To materialize the dialogue shared in Part 1.0, it is relevant to go through a fundamental overview of what miRNA is. Afterwards in Part 2 & 3 we will dive deep into its applications in disease diagnosis and therapies. Because this interlude serves simply as an introduction to this nucleic acid, it’s written for those who are new to micro-biology and simply want to explore. As the reading progresses, the difficulty increments and a more technical vocabulary as well as more complex topics (topics that I do not thoroughly understand either) will be dealt with.
Have you ever wondered why doesn’t your hair grow from head to toe in just one night? How about why doesn’t your nail grow 1 – 2 meters long? In the end, we just need your cells to express a large amount of proteins (such as keratin) and our nails would grow touching the floor in a short period of time. Speaking about cellular expression, we’ve been told that almost all 3.6 billion somatic cells in our body (excluding sexual cells residing in our reproductive organs and few other exceptions) are mostly similar: they have similar genetic code, mostly the same DNA sequence and coiled into chromosomes residing inside the respective nucleus[1]. Indeed, they’re all the same…except they just aren’t: cells circulating in our blood vessels, those that make up the blood vessels, those constituting lung tissue or brain tissue, are clearly different from one another not only in terms of structure, but also in function (neurons transmit electric signals, alveolar cells are in charge of exchanging oxygen and carbon dioxide between lung and blood, white blood cells carry diverse functions from producing antibodies (helper B cells) to inhibiting pathological activity through killing infected host cells (killer T cells), to ingesting pathogens and producing antigens that start an immune response, among others. Nonetheless, it remains true that they all carry the same genetic code. The difference is that each type of cell’s DNA sequence “differentially” expresses its respective genes which translates into different proteins, and these proteins either make up the distinctive structure that distinguishes cells among one another (only neurons have a myelin sheath for example) and represents their distinctive functions (only helper B cells produce antibodies that bind to pathogens’ receptors to inhibit their activities). The “decision” on what cells will constitute brain tissue, which ones the liver tissue, or which ones bone tissue can be traced back at your birth, where there existed a single mother cell (stem cell) that through differential expression of its genes ramified into the formation of different cells, each kind carrying its distinctive structure performing their unique functions.[2]
[1] It’s quiet controversial to suggest that all cells, with few exceptions, carry the same DNA sequence. From (Eric, 2015) in The Patient Will See You Know, we have to take into account that different cells from the same individual might carry mutations in their gene sequences. There is another slight issue which is that there are still genes yet to be identified. But these are details that can be skipped if we want to have a basic understanding of miRNA’s relevance. [2] Alternatively, these stem cells are the ones employed for genetic engineering thanks to them being partially differentiated or completely undifferentiated, which allows them to be used for experimenting as mimics of human organs, called organoids (MIT Technology Review, 2019) , for instance.
So now we sorted why cells are different (their genes express different proteins) but still haven’t answered what mechanisms control which portion and how much of it is expressed, that is, the first questions raised above that concerned why hair or nails don’t grow exponentially all of a sudden. Simply speaking, that’s because there is a mechanism that regulates gene expression, and this is a very complicated and diverse mechanism. Gene expression, also called central dogma, is the process in which a sequence of nucleotides in the DNA is transcribed into mRNA and then translated into amino acids that form a protein, and in summary, it happens as shown in Figure 1:
Figure 1 Central Dogma of Biology: the DNA’s double helix structure inside the nucleus of a cell is unwind by an enzyme called RNA polymerase which then starts manufacturing messenger RNA (mRNA) from the DNA template provided; this process is named as transcription. Such mRNA exits the nucleus to the cytoplasm until encountering a ribosome where transfer RNA (tRNA) will attach, depending on the mRNA nucleotide sequence, amino acids for each triplet of nucleotides, thus forming a protein; this process is names as translation.[2]
In regulation of gene expression, miRNA plays a relevant role.
