we never purified the aberrant cMDH), but Everse published a paper in the Brit J Cancer reporting on finding the enzyme in the serum of a series of cancer patients. If the cMDH had such a high Km for OAA (sitting in the cytoplasmic compartment), the the hydrogen transport into the mitochondrial compartment would be effectively blocked.
the hydrogen transport into the mitochondrial compartment would be effectively blocked — what is the next chemical reaction ?