The length of siRNA is 21-25 bp, they are formed from dsRNA. The source of such RNAs can be viral infections, genetic constructs introduced into the genome, long hairpins in transcripts, and bidirectional transcription of mobile elements.
dsRNA is cut by RNase Dicer into fragments 21-25 bp long. with 3" ends protruding by 2 nucleotides, after which one of the chains is part of RISC and directs the cutting of homologous RNAs. RISC contains siRNAs corresponding to both plus and minus strands of dsRNA. siRNAs do not have their own genes and represent are fragments of longer RNAs. siRNAs direct the cutting of the target RNA, since they are completely complementary to it. In plants, fungi and nematodes, RNA-dependent RNA polymerases are involved in the process of suppressing gene expression, for which siRNAs also serve as primers (seeds for the synthesis of new RNA The resulting dsRNA is cut by Dicer, new siRNAs are formed, which are secondary, thus amplifying the signal. 
RNA interference
In 1998, Craig C. Mello and Andrew Fire published in Nature, which stated that double-stranded RNA (dsRNA) is capable of repressing gene expression. Later it turned out that the active principle in this process is short single-stranded RNA. The mechanism of suppression of gene expression using these RNAs is called
RNA interference, as well as RNA silencing. This mechanism is found in all large taxa of eukaryotes: vertebrates and invertebrates, plants and fungi. In 2006, he received the Nobel Prize for this discovery.
Suppression of expression can occur at the transcriptional level or post-transcriptionally. It turned out that in all cases a similar set of proteins and short (21-32 bp) RNAs are required.
siRNAs regulate gene activity in two ways. As mentioned above, they direct the cutting of target RNAs. This phenomenon is called "suppression" ( quelling) in mushrooms, " post-translational gene silencing"in plants and" RNA interference
"in animals. siRNAs 21-23 bp long are involved in these processes. Another type of effect is that siRNAs can suppress the transcription of genes containing homologous siRNA sequences. This phenomenon was called transcriptional gene silencing
(TGS) and is found in yeast, plants and animals. siRNAs also direct DNA methylation, which leads to heterochromatin formation and transcriptional repression. TGS is best studied in the yeast S. pombe, where siRNAs are found to be integrated into a RISC-like protein complex called RITS. In his case, as in the case of RISC, siRNA interacts with a protein of the AGO family. It is likely that siRNA is able to direct this complex to a gene that contains a homologous siRNA fragment. After this, RITS proteins recruit methyltransferases, as a result of which heterochromatin is formed in the locus encoding the siRNA target gene, and active gene expression ceases. 
Role in cellular processes
What is the significance of siRNA in a cell?
siRNAs are involved in cell protection from viruses, repression of transgenes, regulation of certain genes, and formation of centromeric heterochromatin. An important function of siRNA is suppression of the expression of mobile genetic elements. Such suppression can occur both at the transcriptional level and posttranscriptionally.
The genome of some viruses consists of DNA, while others consist of RNA, and the RNA of viruses can be either single- or double-stranded. The process of cutting foreign (viral) mRNA in this case occurs in the same way as described above, that is, by activating the RISC enzyme complex. However, for greater efficiency, plants and insects have invented a unique way to enhance the protective effect of siRNA. By joining the mRNA strand, a section of siRNA can, with the help of the DICER enzyme complex, first complete the second strand of mRNA and then cut it in different places, thus creating a variety of “secondary” siRNAs. They, in turn, form RISC and carry the mRNA through all the stages discussed above, up to its complete destruction. Such “secondary” molecules will be able to specifically bind not only to the part of the viral mRNA to which the “primary” molecule was directed, but also to other areas, which dramatically increases the effectiveness of cellular defense.
Thus, in plants and lower animal organisms, siRNAs are an important part of a kind of “intracellular immunity” that allows them to recognize and quickly destroy foreign RNA. If an RNA containing a virus has entered the cell, such a protection system will prevent it from multiplying. If the virus contains DNA, the siRNA system will prevent it from producing viral proteins (since the necessary mRNA for this will be recognized and cut), and using this strategy will slow down its spread throughout the body.
Mammals, unlike insects and plants, have a different defense system. When foreign RNA, the length of which is more than 30 bp, enters a “mature” (differentiated) mammalian cell, the cell begins to synthesize interferon. Interferon, by binding to specific receptors on the cell surface, is able to stimulate a whole group of genes in the cell. As a result, several types of enzymes are synthesized in the cell, which inhibit protein synthesis and break down viral RNA. In addition, interferon can also act on neighboring, not yet infected cells, thereby blocking the possible spread of the virus.
As you can see, both systems are similar in many ways: they have a common goal and “methods” of work. Even the names “interferon” and “(RNA) interference” themselves come from a common root. But they also have one very significant difference: if interferon, at the first signs of invasion, simply “freezes” the work of the cell, not allowing (just in case) the production of many, including “innocent” proteins in the cell, then the siRNA system is extremely intelligible : Each siRNA will recognize and destroy only its own specific mRNA. Replacement of just one nucleotide within siRNA leads to a sharp decrease in the interference effect . None of the gene blockers known so far has such exceptional specificity for its target gene.
The discovery of RNA interference has given new hope in the fight against AIDS and cancer. It is possible that by using siRNA therapy along with traditional antiviral therapy, a potentiation effect can be achieved, where the two treatments result in a greater therapeutic effect than the simple sum of each given separately.
In order to use the siRNA interference mechanism in mammalian cells, ready-made double-stranded siRNA molecules must be introduced into the cells. The optimal size of such synthetic siRNA is the same 21-28 nucleotides. If you increase its length, the cells will respond by producing interferon and reducing protein synthesis. Synthetic siRNAs can enter both infected and healthy cells, and a decrease in protein production in uninfected cells would be highly undesirable. On the other hand, if you try to use siRNA smaller than 21 nucleotides, the specificity of its binding to the desired mRNA and the ability to form the RISC complex sharply decrease.
If it is possible to deliver siRNA in one way or another that has the ability to bind to any part of the HIV genome (which, as is known, consists of RNA), one can try to prevent its integration into the DNA of the host cell. In addition, scientists are developing ways to influence various stages of HIV reproduction in an already infected cell. The latter approach will not provide a cure, but it can significantly reduce the rate of virus reproduction and give the cornered immune system a chance to “rest” from the viral attack and try to deal with the remnants of the disease itself. In the figure, the two stages of HIV reproduction in a cell, which scientists hope can be blocked using siRNA, are marked with red crosses (stages 4-5 - integration of the virus into the chromosome, and stages 5-6 - assembly of the virus and exit from the cell). 
Today, however, all of the above relates only to the field of theory. In practice, siRNA therapy encounters difficulties that scientists have not yet been able to overcome. For example, in the case of antiviral therapy, it is the high specificity of siRNA that can play a cruel joke: as is known, viruses have the ability to quickly mutate, i.e. change the composition of its nucleotides. HIV has been especially successful in this, the frequency of changes in which is such that a person infected with one subtype of the virus can, after a few years, develop a completely different subtype. In this case, the modified HIV strain will automatically become insensitive to the siRNA selected at the beginning of therapy.
