RU2468088C1 - Method of estimating efficiency of therapy urinary bladder cancer in humans by method of immunofermentative analysis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of biotechnology, namely to method of estimating efficiency of therapy of urinary bladder cancer in humans by method of immunofermentative analysis. Claimed invention can be used in medicine. Method includes obtaining samples of patient's urine and/or blood. Mixtures of protein components of urine and blood are separated. Performed is reaction of immunofermentative analysis with monoclonal and/or polyclonal antibodies against recombinant protein UBE2C and/or its unique fragments longer than 8 amino acids. Therapy efficiency is determined by determination of UBE2C protein content in analysed samples, conclusion about performed therapy efficiency is made on 2 and more-fold reduction of level of UBE2C protein in urine and/or blood after performed therapy in comparison with initial level of said protein in urine and/or blood, determined before beginning of treatment.

EFFECT: invention makes it possible to diagnose urinary bladder cancer, including at the early stage of tumour transformation progress, with high reliability.

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Description

The invention relates to medicine, in particular oncology and molecular biology, and relates to a method for the diagnosis of bladder cancer (RMP) and a kit for its implementation.

Bladder cancer (RMP) is a common pathology. About 356,000 new cases of RMP are diagnosed annually in the world [Ploeg, M., K.K. Aben and L. A. Kiemeney, The present and future burden of urinary bladder cancer in the world. World J Urol, 2009.27 (3): p. 289-93]. Existing methods for diagnosing RMP are divided into two groups: invasive and non-invasive. Invasive diagnostic methods include cystoscopy, which allows visualization of the tumor and biopsy of the bladder, as well as transurethral ultrasound (transurethral ultrasonography) [Qu, X., X. Huang, L. Wu, et al., Comparison of virtual cystoscopy and ultrasonography for bladder cancer detection: A meta-analysis. Eur J Radiol, 2010]. All invasive methods are associated with discomfort and risk to the patient’s health, high cost and complexity of execution.

Non-invasive ones include detection of RMP markers in physiological body fluids, transabdominal ultrasound tomography, X-ray computed tomography, magnetic resonance imaging, cytological examination of urine or wash fluid [Kenneu, DM, RDGeschwindt, MRKaru, et al., Detection of newly diagnosed bladder cancer, bladder cancer recurrence and bladder cancer in patients with hematuria using quantitative rt-PCR of urinary survivin. Tumour Biol, 2007.28 (2): p. 57-62, Van Tilborg, A.A., C.H. Bangma and E.C. Zwarthoff, Bladder cancer biomarkers and their role in surveillance and screening. Int J Urol, 2009.16 (1); p.23-30].

RMP biomarkers are divided into the following groups:

1) Markers, which are RNA genes differentially expressed in normal and tumor tissues.

2) Protein markers, as well as peptides and products of protein degradation, specifically detected in the urine or blood of the patient.

Various methods are used to evaluate the content of RNA markers of RMP, primarily microarray hybridization and real-time PCR (PCR-RV) [Livak KJ, Flood SJ, Marmaro J., Giusti W., Deets K. 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 4, 357-362]. As RNA markers of RMP use:

- survivin (elevated mRNA levels are found in tumor tissues, urine, and lavage fluid) [Kenney, D.M., R. D. Geschwindt, M. R. Karie, et al. Detection of newly diagnosed bladder cancer, bladder cancer recurrence and bladder cancer in patients with hematuria using quantitative rt-PCR of urinary survivin. Tumour Biol, 2007. 28 (2): p. 57-62];

- cytokeratin 20 (elevated levels of mRNA in urine and wash fluid) [Guo, B., C. Luo, C. Xun, et al., Quantitative detection of cytokeratin 20 mRNA in urine samples as diagnostic tools for bladder cancer by real-time PCR Exp Oncol, 2009. 31 (1): p. 43-7];

- hyaluronidase (mRNA level is elevated in the urine) [Van Tilborg, A.A., C.H. Bangma and B.C. Zwarthoff, Bladder cancer biomarkers and their role in surveillance and screening. Int J Urol, 2009.16 (1): p.23-30];

- telomerase (mRNA level is elevated in urine) [Eissa, S., M.wellam, R. Ali-Labib, et al., Detection of telomerase in urine by 3 methods: evaluation of diagnostic accuracy for bladder cancer. J Urol, 2007. 178 (3 Pt 1): p.1068-72].

However, a significant drawback of all existing RNA markers of RMP is their low prognostic value (less than 20% of cases of RMP), which leads to poor reproducibility of the results and low clinical value of such tests.

For the diagnosis of RMP using protein markers, immunochemical analysis methods based on the reaction of interaction of specific antibodies with a protein marker are used. Using these methods, one can evaluate the quantitative content of the marker protein RMP and its spatial distribution in the tissue under study. The biological sample for analysis of the protein can be immobilized on a solid carrier, for example on a polymer membrane, on which cells, parts of cells or proteins can be immobilized. This carrier is subsequently hybridized with labeled antibodies specific for the test protein. The amount of antibody bound to the carrier is then evaluated. As a rule, primary or secondary antibodies labeled with a chemically bound enzyme are used for this [Voller, A., The enzyme-linked immunosorbent assay (ELISA) (theory, technique and applications). Ric Clin Lab, 1978. 8 (4): p. 289-98, de Savigny, D. and A. Voller, The communication of ELISA data from laboratory to clinician. J Immunoassay, 1980. 1 (1): p.105-28]. The enzyme associated with the antibody reacts with the appropriate substrate, changing its spectral characteristics as a result of the reaction, with the formation of a chemical moiety or compound that can be detected using spectrometric or fluoremetric methods, or visually. In addition, antibodies are also labeled with radioisotopes, metal nanoparticles and other substances. This principle is based on commercially available methods for the diagnosis of RMP, such as NMP-22 (detection in the urine of a nuclear protein released during apoptosis). This diagnostic test system can serve as a prototype of the present invention [Landman J., Chang Y., Kavaler E., Droller MJ, Liu V.S., Sensitivity and specificity of NMP-22, telomerase, and BTA in the detection of human bladder cancer. Urology. 1998 Sep; 52 (3): 398-402]. The following criteria are used as diagnostic features in it: 1) a statistically significant increase in the level of marker content in the RMP sample relative to the upper limit of the norm; 2) pronounced dynamics of a decrease in marker levels with successful treatment of RMP by at least 50%. This system can also be used to detect relapses and predict the future course of the disease. At the same time, this and other available diagnostic systems for RMP, based on the detection of protein markers in physiological fluids, have insufficient sensitivity (less than 25%) for effective use in clinical practice.

