Method for determining lung cancer cell sensitivity to cisplatin applying marker gene expression pattern and set for implementing it

FIELD: medicine.

SUBSTANCE: presented group of inventions refers to medicine, molecular biology and biotechnology. A method for determining lung cancer cell sensitivity to cisplatin involving determining MLH1, ERCC1, DDB2, AKR1B1, FTL genes expression patterns vs. cisplatin IC50 for known cell lines; constructing cisplatin IC50 for these cell lines vs. derived gene expression patterns calibration straight, and hybridising on a microchip. What is also presented is a set of oligonucleotide probes presented by SEQ ID NO: 9-13.

EFFECT: presented group of inventions provides effective aids and methods for determining lung cancer cell sensitivity to cisplatin.

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Area of technology

The invention relates to the field of molecular medicine, molecular biology and biotechnology, namely to the determination of drug resistance of cells and tumors, and to methods of detection of specific RNA targets and biological microarrays.

Lung cancer is one of the first places in the world for morbidity and mortality among all cancers. In some regions of Russia the mortality from lung cancer exceeds the highest worldwide performance, sometimes reaching 44 cases in women and 81 men per 100,000 population per year [Mukeria, A. F. and D. G. Zaridze, Epidemiology and prevention of lung cancer. Bulletin of the RORC. N. N. Blokhin RAMS, 2010. 21(3): 3-13]. The main methods of cancer treatment along with surgery and radiation methods include chemotherapy. It is known that tumor cells often become resistant to drugs (e.g., cisplatin), which prevents effective treatment. Therefore, an important task of modern experimental Oncology is the creation of new molecular biological tests that would allow to explore the mechanisms of chemoresistance tumor cells and on this basis to develop optimal chemotherapy regimens for patients with cancer and in particular lung cancer. There is evidence of correlation stability �notches to cisplatin with the expression level (amount of mRNA) of a number of genes [Dan, S., M. Shirakawa, Y. Mukai, et al., Identification of candidate predictive markers of anticancer drug sensitivity using a panel of human cancer cell lines. Cancer Sci, 2003. 94(12): 1074-82, Dan, S., T. Tsunoda, O. Kitahara, et al., An integrated database of chemosensitivity to 55 anticancer drugs and gene expression profiles of 39 human cancer cell lines. Cancer Res, 2002. 62(4): 1139-47]. One of the most common ways to determine levels of gene expression are methods based on microarrays is solid substrates, which in a certain order covalently immobilized DNA fragments or chemically synthesized oligonucleotides. The present invention is a method of determining the sensitivity of cells of lung cancer to cisplatin based on the expression levels of marker genes AKR1B1, DDB2, ERCC1, FTL, MLH1.

The level of technology

Known direct method of determining the sensitivity of cells to cytostatics MTT method [Alley, M. S., D. A. Scudiero, A. Monks, et al., Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res, 1988. 48(3): 589-601], in which the sensitivity of the cells is determined by the dose of the cytostatic agent in which the living is 50% of the cells in culture (IC50). For use of this method requires special equipment (sterile boxes, disposable sterile tablets and mattresses, equipment for sterilization of reusable instruments, CO2incubators, microscopes, culture media etc.) for the cultivation of eukaryotic cells. Theobromine are not in every laboratory.

It is known that the sensitivity of cells to cisplatin correlates with the expression level (amount of mRNA) of a number of genes. In particular, correlations were shown for genes AKR1B1, DDB2, ERCC1, FTL, MLH1 (table 1) [Almeida, G. M., T. L. Duarte, R. V. Farmer, et al., Multiple end-point analysis reveals cisplatin damage tolerance to be a chemoresistance mechanism in a NSCLC model: implications for predictive testing. Int J Cancer, 2008. 122(8): 1810-9, Dan, S., M. Shirakawa, Y. Mukai, et al., Identification of candidate predictive markers of anticancer drug sensitivity using a panel of human cancer cell lines. Cancer Sci, 2003. 94(12): 1074-82, Dan, S., T. Tsunoda, O. Kitahara, et al., An integrated database of chemosensitivity to 55 anticancer drugs and gene expression profiles of 39 human cancer cell lines. Cancer Res, 2002. 62(4): 1139-47, . K. a., V., P. Surowiak, O. Kiesslich et al., Gene expression profiling of 30 cancer cell lines predicts resistance towards 11 anticancer drugs at clinically achieved concentrations. Int J Cancer, 2006. 118(7): 1699-712, Seve, P. and C. Dumontet, Chemoresistance in non-small cell lung cancer. Curr Med Chem Anticancer Agents, 2005. 5(1): 73-88].

However, the literature does not describe the methods of calculation of IC50 of cisplatin depending on the level of expression of genes AKR1B1, DDB2, ERCC1, FTL, MLH1 (table 1). At the present time to determine the quantity of mRNA in cells using real-time PCR (PCR-RV) and hybridization on microchips.

A method of determining gene expression polymerase chain reaction in real time, coupled with reverse transcription (RT-PCR.), in which the level of gene expression is determined by the change in fluorescence during the accumulation of the reaction product Livak, K. J., S. J. Flood, J. Marmaro, et al., Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl, 1995. 4(6): 357-62]. Depending on the variety method RT-PCR. (SYBR Green® I, TaqMan®, molecular bekesy, "scorpione" samples, etc.) it uses two or more oligonucleotides with a specific structure for each gene [Vanguilder, H. D., K. E. Vrana, and W. M. Freeman, Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques, 2008. 44(5): 619-26]. Shows the correlation of the sensitivity of cells to cisplatin with expression levels radya genes determined by RT-PCR. [boyar, W. A., J. V. Kondrakhin, I. S. Evsen, etc., to predict the sensitivity of small cell lung cancer cells to cisplatin and paclitaxel on the basis of the expression level of marker genes. Molecular Biology, 2011. 45(4): 652-661]. Overall, a significant disadvantage of methods based on RT-PCR. compared with DNA microarrays is limited multiplexity, i.e. the number of genes to be tested in a single reaction. In addition, for determining the expression of one gene requires two or more specific oligonucleotide depending on the variant of PCR-RV. New ways of PCR-RV, based on the molecular Bianco and "scorpionic" samples require significantly more sophisticated design of oligonucleotide probes and less common.