Aging and carcinogenesis
Like any epigenetic factor, siRNAs affect the expression of genes that are silenced. Now there are works that describe experiments on switching off genes associated with tumors. Genes are switched off (knock-down) using siRNA. For example, Chinese scientists used siRNA to turn off the transcription factor 4 (TCF4) gene, whose activity causes Pitt-Hopkins syndrome (a very rare genetic disease characterized by mental retardation and episodes of hyperventilation and apnea) and other mental diseases. In this work, we studied the role of TCF4 in gastric cancer cells. Ectopic expression of TCF4 reduces cell growth in gastric cancer cell lines, knocking out the TCF4 gene using siRNA increases cell migration. Thus, we can conclude that epigenetic switching off (silencing) of the TCF4 gene plays an important role in the formation and development of tumors.
According to research in the Department of Oncology, Albert Einstein Cancer Center, led by Leonard H. Augenlicht, siRNA is involved in turning off the HDAC4 gene, which causes inhibition of colon cancer growth, apoptosis and increased transcription of p21. HDAC4 is a histone deacetylase that is tissue specific, inhibits cell differentiation, and its expression is suppressed during the process of cell differentiation. The work shows that HDAC4 is an important regulator of colon cell proliferation (which is important in the cancer process), and it, in turn, is regulated by siRNA.
The Department of Pathology, Nara Medical University School of Medicine in Japan conducted research on prostate cancer. Replicative cell aging is a barrier against uncontrolled division and carcinogenesis. Short-lived dividing cells (TAC) are part of the prostate cell population from which tumors form. Japanese scientists studied the reasons why these cells overcome aging. Prostate cells in culture were transfected with junB siRNA. These cells exhibit increased expression levels of p53, p21, p16 and pRb, which are detected during aging. Cells in culture that showed reduced levels of p16 were used for the next step. Repeated siRNA transfection into TAC allowed cells to avoid senescence upon p16/pRb inactivation. In addition, silencing of the junB proto-oncogene by junB siRNA causes cell invasion. Based on this, it was concluded that junB is an element for p16 and promotes cellular senescence, preventing TAC malignancy. Thus, junB is a regulator of prostate carcinogenesis and may be a target for therapeutic intervention. And its activity can be regulated using siRNA.
There are a lot of similar studies being carried out. Currently, siRNA is not only an object, but also a tool in the hands of a researcher - doctor, biologist, oncologist, gerontologist. Studying the connection between siRNA and cancer and the expression of age-associated genes is the most important task for science. Very little time has passed since the discovery of siRNA, but many interesting studies and publications related to them have appeared. There is no doubt that their study will be one of humanity’s steps towards victory over cancer and aging...
In a living cell, the flow of information between the nucleus and the cytoplasm never dries up, but understanding all its “swirls” and deciphering the information encoded in it is truly a Herculean task. One of the most important breakthroughs in biology of the last century can be considered the discovery of information (or matrix) RNA (mRNA or mRNA) molecules, which serve as intermediaries carrying information “messages” from the nucleus (from chromosomes) to the cytoplasm. The decisive role of RNA in protein synthesis was predicted back in 1939 in the work of Thorbjörn Kaspersson ( Torbjörn Caspersson), Jean Brachet ( Jean Brachet) and Jack Schultz ( Jack Schultz), and in 1971 George Marbeis ( George Marbaix) triggered hemoglobin synthesis in frog oocytes by injecting the first isolated rabbit messenger RNA encoding this protein.
In 1956–1957 in the Soviet Union, A. N. Belozersky and A. S. Spirin independently proved the existence of mRNA, and also found out that the bulk of RNA in a cell is not template, but ribosomal RNA(rRNA). Ribosomal RNA - the second “main” type of cellular RNA - forms the “skeleton” and functional center of ribosomes in all organisms; It is rRNA (and not proteins) that regulates the main stages of protein synthesis. At the same time, the third “main” type of RNA was described and studied - transfer RNAs (tRNAs), which in combination with two others - mRNA and rRNA - form a single protein-synthesizing complex. According to the fairly popular “RNA world” hypothesis, it was this nucleic acid that lay at the very origins of life on Earth.
Due to the fact that RNA is much more hydrophilic compared to DNA (due to the replacement of deoxyribose with ribose), it is more labile and can move relatively freely in the cell, and therefore deliver short-lived replicas of genetic information (mRNA) to the place where it begins protein synthesis. However, it is worth noting the “inconvenience” associated with this - RNA is very unstable. It is stored much worse than DNA (even inside a cell) and degrades at the slightest change in conditions (temperature, pH). In addition to the “own” instability, a large contribution belongs to ribonucleases (or RNases) - a class of RNA-cleaving enzymes that are very stable and “ubiquitous” - even the skin of the experimenter’s hands contains enough of these enzymes to negate the entire experiment. Because of this, working with RNA is much more difficult than with proteins or DNA - the latter can generally be stored for hundreds of thousands of years with virtually no damage.
Fantastic care during work, tri-distillate, sterile gloves, disposable laboratory glassware - all this is necessary to prevent RNA degradation, but maintaining such standards was not always possible. Therefore, for a long time, they simply did not pay attention to short “fragments” of RNA, which inevitably contaminated solutions. However, over time, it became clear that, despite all efforts to maintain the sterility of the work area, “debris” naturally continued to be discovered, and then it turned out that thousands of short double-stranded RNAs are always present in the cytoplasm, performing very specific functions, and are absolutely necessary for normal development cells and organism.
Principle of RNA interference
Pharmacists have also become interested in the possibility of using siRNA, since the ability to specifically regulate the functioning of individual genes promises unprecedented prospects in the treatment of a host of diseases. Small size and high specificity of action promise high efficacy and low toxicity of siRNA-based drugs; however solve the problem delivery siRNA to diseased cells in the body has not yet been successful - this is due to the fragility and fragility of these molecules. And although dozens of teams are now trying to find a way to direct these “magic bullets” exactly to the target (inside diseased organs), they have not yet achieved visible success. Besides this, there are other difficulties. For example, in the case of antiviral therapy, the high selectivity of the action of siRNA can be a disservice - since viruses quickly mutate, the modified strain will very quickly lose sensitivity to the siRNA selected at the beginning of therapy: it is known that replacing just one nucleotide in siRNA leads to a significant decrease interference effect.
At this point it is worth recalling again - siRNAs were discovered only in plants, invertebrates and unicellular organisms; Although homologues of proteins for RNA interference (Dicer, RISC complex) are also present in higher animals, siRNAs were not detected by conventional methods. What a surprise it was when artificially introduced synthetic siRNA analogues caused a strong specific dose-dependent effect in mammalian cell cultures! This meant that in vertebrate cells, RNA interference was not replaced by more complex immune systems, but evolved along with the organisms, turning into something more “advanced.” Consequently, in mammals it was necessary to look not for exact analogues of siRNAs, but for their evolutionary successors.
Player #2 - microRNA
Indeed, based on the evolutionarily quite ancient mechanism of RNA interference, two specialized systems for controlling the operation of genes appeared in more developed organisms, each using its own group of small RNAs - microRNA(microRNA) and piRNA(piRNA, Piwi-interacting RNA). Both systems appeared in sponges and coelenterates and evolved together with them, displacing siRNA and the mechanism of “naked” RNA interference. Their role in providing immunity is decreasing, since this function has been taken over by more advanced mechanisms of cellular immunity, in particular, the interferon system. However, this system is so sensitive that it also triggers the siRNA itself: the appearance of small double-stranded RNA in a mammalian cell triggers an “alarm signal” (activates the secretion of interferon and causes the expression of interferon-dependent genes, which blocks all translation processes entirely). In this regard, the mechanism of RNA interference in higher animals is mediated mainly by microRNAs and piRNAs - single-stranded molecules with a specific structure that are not detected by the interferon system.