The invention solves the problem of increasing the sensitivity of RMP markers and allows the diagnosis of RMP with a sensitivity of 87%.

The problem is solved by the options for the diagnosis of bladder cancer:

real-time PCR, including biomaterial production and RNA isolation, cDNA synthesis on the RNA matrix, normalization of the concentration of UBE2C cDNA according to the control gene, quantitative PCR amplification of the UBE2C gene fragment and diagnosis by determining the amount of the amplified UBE2C DNA fragment for the biomaterial sample and a kit for the diagnosis of bladder cancer by real-time PCR, including primers with the sequence SEQ ID NO: 1 and 2 with a molar ratio of 1: 1;

enzyme-linked immunosorbent assay, including obtaining urine and blood samples from a patient, isolating a mixture of protein components of urine and blood, carrying out an enzyme-linked immunosorbent assay with monoclonal and / or polyclonal antibodies and / or their fragments against recombinant UBE2C protein and / or its unique fragments of more than 8 in length amino acids and diagnostics by determining the protein content of UBE2C in the studied samples.

In the present invention, products of the UBE2C gene are used as a marker of RMP, which has high specificity of expression in RMP cells, but not in normal bladder tissue. Prior to the present invention, no published data on increased expression of the UBE2C gene in cancer cells compared to normal bladder cells is available. In the present invention, as an indicator of the presence of cancer and / or precancerous changes for RMP, a change in the protein or RNA content of the UBE2C gene in patients with RMP is provided.

In the course of the authors' search for differential transcripts using microarray analysis and identification of differentially expressed genes in normal and tumor samples of bladder tissue at different stages of malignant transformation, a significant increase in the UBE2C gene mRNA was found already in the early stages of malignant transformation of bladder cells. Later, using enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies against the protein product of the UBE2C gene, an increased content of UBE2C protein was also found in the urine and blood of patients with RMP. In total, out of 200 examined tissue samples from patients with RMP, in 174 (87%) the level of protein product UBE2C was observed, twice or more exceeding the highest value in terms of UBE2C for a group of 74 tissue samples from healthy donors.

The present invention relates to a new marker for the diagnosis of bladder cancer (RMP), which is an mRNA of the UBE2C gene and / or UBE2C protein. An increase in the content of the gene mRNA in the tissues of the human bladder allegedly cancerous compared with its content in normal / healthy tissues, as well as an increase in the content of UBE2C protein (the product of the UBE2C gene) in the urine of a patient with human RMP compared to the protein content in the urine of a healthy person serves as a diagnostic marker of RMP.

The present invention relates to a method for diagnosing bladder cancer. This method includes the following stages: obtaining initial samples of biomaterial, for example tissues from a patient; Isolation and purification of RNA preparations from tissue samples; synthesis of cDNA on an RNA matrix; determination of the mRNA concentration of the UBE2C gene by quantitative PCR amplification using a cDNA matrix; normalization of the mRNA concentration of the UBE2C gene according to the control gene, the mRNA content of which is relatively constant in healthy and cancerous human tissues, including the bladder; diagnostics of RMP. For example, an indication for the detection of RMP is the level of the mRNA of the UBE2C gene in the tissues of the bladder in excess of 1% of the level of the human beta-actin gene (ASTV) mRNA in these tissues.

Oligonucleotide primers used for the synthesis of single-stranded or double-stranded cDNA are selected from oligo (dT) -containing primers, random hexamers, or a combination thereof, as well as gene-specific primers.

For amplification of cDNA, oligonucleotide primers and a probe are used that are selected in such a way that they specifically hybridize with cDNA even in the presence of an impurity of genomic DNA. A possible sequence of primers is presented by SEQ ID NO 1-2.

The quantitative amplification reaction of the UBE2C gene fragment is real-time PCR or RT-PCR.

The ASTV gene encoding actin beta is used as a control gene. A possible sequence of primers for determining the concentration of the control gene is presented by SEQ ID NO 3-4. The sequence of the protein-coding DNA of the UBE2C gene is shown in SEQ ID NO 5. The sequence of the UBE2C protein is shown in SEQ ID NO 6.

The set of primers for the implementation of the polymerase chain reaction for determining the content of mRNA of the UBE2C gene have the sequence SEQ ID NO 1-2.

An embodiment of the present invention is a method for diagnosing RMP, comprising the steps of: obtaining a urine and blood sample from a patient for whom a study is performed for the presence of RMP; the allocation of a mixture of protein components of urine and blood; conducting an enzyme immunoassay or other immunological analysis with monoclonal or polyclonal antibodies or their fragments against the recombinant protein UBE2C or its unique fragments with a length of more than 8 amino acids; diagnosing RMP by comparing the UBE2C protein content in the urine and / or blood of the patient under study with the concentration of UBE2C protein in the urine and / or blood of healthy people and patients with RMP.

In this embodiment, chimeric antibodies or antibodies consisting of a single chain or Fab / F (ab ′) ₂ fragments or anti-idiotypic antibodies or epitope-binding fragments can be used.

The present invention provides variants of a method for diagnosing bladder cancer by real-time PCR and enzyme-linked immunosorbent assay (ELISA) based on measuring the mRNA content of the UBE2C gene and / or its protein product, as well as a kit for implementing this method. A reliably detectable difference in the content of UBE2C mRNA in normal and tumor tissues or UBE2C protein in the urine and / or blood of patients with RMP and healthy people can be used to detect RMP in the studied samples.

Biopsy samples, punctate, including material obtained by cystoscopy with direct biopsy, urine and peripheral blood are taken as samples for analysis.