The known method of prediction h�stateliest of cells to cytostatics based on the expression levels of different genes, defined by ProTranslating microarrays [. K. a., V., P. Surowiak, O. Kiesslich et al., Gene expression profiling of 30 cancer cell lines predicts resistance towards 11 anticancer drugs at clinically achieved concentrations. Int J Cancer, 2006. 118(7): 1699-712] [Yatomi, M., Y. Takiguchi, Y. Asaka-Amano, et al., Altered gene expression by cisplatin in a human squamous cell lung carcinoma cell line. Anticancer Res, 2007. 27(5A): 3235-43, Kashkin, K. H., E. A. Musatkina, A. B. Comelico, etc., Genes, potentially associated with resistance cell lung cancer to cisplatin. Reports Of Academy Of Sciences, 2011. 438(6): 829-833]. One gene-expression microarray can contain many different probes spanning the entire transcriptome (the set of all RNA) of an organism. So, microchip Human Gene 1.1 ST Array company Affymetrix (Santa Clara, CA) contains over 750,000 different oligonucleotide probes. The main disadvantages of this method are the high cost, the need for high-tech equipment for the manufacture and processing of microarrays, the complexity of the analysis result and the received redundancy information. These drawbacks limit the practical application ProTranslating microarrays from different manufacturers, for example, firms Affymetrix and Agilent. In fact, the number of genes for which experimentally validated correlation of the expression of drug-resistant tumors, tens. For the analysis of such number of genes using a low density microarray. Their advantage over protrans�reposname chips is they can be produced and analyzed in a small diagnostic laboratory. The low density microarrays allow the study of the expression of the minimum and sufficient set of genes corresponding to a particular task. Despite the fact that the correlation of the expression of some genes with sensitivity to cisplatin is known, is described in the literature methods for calculating IC50 of cisplatin depending on the level of gene expression determined using low-density microarrays.

Known niche low density biochips on the basis of the hydrogel produced in the Institute of molecular biology and the company Biochip-IMB (patent 2157385 RU). These chips are used for the detection of several clinically important mutations and polymorphisms in the genome of various organisms, including humans (see patents 2458131 RU, 2453606 RU, 2453605 RU), as well as to determine the number of antigens by immunological methods [Gryadunov, D., E. Dementieva, V. Mikhailovich, et al., Gel-based microarrays in clinical diagnostics in Russia. Expert Rev Mol Diagn, 2011. 11(8): 839-53]. However, for a quantitative study of the expression of normal genes gel biochips do not apply. The principle drawback of gel microchips is that the size of the gel pores imposes restrictions on the length of the immobilized oligonucleotide probes, and the method of preparation of the test drug. All systems analysis of nucleotide sequence that�new acids on a gel chips require pre-amplification using pnrm severely restricted sections of DNA or RNA. This method requires the synthesis of at least three oligonucleotides for each gene or mutation: primers for PCR and probes for hybridization of the PCR product. Introduction to system analysis each additional gene requires the calculation of at least three oligonucleotides, and in an embodiment, a multiplex PCR recalculation of all oligonucleotides used in the analysis. All this deprives gel microchips advantages over methods using only PCR in different variations. In addition, microchips, in which oligonucleotide probes are immobilized on the surface of a solid substrate (surface microarrays) allow the use of oligonucleotide probes the length of 50-80 or more nucleotides with a sequence that is unique to the human genome. Thus, for analysis of gene expression of any gene can be used only one oligonucleotide probe.

The closest analogs of the method of preparation of the material used at present, are two ways. The first method is applied in the set of Amino Allyl cDNA labeling Kit (Ambion; USA, US patent 5256555, 6586218, 6586219), which is based on the method of double amplification [Van Gelder, R. N., M. E. Von Zastrow, A. Yool, et al., Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc Natl Acad Sci USA, 1990. 87(5): 1663-7]. This method does not provide stage amplification of cDNA: it separately synthesize cDNA, and then the second chain cDNA, after e�CSOs RNA amplification using T7 RNA polymerase, getting ARNK. For one experiment requires at least 10 μg RNA of each cell line or tissue. For smaller amounts of mRNA used recycled double amplification using the same set, which introduces significant variations in the representativeness of the original mRNA molecules in the final product. The second method used in MiniAmp mRNA amplification kit (Arrayit, USA, patent US 8343721 B2), uses SMART technology for cDNA synthesis [Chenchik, A., Y. Y. Zhu, L. Diatchenko, et al., Gene Cloning and Analysis by RT-PCR, 1998. 305-319, Zhu, Y. Y., E. M. Machleder, A. Chenchik, et al., Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques, 2001. 30(4): 892-7], intermediate amplification of cDNA using restricted PCR, synthesis using T7 RNA polymerase of ark, which again translates into cDNA with the introduction of aminoallyl-are. The main drawback of this approach is the extra stage enzymatic copying of material that infringes the representativeness of the original mRNA molecules in the final product.

