Цитирование (постоянный адрес по стандартам ВАКа и ГОСТа)
По стандарту ВАК Республики Беларусь: Nikolai KOLESNIKOV, Sergei TITOV, Igor ZHIMULEV, MICRORNAs IN CANCER DAGNOSTICS [Электронный ресурс]: электрон. данные. - Минск: Белорусская цифровая библиотека LIBRARY.BY, 12 ноября 2021. - Режим доступа: https://library.by/portalus/modules/medecine/readme.php?subaction=showfull&id=1636708766&archive=&start_from=&ucat=& (свободный доступ). – Дата доступа: 23.05.2022.
Nikolai KOLESNIKOV, Sergei TITOV, Igor ZHIMULEV, MICRORNAs IN CANCER DAGNOSTICS / Science in Russia, №6, 2013, C.27-33.
by Nikolai KOLESNIKOV, Dr. Sc. (Biol.), Senior Research Fellow; Sergei TITOV, Cand. Sc. (Biol.), Research Associate; Acad. Igor ZHIMULEV, Director; Institute of Molecular and Cell Biology, RAS Siberian Branch, Novosibirsk, Russia
Early cancer detection is a priority objective of practical medicine: the earlier one does that, the better recovery chances. The recent discovery of a new class of RNA molecules-small noncoding RNAs (microRNAs, or miRs) has revolutionized our ideas of the genomic regulatory potential and spurred new approaches in the early diagnosis of tumor diseases. These molecules can be used as biomarkers permitting to answer many questions concerning the likelihood of progression or regression of this or that type of malignancy, prognostication of its recurrence... hold out a promise of evolving into an essential medication remedy.
Cancer is a sophisticated, subtle disease with a high mortality rate. According to WHO data, it kills as many as 7.5 mln worldwide each year. Here in Russia over 2.6 mln cases were registered early in 2011, with this figure rising to 2,900,629 by the end of that year, or 2.09 percent of the nation's population. Every third cancer victim dies within a year upon diagnosis, for in 60 percent cases malignancies are diagnosed too late, in the third or fourth stage when effective treatment is problematic.
But cancer is not a death warrant, you should fight back; given the present level of medicine, this disease is curable. The main task is to learn how to detect malignant degeneration of cells at the earliest stage.
Unfortunately the available conventional diagnostic methods do not make it possible to do that. Therefore we are searching for more effective methods in attacking this problem proceeding from advanced methods of molecular biology.* The recent discovery of a hitherto unknown class of small noncoding RNA molecules--microRNAs (miRs) orchestrating a great variety of biological functions--is certainly a breakthrough. It has been already demonstrated that deregulation of the expression of individual miRs or their groups, or else the breakdown of their performance entails pathologies, tumors including.
"SMART AS A WHIP"
We know of several classes of RNA molecules different in structure and dimensions, and different in their cell functions as well. Thus, informational (messenger) RNAs (mRNAs) act as intermediaries carrying "messages" from the cell nucleus to the cytoplasm, while ribosomal RNAs (rRNAs) are in control of the basic stages of protein synthesis. The miRs are far smaller, they have but one strand comprising ~22 nucleotides. Encoded by small DNA sites, miRs have been identified in genomes of animals, plants and viruses. In man as many as 1,600 miR genes have been identified; they are capable of controlling the work of 60 percent protein-coding genes. Their function--regulate the expression of protein-coding genes at the transcriptional and posttranscriptional levels**--miRs are carrying out via complementary interactions with mRNA sites that knocks out corresponding genes.
MicroRNAs were discovered in 1993 by Professor Ambros of the University of Massachusetts in the nematode (round worm) as genes implicated in regulating growth processes. At first, however, this discovery was not given its due. Yet in 2002, according to the Science journal, miR was recognized as a "molecule of the year*** (the same year the world scientific community received a draft version of human genome decoding). The discovery of the new class of RNA molecules led to the detection of the "dark" part of the genome, quite tiny (just 0.001 percent of the genome), but playing the leading part in processes of regulation both in individual cells and in the organism at large. MiRs operate as "global switches" of the genome, coordinating and controlling multiple metabolic pathways from the moment of conception. In fact more than 1,000 miRs are expressed in one cell alone. A large body of evidence attests to the critical role of these molecules in the
* See: F. Kiselev, "New Trends in Molecular Diagnosis of Cancer", Science in Russia, No. 1, 2008.--Ed.