Various methods can be used to isolate RNA. In classical RNA isolation methods, strong chaotropic agents are used, such as guanidinochloride and guanidinisothiocyanate, dissolving proteins, and sequential extraction with phenol and chloroform to denature and remove proteins [Sambrook J., Fritsch EF, Maniatis T., Molecular Cloning. A laboratory manual. 2 ^nd Edition ed. 1989, Cold Spring Harbor: CSHL Press]. The Trizol reagent method [GIBCO / Life Technologies] is also widely used.

In order to prevent RNA degradation by RNase enzymes, inhibitors are used, such as an RNAsin inhibitor of placental or recombinant origin or, for example, a vanadyl riboside complex, a non-specific broad-spectrum RNAse inhibitor.

Rapid and high-quality RNA isolation is also carried out using a number of commercially available kits (Clonogen, St. Petersburg; RNeasy kits (Qiagen); SV Total RNA Isolation System, Promega (USA), etc.). The use of various devices, for example, QuickGene-810 (Life Science, Japan), allows one to minimize work with aggressive agents and media, accelerate and simplify RNA isolation.

The reverse transcription reaction, as a result of which a single-stranded cDNA chain is synthesized on an RNA matrix, if necessary, with the completion of the second chain, allows us to switch from unstable RNA molecules to more stable DNA molecules. PCR amplification allows the use of small amounts of the original RNA (at the level of several nanograms), which corresponds to the minimum amounts of tissue under study. The reverse transcription reaction is carried out using commercially available reverse transcriptase preparations, such as Moloney mouse leukemia virus (M-MLV), avian myeloblastosis virus (AMV) reverse transcriptase, PowerScript (M-MLV-RT point mutation), C. Therm Polymerase, MINT -polymerase and others. For reverse transcription using different primers, for example:

1) Oligo (dT) n-containing primers bind to the poly (A) tail at the 3′-end of the mRNA (the number n is usually 12-18, but can reach a larger value). These primers are used to obtain full-size cDNA.

2) Sets of random hexanucleotide primers (statistical primers) hybridize with RNA in numerous sites. When reverse transcription with these primers receive cDNA, shortened relative to the size of the original RNA. Random hexamers are used to overcome the difficulties associated with reverse transcriptase passage of the secondary RNA structure; they are more effective in reverse transcription of 5'-regions of mRNA.

3) Hexamers or other short oligonucleotides of random composition (up to 12 nucleotides) can be used in combination with oligo (dT) -containing primers;

4) Specific oligonucleotide primers are used for reverse transcription of the mRNA region of interest for the study.

Gene transcription analysis is performed using cDNA as a template for quantitative PCR.

For the selection of specific primers and probes, special programs are used, many of which are freely available on the Internet. Among these programs are Oligo (version 6.42), PrimerSelect from the Lasergene package (www.dnastar.com). Primer Express (Applied Biosystems, USA), Primer Designer (IMB), FastPCR (http://www.biocenter.helsinki.fi/bi/Programs/fastpcr.htm), PrimerQuest (http://scitools.idtdna.com/ Primerquest /), etc., as well as programs such as Vector NTI (Informax), Gene Runner, etc. In addition, it is possible to select primers without using special software.

In a preferred embodiment, primers with the sequence SEQ ID NO 1-2 are used.

All variants of detection of PCR products can be divided into specific and non-specific to a specific DNA sequence. Non-specific systems can be divided into systems using intercalating dyes and systems with primer labeling with fluorescent dyes.

The most inexpensive, but at the same time highly sensitive system is PCR in the presence of intercalating dyes, for example ethidium bromide, YOYO [Srinivasan, K., SC Morris, JEGirard, et al., Enhanced detection of PCR products through use of TOTO and YOYO intercalating dyes with laser induced fluorescence-capillary electrophoresis. Appl Theor Electrophor, 1993. 3 (5): p.235-9], YO-PRO-1 [Ishiguro, T., J. Saitoh, H. Yawata, et al., Homogeneous quantitative assay of hepatitis C virus RNA by polymerase chain reaction in the presence of a fluorescent intercalater. Anal Biochem, 1995. 229 (2): p.207-13], SYBR Green I [Morrison, T. B., J. J. Weis and C. T. Wittwer, Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques, 1998. 24 (6): p.954-8, 960, 962], SYBR Gold, Eva Green and others. These dyes integrate into a double-stranded DNA molecule, change the spectral characteristics of the dyes, which is reflected in a change in the fluorescent signal, amplified by as PCR product accumulates.

High specificity of PCR-RV can be achieved by using a probe (TaqMan system) containing a fluorophore or fluorescent dye at the 5′-end (for example, FAM - 6-carboxy-fluoroscein), and at the 3′-end, the so-called quencher (e.g. DABCYL - 4- (dimethylammoazo) benzene-4-carboxylic acid). During PCR, the interaction of FAM and DABCYL is disrupted due to the cleavage of the Taq probe with DNA polymerase due to its 5′-exonuclease activity, and fluorescence emission recorded by the device occurs [Holland, PM, RDAbramson, R. Watson, et al., Detection of specific polymerase chain reaction product by utilizing the 5 ′ ---- 3 ′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA, 1991. 88 (16): p. 7276-80]. The selection of probes for PCR-RV is carried out in accordance with standard recommendations and the requirements of the method (protocols of Applied Biosystems, http://docs.appliedbiosystems.com). In addition, other modifications of this method can be used, such as displacing probes, molecular beacons, adjoining samples [Solinas, A., LJBrown, C. McKeen, et al., Duplex Scorpion primers in SNP analysis and FRET applications. Nucleic Acids Res, 2001.29 (20): p.E96, Li, Q., G. Luan, Q. Guo, et al., A new class of homogeneous nucleic acid probes based on specific displacement hybridization. Nucleic Acids Res, 2002.30 (2): p.E5, Tyagi, S. and F.R. Kramer, Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol, 1996.14 (3): p.303-8, Didenko, V.V., DNA probes using fluorescence resonance energy transfer (FRET): designs and applications. Biotechniques, 2001. 31 (5): p. 1106-16, 1118, 1120-1].

