In the last session of my talk I would like to talk to you about microRNA in toxicology, or why I believe microRNAs are important in toxicology field and can be used at the biomarkers or end point of toxicological measurements. The next two slides summarize the knowledge of microRNA biology and functions that I already presented to you in a previous section, but in terms of how we can use it in toxicology. For example, microRNAs are conserved trough the phyla. It's mean that silly guns, and flies and mouse have part of microRNAs which are the same as humans. So this makes it easier to extrapolate interspecies differences in toxicological studies. So if you perform studies with worms and mice, we can be sure that the same microRNAs are present in the human and most probably it should be rigged the same way. Another important point to mention is that microRNA's number is much, much lower than the protein coding genes. So far in microRNA database we have 2,578 microRNAs entries for humans, which is ten times less than protein coding genes. That means that analyzing the microRNA profiles make it much easier for the researchers to make the conclusion about the effects rather than analyze 25,000 different genes. And as I already said before, 60% of all messenger RNA are regulated by microRNAs. So microRNAs are very important in gene expression regulation. Then they may regulate hundred different messenger RNA. And it was shown that microRNA targets are twice as likely to be affected by chemicals than those messenger RNA which like microRNA binding sites. I also told you already that microRNAs are spatial-specific and temporalyl-specific regulated. That's mean that certain microRNAs are expressed predominantly in certain organs. For example, like mir-9 and mir-124 is expressed in the brain, mir-1 is expressed in heart, and mir-122 is specific for liver. The same as I already described before, microRNA regulate the important process in the development and the perturbations of those processes by toxicological insult can be studied by studying microRNA profiling. Recently MicroRNAs were discovered in biofluids. In the blood, saliva, urine. And this make them perfect biomarkers for different diseases. For cancer, for liver toxicity or injury, for heat injury, neurotoxicity, and central nervous system diseases. MicroRNA in biofluids are very stable because they are either bound to the protein complexesor are encapsuled in the vesicles, which makes, again, them very attractive to other biomarkers. So far, two microRNAs are more studied in the sense of the biomarkers. For example, mir-122, which is specific for liver, is used as a biomarker for liver injury and toxicity. However, mir-1, which is specific for heart, is used as a biomarker for myocardial infarction, for example. So basically we can detect, take a blood or urine sample from the patient and detect those microRNAs level in the blood and predict certain disease outcome. As I have just said, microRNA are much more stable than messenger RNA. First of all, because of their size. Second of all, because their in biofluids are encapsulated in the microvesicles or bound to the proteins. And this makes it attractive to use microRNA profiling, for example, in archive samples and degraded samples and formaldehyde fixed samples, which is usually obstacle to study messenger RNA. So it's the stability of micro RNA make them an effective tool of the biomarkers in different variety of difficult samples. So all together those points make clear that MicroRNA rapidly responds to the toxicant treatment and can be used in toxicological study to indentify mechanism of different toxicants in different organism organs and self. This table just to show you some examples how environmental toxicants may perturb microRNA expression and what's target in the function and the pathways are associated with these perturbations. On the last slide I will use an example of my own research how we can use microRNA to study the neurotoxicity and the environmental neurotoxicity. In this experiment we used embryonic stem cells which were differentiated to neurons and astrocytes, and the exposure to two well known developmental toxicants, valproic acid, or VPA, and arsenic. The cells were differentiated from day 1 to day 16 of differentiation. And VPA is an antiepileptic drug which is used to treat bipolar disorder, cancer, and migraine. And this substance has potency. It leads to heart malformations and neural tube closure defects. VPA is a non histone deacetylase inhbitor, but molecular mechanism of its is not well understood. Arsenic is very toxic and environmental pollutant. It's thermogenic and embryotoxic. Its toxic and thermogenic activity could be mediated by epigenetic mechanisms such as DNA hypomethylation and alternation of distal methylation. So after the treatment of our newly differentiated tempering stems cells to those two compounds, we analyze the viability of the cells and then we perform microRNA profiling by using microarrays. And we confirm these profiles with real-time PCR. And this slide summarized the result from this study, where you can see two volcano plots for valproic acid and arsenic samples. And basically you can see the fold change of different microRNA per chunk by VPA. To the right of the red line, you can see all microRNAs which were up regulated. And to the left of the red line, you can see all microRNAs which were down regulated. And as higher them dots of those microRNAs are on the y axis, as more significant is their effect. And surprisingly we found out that exposure of neurally differentiated embryonic stem cell to VP cells, to VPA lead to induction of microRNAs which are involved in muscle differentiation. While neural specific mircoRNAs were down regulated, we couldn't observe this effect with arsenic samples. This means that VPA exposure during differentiation induced myogenesis in our neuronal cultures. We confirm this VPA effect on the neural differentiation morphologically. Upper panel shows the samples which were exposed to VPA, and lower panel is a control. And you see both up and low panel are positive for which is the neuronal marker, but in VPA samples we could also observe some muscles which were positive for alpha actinin, what we couldn't find in control samples. So taken together, microRNAs, which are neuro-specific, are responsible for transition from embryonic stem cell stage to the neurons. But on the valproic acid exposure, cells decide to make a detour and become muscles, and probably micro RNAs which are specific for muscles like myoMiRs are involved in this process. And the take home message from my talk, you probably understood that epigenetic marks and non-coding RNAs fine-tune gene expression and may act on interface between genetics and environment. Physiologically, they may buffer altered noisy gene expression and thus maintain steady-state of the biological system. But the alterations of epeginome or microRNAs may lead to adverse outcome in later stages to diseases. In this point, I would like to thank you for your attention. And I hope you enjoyed my lecture and learned a lot new things about microRNAs and their role in toxicology. And I will be happy to hear any feedback about the lecture, how I can improve it. And we will talk to you further during our assignments and exam classes. Thank you very much for your attention. [MUSIC]