The present invention solves the problem of determining the sensitivity (calculation of IC50) of human cells of lung cancer to cisplatin - drug medication used in practical Oncology for the treatment of patients with lung cancer. The invention can be used in cases when it is not possible to determine the sensitivity of cells to cisplatin other known methods,�reamer MTT method [Alley, M. S., D. A. Scudiero, A. Monks, et al., Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res, 1988. 48(3): 589-601], in particular, in laboratories that do not have the equipment for cultivation of animal cells. The problem is solved by determining the expression levels of marker genes AKR1B1, DDB2, ERCC1, FTL, MLH1 (table 1) provided that their expression level is not lower than ten molecules of the marker gene mRNA in the cell. The method includes receiving biological material (cells in culture), isolation of total RNA from cells, SMART-cDNA synthesis followed by amplification of RNA (arnc) and include fluorescent labels, their hybridization with a set of unique oligonucleotide probes to the genes (table 1, table 2), which are immobilized on a solid support, washing of unbound labeled drug ARNK, scanning the substrate with a laser scanner, the processing of the received image and conducting a quantitative analysis result, and a set of oligonucleotides SEQ ID NO: 1-13 (table 2) for the implementation of the proposed method. In the proposed method used SMART cDNA synthesis and its limited amplification (PCR) using an inexpensive kit for the amplification of cDNA firm Evrogen (Russia), followed by linear amplification of RNA using T7 RNA polymerase. This approach, on the one hand, ensures effective Shin�ez full-size cDNA, and with another - allows to reduce the stage of enzymatic copying of the original mRNA while maintaining the representativeness of the transcripts. Another advantage of this method is that the need can infinitely copy of the intermediate material (cDNA) by PCR. For setting up experiments on a microchip using standard equipment available in every molecular biology laboratory. Choose a substrate with oligonucleotide probes (microarray) can be produced and analyzed either in the same laboratory with the use of fiscal printers (Xact Xpress Line) and scanner (DITABIS MaRS), or equipment for mass printing of microarrays in specialized technology parks - for example, using the stations for printing ArrayIt NanoPrint™ Workstations and scanners GenePix A Microarray Scanner with automatic loader GenePix SL50. After image processing and normalization values of fluorescence on hybridization results and the known IC50 of cisplatin for other cells of lung cancer calculated regression equations and/or build direct calibration for predicting IC50 of cisplatin depending on the fluorescence signals of the probes AKR1B1 (SEQ ID NO: 11), DDB2 (SEQ ID NO: 12), ERCC1 (SEQ ID NO: 9), FTL (SEQ ID NO: 10), MLH1 (SEQ ID NO: 13) using Statistica (Statsoft, Inc.) or other mathematical apparatus. In the same way determine the level�and the expression of these genes for the studied cells, the results are averaged and compared with a calibration straight line, thus determining the IC50 of cisplatin for the studied cells.

Thus, the proposed method of determining the sensitivity of cells to cisplatin-based measurements of the expression levels (mRNA) of marker genes comprises the following advantages of PCR-RV, surface ProTranslating microarrays and microchips low density:

1) the minimum amount of starting material - 1-2 µg total RNA;

2) a universal method for preparation of labeled material from the total RNA, based on cheap domestic set for amplification of cDNA firm Evrogen. Method of preparation does not depend on the set of genes to be investigated;

3) the opportunity for unlimited reproduction of the intermediate material for analysis by cDNA amplification;

4) the use of only one oligonucleotide probe for each gene;

5) using a limited set of oligonucleotide probes immobilized on a solid support, sufficient for the task, if necessary, the set can be expanded;

6) relatively low cost microchips due to the small number of immobilized probes;

7) the possibility of printing, processing and microarray analysis on low-end hardware in the researcher�coy laboratories, and equipment for mass printing of microarrays in terms of specialized industrial parks;

8) reduction of analysis in the mass production of microchips.

The combination used in the present invention genes, specific sequences of oligonucleotides, the method of preparation of the material for hybridization and experimental approach using low-density microarrays to determine the IC50 of cisplatin for cells of lung cancer in the literature and in practice is not used.

The technical result

The technical result of the invention is defined in the experiment figure IC50 of cisplatin cell lung cancer based on the analysis of the expression of marker genes AKR1B1, DDB2, ERCC1, FTL, MLH1 (table 1), increasing the reliability of determining the sensitivity of cells to cisplatin lung cancer and additional confirmation IC50 of cisplatin cell lung cancer, certain other methods.

The implementation of the invention

To the invention, the isolated total RNA from cells of lung cancer grown in culture. RNA isolated using different methods, for example through chaotropic agents [Chomczynski, P. and Sacchi N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem, 1987.162(1): 156-9], using Trizol reagent (Gibco/Life Technologies, USA) or commercial�their kits for RNA extraction type RNAEasy Kit (Quiagen, USA) or ExtracrRNA (Evrogen, Russia). Required the quality of the drug RNA is evaluated by the ratio of the fractions of 28S to 18S ribosomal RNA, which is close to 2, as assessed by electrophoresis of total RNA [Sambrook, J. and D. W. Russell, Molecular cloning: a laboratory manual. 3d Ed. 2001], or by index RIN, close to 10, defined by the instrument, similar to a Bioanalyzer Agilent 2100 Bioanalyzer. From the total RNA of cells using reverse transcription using reverse transcriptase enzyme and SMART technology to prepare the total cDNA preparations. This cDNA amplificateur with limited polymerase chain reaction. Of the drugs amplified cDNA using RNA polymerase prepare medications amplified RNA (arnc), which are labeled with fluorescent dyes differing in absorption spectra and emission - for example, SS3 (drug control) and Cy5 (the study drug). To get the control of drug ARNK use cell adenocarcinoma of the lung A or other cells. Labeled with two different dyes of the control and investigational drugs ARNK mixed in equal proportions and conduct hybridization with oligonucleotide probes homologous to mRNA of genes AKR1B1 (oligonucleotide probe SEQ ID NO: 11), DDB2 (SEQ ID NO: 12), ERCC1 (SEQ ID NO: 9), FTL (SEQ ID NO: 10), MLH1 (SEQ ID NO: 13), which are immobilized on a solid support (DNA microarray) together with the control oligonucleotide�governmental probes intended for normalization of the results (table 1, table 2). Unbound part of the drugs ARNK washed with saline. After hybridization and washing, the substrate is scanned with a scanner for microarray (Perkin Elmer ScanArray GL Plus, DITABIS MaRS or similar) at two wavelengths corresponding to the maximum emission of fluorescent dyes. The obtained images are combined and processed using software for image processing (Perkin Elmer ScanArray Express, Imaging Research ArrayVision 7.0, Axon GenePix Pro or other). The ratio of the fluorescence signals of the two dyes for each probe to determine the levels of gene expression in the test cells relative to control. When using cells A as control and the Imaging Research ArrayVision 7.0 for image processing IC50 of cisplatin was determined by the following equations:

IC50 (μm)=2,30+11,33*"MLH1" (p<0.004 percent);

IC50 (μm)=2,66+2,81*"ERCC1" (p<0,011);

IC50 (μm)=2,81+3,20*"DDB2" (p<0,05);

IC50 (μm)=4,10+4,01*"AKR1B1" (p<0,05);

IC50 (μm)=1,79-3,01*"FTL" (p<0,03)

IC50 (μm)=3,16+0,89*"DDB2"+1,77*"ERCC1"+1,46*"AKR1B1" (p=0,013).

In the equations using normalized indicators SS3/Cy5 for the respective probes "Ratio (cnLogARMDens): Ctrl:Data" (shown in brackets); the more accurate the coefficients are indicated in Fig. 1. These equations build direct calibration based IC50 of cisplatin on the levels of expression are suitable� genes. Closest to the IC50 value obtained as an average value of values obtained by these equations.

In the case of other control cells, other image-editing programs or other parameters of hybridization and normalization of the hybridization results and the known IC50 of cisplatin for other cells of lung cancer calculated regression equations and build direct calibration for predicting IC50 of cisplatin depending on the fluorescence signals of the probes AKR1B1 (SEQ ID NO: 11), DDB2 (SEQ ID NO: 12), ERCC1 (SEQ ID NO: 9), FTL (SEQ ID NO: 10), MLH1 (SEQ ID NO: 13) using Statistica (Statsoft, Inc.) or other mathematical apparatus. In the same way determine the levels of expression of these genes for the studied cells, the results averaged and compared with a calibration straight line, thus determining the IC50 of cisplatin for the studied cells.

The method is illustrated by the following examples.

Example 1.

a) Synthesis of oligonucleotides SEQ ID NO: 1-13 carried out using standard potamididae procedures on an automatic synthesizer ABI 3900 ("Applied Biosystems", USA). At the 3'-end of oligonucleotides SEQ ID NO: 2-13 synthesis when administered spacer with a free amino group by using 3'-Amino-Link"Glen Reseach, USA).

b) For the immobilization of oligonucleotide probes on a solid substrate using the printer for contact printing Xact Xpress Lane and the substrate VALS-25 � activated surface (CEL Associates, Inc., USA) under conditions recommended by the manufacturers of the substrate and the printer. The probes applied in a certain order three times.

b) Cell lung cancer lines A, NCI-H23, NCI-H292, NCI-H322, NCI-N, NCI-H1299 and NCI-H460 grown in 25 cm2flasks in DMEM/F12 (1:1) containing 10% fetal bovine serum with the addition of streptomycin to a concentration of 10 μg/ml and penicillin to a concentration of 10 units/ml at 37°C in a CO2incubator at a relative humidity of 96%.

d) Total RNA from cells isolated by a standard method using guanidine of isothiocyanate and phenol [Chomczynski, P. and Sacchi N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem, 1987. 162(1): 156-9], further purified using the RNeasy kit RNA Mini Kit (Qiagen, Valencia, CA, USA) and treated with Dnazol I. the Quality of RNA was evaluated by electrophoresis in agarose gel with ethidium bromide [Sambrook, J. and D. W. Russell, Molecular cloning: a laboratory manual. 3d Ed. 2001]. The ratio of the fractions of 28S:18S ribosomal RNA, which is close to 2, indicates the high quality of the drug RNA, which is a prerequisite for the determination of gene expression. A more reliable indicator of good quality is the index of the RIN (RNA Integrity Number), close to 10. Index RIN is determined using the Agilent Bioanalyser 2100.

d) Synthesis of labeled preparations of ARNK carried out in the following manner. cDNA synthesized from 2 µg total RNA using neb�RA Mint (Evrogen, Russia) according to the manufacturer's recommendations, using the modified priming oligonucleotide SEQ ID NO: 1 and a standard Plug-oligonucleotide to switch the circuit. Priming oligonucleotide SEQ ID NO: 1 represents a modification of the standard 3'-primer Mint (Evrogen) and further comprises a promoter RNA polymerase of phage T7. Next spend 18-21 cycle PCR with primer M1 for amplification of cDNA under conditions recommended by Evrogen. The preparation of cDNA is purified on spin columns Clean-up (Evrogen, Russia) or equivalent according to manufacturer's instructions. 100 ng amplified cDNA used for the synthesis of ARNK RNA using RNA polymerase of bacteriophage T7 (Ambion, Austin, TX, USA or Fermentas, Lithuania) in a volume of 40 μl in the presence of ATP, GTP, CFT (7.5 mm), UTP and aminoallyl-UTP (Fermentas, Lithuania; 3.75 mm). The reaction is conducted at 37°C for 4-8 hours, then stopped by dilution bezoglyadnoi water to 100 μl. Product of synthesis (arnc) purified on spin columns (RNAEasy Kit (Quiagen, USA) or equivalent according to manufacturer's instructions. Received by ARNK labeled by reaction with Succinimidyl esters of fluorescent dyes dyes SS3 or Cy5. This is done using Amino Allyl MessageAmp II aRNA Amplification kit (Ambion, USA, #1797) or su-Dye Post-Labelling Reactive Dye Pack (Amersham/GE Healthcare, USA, #RPN 5661) or similar products from other manufacturers. One bottle of dye per 1 reaction is diluted in 11 ál LCA�. 10-20 µg of ARNK lyophilizer and dissolved in 9 μl of buffer for conjugation (Coupling buffer, Ambion, USA), then mixed with a solution of the dye in DMSO and gently stirred. The mixture was incubated for 30 minutes in the dark, then added to 4.5 μl of 4M hydroxylamine, mixed and incubated for 15 minutes in the dark. Reaction volume is adjusted to 30 μl. Labeled aminoallyl ARNK purified on spin columns for removal of dye from Ambion kit #1797 or by using the RNAEasy Kit (Quiagen, USA) or equivalent according to manufacturer's instructions.