A quantitative assessment of the mRNA content is achieved using parallel PCR-RV with the test and control samples (Figure 2). As an internal control, relative to which the amplification products of the studied UBE2C gene are normalized, the “housekeeping gene”, ASTV, encoding the main beta-actin cytoskeleton protein, was selected. According to the preferred form of quantifying the mRNA content, it is necessary to select a control gene having a low variability of the mRNA content in tumor and normal tissues of the bladder compared with the variability of the mRNA content of the studied genes [Radonic A., Thuike S., Mackay IM, Landt O., Siegert W., Nitsche A. 2004. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 313, 856-862]. As a rule, “housekeeping” genes, expressed at a high level in each cell of the body, are chosen as control genes, although any gene with a relatively constant mRNA content in the studied samples can be used for this purpose. The decision to select a gene as a control depends on the degree of accuracy selected / required. In a preferred embodiment of the present invention, a traditional and frequently used control gene for ASTV is selected.

Assessment of the mRNA content of genes can be based on the absolute and relative measurement of the number of copies of the studied transcripts - the absolute method (standard curve method) and the relative measurement method (ΔΔCt - method, http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf). According to a preferred form of quantifying the mRNA content of genes, the second of them is selected as the main measurement method. This method allows a double comparison of the data for the studied and control genes in the tumor and normal and does not require alignment of the concentrations of tumor and normal RNA / cDNA samples, which is necessary when using other methods, for example, RT-PCR.

According to the preferred form of quantification of the mRNA content of the genes, it is important to verify the suitability of the reference samples, in this case conditional norms. A “conventional norm” is considered to be a sample of bladder tissue with missing macro- and microscopic signs of tumor growth or a sample of venous blood of a patient. In addition, additional comparison samples obtained from conditionally healthy donors after death (donors who did not suffer bladder cancer during their lifetime) were used.

Since not all tumor samples had suitable paired conditional norms, the calculations of the relative content of the mRNA of the UBE2C gene were carried out using different comparison samples — paired conditional norms, if any, and norms from conditionally healthy donors, if there were no paired norms.

The enzyme-linked immunosorbent assay and its most commonly used modification (enzyme-linked immunosorbent assay, ELISA) are based on the principle of specific interaction between an antigen and its corresponding antibody. Identification of the resulting complex is carried out using a conjugate, which is a complex of a secondary antibody connected to an enzyme label (usually horseradish peroxidase or other peroxidases are used) or otherwise labeled. The conjugate can be obtained using polyclonal antibodies (for example, rabbit antibodies against human immunoglobulins) or monoclonal antibodies directed against human immunoglobulins of a certain class (M, G, A). The present invention contemplates the use of monoclonal and / or polyclonal antibodies and / or fragments thereof obtained against the recombinant protein UBE2C and / or its unique fragments of more than 8 amino acids in length.

There are several methods for setting the reaction, however, at present, the following scheme is most often used to detect specific antibodies (the so-called ELISA sandwich method):

1) the antigen is fixed on the wells of the test tablet, which is incubated with the test serum or blood plasma. In the presence of specific antibodies in them, they bind to form an antigen-antibody complex;

2) in the future, upon incubation of this complex with a conjugate of a secondary antibody and horseradish peroxidase, the antiantibodies attach to the existing antigen-antibody complexes. An enzymatic reaction (color reaction) takes place in the presence of hydrogen peroxide and a substrate represented by an unpainted compound, which, during the peroxidase reaction (or other enzymatic reaction), is oxidized to a colored product at the final stage of the study. Staining intensity directly correlates with the number of specific antibodies detected;

3) the result of the determination is evaluated spectrophotometrically or visually (Figure 2).

In the present invention, the UBE2C protein acts as a marker of RMP, the content of which is increased in the urine of patients with RMP compared to healthy donors who do not have malignant neoplasms.

The present invention is illustrated with reference to specific examples representing the most preferred embodiments of the invention. The invention is not limited to the described embodiments, but includes any alternatives, modifications, or equivalents that are acceptable given the nature and scope of the invention.

The invention is illustrated by graphic materials.

Figure 1. Schematic representation of the structure of the UBE2C gene, cDNA of UBE2C and the resulting amplification product using PCR and / or PCR-RV.

Figure 2. Scheme of immunofluorescence analysis.

Figure 3. Electrophoretic separation in a 1.5% agarose gel of the PCR-PB product (35 PCR cycles) - transcript of the UBE2C gene (size 158 bp), 1st lane - marker of molecular weight of DNA from 100 to 1500 p .n., 2nd and 3rd tracks - transcript of the UBE2C gene of the expected size in tumor tissues, 4th and 5th tracks - in normal tissues of the bladder.

Figure 4. The relative level of expression of the UBE2C gene in tumor and normal tissues of the bladder, measured by PCR-PB after normalizing its expression relative to the expression of the ASTV gene in the same tissue samples. Samples No. 1-9 - tumor tissue of the bladder, 10-12 - normal (without malignant transformation) tissue of the bladder.

The invention is illustrated by examples.

Example 1. Determination of the mRNA level of the UBE2C gene in normal and cancerous tissues of the bladder.

1) Tissue samples

Tissue samples of different histological types of PRMP (T), morphologically normal tissues adjacent to tumors (the so-called conditional norms (N), as well as normal tissues of the bladder (NB) from suddenly died donors were collected and characterized independently by two different groups of FSI employees MNIII named after P.A. Herzen of Rosmedtechnology and the faculty of fundamental medicine of Moscow State University named after M.V. Lomonosov). The clinical diagnosis was established taking into account data from a retrospective analysis of the primary medical documentation of patients, cytological and histological studies of biopsy specimens obtained by cystoscopy, as well as samples of bladder tissue obtained after transurethral resection and surgical material. Bladder tissue samples (tumor, conditionally normal tissue) were obtained immediately after removal of the tumor. Each sample was stored in an RNALater Reagent (QIAGEN) solution stabilizing RNA molecules at a temperature of -70 ° C. All tumor samples are characterized according to the international system of clinical and morphological classification of TNM tumors, where T (tumor) are T0-T4 categories reflecting an increase in the size and / or local distribution of the primary tumor, N (nodules) are N0-N3 categories reflecting different the degree of damage to regional lymph nodes metastases; M (metastasis) - M0-M1 - characterizes distant metastases. All possible combinations of TNM are combined into larger groups - stages that reflect the course of the tumor process.