MiRNA is also a byproduct of DNA transcription (as explained later in this section), however it doesn’t undergo translation like mRNA; instead it inhibits the completion of the central dogma by pairing and inhibiting mRNA so that it’s unable to bind with tRNA anymore or degrades in the cytoplasm. Because it’s not translated, miRNA is also called noncoding RNA (nc-RNA).
So, when there is a balanced concentration of miRNA in the cell’s cytoplasm (not necessarily in the nucleus), mRNA translation can be regulated; conversely, if it isn’t, then this deregulation of concentration might decay into several diseases such as cancer, as dysregulation might mean the lack of proteins essential to maintain cell survival (such as pathways that induce apoptosis to prevent tumorigenesis) or the excess of proteins harmful for the cell, such as those that induce cell proliferation or prevent apoptosis.
So back to the original (strange) question proposed as a starting point to introduce miRNAs, the possible reason that may explain why our hairs or nails don’t grow exponentially, or as in general why our cells don’t over-produce proteins or undergo excessive replication (unless it’s part of a disease like cancer), it’s partly due to the balanced concentration of miRNAs that are constantly regulating gene expression.
With the above introduction, it is pertinent to talk about the biogenesis of miRNA. This is because it will serve as a premise for understanding how biomedical researchers design drugs used to target or mimic miRNAs as a viable treatment for diverse diseases. Biogenesis concerns pathways that lead to the formation of mature miRNA, and drugs are designed to inhibit such pathways for a downregulation of miRNA or imitate the structure of miRNA for upregulation.
MiRNA biogenesis
Simulation
Source: https://vimeo.com/241703528
MiRNAs are small ncRNAS that are 18-25 nucleotides long (Deng & Guangchao Sui1, 2013). This usually refers to mature miRNAs, which leads to the interrogative of how they are formed.
Figure 2: miRNA biogenesis pathway
As stated above, miRNAs are also byproducts of transcription (however they apparently lack open reading frames, so they don’t get translated). Such transcription starts with RNA Polymerase III transcribing from the DNA primary miRNAs (pri-miRNA) that have hairpin structures. Afterwards, it is processed by a protein called DROSHA forming precursor miRNA (pre-miRNA), which are 70-100 nucleotides long. A protein called Exportin 5 recognizes this pre-miRNA and mediates its translocation to the cytoplasm (Marilena & Carlo, 2012), where DICER protein complex and Argonaute 2 (AGO2) attaches to it and unwinds one of its helix: the complementary (3’ – 5’) strand, called 3p strand. The remaining (5’ – 3’) strand, called 5p strand, or guide strand, together form the RISC complex and target particulars mRNA depending on its sequence specific complementarity to inhibit translation of such mRNA. (Marco, November, 2014) . The 5p strand generally does this in the RISC complex by binding to the 3’ untranslated region (UTR) of the target mRNAs.
The description above corresponds to the canonical pathway (Figure 3a is the same as Figure 2), however, researchers have also found alternative pathways as displayed below.
Figure 3: (b) and (c) are alternative pathways for miRNA biogenesis
Source: miRNomics: MicroRNA Biology and Computational Analysis, 2014 by Malik & Jens
In Figure 3b, intronic microRNAs, also called mirtrons, are processed by another protein in the nucleus called spliceosome, an alternative to Drosha. These processed mirtrons, pre-miRNA, follows afterwards a similar path as above, that is, recognition by Exportin-5, and then processed into the RISC complex. Figure 3c describes simtrons, which in contrast to mirtrons, may require Drosha but not DICER protein complex processing (Malik & Jens, 2014).