** Process by which DNA gives rise to RNA serving as template for the new polynucleotide. RNA formed in this way to act as a program for synthesis of protein is known as messenger (informational) RNA.--Ed.
*** In 2008 Victor Ambros as well as Gary Ruvkun, professor of genetics at Harvard University (USA), and David Balcombe, professor of botanics at Cambridge University (Britain), merited prestigious Albert Lasker award for Basic medical research.--Ed.
miRNA-146 implicated in immune system and inflammations regulation. Different colors denote conservity of a nucleotide sequence: red-most conserved nucleotides, i.e. preserved in millions of years of evolution.
Detection of miR by reverse transcription and polimerase chain reaction (PCR) in a patient (in real times). RFU-relative fluorescent units; U6--small RNA as a marker to determine the level of miRNAs studied; reversion--chemical reaction for a reverse DNA-RNA loop; cDNA--complementary DNA synthesized on mRNA.
key processes of embryonic growth, in cell proliferation and differentiation, in ageing and in immune and stress responses. Our further studies show that miRs are capable of performing not only as posttranslational negative regulators kind of "switching out" gene activity but also possibly as transcription and translation activators.*
Deregulation, i.e. miR dysfunctions (compared with the norma), takes place in a variety of diseases, in oncological diseases, too. Many experiments demonstrate that each type of tumors is characterized by a unique set of miRs, a kind of bar code or signature, which could be detected by sensitive methods based on the polymerase chain reaction (PCR). Yet it has been shown on the other hand that, though there are as many as 250 types of cancer, there exist both "specific" and "gen-
* Translation--process of protein synthesis from amino acids on the template of informational (messenger) RN (mRNA).--Ed.
eral" miRs--deregulation of the latter is registered in every type of malignant cells. Identified to date are 84 "general" oncoworld-related cells, while their expression profiles are thought to be correlating with a diagnostic stage (progression) of the tumor or a prognosis of carcinomas yet to be. The latest findings reveal miRs to be associated with different tumors, and not only that: they can act both as oncogenes and suppressors of neoplasms, and trigger malignant degenerations and gene mutations.
Yet another two factors make miRs an attractive proposition for further research. The first one is of practical value related as it is to the stability of miRs (unlike mRNAs) making it possible to isolate them from biosamples (surgical material, biological fluids) and also from fixed preparations; this opens up wide opportunities for comparative studies. The other factor is connected with stable miRs detected in the blood channel; these molecules are in a free state, that is outside the cell. Furthermore, it has been demonstrated that cancer cells liberate miRs--they can be found in blood plasma or serum within exosomes (heterogeneous extracellular structures). However, the role of miRs discharged by a cancer cell and circulating in blood is yet to be elucidated. It is not quite clear either whether such molecules are decomposition products of malignant cells upon their death or there are specific miRs secreted by cancer cells during intracell communication. This proposition is supported by experimental results obtained in search of methods of early noninvasive diagnostics of oncological diseases through detection in blood plasma or serum of oncomiRs specific for definite kinds of cancer.
Consequently the data thus obtained allow to regard miRs as ideal biomarkers (stable, tissue-specific, quantifiable) for diagnosis of different malignancies, their
Comparison of miR expression in thyroid papillary cancer and thyroid gland colloidal node.
progression and in searching for effective therapeutic strategies. Such cells can be also used as targets to suppress carcinomas. True, we have to admit that experimental work in this field is finding adequate financial support and making progress only abroad by and large. In our country these explorations are off and on both within institutions of the Russian Academy of Sciences and the Russian Academy of Medicine.