2) Isolation of RNA preparations from tissues

Total RNA was isolated from frozen, stored in RNALater Reagent (QIAGEN) solution samples of tumor and normal tissue using TRIZOL Reagent (Invitrogen). The main steps include:

a) the destruction of tissue frozen in liquid nitrogen, using a microdesembler ("Sartorius", Germany) and the homogenization of the destroyed sample in a lysis solution containing guanidine isothiocyanate and phenol;

b) the addition of chloroform and centrifugation for 10 minutes at ≤12000 × g at a temperature of 4 ° C;

c) precipitation of RNA from the aqueous phase formed after centrifugation by adding isopropyl alcohol and centrifuging again for 10 minutes at ≤12000 × g at 4 ° C.

Next, the RNA pellet after repeated centrifugation is washed with aqueous 75% ethanol, dried and dissolved in water purified from nucleases.

The concentration of RNA is determined spectrophotometrically. The quality of the isolated RNA was checked by electrophoresis in 1% agarose in the presence of ethidium bromide and spectrophotometrically (Nanodrop, USA).

3) reverse transcription reaction

Single-stranded cDNA is synthesized on an RNA template isolated as described in paragraph 2). For the reverse transcription reaction, 1 μg of RNA obtained by one of the two methods described above using Trizol Reagent reagent kits (Invitrogen) or RNeasy Mini Kit reagents (Qiagen) pretreated with RNase-free DNase I (Sigma-Aldrich) and incubated 5 is taken. min at 70 ° C, then placed in ice.

For each sample, 20 μl of a mixture containing:

5 mM MgCl ₂ ;

1 × AMV reverse transcriptase buffer containing 250 mM Tris-HCl (pH 8.3, 25 ° C), 250 mM KCl, 50 mM MgCl ₂ , 2.5 mM spermidine and 50 mM DTT (Promega);

100 ng hexanucleotide primers;

1 mM dNTP (Evrogen);

200 units of AMV reverse transcriptase (Promega).

RNA and water are added to the mixture to a final volume of 20 μl and the reaction is carried out at the following temperature conditions: 25 ° C for 10 minutes, 42 ° C for 60 minutes, 70 ° C for 10 minutes.

4) Selection of conditions for determining the content of mRNA of the UBE2C gene in tissue samples of the bladder

For RT-PCR and PCR-PB, the same primers UBE2C_F (SEQ ID NO: 1) and UBE2C_R (SEQ ID NO: 2) are used with their molar ratio 1: 1, specific for different exons of the UBE2C gene, one of the primers overlapping the boundary between exons. Amplicon size - 215 bp

The sequence of the selected primers for the control gene: SEQ ID NO 3-4, with their molar ratio of 1: 1, the size of the amplicon is 145 bp

For PCR-PB select the optimal concentration of primers of the studied and control genes (Figure 3).

5) Quantification of the mRNA content of the UBE2C gene

For quantitative measurements using the device Stratagene MX3000P (USA).

Protocol for determining the mRNA content by PCR-RV using the kit “Set of reagents for PCR-RV in the presence of SYBR Green I” or “Set of reagents for PCR-RV in the presence of EVA Green” (Syntol, Russia):

1. Prepare reaction mixtures for the UBE2C and ASTV genes, carefully mixing all the components of the reaction except the template (cDNA), at the rate of 1 reaction with a volume of 25 μl for each sample + 1 additional reaction of 25 μl.

Table 1 The composition of the reaction mixture for the ASTV gene: Reagent The concentration of stock solutions Volume per reaction (μl) The final concentration in the reaction PCR Buffer B 10X 2.5 1X dNTPs 2.5 mm 2.5 250 μM MgCl ₂ 25 mm 2.5 2.5 mm Primer ACTB_F 10 μM one 0.4 μM Primer ACTB_R 10 μM one 0.4 μM Taq DNA polymerase 5 units / μl 0.5 0.1 units / μl H ₂ O 13,2 cDNA 2

table 2 The composition of the reaction mixture for the UBE2C gene: Reagent The concentration of stock solutions Volume per reaction (μl) The final concentration in the reaction PCR Buffer B 10X 2.5 1X dNTPs 2.5 mm 2.5 250 μM MgCl ₂ 25 mm 2.5 2.5 mm Primer UBE2C_F 10 μM one 0.4 μM Primer UBE2C_R 10 μM one 0.4 μM Taq DNA polymerase 5 units / μl 0.5 0.1 units / μl H ₂ O 13,2 cDNA 2

2. In 0.2 ml tubes designed for PCR-PB, add 23 μl of the prepared reaction mixtures without a matrix.

3. Pipette 2 μl of the matrix, tightly close the tubes with caps.

4. Place the tubes in the instrument compartment, set the cell names, temperature for 35 cycles: 95 ° С - 5 min - denaturation and enzyme activation, 95 ° С - 20 s - denaturation, 60 ° С - 20 s - probe annealing and polymerization , 72 ° С - 30 s - elongation of chains.

5. The reactions are carried out in the mode of relative quantitative measurements (software Stratagene MxPro, USA).

6. After completion of the amplification reaction, data is saved.

7. The reaction products are analyzed by gel electrophoresis in TBE buffer containing 10 mg / l ethidium bromide. For this, 5 μl of amplification products (40 cycles) are taken, 1.8% agarose gel containing 0.5 mg / L ethidium bromide samples and a molecular weight marker for DNA are used to evaluate the size of the amplification products (SibEnzyme NPO).

6) Mathematical processing of PCR-RV data

PCR-RV data is transferred as a text file to Microsoft Excel and mathematical processing of the data is carried out. The relative content of mRNA of the UBE2C-R _cDNA gene (hereinafter R) in tumor and normal tissues of the bladder is normalized relative to the content of mRNA of the ASTV gene according to the formula:

R = 2 ^ (Ct (ACTB) -Ct (UBE2C), where Ct is the threshold cycle.