(e) as a control drug use of arnc from cells A. Experimental (Cy5 labeled) and control (labeled SS3) drugs of ark mixed in a weight ratio of 1:1, the fragment with the buffer for fragmentation (AM, Ambion) according to the manufacturer's recommendations and added to the hybridisation buffer (RPK0325, GE Healthcare) and applied to a DNA microarray for hybridization. The microchips were incubated over night at 37°C, then washed citrate buffer with decreasing salt concentrations and dried. Washing is carried out in the following mode: 2×SSC, 2×15 min at 20°, 1×SSC, 15 min at 50°, in 0.1×SSC, 5 min at 20° C, the addition of 0.1% Tween 20, rinsed in 0.1×SSC without Tween 20 and dried in dust-free conditions.

g) the Microarray is scanned with a scanner ScanArray GL Plus (Perkin Elmer, USA). Image analyzed by the program ArrayVision 7.0 (Imaging Research, USA). The background correction wire�t-fluorescence-free oligonucleotides region of the chip, and residual fluorescence (control washing) - the probe SEQ ID NO: 3. The results normalized by control signals of probes homologous to mRNA of genes of the 'household' (SEQ ID NO: 4-8). Differential expression of fix in cases not less than two-fold differences normalized relations from SS3/Cy5 (figure cnLogARMDens >0,3) when the ratio signal/noise S/N ≥3. To control the result, carried out the microarray hybridization with oppositely labeled matrices (Cy5/SS3), the results are averaged.

h) When using cells A as control IC50 of cisplatin for the investigated cell line cancer of the lung is determined on the basis of its direct correlation with the signals of probes "MLH1" (SEQ NO: 13), "ERCC1" (SEQ NO: 9), "DDB2" (SEQ NO: 12) and "AKR1B1" (SEQ NO: 11) and inverse correlation with the signal probe "FTL" (SEQ NO: 10) in accordance with the following equations (a more precise coefficients are shown in Fig. 1):

IC50 (μm)=2,30+11,33*"MLH1" (p<0.004 percent);

IC50 (μm)=2,66+2,81*"ERCC1" (p<0,011);

IC50 (μm)=2,81+3,20*"DDB2" (p<0,05);

IC50 (μm)=4,10+4,01*"AKR1B1" (p<0.05);

IC50 (μm)=1,79-3,01*"FTL" (p<0,03)

IC50 (μm)=3,16+0,89*"DDB2"+1,77*"ERCC1"+1,46*"AKR1B1" (p=0,013).

In the equations using normalized indicators SS3/Cy5 for the respective probes "Ratio (cnLogARMDens): Ctrl:Data" (shown in quotes), defined as described in Example 1G. Closest to the IC50 value of cisplatin (study of cells obtained in the form �redna magnitude of the values obtained according to the equations. These equations build direct calibration based IC50 of cisplatin on the levels of expression of the corresponding genes.

In the case of other control cells, other image-editing programs or other parameters of hybridization and normalization of the hybridization results and the known IC50 of cisplatin for other cells of lung cancer calculated regression equations and build direct calibration for predicting IC50 of cisplatin depending on the fluorescence signals of the probes AKR1B1 (SEQ ID NO: 11), DDB2 (SEQ ID NO: 12), ERCC1 (SEQ ID NO: 9), FTL (SEQ ID NO: 10), MLH1 (SEQ ID NO: 13). In the same way determine the levels of expression of these genes for the studied cells, the results averaged and compared with a calibration straight line, thus determining the IC50 of cisplatin for the studied cells.

Example 2. Determination of IC50 of cisplatin for cell line NCI-H460 when used as a control cell line A (values are rounded to two decimal places)

The average value of the calculated IC50 is equal to 2.30 μm, which is close to the value obtained in direct experiment (2,07 m) MTT method [boyar, W. A., J. V. Kondrakhin, I. S. Evsen, etc., to predict the sensitivity of small cell lung cancer cells to cisplatin and paclitaxel based on the level of EC�of depression of marker genes. Molecular Biology, 2011. 45(4): 652-661, Kashkin, K. N., E. A. Musatkina, A. W. Comelico, etc., Genes, potentially associated with resistance cell lung cancer to cisplatin. Reports Of Academy Of Sciences, 2011. 438(6): 829-833]. Even more accurate IC50 value gives the averaging of the calculated IC50 after discarding extreme values (2,19 μm) that is different from the IC50 determined by MTT method, only 5.8%. Similarly can be determined IC50 of cisplatin for other cells (Fig. 1).

