), preventing the translation of mRNA on ribosomes into the protein it encodes. Ultimately, the effect of small interfering RNA is identical to that of simply reducing gene expression.
Small interfering RNAs were discovered in 1999 by David Baulcombe's group in the UK as a component of a post-transcriptional gene silencing system in plants. PTGS, en:post-transcriptional gene silencing). The team published their findings in the journal Science.
Double-stranded RNA can enhance gene expression through a mechanism called RNA-dependent gene activation. RNAa, small RNA-induced gene activation). It has been shown that double-stranded RNAs complementary to the promoters of target genes cause activation of the corresponding genes. RNA-dependent activation upon administration of synthetic double-stranded RNA has been demonstrated for human cells. It is not known whether a similar system exists in the cells of other organisms.
By providing the ability to turn off essentially any gene at will, small interfering RNA-based RNA interference has generated enormous interest in basic and applied biology. The number of broad-based RNAi-based tests to identify important genes in biochemical pathways is growing. Since the development of diseases is also determined by the activity of genes, it is expected that in some cases, switching off a gene using small interfering RNA may have a therapeutic effect.
However, the application of small interfering RNA-based RNA interference to animals, and especially to humans, faces many difficulties. Experiments have shown that the effectiveness of small interfering RNA is different for different types of cells: some cells easily respond to the influence of small interfering RNA and demonstrate a decrease in gene expression, while in others this is not observed, despite effective transfection. The reasons for this phenomenon are still poorly understood.
Results from phase 1 trials of the first two RNAi therapeutics (intended to treat macular degeneration), published in late 2005, show that small interfering RNA drugs are easily tolerated by patients and have acceptable pharmacokinetic properties.
Preliminary clinical trials of small interfering RNAs targeting the Ebola virus indicate that they may be effective for post-exposure prophylaxis of the disease. This drug allowed the entire group of experimental primates to survive after receiving a lethal dose of the Zaire Ebolavirus
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.
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Scientists believe that incorrect expression of small RNAs is one of the causes of a number of diseases that seriously affect the health of many people around the world. These diseases include cardiovascular 23 and cancer 24 . As for the latter, this is not surprising: cancer indicates abnormalities in the development of cells and their fate, and small RNAs play a critical role in the corresponding processes. Here is one of the most significant examples of the enormous impact that small RNAs have on the body during cancer. We are talking about a malignant tumor, which is characterized by incorrect expression of those genes that act during the initial development of the organism, and not in the postnatal period. This is a type of childhood brain tumor that usually appears before the age of two. Unfortunately, this is a very aggressive form of cancer, and the prognosis here is unfavorable even with intensive treatment. The oncological process develops due to improper redistribution of genetic material in brain cells. A promoter that normally drives strong expression of one of the protein-coding genes undergoes recombination with a specific cluster of small RNAs. Then this entire rearranged region undergoes amplification: in other words, many copies of it are created in the genome. Consequently, small RNAs located “downstream” of the relocated promoter are expressed much more strongly than they should be. The level of active small RNAs is approximately 150-1000 times higher than normal.
Rice. 18.3. Small RNAs activated by alcohol can combine with messenger RNAs that do not affect the body's resistance to the effects of alcohol. But these small RNAs do not bind to the messenger RNA molecules that promote such resistance. This results in a relative predominance of the proportion of messenger RNA molecules encoding protein variations associated with alcohol tolerance.
This cluster encodes more than 40 different small RNAs. In fact, this is generally the largest of such clusters found in primates. It is usually expressed only early in human development, in the first 8 weeks of embryonic life. Its strong activation in the infant brain leads to catastrophic effects on genetic expression. One consequence is the expression of an epigenetic protein that adds modifications to the DNA. This leads to widespread changes in the entire pattern of DNA methylation, and therefore to abnormal expression of all sorts of genes, many of which should only be expressed when immature brain cells divide during the early stages of development. This is how the cancer program starts in the baby's cells 25.
Such communication between small RNAs and the epigenetic machinery of the cell can have a significant impact on other situations when cells develop a predisposition to cancer. This mechanism likely results in the effect of disruption of small RNA expression being enhanced by changes in epigenetic modifications that are transmitted to daughter cells from the mother. This can create a pattern of potentially dangerous changes in the pattern of gene expression.
So far, scientists have not figured out all the stages of the interaction of small RNAs with epigenetic processes, but they can still get some hints about the features of what is happening. For example, it turned out that a certain class of small RNAs, which enhance the aggressiveness of breast cancer, targets certain enzymes in messenger RNAs that remove key epigenetic modifications. This alters the pattern of epigenetic modifications in the cancer cell and further disrupts genetic expression 26 .
Many forms of cancer are difficult to track in a patient. Oncological processes can occur in hard-to-reach places, which complicates the sampling procedure. In such cases, it is not easy for the doctor to monitor the development of the cancer process and the response to treatment. Often doctors are forced to rely on indirect measurements - say, a tomographic scan of a tumor. Some researchers believe that small RNA molecules could help create a new technique for monitoring tumor development, which could also study its origin. When cancer cells die, small RNAs leave the cell when it ruptures. These small junk molecules often form complexes with cellular proteins or are wrapped in fragments of cell membranes. Due to this, they are very stable in body fluids, which means that such RNAs can be isolated and analyzed. Since their quantities are small, researchers will have to use very sensitive analysis methods. However, nothing is impossible here: the sensitivity of nucleic acid sequencing is constantly increasing 27 . Data have been published confirming the promise of this approach for breast cancer 28 , ovarian cancer 29 and a number of other cancers. Analysis of small circulating RNAs in lung cancer patients has shown that these RNAs help distinguish between patients with a solitary pulmonary nodule (not requiring therapy) and patients who develop malignant tumor nodules (requiring treatment) 30 .
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
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