It is worth noting that previously it was thought that the unzipped 3p strand would have degraded in the cytoplasm, so its up or down-regulation was irrelevant for the study of the progression of diseases like cancer. Nonetheless, due to recent studies, it’s suggested that the 3’ complementary chain, or 3p strand, doesn’t necessarily degrades, but also plays a crucial role together with its complementary 5p strand in regulating gene expression. For example, the 5p and 3p strands of miR-582 and miR-28 are downregulated in bladder and colorectal cancer, respectively. However, experimental forced expression of miR-582-5p and miR-582-3p to achieve its upregulation inhibited bladder cancer cell proliferation and tumor growth, whereas forced expression of miR-28-5p, but not miR28-3p, reduced colorectal cancer cell proliferation (Ramkrishna, Clare, Jiang, Evan, & Christine, 2020). This kind of interdependence suggests that miRNA 5p/3p pairs cooperatively modulate cancer cell growth. If we go through the literature on miRNA studies, we’d tend to find authors employing names like miR-122, miR-582, among others, as the default miRNA for studies. Such conventional way of calling miRNAs usually refers to the 5p strand, but since recent studies show how both strands play relevant roles in regulating cell cycle pathways, cancer proliferation, or inhibition of tumorigenesis, it’s worth keeping an eye on in further papers whether researchers also focus on the 3p strands, as these may also play relevant roles in searching for diagnostic and therapeutic targets.
Despite given the above, it ought to be clarified that the study of these small molecules are still a developing field, so, for instance, the precise mechanisms involved in the miRNA transcription is not known (Ardekani & Naeini, 2010). As more research is done, some new findings may contradict previous conceptions. One such example is the 5p/3p strands’ cooperativity; another controversial finding suggests that miRNAs seem to also promote translation, despite the term “regulation” conveying a sense that miRNAs should solely inhibit mRNAs, for example, miR-10a was shown to bind to the 5’ UTR of ribosomal protein mRNAs and enhance their translation (Malik & Jens, 2014). Regardless of such uncertainty, with what we currently know and will find out about miRNAs can already be used for diving deep into how it can act as a biomarker for the diagnosis (or maybe prognosis) of diverse diseases and afterwards for targeted therapy of such.
Voyage for a better health
Far away on the red sea, a big ship with a flag displaying “AGO – 2” carrying brave Argonauts is seemingly wandering aimlessly.
Argonaut #1: Captain, when are we going to see some action? It’s been too long since we’ve performed a heroic deed in the faraway Red Sea.
Captain of AGO – 2: no rush my brothers! Sooner we’ll come across a defective ship with a defective crew to stop them. Look, I can now see a ship from afar. Give me my binoculars, I need to see whether they’re acting normally or are they problematic.
The Captain stands from tip of the AGO – 2 and through the binoculars. The Captain sees that the crew members of that ship seem to be working as normal.
In the other ship:
Ship’s Boss: Alright, keep replacing parts of the ship and polish what’s dirty so we can sail further into the Red Sea! Remember, we have to deliver the cargo by the end of today!
Crew members (shouting monotonously): Roger!
Crew members A, U, G (shouting): Boss, we’ll start changing the mast, polish the anchor, clean the deck, and lift new sails!
Ship’s Boss: That’s the spirit! Let our ship navigate further and more stable.
(After working for a while by replacing the defective parts of ship and performing other functions)
Crew members U, G, A (shouting): Alright, stop! That’s it, anymore polishing or adding new parts is going to do more harm than good. Let’s just keep sailing further. Well done for today.
Back to AGO – 2:
Captain of AGO – 2: Hmm. That one seems to be working properly
Argonaut #2 (suddenly rushing): Cap’n! We’ve just spotted a ship whose crew members are working ferociously. They’ve already broken the mast and added new ones several times, polished the deck till it broke apart, built so many anchors and released them that the ship is now stuck. They’re crazy! They’ve added so many sails yet tore apart many too. Now their members are fighting each other and nobody seems to stop.
Captain of AGO – 2: Oh my gracious! What’s gotten into their mind! From where do they derive such madness?
Argonaut #3 (looking from the deck): who knows? Such kind of reckless behavior seems to be probabilistic and happens quiet often.
In the maddened ship:
Corrupted member #1: I have to built anchors! Built more anchors! More anchors!
Corrupted member #2: add more sails! Add more sails! There is just not enough sails!