As the genome became more complex, microRNAs and piRNAs became increasingly involved in the regulation of transcription and translation. Over time, they turned into an additional, precise and subtle system of genome regulation. Unlike siRNA, microRNA and piRNA (discovered in 2001, see Box 3) are not produced from foreign double-stranded RNA molecules, but are initially encoded in the host genome.
Meet: microRNA
The microRNA precursor is transcribed from both strands of genomic DNA by RNA polymerase II, resulting in the appearance of an intermediate form - pri-microRNA - which carries the features of ordinary mRNA - m 7 G-cap and polyA tail. This precursor forms a loop with two single-stranded “tails” and several unpaired nucleotides in the center (Fig. 3). Such a loop undergoes two-stage processing (Fig. 4): first, the endonuclease Drosha cuts off single-stranded RNA “tails” from the hairpin, after which the excised hairpin (pre-microRNA) is exported to the cytoplasm, where it is recognized by Dicer, who makes two more cuts (a double-stranded section is cut out , indicated by color in Fig. 3). In this form, the mature microRNA, similar to siRNA, is included in the RISC complex.
Figure 3. Structure of a double-stranded microRNA precursor molecule. Main features: the presence of conserved sequences that form a hairpin; the presence of a complementary copy (microRNA*) with two “extra” nucleotides at the 3′ end; a specific sequence (2–8 bp) that forms a recognition site for endonucleases. The microRNA itself is highlighted in red - this is what Dicer cuts out.
The mechanism of action of many microRNAs is similar to the action of siRNAs: a short (21–25 nucleotides) single-stranded RNA as part of the RISC protein complex binds with high specificity to the complementary site in the 3′ untranslated region of the target mRNA. Binding leads to the cleavage of the mRNA by the Ago protein. However, the activity of microRNA (compared to siRNA) is already more differentiated - if the complementarity is not absolute, the target mRNA may not be degraded, but only reversibly blocked (there will be no translation). The same RISC complex can also be used artificially introduced siRNA. This explains why siRNAs made by analogy with protozoa are also active in mammals.
Thus, we can complement the illustration of the mechanism of action of RNA interference in higher (bilaterally symmetric) organisms by combining in one figure the action diagram of microRNAs and biotechnologically introduced siRNAs (Fig. 5).
Figure 5. Generalized scheme of action of artificial microRNAs and siRNAs(artificial siRNAs are introduced into the cell using specialized plasmids - targeting siRNA vector).
Functions of microRNA
The physiological functions of microRNAs are extremely diverse - in fact, they act as the main non-protein regulators of ontogenesis. microRNAs do not cancel, but complement the “classical” scheme of gene regulation (inducers, suppressors, chromatin compaction, etc.). In addition, the synthesis of microRNAs themselves is complexly regulated (certain pools of microRNAs can be turned on by interferons, interleukins, tumor necrosis factor α (TNF-α) and many other cytokines). As a result, a multi-level network of tuning an “orchestra” of thousands of genes emerges, amazing in its complexity and flexibility, but this does not end there.
microRNAs are more “universal” than siRNAs: “ward” genes do not have to be 100% complementary - regulation is also carried out through partial interaction. Today, one of the hottest topics in molecular biology is the search for microRNAs that act as alternative regulators of known physiological processes. For example, microRNAs involved in the regulation of the cell cycle and apoptosis in plants, Drosophila and nematodes have already been described; in humans, microRNAs regulate the immune system and the development of hematopoietic stem cells. The use of biochip-based technologies (micro-array screening) has shown that entire pools of small RNAs are switched on and off at different stages of cell life. Dozens of specific microRNAs have been identified for biological processes, the expression level of which under certain conditions changes thousands of times, emphasizing the exceptional controllability of these processes.
Until recently, it was believed that microRNAs only suppress - completely or partially - the work of genes. However, it recently turned out that the action of microRNAs can differ radically depending on the state of the cell! In an actively dividing cell, microRNA binds to a complementary sequence in the 3′ region of the mRNA and inhibits protein synthesis (translation). However, in a state of rest or stress (for example, when growing in a poor environment), the same event leads to the exact opposite effect - increased synthesis of the target protein!
Evolution of microRNA
The number of microRNA varieties in higher organisms has not yet been fully established - according to some data, it exceeds 1% of the number of protein-coding genes (in humans, for example, they say there are 700 microRNAs, and this number is constantly growing). microRNAs regulate the activity of about 30% of all genes (the targets for many of them are not yet known), and there are both ubiquitous and tissue-specific molecules - for example, one such important pool of microRNAs regulates the maturation of blood stem cells.
The wide expression profile in different tissues of different organisms and the biological prevalence of microRNAs indicate an evolutionarily ancient origin. MicroRNAs were first discovered in nematodes, and for a long time it was believed that these molecules appear only in sponges and coelenterates; however, they were later discovered in unicellular algae. Interestingly, as organisms become more complex, the number and heterogeneity of the miRNA pool also increases. This indirectly indicates that the complexity of these organisms is provided, in particular, by the functioning of microRNAs. The possible evolution of miRNAs is shown in Figure 6.
Figure 6. MicroRNA diversity in different organisms. The higher the organization of the organism, the more microRNAs are found in it (the number in parentheses). Species in which they were found are highlighted in red. single microRNA.
A clear evolutionary connection can be drawn between siRNA and microRNA, based on the following facts:
- the action of both types is interchangeable and is mediated by homologous proteins;
- siRNAs introduced into mammalian cells specifically “turn off” the desired genes (despite some activation of interferon protection);
- microRNAs are being discovered in more and more ancient organisms.
These and other data suggest the origin of both systems from a common “ancestor”. It is also interesting to note that “RNA” immunity as an independent precursor of protein antibodies confirms the theory of the origin of the first forms of life based on RNA, and not proteins (recall that this is the favorite theory of Academician A.S. Spirin).
The further you go, the more confusing it becomes. Player #3 - piRNA
While there were only two “players” in the arena of molecular biology - siRNA and microRNA - the main “purpose” of RNA interference seemed completely clear. Indeed: a set of homologous short RNAs and proteins in different organisms carries out similar actions; As organisms become more complex, so does functionality.
However, in the process of evolution, nature created another, evolutionarily latest and highly specialized system based on the same successful principle of RNA interference. We are talking about piRNA (piRNA, from Piwi-interaction RNA).
The more complex the genome is organized, the more developed and adapted the organism is (or vice versa? ;-). However, the increase in genome complexity also has a downside: a complex genetic system becomes unstable. This leads to the need for mechanisms responsible for maintaining the integrity of the genome - otherwise spontaneous “mixing” of DNA will simply disable it. Mobile genetic elements ( MGE) - one of the main factors of genome instability - are short unstable regions that can be autonomously transcribed and migrate throughout the genome. Activation of such transposable elements leads to multiple DNA breaks in chromosomes, which can have lethal consequences.