Studies into the role of miRs and elaboration of approaches in using them as diagnostic biomarkers in oncological diseases--such are the main lines of research at the Institute of Molecular and Cell Biology of the RAS Siberian Branch. We are cooperating in this field with the Vector-Best, Ltd., Company, with medical institutions of Novosibirsk and the Novosibirsk administrative region (Novosibirsk Clinical Hospital No. 1, Regional Oncologic Dispensary, Neurosurgery Clinic affiliated with the Research Institute for Traumatology and Orthopedics of the federal Health Ministry) and also with the Laboratory of Molecular Mechanisms of Carcinogenesis headed by Dr. Lyudmila Gulyayeva (Research Institute for Molecular Biology and Biophysics, Siberian Branch, Russian Academy of Medicine).
We selected miRs for our studies on the basis of bioinformational analysis of numerous databases and literary evidence. The criterion of our research was implication and even the lead role of miRs in processes characterizing the distinguishing features of cancer cells and their behavior, such as avoidance of apoptosis (natural death), runaway proliferation (tumor growth), invasions and secondary cancers, angiogenesis (formation of new blood vessels). At first five miRs were chosen--those expressed in different types of tumors: miR-21, miR-221, miR-222, miR-155, and miR-205.* Thereupon we took yet another 15 oncogenic miRs to be used for screening different histotypes of tumorsthyroid gland and breast cancers as well as carcinoma of the brain--in our search for bimarkers. The surgical material with an official statement on histology analysis was supplied by the Novosibirsk Clinical Hospital No. 1. We took care to honor the ethical standards enforced by the federal legislation: the use of biological material for scientific purposes is permitted only anonymously and with a patient's consent. Malignant and normal tissues were donated by 180 patients afflicted with different types of thyroidal tumors: follicular adenoma and carcinoma, papillary carcinoma, tumoral lesions... We used a quantitative method of PCR analysis realized by the CFX96 amplificator (Bio-Rad Laboratories, USA). To isolate miRs from the operative material we made use of the home-made set of reagents RealBest extraction 100 (Vector Best, Novosibirsk).
DETECTION OF THYROIDAL CANCER (STRUMA MALIGNA)
The thyroid gland (glandula thyroidea), the largest among the endocrine glands of the organism, synthe-
* In keeping with the nomenclature of international miR databases, the figures denote an identifier of the miR type in the genome of man or animals.--Ed.
Comparison of miR expression in follicular adenoma and follicular cancer.
sizes such essential hormones as thyroxine, triiodthyronine and thyreocalcytonin. These hormones are secreted into blood and thus into all tissues; they regulate metabolism, body temperature, cerebral activity as well the activity of the nervous system, of the genital and mammary glands; that of the heart, muscles and other organs. Thyroidal malfunctions trigger many diseases of millions who often do not even suspect the true cause of their maladies.
Epithelial tumors of the thyroid glad are broken down into benign (innocent) and malignant ones. The most frequent among thyroidal malignancies is papillary cancer (80 percent of the cases) that occurs at any age but three times as often among women. Benign tumors (multinodular goiter or struma, autoimmune thyroiditis, nodules) give rise to malignancies (30 to 50 percent cases). Such pathologies should be diagnosed accurately and told apart from malignancies, especially if cytological assays of the samples are dubious (15-30 percent of biopsies). Another essential consideration: whether surgery is really necessary and justified.
We sought to find potential biomarkers of thyroid gland tumors. To do that we had to address several problems, above all determine the level of expression of mature microRNAs by using the RT-PCR method (the method of polymerase chain reaction and reverse transcription) on surgical material in real time. We had to compare miR expression in pathomorphological types of most common tumors different in their malignancy. And last, we had to define strategies in creating a diagnostic panel, or a set of biomarkers based on miRs and separate somatic mutations.