The range of extreme values of R is calculated taking into account the calculated deviations E for the Ct values:

E = ((Σ (xx _cp ) ² ) / (n-1)) ^1/2 ,

where x is the values obtained as a result of the measurement (number 1, number 2 ...),

X _cf = Σx / n,

n is the sample size (the number of repetitions of the reaction).

The reliability of the observed changes is estimated based on the normal distribution of data. Data is considered reliable at P <0.05, where P is an indicator of the statistical significance of the data (Figure 4).

7) Results

As a result, for all the studied RMP samples (n = 26), a 5-64-fold increase in the mRNA level of the UBE2C gene was revealed relative to normal samples of bladder tissue (n = 47).

Example 2. Determination of the level of protein UBE2C in normal and cancerous tissues of the bladder by enzyme-linked immunosorbent assay.

1) Tissue samples

Tissue samples of various histological types of RMP (T), morphologically normal tissues adjacent to tumors (the so-called conditional norms (N), as well as normal bladder tissue (NB) from suddenly died donors were collected and characterized independently by two different groups of FSI employees MNIII named after P.A. Herzen of Rosmedtechnology and the faculty of fundamental medicine of Moscow State University named after M.V. Lomonosov). The clinical diagnosis was established taking into account data from a retrospective analysis of the primary medical documentation of patients, cytological and histological studies of biopsy specimens obtained by cystoscopy, as well as samples of bladder tissue obtained after transurethral resection and surgical material. Bladder tissue samples (tumor, conditionally normal tissue) were obtained immediately after removal of the tumor. Each sample was stored in an RNALater Reagent (QIAGEN) solution stabilizing RNA molecules at a temperature of -70 ° C. All tumor samples were characterized according to the international system of clinical and morphological classification of TNM tumors, where T (tumor) - T0-T4 - categories reflecting an increase in the size and / or local distribution of the primary tumor, N (nodules) - N0-N3 - categories reflecting different the degree of defeat by metastases of the regional lymph nodes; M (metastasis) - M0-M1 - characterizes distant metastases. All possible combinations of TNM are combined into larger groups - stages that reflect the course of the tumor process.

3) Isolation of total protein from bladder tissue samples

1) The total protein is isolated from frozen samples of the bladder by homogenizing pieces of tissue, punctures or biopsies in a lysis buffer. The composition of the lysis buffer: 1 · PBS (pH 7.6) buffer (1.7 mM KH ₂ PO ₄ , 5.2 mM Na ₂ HPO ₄ , 150 mM NaCl), 8 M urea. To analyze the content of the studied protein in the blood, a plasma fraction is used. Whole urine is used to analyze the protein content in the urine.

2) To bind antigens (UBE2C protein or its fragments with a length of more than 8 amino acids) to the solid phase (carrier), the antigen is incubated with the carrier for 14 hours at 4 ° C at the rate of 100 μl of antigen solution per cell in a 96-well plate.

3) Remove unbound antigens and wash the plate 3 times with 1 · PBS.

4) Block non-specific binding by adding 1% BSA (100 μl / well) in 1 · PBS for at least 1 hour at 37 ° C.

5) BSA is removed and washed 3 times with 1 × PBS containing 0.01% tween (polysorbate).

6) Add antiserum diluted in 1 × PBS (if necessary) and incubate for 2 hours at 37 ° C.

7) Wash the plate 4 times with 1 × PBS containing 0.01% tween (polysorbate).

8) A conjugate of peroxidase (or another enzyme) (50 μl / well) diluted 1: 1000 in 1% BSA is added and incubated for 1 hour at 37 ° C.

9) Wash the plate 4 times with 1 × PBS containing 0.01% tween (polysorbate).

10) Add substrate (100 μl / well).

11) Incubate 1-5 minutes in the dark. Record a color change (changes to blue during the course of the reaction.

12) Stop the reaction by adding 25 μl of 0.1 M H ₂ SO ₄ / well.

13) Analyze the results using a spectrophotometer at a wavelength of 450 nm (for tetramethylbenzidine substrate - TMB).

The composition of the buffers used:

1 × PBS (Phosphate-Buffered Saline):

NaCl 8 g

KCl 0.2 g

Na ₂ PO ₄ (anhydrous) 1.15 g

KH ₂ PO ₄ (anhydrous) 0.2 g

dd H ₂ O - up to 100 ml.

PBS for washing dies:

100 ml 1 × PBS

10 μl of tween (to a final concentration of 0.01%)

Citrate / Acetate Buffer (pH 6.0):

1.500 ml of 0.1 M sodium acetate (sodium acetate - 4.1 g, dd H ₂ O - up to 500 ml)

2.100 ml of 0.1 M citric acid (citric acid - 2.1 g, dd H ₂ O - up to 100 ml)

Titrate solution 1 with solution 2 to a final pH of 6.0. Store the resulting buffer at -20 ° C.

A solution of TMB - 10 mg / ml in DMSO (store at 4 ° C).

Add 3 μl of H ₂ O ₂ to 20 ml of citrate / acetate buffer. For every 5 ml of buffer, 50 μl of TMB solution is added. The color of the solution changes from colorless to blue.

4) Results

As a result, for all the studied tumor samples (n = 26), a 7-53-fold excess of the level of UBE2C protein was revealed relative to normal samples of bladder tissue (n = 47).

Example 3. Determination of the concentration of mRNA of the UBE2C gene in the urine and blood of patients with RMP and healthy people.

1) Urine and blood samples

Blood and urine samples from patients diagnosed with RMP, as well as from healthy donors, were collected and characterized by a group of employees of FGU MNIII im. P.A. Herzen of the Russian Medical Technologies. The clinical diagnosis was established taking into account data from a retrospective analysis of the primary medical documentation of patients, cytological and histological studies of biopsy specimens obtained by cystoscopy, as well as samples of bladder tissue obtained after transurethral resection and surgical material. Blood is mixed with a solution of an anticoagulant (for example, sodium citrate, EDTA, etc.) in a ratio of 9: 1 (9 parts of blood and 1 part of an anticoagulant), the plasma is separated from blood cells by centrifugation for 15 minutes at 1500 × g and immediately mixed with a solution that stabilizes RNA molecules (RNAlater Reagent, QIAGEN). The resulting solution was stored at a temperature of -20 ° C. Urine samples are mixed with a solution of RNAlater Reagent, QIAGEN, frozen and stored at a temperature of -20 ° C.