The number of MGEs increases nonlinearly with genome size, and their activity must be contained. To do this, animals, starting with coelenterates, use the same phenomenon of RNA interference. This function is also performed by short RNAs, but not those that have already been discussed, but a third type of them - piRNAs.
“Portrait” of piRNA
Functions of piRNA
The main function of piRNA is to suppress MGE activity at the level of transcription and translation. It is believed that piRNAs are active only during embryogenesis, when unpredictable genome shuffling is especially dangerous and can lead to the death of the embryo. This is logical - when the immune system has not yet started working, the cells of the embryo need some simple but effective protection. The embryo is reliably protected from external pathogens by the placenta (or egg shell). But in addition to this, defense is also necessary from endogenous (internal) viruses, primarily MGE.
This role of piRNA has been confirmed by experience - “knockout” or mutations of the Ago3, Piwi or Aub genes lead to serious developmental disorders (and a sharp increase in the number of mutations in the genome of such an organism), and also cause infertility due to disruption of the development of germ cells.
Distribution and evolution of piRNAs
The first piRNAs are already found in sea anemones and sponges. Plants apparently took a different path - Piwi proteins were not found in them, and the role of a “muzzle” for transposons is performed by the endonuclease Ago4 and siRNA.
In higher animals - including humans - the piRNA system is very well developed, but it can only be found in embryonic cells and in the amniotic endothelium. Why the distribution of piRNA in the body is so limited remains to be seen. It can be assumed that, like any powerful weapon, piRNAs are beneficial only under very specific conditions (during fetal development), and in the adult body their activity will cause more harm than good. Still, the number of piRNAs is an order of magnitude greater than the number of known proteins, and the nonspecific effects of piRNAs in mature cells are difficult to predict.
| siRNA | microRNA | piRNA | |
|---|---|---|---|
| Spreading | Plants, Drosophila, C. elegans. Not found in vertebrates | Eukaryotes | Embryonic cells of animals (starting with coelenterates). Not in protozoa and plants |
| Length | 21–22 nucleotides | 19–25 nucleotides | 24–30 nucleotides |
| Structure | Double-stranded, 19 complementary nucleotides and two unpaired nucleotides at the 3′ end | Single-chain complex structure | Single-chain complex structure. U at 5′ end, 2′ end O-methylated 3′ end |
| Processing | Dicer-dependent | Dicer-dependent | Dicer-independent |
| Endonucleases | Ago2 | Ago1, Ago2 | Ago3, Piwi, Aub |
| Activity | Degradation of complementary mRNAs, acetylation of genomic DNA | Degradation or inhibition of translation of target mRNA | Degradation of mRNA encoding MGE, regulation of MGE transcription |
| Biological role | Antiviral immune defense, suppression of the activity of one’s own genes | Regulation of gene activity | Suppression of MGE activity during embryogenesis |
Conclusion
In conclusion, I would like to provide a table illustrating the evolution of the protein apparatus involved in RNA interference (Fig. 9). It can be seen that protozoa have the most developed siRNA system (protein families Ago, Dicer), and as organisms become more complex, the emphasis shifts to more specialized systems - the number of protein isoforms for microRNA (Drosha, Pasha) and piRNA (Piwi, Hen1) increases. At the same time, the diversity of enzymes that mediate the action of siRNA decreases.
Figure 9. Diversity of proteins involved in RNA interference(numbers indicate the number of proteins of each group). Blue elements characteristic of siRNA and microRNA are highlighted, and red- protein And piRNA-related.
The phenomenon of RNA interference began to be used by the simplest organisms. Based on this mechanism, nature created a prototype of the immune system, and as organisms become more complex, RNA interference becomes an indispensable regulator of genome activity. Two different mechanisms plus three types of short RNAs ( cm. tab. 1) - as a result, we see thousands of fine regulators of various metabolic and genetic pathways. This striking picture illustrates the versatility and evolutionary adaptation of molecular biological systems. Short RNAs again prove that there are no “little things” inside the cell - there are only small molecules, the full significance of whose role we are only beginning to understand.
(True, such fantastic complexity rather suggests that evolution is “blind” and acts without a pre-approved “master plan””;
Small RNAs that form hairpins, or short RNAs that form hairpins (shRNA short hairpin RNA, small hairpin RNA) molecules of short RNAs that form dense hairpins in the secondary structure. ShRNAs can be used to turn off expression... ... Wikipedia
RNA polymerase- from a T. aquaticus cell during replication. Some elements of the enzyme are made transparent, and the RNA and DNA chains are more clearly visible. The magnesium ion (yellow) is located at the active site of the enzyme. RNA polymerase is an enzyme that carries out ... ... Wikipedia
RNA interference- Delivery of small RNAs containing hairpins using a lentivirus-based vector and the mechanism of RNA interference in mammalian cells RNA interference (a ... Wikipedia
RNA gene- Non-coding RNA (ncRNA) are RNA molecules that are not translated into proteins. The previously used synonym, small RNA (smRNA, small RNA), is no longer used, since some non-coding RNAs can be very ... ... Wikipedia
Small nuclear RNAs- (snRNA, snRNA) a class of RNA that is found in the nucleus of eukaryotic cells. They are transcribed by RNA polymerase II or RNA polymerase III and are involved in important processes such as splicing (removal of introns from immature mRNA), regulation ... Wikipedia
Small nucleolar RNAs- (snoRNA, English snoRNA) a class of small RNAs involved in chemical modifications (methylation and pseudouridylation) of ribosomal RNA, as well as tRNA and small nuclear RNA. According to the MeSH classification, small nucleolar RNAs are considered a subgroup... ... Wikipedia
small nuclear (low molecular weight nuclear) RNAs- An extensive group (105,106) of small nuclear RNAs (100,300 nucleotides), associated with heterogeneous nuclear RNA, are part of small ribonucleoprotein granules of the nucleus; M.n.RNAs are a necessary component of the splicing system... ...
small cytoplasmic RNAs- Small (100-300 nucleotide) RNA molecules localized in the cytoplasm, similar to small nuclear RNA. [Arefyev V.A., Lisovenko L.A. English-Russian explanatory dictionary of genetic terms 1995 407 pp.] Topics genetics EN scyrpssmall cytoplasmic... ... Technical Translator's Guide
class U small nuclear RNAs- A group of protein-associated small (from 60 to 400 nucleotides) RNA molecules that make up a significant part of the contents of the splicome and are involved in the process of excision of introns; in 4 of the 5 well-studied Usn types, U1, U2, U4 and U5 RNAs are 5... ... Technical Translator's Guide
RNA biomarkers- * RNA biomarkers * RNA biomarkers a huge number of human transcripts that do not encode protein synthesis (nsbRNA or npcRNA). In most cases, small (miRNA, snoRNA) and long (antisense RNA, dsRNA and other types) RNA molecules are... ... Genetics. encyclopedic Dictionary
Books
- Buy for 1877 UAH (Ukraine only)
- Clinical genetics. Textbook (+CD), Bochkov Nikolay Pavlovich, Puzyrev Valery Pavlovich, Smirnikhina Svetlana Anatolyevna. All chapters have been revised and supplemented in connection with the development of medical science and practice. The chapters on multifactorial diseases, prevention, treatment of hereditary diseases,…
Destruction of the target mRNA can also occur under the influence of small interfering RNA (siRNA). RNA interference is one of the new revolutionary discoveries in molecular biology, and its authors received the Nobel Prize for it in 2002. Interfering RNAs are very different in structure from other types of RNA and are two complementary RNA molecules approximately 21-28 nitrogen bases long, which are connected to each other like strands in a DNA molecule. In this case, two unpaired nucleotides always remain at the edges of each siRNA chain. The impact is carried out as follows. When a siRNA molecule finds itself inside a cell, at the first stage it binds into a complex with two intracellular enzymes - helicase and nuclease. This complex was called RISC ( R NA- i induced s ilencing c complex; silence - English be silent, shut up; silencing - silencing, this is how the process of “turning off” a gene is called in English and specialized literature). Next, the helicase unwinds and separates the siRNA strands, and one of the strands (antisense in structure) in complex with the nuclease specifically interacts with the complementary (strictly corresponding to it) region of the target mRNA, which allows the nuclease to cut it into two parts. The cut sections of mRNA are then exposed to the action of other cellular RNA nucleases, which further cut them into smaller pieces.