Mad captain: Stop adding more things to the ship! At this pace it’s going to sink!
Mad captain: then jump off the ship!
Mad captain: Bah! Humbug! Where is member #53 when you most need it?
Corrupted members #1 & #2 (building more things which add more weight to the sinking ship): we don’t know!
Back to the AGO-2 ship:
Captain of AGO – 2: Drive AGO – 2 to the tail of that ship and let our members in. We have to stop this madness as soon as possible otherwise this ship is going to sink to the Red Sea and contaminate it.
Argonauts: Roger!
The tip of AGO – 2 hits the tail of the ship serving as an anchor to keep both ships joint together and allow the Argonauts jump to the corrupted ship. The Argonauts jump the ship and start mediating the reckless behaviors of the corrupted members. Details are omitted.
After a while:
Captain of AGO – 2 (looking from a binoculars from the deck of AGO – 2): It seems that the ship is almost back to normal. The corrupted members’ behaviors have been regulated.
Argonaut #1: how did they do it?
Captain of AGO – 2: Well, can’t you see that “details are omitted”? Plus, I couldn’t even see clearly through my binoculars, since they were predesigned to be able to track the actions of only Argonaut #1301, who was in charge of convincing some of the corrupted members to commit suicide, and Argonaut #126, who seemed to have stopped such reckless behavior from spreading to other crew members. As for the specific, very specific details of how they did it, I can’t say for certain and I hardly will, nonetheless, knowing that my fellow Argonauts can prevent the spread of such catastrophe is enough for me to ask of you to recruit more Argonauts like them.
Afterthoughts
Like before, I’d love to share some of my thoughts regarding the crafting of such section as well as the dialogue annexed to it. To begin, I do clarify that this was just an Interlude, meaning that it was just a “side story” to introduce you the concept of miRNAs and how they are created, in other words, their biogenesis, as this would serve as a basis for understanding how it can act as a biomarker for diverse diseases ranging from cancer to neurological diseases like Alzheimer. However, I didn’t, and won’t dive deeper into the specific details surrounding miRNA, like their structure, how “precisely” they work, partly because I’d like you to read it yourself from papers available online; the main source I consult is PubMED from NCBI. Another source, which I’ve mentioned in the bibliography is called “miRNomics: MicroRNA Biology and Computational Analysis” by Malik Yousef and Jens Allmer, which you can search online for a thorough explanation of miRNAs. As for my produced content, I’d instead focus on introducing to the reader what are the possible prospects to look up to in this emerging and amazing field in a way that’s understandable and creative.
Now, proceeding to the Dialogue, AGO – 2 is basically referring to the AGO 2 protein complex that carries the guide strand of miRNA, represented by Argonauts. They are sailing on the Red Sea which is analogous to miRNAs flowing in our blood. The first ship the Captain encountered was supposed to represent the moderated translation of mRNA, resembling the modulated growth of our hair, nails, or, simply speaking, not developing cancer, as shown by how crew members worked properly and didn’t proliferate the parts of the ship like the mast, sails or anchors. A little detail that you may have noticed is how crew members A, U, G signaled the start of the crew’s activity, which was an analogy to how the start codon signaled the beginning of mRNA transcription. Similarly, crew members U, G, A represented one of the 3 stop codons that ended mRNA transcription. AGO – 2’s crew probably didn’t intervene as they seemed to work ordinarily. Conversely, the next ship was an example of a cancer. Its crew members started acting recklessly, building at an uncessarily large scale parts of the ship that were doing more harm than good, which you can view it as tumorigenesis or angiogenesis. The captain of this corrupted ship asked why crew member #53 wasn’t here to stop the progression of this cancer, which resembles the lack of p53’s activity during tumorigenesis, where p53 is a tumor-suppressor protein that induces apoptosis in cancerous cells. It’s of course of great interest to understand why do crew members go reckless, but I answered it through Argonaut #3’s comment, which is that such occurrence, given that’s caused by genetic mutations, is probabilistic by nature. What happens afterwards till the end is analogous to what was explained, AGO – 2 anchoring itself to the corrupted ship resembles miRNA binding to the 3’ UTR of the target mRNA and, in this case, inhibiting its activity. The two Argonauts correspond respectively to miR–1301, which promotes apoptosis, and miR-126, which inhibits metastasis (Simon, 2019).