Example 3. In the case of other control cells, other image-editing programs or other parameters of hybridization and normalization of the hybridization results and the known IC50 of cisplatin for other cells of lung cancer calculated regression equations and based on them build direct calibration for predicting IC50 of cisplatin depending on the fluorescence signals of the probes AKR1B1 (SEQ ID NO: 11), DDB2 (SEQ ID NO: 12), ERCC1 (SEQ ID NO: 9), FTL (SEQ ID NO: 10), MLH1 (SEQ ID NO: 13) using the Statistics program 8.0 (Statsoft Inc., OK, USA) or other mathematical apparatus. In the same way determine the levels of expression of these genes for the studied cells, the results averaged and compared with a calibration straight line, thus determining the IC50 of cisplatin for the studied cells.

The invention is illustrated graphic materials.

Fig. 1 shows the correspondence between the IC50 of Cipla�ina cell lung cancer, defined by the MTT (observed IC50, horizontally), and IC50 calculated on the basis of the expression of marker genes AKR1B1, DDB2, ERCC1, FTL, MLH1 (vertical) when used as a control cell line A.

Industrial applicability

The proposed method allows to determine the IC50 of cisplatin to cancer cells of the lung in cases when other methods of determining the sensitivity to the cytostatic inapplicable. IC50 of cisplatin to cancer cells of the lung is determined on the basis of the expression levels of marker genes AKR1B1, DDB2, ERCC1, FTL, MLH1 (table 1), provided their expression at the level of at least ten molecules of mRNA of each gene on the cell. The method can be used as an alternative to existing methods of determining the IC50 of cisplatin or Supplement thereto.

1. The method of determining the sensitivity of cells of lung cancer to cisplatin, comprising determining the expression level of genes MLH1, ERCC1, DDB2, AKR1B1, FTL, depending on the IC50 of cisplatin for known cell lines, further carry out the construction of calibration direct correlation with IC50 of cisplatin in these cell lines obtained from the level of expression of these genes, then conduct the selection of the investigated cell lung cancer followed by isolation of total RNA of these cells, SMART-�the Intesa cDNA followed by amplification of RNA by adding fluorescently-labeled probes and hybridization is carried out on a microchip containing a set of oligonucleotide probes to genes MLH1 (SEQ ID NO: 13), ERCC1 (SEQ ID NO: 9), DDB2 (SEQ ID NO: 12), AKR1B1 (SEQ ID NO: 11), FTL (SEQ ID NO: 10) with subsequent determination of the level of expression of these genes relative to control, these levels are averaged and compared with a calibration straight line, thus determining the IC50 of cisplatin for the studied cells.

2. A set of oligonucleotide probes represented by SEQ ID NO: 9-13 designed to determine the sensitivity of cells of lung cancer to cisplatin according to claim 1.



 

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FIELD: medicine.

SUBSTANCE: invention can be used for treating early breast cancer (BC) involving a radical mastectomy. To this effect, blood in an amount of 8-10 ml is sampled before the surgical intervention and on the first day following the surgical intervention to detect tumour cells. If the blood is found to contain circulating tumour cells on the first postoperative day with a zero level, the therapeutic setting is added with early postoperative FAC-based polychemotherapy.

EFFECT: invention enables assessing the activity of the tumour process accompanying BC and planning the further therapeutic approach.

1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to a method for determining a response to an anti-oestrogen therapy in the individuals suffering diagnosed breast cancer. Substance of the method for determining the response to the anti-oestrogen therapy in the individual suffering breast cancer consists in calculating an endocrine therapeutic index of circulating immune complexes (ETI-COC). If the ETI-COC falls within the range of 0-3, the favourable response to the anti-oestrogen therapy is stated, while the value of 4-6 shows the moderate response, and the value of 7-14 indicates the weak response.

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10 cl, 7 tbl, 8 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention aims at detecting benign and malignant new growths in human thyroid. Involved thyroid and reference adjacent intact tissues are sampled; micro-RNA is recovered from the samples; that is followed by conducting a reverse transcription reaction, measuring an expression level of microRNA-21, -221, -222, -155, -205 by real-time RNA followed by a comparative analysis of the microRNA expression according to the norm and thyroid tumour involvement, and stating the presence and type of the new growth. If the above microRNA expression varies by no more than 4 times to the higher and lower figures of expression in relation to the reference, the benign new growth is stated. The malignant new growth is shown by the measured microRNA expression by more than 4 times.

EFFECT: effective detection of the benign and malignant thyroid new growths that promotes improving the further therapeutic approach.

5 dwg, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: present invention refers to medicine, namely to a method for detecting patients with progressing leukaemia and/or lymphoma and a risk of developing side effects of administering the CD19×CD3 bispecific antibody. A B:T cell ratio is measured in the patients; the ratio of 1:9 or less indicates the risk of possible side effects in the above patient. A dose schedule of the CD19×CD3 bispecific antibody provides: (a) administering the first dose of the CD19×CD3 bispecific antibody for the first time period; and then (b) administering the second dose of the above antibody for the second time period; the above second dose exceeds the above first dose.

EFFECT: using the given method enables facilitating the clinical course or preventing any side effect caused by administering the above bispecific antibody when treating the patients with leukaemia and/or lymphoma.

34 cl, 2 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to oncology, and aims at sub-typing breast cancer. RIL (PDLIM4) expression is measured in a patient's tumour tissue sample by the sequencing technology of the following NGS generation or by northern hybridisation methods and real-time PCR. If the RIL (PDLIM4) expression tends to decrease twice as much as its level in the normal tissues, a sub-type of a malignant breast new growth is diagnosed.

EFFECT: invention provides effective diagnostic technique for breast cancer subtype - tumours characterised by incomplete epithelial-mesenchymal transition and relatively minimally invasive.