SiRNAs found in plants and lower animal organisms (insects) are an important part of a kind of “intracellular immunity” that allows them to recognize and quickly destroy foreign RNA. If an RNA containing a virus has entered the cell, such a protection system will prevent it from multiplying. If the virus contains DNA, the siRNA system will prevent it from producing viral proteins (since the necessary mRNA for this will be recognized and cut), and using this strategy will slow down its spread throughout the body. It has been established that the siRNA system is extremely discriminating: each siRNA will recognize and destroy only its own specific mRNA. Replacement of just one nucleotide within siRNA leads to a sharp decrease in the interference effect. None of the gene blockers known so far has such exceptional specificity for its target gene.
Currently, this method is used mainly in scientific research to identify the functions of various cellular proteins. However, it could potentially also be used to create drugs.
The discovery of RNA interference has given new hope in the fight against AIDS and cancer. It is possible that by using siRNA therapy in conjunction with traditional antiviral and anticancer therapies, a potentiation effect can be achieved, where the two treatments result in a greater therapeutic effect than the simple sum of each given alone.
In order to use the siRNA interference mechanism in mammalian cells for therapeutic purposes, ready-made double-stranded siRNA molecules must be introduced into the cells. However, there are a number of problems that currently do not allow this to be done in practice, much less to create any dosage forms. Firstly, in the blood they are affected by the first echelon of the body’s defense, enzymes - nucleases, which cut potentially dangerous and unusual double strands of RNA for our body. Secondly, despite their name, small RNAs are still quite long, and, most importantly, they carry a negative electrostatic charge, which makes their passive penetration into the cell impossible. And thirdly, one of the most important questions is how to make siRNA work (or penetrate) only in certain (“sick”) cells, without affecting healthy ones? And finally there is the issue of size. The optimal size of such synthetic siRNA is the same 21-28 nucleotides. If you increase its length, the cells will respond by producing interferon and reducing protein synthesis. On the other hand, if you try to use siRNA smaller than 21 nucleotides, the specificity of its binding to the desired mRNA and the ability to form the RISC complex sharply decrease. It should be noted that overcoming these problems is critical not only for siRNA therapy, but also for gene therapy in general.
Some progress has already been made in solving them. For example, scientists are trying to make siRNA molecules more efficient by chemical modifications. lipophilic, that is, capable of dissolving in the fats that make up the cell membrane, and thus facilitating the penetration of siRNA into the cell. And in order to ensure specificity of work within only certain tissues, genetic engineers include in their constructs special regulatory sections, which are activated and trigger the reading of the information contained in such a construct (and therefore siRNA, if it is included there), only in certain cells fabrics.
So, researchers from the University of California, San Diego School of Medicine have developed a new effective system for delivering small interfering RNA (siRNA), which suppresses the production of certain proteins, into cells. This system should become the basis for technology for specific drug delivery to various types of cancer tumors. “Small interfering RNAs, which carry out a process called RNA interference, have incredible potential for treating cancer,” explains Professor Steven Dowdy, who led the research: “and although we still have a lot of work to do, we have now developed the technology delivering drugs to a population of cells – both the primary tumor and metastases, without damaging healthy cells.”
For many years, Dowdy and his colleagues have been studying the anticancer potential of small interfering RNAs. However, conventional siRNAs are tiny, negatively charged molecules that, due to their properties, are extremely difficult to deliver into cells. To achieve this, scientists used a short signaling protein PTD (peptide transduction domain). Previously, more than 50 “hybrid proteins” were created with its use, in which PTD was combined with tumor suppressor proteins.
However, simply connecting siRNA to PTD does not lead to delivery of RNA into the cell: siRNA is negatively charged, PTD is positively charged, resulting in the formation of a dense RNA-protein conglomerate that is not transported across the cell membrane. So the researchers first coupled the PTD to a protein RNA-binding domain that neutralized the negative charge of the siRNA (resulting in a fusion protein called PTD-DRBD). Such an RNA-protein complex easily passes through the cell membrane and enters the cell cytoplasm, where it specifically inhibits the messenger RNA proteins that activate tumor growth.
To test the ability of the PTD-DRBD fusion protein to deliver siRNA into cells, the scientists used a cell line derived from human lung cancer. After treating cells with PTD-DRBD-siRNA, it was found that tumor cells were most susceptible to siRNA, while in normal cells (T cells, endothelial cells and embryonic stem cells were used as controls), where there was no increased production of oncogenic proteins, no toxic effects were observed.
This method can be subjected to various modifications using different siRNAs to suppress different tumor proteins - not only those produced in excess, but also mutant ones. It is also possible to modify therapy in case of relapse of tumors, which usually become resistant to chemotherapy drugs due to new mutations.
Oncological diseases are very variable, and the molecular characteristics of tumor cell proteins are individual for each patient. The authors of the work believe that in this situation, the use of small interfering RNA is the most rational approach to therapy.
A.M. Deichman, S.V. Zinoviev, A.Yu. Baryshnikov
GENE EXPRESSION AND SMALL RNAS IN ONCOLOGY
GU RONC im. N.N.Blokhin RAMS, Moscow
SUMMARY
The article presents the role of small RNAs that control most of the vital functions of the cell and body, and their possible connection, in particular, with oncogenesis and other (including hypothetical) intracellular mechanisms of genomic expression.
Keywords: small RNAs, RNA interference (RNAi), double-stranded RNA (dsRNA), RNA editing, oncogenesis.
A.M. Deichman, S.V.Zinoviev, A.Yu.Baryshnikov.
THE GENE EXPRESSION AND SMALL RNAS IN ONCOLOGY
N.N. Blokhin Russian Cancer Research Center RAMS, Moscowow
ABSTRACT
In the paper role of small RNAs supervising the majority vital functions of cell and organism and possible connection of them in particular with oncogenesis and others (including hypothetical) intracellular mechanisms of genome expression is submitted.
Key words: Small RNAs, interference RNAs (RNAi), double strand RNAs (dsRNAs), RNA editing, tumorogenesis.
Introduction
The expression of individual genes and entire eukaryotic genomes, including processing, various types of transcription, splicing, rearrangements, RNA editing, recombination, translation, RNA interference, is regulated by certain proteins (products of regulatory, structural, homeotic genes, transcription factors), mobile elements, RNA and low molecular weight effectors. Among the processing RNAs are rRNA, tRNA, mRNA, some types of regulatory RNA and small RNA.