2) Isolation of RNA preparations from tissues

Total RNA was isolated from frozen, stored in RNAlater Reagent (QIAGEN) solution samples of urine and blood obtained from patients and healthy donors using TRIZOL Reagent (Invitrogen). The main steps include:

a) Treatment of blood and urine with a lysing solution containing guanidine isothiocyanate and phenol;

b) the addition of chloroform and centrifugation for 10 minutes at 12000 × g at a temperature of 4 ° C;

c) precipitation of RNA from the aqueous phase formed after centrifugation by adding isopropyl alcohol and centrifuging again for 10 minutes at 12000 × g at 4 ° C.

Next, the RNA pellet after repeated centrifugation is washed with aqueous 75% ethanol, dried and dissolved in water purified from nucleases.

The concentration of RNA is determined spectrophotometrically. The quality of the isolated RNA was checked by electrophoresis in 1% agarose in the presence of ethidium bromide and spectrophotometrically (Nanodrop, USA).

3) reverse transcription reaction

Single-stranded cDNA is synthesized on an RNA template isolated as described in paragraph 2). For the reverse transcription reaction, 1 μg of RNA obtained by one of the two methods described above using Trizol Reagent reagent kits (Invitrogen) or RNeasy Mini Kit reagents (Qiagen) pretreated with RNase-free DNase I (Sigma-Aldrich) and incubated 5 is taken. min at 70 ° C, then placed in ice.

For each sample, 20 μl of a mixture containing:

5 mM MgCl ₂ ;

1 × AMV reverse transcriptase buffer containing 250 mM Tris-HCl (pH 8.3, 25 ° C), 250 mM KCl, 50 mM MgCl ₂ , 2.5 mM spermidine and 50 mM DTT (Promega);

100 ng hexanucleotide primers;

1 mM dNTP (Evrogen);

200 units of AMV reverse transcriptase (Promega).

RNA and water are added to the mixture to a final volume of 20 μl and the reaction is carried out at the following temperature conditions: 25 ° C for 10 minutes, 42 ° C for 60 minutes, 70 ° C for 10 minutes.

4) Selection of conditions for determining the content of mRNA of the UBE2C gene

For RT-PCR and PCR-PB, the same primers UBE2C_F (SEQ ID NO: 1) and UBE2C_R (SEQ ID NO: 2), specific for different exons of the UBE2C gene, are used, with their molar ratio 1: 1, one of the primers overlapping the border between exons. Amplicon size - 215 bp

The sequence of the selected primers for the control gene: SEQ ID NO 3-4, the size of the amplicon - 145 BP

For PCR-RV, optimal primer concentrations of the studied and control genes are selected.

5) Quantification of the mRNA content of the UBE2C gene

For quantitative measurements using the device Stratagene MX3000P (USA).

Protocol for determining the mRNA content by PCR-RV using the kit “Set of reagents for PCR-RV in the presence of SYBR Green I” or “Set of reagents for PCR-RV in the presence of EVA Green” (Syntol, Russia):

1. Prepare reaction mixtures for the UBE2C and ASTV genes, carefully mixing all the components of the reaction except the template (cDNA), at the rate of 1 reaction with a volume of 25 μl for each sample + 1 additional reaction of 25 μl.

Table 3 The composition of the reaction mixture for the ASTV gene: Reagent The concentration of stock solutions Volume per reaction (μl) The final concentration in the reaction PCR Buffer B 10X 2.5 1X dNTPs 2.5 mm 2.5 250 μM MgCl ₂ 25 mm 2.5 2.5 mm Primer ACTB_F 10 μM one 0.4 μM Primer ACTB_R 10 μM one 0.4 μM Taq DNA polymerase 5 units / μl 0.5 0.1 units / μl H ₂ O 13,2 cDNA 2

Table 4 The composition of the reaction mixture for the UBE2C gene: Reagent The concentration of stock solutions Volume per reaction (μl) The final concentration in the reaction PCR Buffer B 10X 2.5 1X dNTPs 2.5 mm 2.5 250 μM MgCl ₂ 25 mm 2.5 2.5 mm Primer UBE2C_F 10 μM one 0.4 μM Primer UBE2C_R 10 μM one 0.4 μM Taq DNA polymerase 5 units / μl 0.5 0.1 units / μl H ₂ O 13,2 cDNA 2

2. In 0.2 ml tubes designed for PCR-PB, add 23 μl of the prepared reaction mixtures without a matrix.

3. Pipette 2 μl of the matrix, tightly close the tubes with caps.

4. Place the tubes in the instrument compartment, set the cell names, temperature for 35 cycles: 95 ° С - 5 min - denaturation and enzyme activation, 95 ° С - 20 s - denaturation, 60 ° С - 20 s - probe annealing and polymerization , 72 ° С - 30 s - elongation of chains.

5. The reactions are carried out in the mode of relative quantitative measurements (software Stratagene MxPro, USA).

6. After completion of the amplification reaction, data is saved.

7. The reaction products are analyzed by gel electrophoresis in TBE buffer containing 10 mg / l ethidium bromide. For this, 5 μl of amplification products (40 cycles) are taken, 1.8% agarose gel containing 0.5 mg / L ethidium bromide samples and a molecular weight marker for DNA are used to evaluate the size of the amplification products (SibEnzyme NPO).

6) Mathematical processing of PCR-RV data

PCR-RV data is transferred as a text file to Microsoft Excel and mathematical processing of the data is carried out. The relative content of mRNA of the UBE2C-R _cDNA gene (hereinafter R) in tumor and normal tissues of the bladder is normalized relative to the content of mRNA of the ASTV gene according to the formula:

R = 2 ^ Ct (ACTB) -Ct (UBE2C), where Ct is the threshold cycle.

The range of extreme values of R is calculated taking into account the calculated deviations E for the Ct values:

E = ((Σ (x-x _sr ) ² ) / (n-1)) ^1/2 ,

where x is the values obtained as a result of the measurement (number 1, number 2 ...),

x _cp = Σx / n,

n is the sample size (the number of repetitions of the reaction).