2 dwg, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, pharmaceutics and biochemistry, and concerns a metastatic melanoma grade marker containing the amino acid sequence SEQ ID NO:1. There are also declared a recovered antibody and a monoclonal antibody, which recognise human TM9SF4 protein by binding to a peptide fragment of SEQ ID NO:1, as well as a kit for determining a tumour grade comprising the above antibody, using SEQ ID NO:1 for determining the metastatic pattern of melanoma.

EFFECT: group of inventions provides the faster detection of melanoma metastasis.

10 cl, 8 ex, 2 tbl, 10 dwg

FIELD: medicine.

SUBSTANCE: claimed group of inventions relates to the field of medicine, in particular to oncology and molecular biology. Claimed are a method and a set of primers and a probe with sequences SEQ ID NO: 1, 2 and 3 for the realisation of a polymerase chain reaction in a real time mode to diagnose clear cell renal cell carcinoma (CCRCC). The quantitative content of mRNA of the NETO2 gene is evaluated. In case of an increased content of mRNA in a supposedly cancer-affected human tissue in comparison with the quantity of mRNA in a healthy tissue, CCRCC is diagnosed.

EFFECT: claimed group of inventions makes it possible to diagnose CCRCC with high reliability, including an early stage of the tumour disease.

4 cl, 1 dwg, 5 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: claimed group of inventions relates to the field of medicine, in particular to oncology and molecular biology. Claimed are a method and a set of primers and a probe with sequences SEQ ID NO: 1, 2 and 3 for the realisation of a polymerase chain reaction in a real time mode to diagnose clear cell renal cell carcinoma (CCRCC). The quantitative content of mRNA of the ACY1 gene is evaluated. In case of a reduced content of mRNA in a supposedly cancer-affected human tissue in comparison with the quantity of mRNA in a healthy tissue, CCRCC is diagnosed.

EFFECT: claimed group of inventions makes it possible to diagnose CCRCC with high reliability, including an early stage of the tumour disease.

4 cl, 1 dwg, 5 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to immune therapy, and can be used to assess the efficacy of treating the patients suffering cancer. That is ensured by administering an immunogenic composition, which contains a recombinant viral vector expressing in vivo the whole MUC-1 antigen or a portion thereof. A method for analysing preparing a biological sample from a patient after the above immunogenic composition has been introduced, and measuring interferon γ in the sample. If interferon γ is more than approximately 4 pg/ml the patient is suggested to show the favourable clinical outcome.

EFFECT: invention assessing the clinical response to the cancer treatment in the patient.

15 cl, 3 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to qualitative differential instant diagnostic technique for benign and malignant periglottis new growths as shown by oral fluid biomarkers. Substance of the method consists in measuring a quantity of matrix metalloproteinase 2 (MMP 2) in patient's oral fluid; the clinical reference is the level of 1.7-2.9 ng/ml; if the MMP 2 content is 14.4-24.3 ng/ml, patient's periglottis papilloma is diagnosed; if the patient's oral fluid MMP 2 content is 4.1-6.8 ng/ml, periglottis cancer is diagnosed. A biomarker for the qualitative differential instant diagnosis of the periglottis new growths is a tissue inhibitor of metalloproteinase 2 (TIMP 2); the clinical reference is a level of 6.44-11.23 ng/ml; if the TIMP 2 content 29.25-48.75 ng/ml, patient's periglottis papilloma is diagnosed; the TIMP 2 content being 57.23-95.03 ng/ml, periglottis cancer is diagnosed.

EFFECT: using the declared technique enables providing more accurate differential diagnosis of the benign and malignant periglottis new growths.

2 cl, 6 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of biochemistry, in particular to method of identifying changes in gene expression, characteristic of ageing, in selected tissue. Selected tissue represents cardiac, muscular, cerebral or adipose tissue. Method includes selection of one or several genes, differentially expressed in tissue of old subjects in comparison with young subjects, with application of two criteria. Said criteria are: change of expression in at least 50% of lines, breeds or ethnic groups of tested species at preliminarily determined significance level p<0.10, as well as at least partial reversion of changes in gene expression by restriction in caloric content.

EFFECT: invention provides obtaining reliable tissue-specific biomarkers of ageing.

7 cl, 11 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention refers to molecular biology and can be used in diagnosing cardiomyopathies of various origin Presented is a set of synthetic oligonucleotides for identifying mutations of a coding part of desmin (DES) gene associated with cardiomyopathies. The mutations are identified by detecting a full sequence of the coding part of DES gene. The coding part of DES gene is amplified by means of 7 pairs of synthetic oligonucleotides at the same temperature and annealing time, and the prepared amplification products are sequenced by means of one pair of universal primers.

EFFECT: presented invention enables the sensitive and specific detection of DES gene mutations, with reducing the amplification reaction time, the number of manipulations, the time of agent application for the sequencing reaction, and reducing a probability of a reaction error.

3 cl, 1 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to AXL signalling pathway inhibitors, and can be used in medicine. What is produced is a soluble AXL polypeptide version free from AXL transmembrane domain, which contains at least one amino acid modification in position No. n, wherein n is specified in 32, 72, 87, 92 or 127 or a combination thereof, wherein n+7 is described by numbering SEQ ID NO: 1 that are wild-type AXL sequences, wherein the above modification increases a AXL polypeptide binding affinity to protein 6 specifically inhibiting the growth (GAS6), which is twice as strong as the wild-type AXL polypeptide affinity. The polypeptide can be fused with Fc fragment and used in a method of treating, reducing or preventing tumour dissemination and invasion in a mammalian patient.

EFFECT: invention enables inhibiting the AXL/GAS6 signalling pathways effectively.