It is now known that small RNAs do not encode proteins, often number in the hundreds per genome, and are involved in the regulation of the expression of various eukaryotic genes (somatic, immune, germinal, stem cells). The processes of differentiation (hematopoiesis, angiogenesis, adipogenesis, myogenesis, neurogenesis), morphogenesis (including embryonic stages, development/growth, physiological regulation), proliferation, apoptosis, carcinogenesis, mutagenesis, immunogenesis, aging (life extension), epigenetic silencing are under control ; cases of metabolic regulation (for example, glycosphingolipids) have been noted. A wider class of non-coding RNAs of 20-300/500 nucleotides and their RNPs are found not only in the nucleus/nucleolus/cytoplasm, but also in DNA-containing cellular organelles (animal mitochondria; microRNAs and small consensus sequences for chloroplast transcripts have been found in plants RNA).
For control and regulation of v.n. processes, it is important: 1. that small-sized natural/artificial RNAs (small RNAs, tRNAs, etc.) and their complexes with proteins (RNPs) are capable of transmembrane cellular and mitochondrial transport; 2. that after the breakdown of mitochondria, part of their contents, RNA and RNP, may end up in the cytoplasm and nucleus. The listed properties of small RNAs (SRNAs), the functionally significant role of which is only increasing in the process of study, obviously have a connection with the alertness factor for cancer and other genetic diseases. At the same time, the high significance of epigenomic modifications of chromatin in the occurrence of tumors became clear. We will consider only a very limited number of cases out of many similar ones.
Small RNAs
The mechanism of action of small RNAs is their ability to bind almost complementarily to the 3"-untranslated regions (3"-UTRs) of target mRNAs (which sometimes contain DNA/RNA transposing MIR/LINE-2 elements, as well as conservative Alu repeats ) and cause RNA interference (RNAi=RNAi; in particular, during an antiviral response). The complication, however, is that in addition to cellular ones, there are also virus-encoded small RNAs (herpes, SV40, etc.; EBV, for example, contains 23, and KSHV - 12 miRNAs) that interact with transcripts of both the virus and the host. More than 5 thousand cellular/viral miRNAs alone are known in 58 species. RNAi initiates either degradation (with the participation of the RISC complex, RNA-Induced Silencing Complex) along nuclease-vulnerable fragments of continuous lncRNA helices (double-stranded RNA mRNA, etc.), or partially reversible inhibition of discontinuously helical lncRNAs during translation of target mRNAs. Mature small RNAs (~15-28 nucleotides) are formed in the cytoplasm from their nuclear-processed precursors of varying lengths (tens and hundreds of nucleotides). In addition, small RNAs are involved in the formation of the silent chromatin structure, regulation of transcription of individual genes, suppression of transposon expression, and maintenance of the functional structure of extended regions of heterochromatin.
There are several main types of small RNAs. The most well studied are microRNAs (miRNAs) and small interfering RNAs (siRNAs). In addition, among small RNAs, the following are being studied: piRNAs active in germinal cells; small interfering RNAs associated with endogenous retrotransposons and repeating elements (with local/global heterochromatization - starting from the early stages of embryogenesis; maintain the telomere level), Drosophila rasiRNAs; often encoded by introns of protein genes and functionally important in translation, transcription, splicing (de-/methylation, pseudouridylation of nucleic acids) small nuclear (snRNAs) and nucleolar (snoRNAs) RNAs; small modulator RNAs, smRNAs, with little-known functions, complementary to the DNA-binding NRSE (Neuron Restrictive Silencer Element) motifs; plant transactivating small interfering RNAs, tasiRNAs; short hairpin RNAs, shRNAs, providing long-term RNAi (stable gene silencing) of long lncRNA structures during the antiviral response in animals.
Small RNAs (miRNAs, siRNAs, etc.) interact with newly synthesized transcripts of the nucleus/cytoplasm (regulating splicing, translation of mRNA; methylation/pseudouridylation of rRNA, etc.) and chromatin (during temporary local and epigenetically inherited heterochromatinization of dividing somatic germ cells). Heterochromatinization, in particular, is accompanied by DNA de-/methylation, as well as methylation, acetylation, phosphorylation and ubiquitination of histones (modification of the “histone code”).
The first among small RNAs were the miRNAs of the nematode Caenorhabditis elegans (lin-4), their properties and genes, and somewhat later the miRNAs of the plant Arabidopsis thaliana. Currently, they are associated with multicellular organisms, although they are shown in the unicellular alga Chlamydomonas reinhardtii, and RNAi-like silencing pathways, in connection with antiviral/like protection involving the so-called. psiRNAs, discussed for prokaryotes. The genomes of many eukaryotes (including Drosophila and humans) contain several hundred miRNA genes. These stage-/tissue-specific genes (as well as their corresponding target mRNA regions) are often highly homologous in phylogenetically distant species, but some of them are lineage-specific. miRNAs are contained in exons (protein-coding, RNA genes), introns (most often pre-mRNA), intergenic spacers (including repeats), have a length of up to 70-120 nucleotides (or more) and form hairpin loop/stem structures. To determine their genes, not only biochemical and genetic approaches are used, but also computer approaches.
The most typical length of the “working region” of mature miRNAs is 21-22 nucleotides. These are perhaps the most numerous of the non-protein-coding genes. They can be located in the form of single copies (more often) or clusters containing many similar or different miRNAs genes, transcribed (often from autonomous promoters) as a longer precursor, processed in several stages to individual miRNAs. It is believed that there is a miRNA regulatory network that controls many fundamental biological processes (including tumorigenesis/metastasis); probably at least 30% of human expressed genes are regulated by miRNAs.
This process involves the lncRNA-specific RNase III-like enzymes Drosha (nuclear ribonuclease; initiates the processing of intronic pre-miRNAs after splicing of the main transcript) and Dicer, which functions in the cytoplasm and cleaves/degrades, respectively, hairpin pre-miRNAs (to mature miRNAs ) and hybrid miRNAs/mRNA structures formed later. Small RNAs, together with several proteins (including vn RNases, AGO-family proteins, transmethylases/acetylases, etc.) and with the participation of the so-called. RISC- and RITS-like complexes (the second one induces transcriptional silencing) are capable, respectively, of causing RNAi/degradation and subsequent gene silencing at the RNA (before/during translation) and DNA (during transcription of heterochromatin) levels.
Each miRNA potentially pairs with multiple targets, and each target is controlled by a number of miRNAs (reminiscent of gRNAs-mediated pre-mRNA editing in trypanosome kinetoplasts). In vitro analysis has shown that miRNA regulation (as well as RNA editing) is a key post-transcriptional modulator of gene expression. Similar miRNAs competing for the same target are potential transregulators of RNA-RNA and RNA-protein interactions.
In animals, miRNAs are best studied in the nematode Caenorhabditis Elegans; more than 112 genes have been described. Thousands of endogenous siRNAs (no genes; associated, in particular, with spermatogenesis-mediated transcripts and transposons) are also found here. Both small RNAs of metazoans can be generated by RNA polymerases that exhibit the activity (not homology) of RdRP-II (as for most other RNAs) and RdRP-III types. Mature small RNAs are similar in composition (including terminal 5"-phosphates and 3"-OH), length (usually 21-22 nucleotides) and function, and can compete for the same target. However, RNA degradation, even with complete target complementarity, is more often associated with siRNAs; translational repression, with partial, usually 5-6 nucleotides, complementarity - with miRNAs; and the precursors, respectively, are exo-/endogenous (hundreds/thousands of nucleotides) for siRNAs, and usually endogenous (tens/hundreds of nucleotides) for miRNAs and their biogenesis is different; however, in some systems these differences are reversible.