The reliability of the observed changes is estimated based on the normal distribution of data. Data is considered reliable at P <0.05, where P is an indicator of the statistical significance of the data.

7) Results

As a result, for all studied urine and blood samples obtained from patients with RMP (n = 26), a 10-48-fold excess of the concentration of mRNA of the UBE2C gene was revealed relative to urine and blood samples obtained from healthy donors (n = 70).

Example 4. Determination of the level of UBE2C protein in the urine and blood of patients with RMP and healthy people by enzyme-linked immunosorbent assay.

1) Urine and blood samples

Blood and urine samples from patients diagnosed with RMP, as well as from healthy donors, were collected and characterized by a group of employees of FGU MNIII im. P.A. Herzen of the Russian Medical Technologies. The clinical diagnosis was established taking into account data from a retrospective analysis of the primary medical documentation of patients, cytological and histological studies of biopsy specimens obtained by cystoscopy, as well as samples of bladder tissue obtained after transurethral resection and surgical material. Blood is mixed with a solution of an anticoagulant (for example, sodium citrate, EDTA, etc.) in a ratio of 9: 1 (9 parts of blood and 1 part of an anticoagulant) and stored at + 4 ° C or the plasma is separated from blood cells by centrifugation for 15 minutes at 1500 × g and stored at a temperature of -20 ° C. Urine samples are frozen and stored at -20 ° C.

To analyze the content of the studied protein in the blood, a plasma fraction is used. Whole urine is used to analyze the protein content in the urine.

2) To bind antigens (UBE2C protein or its fragments with a length of more than 8 amino acids) to the solid phase (carrier), the antigen is incubated with the carrier for 14 hours at 4 ° C at the rate of 100 μl of antigen solution per cell in a 96-well plate.

3) Remove unbound antigens and wash the plate 3 times with 1 × PBS.

4) Block non-specific binding by adding 1% BSA (100 μl / well) in 1 × PBS for at least 1 hour at 37 ° C.

5) BSA is removed and washed 3 times with 1 × PBS containing 0.01% tween (polysorbate).

6) Add antiserum diluted in 1 × PBS (if necessary) and incubate for 2 hours at 37 ° C.

7) Wash the plate 4 times with 1 × PBS containing 0.01% tween (polysorbate).

8) A conjugate of peroxidase (or another enzyme) (50 μl / well) diluted 1: 1000 in 1% BSA is added and incubated for 1 hour at 37 ° C.

9) Wash the plate 4 times with 1 × PBS containing 0.01% tween (polysorbate).

10) Add substrate (100 μl / well).

11) Incubate for 5 minutes in the dark. Record a color change (changes to blue during the course of the reaction.

12) Stop the reaction by adding 25 μl of 0.1 M H ₂ SO ₄ / well.

13) Analyze the results using a spectrophotometer at a wavelength of 450 nm (for tetramethylbenzidine substrate - TMB).

The composition of the buffers used:

1 × PBS (Phosphate-Buffered Saline):

NaCl 8 g

KCl 0.2 g

Na ₂ PO ₄ (anhydrous) 1.15 g

KH ₂ PO ₄ (anhydrous) 0.2 g

ddH ₂ O - up to 100 ml.

PBS for washing dies:

100 ml 1 × PBS

10 μl of tween (to a final concentration of 0.01%)

Citrate / Acetate Buffer (pH 6.0):

1.500 ml of 0.1 M sodium acetate (sodium acetate - 4.1 g, dd H ₂ O - up to 500 ml)

2.100 ml of 0.1 M citric acid (citric acid - 2.1 g, dd H ₂ O - up to 100 ml)

Titrate solution 1 with solution 2 to a final pH of 6.0. Store the resulting buffer at -20 ° C.

A solution of TMB - 10 mg / ml in DMSO (store at 4 ° C).

Add 3 μl of H ₂ O ₂ to 20 ml of citrate / acetate buffer. For every 5 ml of buffer, 50 μl of TMB solution is added. The color of the solution changes from colorless to blue.

Results.

As a result, for all investigated urine and blood samples obtained from patients with RMP (n = 26), a 4-38-fold excess of the level of UBE2C protein was revealed relative to urine and blood samples obtained from healthy donors (n = 70).

Example 5

Evaluation of the effectiveness of the treatment of RMP by changing the expression level of the UBE2C gene and / or UBE2C protein in the urine and / or blood of the examined patients.

Evaluation of the effectiveness of the treatment of RMP is carried out by analyzing the content of the UBE2C protein and / or the expression level of the UBE2C gene in the urine and / or blood of patients with RMP before treatment and after treatment by the methods described in examples 3 and 4. As a result, for all examined patients ( n = 10) with a diagnosis of RMP, a decrease in the level of expression of the UBE2C gene and the level of UBE2C protein in urine and blood was 2-8 times after the treatment compared with the initial level of expression of this gene or the level of protein in urine and blood detected before cross section. This indicates the possibility of using the present invention in monitoring the effectiveness of anti-cancer therapy, and analysis by the methods of the present invention should be carried out before and after the course of treatment, and also, if necessary, during the course of treatment. A decrease in the content of the mRNA of the UBE2C gene and / or UBE2C protein during or at the end of the course of treatment may indicate positive changes in treatment. A further increase in the mRNA content of the UBE2C gene and / or UBE2C protein indicates a low treatment efficiency.

Claims (1)

A method for evaluating the effectiveness of the treatment of human bladder cancer by enzyme immunoassay, including obtaining urine and / or blood samples from a patient, isolating a mixture of protein components of urine and blood, conducting an enzyme immunoassay reaction with monoclonal and / or polyclonal antibodies against recombinant protein UBE2C and / or its unique fragments with a length of over 8 amino acids and determining the effectiveness of therapy by determining the content of UBE2C protein in the studied samples, while on the effectiveness of therapy is judged by a decrease in the level of UBE2C protein in the urine and / or blood by 2 or more times after the treatment compared with the initial level of this protein in the urine and / or blood detected before treatment.

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