The last comment by the Captain restates an idea shared previously, which is that we don’t know precisely how miRNA works despite they playing an essential role in regulating gene expression which correlates with cancer progression, so further studies are required; in the end, science is the art of reducing uncertainty; the use of a ship sailing to the Red Sea to convey the functioning of miRNAs is to transmit the underlining idea that science is a never-ending voyage of discovery... Nonetheless, with the current knowledge we already have, and researches being carried out, we already have at our disposal useful knowledge that can be put to practice in order to diagnose and treat diseases that involve miRNA deregulation.
So, for the next section we’ll focus on how miRNA can act as biomarker for diverse diseases and some thoughts about how artificial intelligence can play a relevant role for this field’s development.
Bibliography
Alan, D., Randy, M., Patricia, T., & William, W. (2014). HIGHER LEVEL Biology 2nd Edition. Essex: Pearson Education Limited .
Ardekani, A. M., & Naeini, M. M. (2010). The Role of MicroRNAs in Human Diseases . Review Article, Avicenna J Med Biotechx, 2(4): 161-179.
Deng, G., & Guangchao Sui1, *. (2013). Noncoding RNA in Oncogenesis: A New Era of Identifying Key Players. International Journal of Molecular Sciences, 18319–18349. .
Editors: Malik, Y., & Jens, A. (2014). miRNomics: MicroRNA Biology and Computational Analysis. Springer New York Heidelberg Dordrecht London.
Eric, T. (2019). Deep Medicine: How Artificial Intelligence Can Make Healthcare Human Again. New York: Hachette Book Group.
Marco, F. (November, 2014). Drug target miRNAs: chances and challenges. Review Cell Press, Trends in Biotechnology,, 578-586.
Marilena, V. I., & Carlo, M. C. (2012). Causes and Consequences of microRNA Dysregulation. Cancer J , 18(3): 215–222.
MIT Technology Review. (2019). 科技之巅3 (Eng. The Epitome of Science and Technology III). Beijing : Posts & Telecom Press.
NCBI. (2007, 05 11). Molecular Biology Review. Retrieved 8 6, 2020, from Central Dogma of Biology: Classic View: https://www.ncbi.nlm.nih.gov/Class/MLACourse/Modules/MolBioReview/central_dogma.html
Ramkrishna, M., Clare, M. A., Jiang, W., Evan, G., & Christine, M. E. (2020). Pan-cancer analysis reveals cooperativity of bothstrands of microRNA that regulate tumorigenesisand patient survival. NATURE COMMUNICATIONS, Journal Article 1 - 15.
Simon, C. (2019, September 02). Cancer Genetics Web. Retrieved August 08, 2020, from MicroRNAs: http://www.cancerindex.org/geneweb/MicroRNAs.html
[1]It’s quiet controversial to suggest that all cells, with few exceptions, carry the same DNA sequence. From (Eric, 2015) in The Patient Will See You Know, we have to take into account that different cells from the same individual might carry mutations in their gene sequences. There is another slight issue which is that there are still genes yet to be identified. But these are details that can be skipped if we want to have a basic understanding of miRNA’s relevance.
[2] Alternatively, these stem cells are the ones employed for genetic engineering thanks to them being partially differentiated or completely undifferentiated, which allows them to be used for experimenting as mimics of human organs, called organoids (MIT Technology Review, 2019) , for instance. [3] To gain a deeper understating from such brief explanation, please visit https://phet.colorado.edu/en/simulation/gene-expression-essentials for an interactive simulation, as well as read further on the literature about gene expression.
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