RNAi, mediated by siRNAs and miRNAs, has a variety of natural roles: from the regulation of gene expression and heterochromatin to genome protection against transposons and viruses; but siRNAs and some miRNAs are not conserved between species. In plants (Arabidopsis thaliana) the following were found: siRNAs corresponding to both genes and intergenic (including spacers, repeats) regions; a huge number of potential genome sites for various types of small RNAs. Nematodes also have so-called variable autonomously expressed 21U-RNAs (dasRNAs); they have a 5"-Y-monophosphate, comprise 21 nucleotides (20 of them are variable), and are located between or inside the introns of protein-coding genes at more than 5700 sites in two regions of chromosome IV.
MiRNAs play an important role in gene expression in health and disease; in humans there are at least 450-500 such genes. Usually binding to the 3"-UTR regions of mRNA (other targets), they can selectively and quantitatively (in particular, when removing products of low-expressed genes from circulation) block the work of some genes and the activity of other genes. It turned out that sets of profiles of expressed micro- RNAs (and their targets) change dynamically during ontogenesis, cell and tissue differentiation.These changes are specific, in particular, during cardiogenesis, the process of optimizing the size of the length of dendrites and the number of synapses of a nerve cell (with the participation of miRNA-134, other small RNAs). development of many pathologies (oncogenesis, immunodeficiencies, genetic diseases, parkinsonism, Alzheimer's disease, ophthalmological disorders (retinoblastoma, etc.) associated with infections of various nature). The total number of detected miRNAs is growing much faster than the description of their regulatory role and connection with specific targets .
Computational analysis predicts hundreds of mRNA targets for individual miRNAs and the regulation of individual mRNAs by multiple miRNAs. Thus, miRNAs can serve the purpose of eliminating transcripts of target genes or fine-tuning their expression at the transcriptional/translational levels. Theoretical considerations and experimental results support the existence of diverse roles of miRNAs.
A more complete list of aspects related to the fundamental role of small RNAs in eukaryotes in growth/development processes and in some pathologies (including cancer epigenomics) is reflected in the review.
Small RNAs in Oncology
The processes of growth, development, progression and metastasis of tumors are accompanied by many epigenetic changes that develop into rarer, persistently heritable genetic changes. Rare mutations, however, can have great weight (for a specific individual, nosology), because in relation to individual genes (for example APC, K-ras, p53) the so-called “funnel” effect associated with almost irreversible development/consequences of cancer. The tumor-specific heterogeneity of progenitor cells in terms of the expression profile of various genes (proteins, RNAs, small RNAs) is determined by associated variations in restructured epigenomic structures. The epigenome is modulated by methylation, post-translational modifications/replacements of histones (with non-canonical ones), remodeling of the nucleosomal structure of genes/chromatin (including genomic imprinting, i.e. dysfunction of the expression of alleles of parental genes and X chromosomes). All this, and with the participation of RNAi regulated by small RNAs, leads to the appearance of defective heterochromatic (including hypomethylated centromeric) structures.
The formation of gene-specific mutations may be preceded by the known accumulation of hundreds of thousands of somatic clonal mutations in simple repeats or microsatellites of a non-coding (rarely coding) region - at least in tumors with a microsatellite mutator phenotype (MMP); they make up a significant part of colorectal cancers, as well as lung, stomach, endometrial, etc. Unstable mono-/heteronucleotide microsatellite repeats (poly-A6-10, similar) are contained many times more often in regulatory non-coding genes that control the expression (introns, intergenic) than in the coding (exon) regions of the genome of microsatellite-unstable, MSI+, tumors. Although the nature of the appearance and mechanisms of localization of MS-stable/unstable regions are not completely clear, the formation of MS instability correlated with the frequency of mutations of many genes that were not previously mutated in MSI+ tumors and probably channeled the pathways of their progression; Moreover, the frequency of MSI repeat mutations in these tumors increased by more than two orders of magnitude. Not all genes have been analyzed for the presence of repeats, but their degree of mutability in coding/non-coding regions is different, and the accuracy of methods for determining the frequency of mutations is relative. It is important that non-coding regions of MSI-mutable repeats are often biallelic, while coding regions are monoallelic.
A global decrease in methylation in tumors is typical for repeats, transposable elements (TEs; their transcription increases), promoters, CpG sites of tumor suppressor miRNA genes and correlates with hypertranscription of retrotransposons in progressive cancer cells. Normally, fluctuations in the “methylome” are associated with parent-/stage-/tissue-specific “methylation waves” and strong methylation of centromeric satellite regions of heterochromatin, regulated by small RNAs. When satellites are undermethylated, the resulting chromosome instability is accompanied by increased recombination, and disruption of ME methylation can trigger their expression. These factors favor the development of a tumor phenotype. Small RNA therapy can be highly specific, but must be controlled because targets may be not only individual, but also many mRNA/RNA molecules, and newly synthesized RNAs of various (including non-coding intergenic repeats) regions of chromosomes.
Most of the human genome is made up of repeats and MEs. Retrotransposon L1 (LINE element) contains, like endogenous retroviruses, reversease (RTase), endonuclease and is potentially capable of transferring non-autonomous (Alu, SVA, etc.) retroelements; silencing of L1/like elements occurs as a result of methylation at CpG sites. Note that among the CpG sites of the genome, the CpG islands of gene promoters are weakly methylated, and 5-methylcytosine itself is a potentially mutagenic base, deaminated into thymine (chemically, or with the participation of RNA/(DNA) editing, DNA repair); however, some of the CpG islands are subject to excessive aberrant methylation, accompanied by repression of suppressor genes and cancer development. Next: the RNA-binding protein encoded by L1, interacting with the proteins AGO2 (Argo-naute family) and FMRP (fragile mental retardation protein, protein of the effector RISC complex), promotes the movement of the L1 element - which indicates a possible mutual regulation of the systems RNAi and retroposition of human LINE elements. It is important, in particular, that Alu repeats are able to move into the intron/exon region of genes.
These and similar mechanisms can enhance the pathological plasticity of the tumor cell genome. Suppression of RTase (encoded, like endonuclease, by L1 elements; RTase is also encoded by endogenous retroviruses) via the RNAi mechanism was accompanied by a decrease in proliferation and increased differentiation in a number of cancer cell lines. When the L1 element was introduced into a proto-oncogene or suppressor gene, DNA double-strand breaks were observed. In germline tissues (mice/human), the expression level of L1 was increased, and its methylation depended on the piRNAs-(26-30-bp)-associated silencing system, where PIWI proteins are variants of the large Argo-naute protein family, mutations in which they lead to demethylation/derepression of L1/like elements with long terminal repeats. PIWI proteins, to a greater extent than Dicer-1/2 and Ago proteins, are associated with rasiRNAs silencing pathways. The silencing pathways mediated by piRNAs/siRNAs are realized through intranuclear bodies containing large evolutionarily conserved multiprotein PcG complexes, the functions of which are often impaired in tumor cells. These complexes are responsible for long-range action (across more than 10 kb, between chromosomes) and regulate the cluster of HOX genes responsible for the body plan.