8 cl, 15 dwg, 6 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to a set of oligonucleotide primers and fluorescently-labelled probe for identification of Burkholderia pseudomallei and differentiation of the actinobacillus mallei by the method of polymerase chain reaction with fluorescence detection.

EFFECT: invention enables in a short time with high sensitivity and specificity to detect the melioidosis agent and differentiate it from the actinobacillus mallei in samples of pure cultures and biological material.

1 dwg, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, molecular biology and biotechnology. What is presented is a method for determining the polymorphism of human GP6 gene coding glycoprotein VI by the polymorphous position rs 1613662 based on recording melting curves with fluorescence-labelled allele-specific oligonucleotide tests.

EFFECT: due to the more reliable determination of variable positions and a possibility to detect in one test tube with the use of standard equipment, the method can be effectively used to diagnose the individual's inherited predisposition with the recording the real-time PCR results.

1 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, namely to internalisation of therapeutic molecules into cell, and can be applied in medicine. Obtained is composition for delivering molecules of nucleic acids into cells, containing at least one peptide with at least 92% identity to GAAEAAARVYDLGLRRLRQRRRLRRERVRA (SEQ ID NO: 2); IREIMEKFGKQPVSLPARRLKLRGRKRRQR (SEQ ID NO: 3); or YLKVVRKHHRVIAGQFFGHHHTDSFRMLYD (SEQ ID NO: 4), bound to one or several molecules of nucleic acids.

EFFECT: invention makes it possible to increase efficiency of delivery of molecules of nucleic acids into mammalian cell due to peptide, capable of internalisation into mammalian cell with efficiency, constituting at least 200% of efficiency of internalisation of peptide TAT, which has amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 1).

8 cl, 16 dwg, 1 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, namely to a method for analysing the applicability of RNA extracted from a tissue or a cell (cells) fixed by a fixative for analysing gene expression. The method involves performing electrophoresis with the above RNA. The method implies stating, if the above RNA complies with the following equation: B/A≤1, wherein A represents the mass ratio (%) of RNA falling within the range from 1,000 to 4,000 nucleotides to the total mass of RNA that is determined by electrophoresis, while B represents the mass ratio (%) of RNA falling within the range from more than 4,000 nucleotides to the total mass of RNA that is determined by electrophoresis. If the above RNA extracted from the tissue or cell (cells) complies with the above equation, it is considered to be applicable for analysing gene expression.

EFFECT: presented invention enables the fast and high-effective determination of RNA applicability for analysing gene expression.

7 cl, 3 dwg, 11 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to cell lysis. A method for selective lysis of animal cells and a device for detecting microorganisms are disclosed. A method for selective lysis of animal cells in a water sample with animal cells, containing or possibly containing microorganisms, includes steps of providing a water sample with animal cells, containing or possibly containing microorganisms, adding to said sample a nonionic detergent and a buffer solution to obtain a solution with pH of about 9.5 or higher, incubating said solution for a period sufficient for lysis of animal cells. The device for detecting microorganisms in the sample consists of a lysis chamber for receiving a water sample with animal cells, a vessel with an alkaline buffer solution with pH 9.5 or higher and a nonionic detergent, or a vessel with an alkaline buffer solution with pH of about 9.5 or higher and a vessel with a nonionic detergent, a filter connected to the lysis chamber for filtering the sample after lysis of the animal cells, an indication chamber for analysing presence of DNA of microorganisms.

EFFECT: present inventions enable to process a sample without considerable dilution thereof and without using enzymatic breakdown of DNA or heat treatment, and also enables to process considerably larger sample volumes and determine lower concentrations of pathogenic microorganisms in a sample.

13 cl, 12 dwg, 8 ex

FIELD: biotechnology.

SUBSTANCE: characterised method comprises carrying out of PCR with use of specific primers to genes vc0497, vc0502 and vc0514 from the island of pandemicity VSP-II. The characterised test system comprises the components for isolation of DNA, the components for carrying out PCR, including, in particular, a primer mix VSPIIreg-F - 5'-TGGAAAGAAGAGCGTTACTGC-3', VSPIIreg-R - 5'-CCCTGTTGATGATGTGATTTG-3' to the gene vc0497, VSPIIpilin-F - 5'-CTGTGATTCGGGCTTTATCGG-3', VSPIIpilin-R - 5'-GCGTAAACTGAGCCAATAAGC-3' to the gene vc0502, VSPIIchem-F - 5'-CTTGATGGAGCGGAGAAAAC-3', VSPIIchem-R - 5'-CGATGAATAGCCTGTTGAAC-3' to the gene vc0514, taken in the ratio 1:1:1:1:1:1, respectively.

EFFECT: inventions enable to differentiate quickly and reliably the toxigenic genetically modified strains to genovariants with low and high epidemic potential.

2 cl, 1 dwg, 2 tbl

FIELD: biotechnology.

SUBSTANCE: method comprises isolation of DNA from lymphocytes in peripheral blood by the method of phenol-chloroform extraction, carrying out of PCR, amplification of 18 parts of gene MYO7A, detection in the denaturing acrylamide gel and sequencing. PCR is carried out using specially selected sequences of oligonucleotides flanking regions of 18 exons of the gene MYO7A with possible content of different mutations.

EFFECT: invention enables to simplify the method and to improve the accuracy of determining mutations of the gene MYO7A, to reduce the time of the study.

3 dwg, 1 ex

FIELD: medicine, psychiatry.

SUBSTANCE: one should isolate DNA out of lymphocytes of peripheral venous blood, then due to the method of polymerase chain reaction of DNA synthesis one should amplify the fragments of hSERT locus of serotonin carrier gene and at detecting genotype 12/10 one should predict the risk for the development of hallucino-delirious forms of psychoses of cerebro-atherosclerotic genesis.

EFFECT: more objective prediction of disease development.

3 ex

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