And so, first, we made a comparative study of expression levels in 12 onco- and tumorsuppressor miRs (tissue growth suppressors) in different histopathological types of thyroid tumors, benign and malignant alike. Second, we identified specific miRNA profiles characteristic of tumoral lesions, adenomas and autoimmune pathologies destroying organs and tissues under the effect of the autoimmune system; of papillary, follicular and medullary carcinomas as well as anaplastic thyroidal cancer. The expression of miR-21, miR-221 is positively different in benign nodular neoplasms and carcinomas compared with the putatively normal adjacent tissues. Cancer causes a rise in the level of the same miRs (from 4- to 100fold compared with the norma); the same is true of miR-1466, or the prognostic microRNA. Insignificant changes in the levels of these miRs take place in the colloidal node. However, a wide range of changes occurs in the case of follicular neoplasms. And thirdly, the samples were identified for the presence of a somatic mutation in the protoncogene BRAF V600E detected in 45-60 percent of the cases, according to literary data, in thyroid papillary cancer
Using miRNA in diagnosing malignancies and in their progression prediction. Red--danger (miRNA deregulation); gray--changes within norma.
but not found in thyroidal follicular carcinoma and benign nodules.
Then we made a statistical study of our data with the aim of developing a diagnostic algorithm, and checking up on its accuracy, specificity and sensitivity. We had to learn it for sure: cancer or no cancer with re-
spect to two groups of cells--malignant and benign. We studied several groups of cells and succeeded in such identification. For instance, the carcinoma-affected group of cells is reliably different from one having tumoral lesions in the level of miR-21, miR-221, and miR-222; it differs from the adenoma group also in the level of miR-205. No oncogene BRAF mutations were detected in the group of follicular adenomas, while in the group of thyroid papillary cancer mutations totaled 81 percent. This shows that BRAF mutations point to the higher accuracy of telling benign tumors from malignancies.
To optimize the algorithm so as to make a clear distinction between "cancer" and "no-cancer" miR groups, we made a comparative study of our results using a set of specialized programs. And thus we identified specific profiles of miR expressions characteristic of different tumor histotypes also different in the presence of a somatic mutation in the BRAF oncogene. Thanks to our algorithm we can now diagnose the case exactly--cancer or no cancer. But how to make use of our results in practical medicine, by clinicians? No easy task! However, we have mapped out strategies toward a miR-based diagnostic and prognostic panel.
DIAGNOSIS OF MAMMARY CANCER AND BRAIN TUMORS
Breast cancer is a widespread tumor among women. But this disease takes on a wide variety of phenotypical as well as pathological and molecular forms and characteristics. MiRs help to improve diagnostic accuracy. We have demonstrated the difference between their expression in infiltrating cancer of mammary ducts and in fibroadenomas (benign tumors). We have also shown that miRs can be used for control of neoadjuvant (new auxiliary) therapy. We have identified a unique profile of expression for the entire set of miRs depending on the kind and intensity of preoperative treatment. Among other things, we have observed different miR indicators in radio- and chemotherapy, and also in combined treatment.
And last, brain tumors. These are a heterogeneous group of neoplasms of different cell composition and origin. They are subdivided into benign and malignant tumors. The commonest--gliomas, meningiomas and neurinomas--were the object of our comparative analysis. The surgical material--tumoral and normal tissues--was obtained from 70 patients of different pathology.
We made a comparative study of expression profiles of a set of seven miRs from brain tumors and normal tissues, and determined specific characteristics for our miR panel so as to identify different histotypes (tissue types). We demonstrated that characteristic of anaplastic astrocytomas is a lower level of miR-221 and miR-222 compared with conventionally normal tissues, while in meningiomas qualitative changes of these miRs differ but little from the norma. The most significant changes of the level on the plus or the minus side were determined for miR-155 and miR-451, depending on the degree of malignancy; this may be of prognostic significance for progression of the disease.
Thus, we have determined specific profiles of miR expression relative to different tumor histotypes depending on the progression of a malignancy. We have mapped out strategies toward a diagnostic and prognostic panel based on microRNAs. Such an approach is imperative because of the absence of screening methods that could be adopted in clinical practice. This would have made it possible to start treatment in good time, be it surgery or adjuvant (auxiliary) therapy, and lengthen a patient's life and improve the quality of his or her life. An approach like that might give an evolutionary impulse in cancer treatment.
The results of our research may be applied in a variety of practical areas. First, in early detection of the disease. In identification of the tumoral histotype and tumor progression. In determining the probability of secondary cancers. Thus, we shall be able to monitor the effect medication (chemo- and radiotherapy) and predict survival chances.