New principles of antisense therapy can be developed taking into account knowledge about more highly specific (than histone-modifying inhibitors of DNA/protein methylation) antitumor epigenomic agents, the fundamental principles of epigenomic RNA silencing and the role of small RNAs in carcinogenesis.
Micro-RNA in Oncology
It is known that increased tumor growth and metastasis can be accompanied by an increase in some and a decrease in the expression of other individual/sets of miRNAs (Table 1). Some of them may have a causative role in tumorigenesis; and even the same miRNAs (like miR-21/-24) in different tumor cells can exhibit both oncogenic and suppressive properties. Each type of human malignant tumor is clearly distinguishable by its “miRNA fingerprint,” and some miRNAs can function as oncogenes, tumor suppressors, initiators of cell migration, invasion, and metastasis. In pathologically altered tissues, reduced amounts of key miRNAs likely involved in anticancer defense systems are often found. The miRNAs (miRs) involved in oncogenesis have formed the idea of the so-called. “oncomirah”: analysis of the expression of more than 200 miRNAs in over 1000 samples of lymphomas and solid cancers made it possible to successfully classify tumors into subtypes according to their origin and stage of differentiation. The functions and role of miRNAs have been successfully studied using: anti-miR oligonucleotides modified (to increase lifetime) at 2"-O-methyl and 2"-O-methoxyethyl groups; as well as LNA oligonucleotides, in which the ribose oxygen atoms in positions 2" and 4" are connected by a methylene bridge.
(Table 1)……………….
Tumor | miRNAs |
Lungs' cancer | 17-92 , let-7↓ , 124a↓ , 126 ↓ , 143 ↓ , 145 ↓ , 155 , 191 , 205 , 210 |
Mammary cancer | 21 , 125b↓ , 145 ↓ , 155 |
Prostate cancer | 15a ↓ , 16-1 ↓ , 21 , 143 ↓ ,145 ↓ |
Bowel cancer | 19a , 21 , 143 ↓ , 145 ↓ |
Pancreas cancer | 21 , 103 , 107 , 155 v |
Ovarian cancer | 210 |
Chronic lymphocytic leukemia | 15a ↓ , 16-1 ↓ , 16-2 , 23 b , 24-1 , 29 ↓ , 146 , 155 , 195 , 221 , 223 ↓ |
Table 1 .
miRNAs whose expression increases () or decreases ( ↓ ) in some of the most common tumors compared to normal tissues (see, and also).
It is believed that the regulatory role of expression, disappearance and amplification of miRNA genes in the susceptibility to initiation, growth and progression of most tumors is significant, and mutations in miRNA/target mRNA pairs are synchronized. The expression profile of miRNAs can be used for classification, diagnosis and clinical prognosis in oncology. Changes in the expression of miRNAs can affect the cell cycle, the cell's survival program. Mutations of miRNAs in stem and somatic cells (as well as the choice of polymorphic variants of mRNA targets) may contribute to, or even play a critical role in the growth, progression and pathophysiology of many (if not all) malignancies. With the help of miRNAs, correction of apoptosis is possible.
In addition to individual miRNAs, clusters of them were discovered, acting as an oncogene that provokes the development, in particular, of hematopoietic tissue cancer in experimental mice; miRNA genes with oncogenic and suppressor properties can be located in the same cluster. Cluster analysis of miRNAs expression profiles in tumors makes it possible to determine its origin (epithelium, hematopoietic tissue, etc.) and classify different tumors of the same tissue with non-identical transformation mechanisms. Assessment of the expression profile of miRNAs can be carried out using nano-/microarrays; The accuracy of such classification, when developing the technology (which is not easy), turns out to be higher than using mRNA profiles. Some of the miRNAs are involved in the differentiation of hematopoietic cells (mouse, human), initiation of cancer cell progression. Human miRNA genes are often located in the so-called. “fragile” sites, areas with a predominance of deletions/insertions, point breaks, translocations, transpositions, minimally deleted and amplified regions of heterochromatin involved in oncogenesis.
Angiogenesis . The role of miRNAs in angiogenesis is likely significant. Increased angiogenesis in some Myc-activated human adenocarcinomas was accompanied by changes in the expression pattern of some miRNAs, and gene knockdown of other miRNAs led to weakening and suppression of tumor growth. Tumor growth was accompanied by mutations in the K-ras, Myc and TP53 genes, increased production of the angiogenic VEGF factor and the degree of Myc-associated vascularization; while the antiangiogenic factors Tsp1 and CTGF were suppressed by miR-17-92 and other cluster-associated miRNAs. Tumor angiogenesis and vascularization were enhanced (particularly in colonocytes) by coexpression of two oncogenes rather than one.
Neutralization of the antiangiogenic factor LATS2, an inhibitor of animal cyclin-dependent kinase (CDK2; human/mouse), with miRNAs-372/373 (“potential oncogenes”) stimulated testicular tumor growth without damaging the p53 gene.
Potential modulators of angiogenic properties (in-vitro/in-vivo) are miR-221/222, the targets of which, c-Kit receptors (others), are factors of angiogenesis of endothelial venous HUVEC cells of the umbilical cord, etc. These miRNAs and c-Kit interact as part of a complex cycle that controls the ability of endothelial cells to form new capillaries.
Chronic lymphocytic leukemia (CLL). In B-cell chronic lymphocytic leukemia (CLL), a reduced level of gene expression miR-15a/miR-16-1 (and others) is noted in the 13q14 region of the human chromosome - the site of the most common structural abnormalities (including deletions of the 30kb region), although the genome expressed hundreds of human mature and pre-miRNAs. Both miRNAs, potentially effective in tumor therapy, contained antisense regions of the antiapoptotic protein Bcl2, suppressed its over-expression, stimulated apoptosis, but were almost/completely absent in two-thirds of the “deviant” CLL cells. Frequent mutations of sequenced miRNAs in stem/somatic cells were identified in 11 of 75 patients (14.7%) with a familial predisposition to CLL (mode of inheritance unknown), but not in 160 healthy patients. These observations raise speculation about the direct functioning of miRNAs in leukemogenesis. Currently, not everything is known about the relationship between the gene expression levels of miRNAs (and their functions) and other genes in normal/tumor cells.
DocumentRelevance. Dysfunction of the facial nerve during surgery on the parotid salivary gland is one of the current problems and is determined by both the prevalence of the disease and the significant frequency
Dawson Church - the genius is in your genes epigenetic medicine and the new biology of intentions book from the library www e - puzzle ru book from the library www e - puzzle ru table of contents
BookEthics spirituality oncology hiv p garyaev* a enfi summary
DocumentThis article reflects a new look at the problem of oncology and HIV infection in the light of Linguistic-Wave Genetics (LWG) and the Essence Coding Theory (ESC) based on Russian and other socio-cultural realities.
Oncological Research Center and Blokhina Odintsova Anastasia Sergeevna new chemotherapy regimens for advanced and recurrent cervical cancer 01/14/12 – oncology
Thesis4.4. Determination of the uridine glucoronyl transferase isoenzyme gene (UGT1A1) in the blood serum of patients with cervical cancer who received first-line chemotherapy with irinotecan with platinum derivatives 105