RU2395234C1 - DETERMINATION OF GENE ZG16 mRNK LEVEL REDUCTION AS METHOD OF LARGE INTESTING CANCER DIAGNOSTICS AND SET OF ITS REALISATION - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: claimed invention relates to field of medicine, in particular to oncology and molecular biology and can be used for diagnostics of large intestine cancer. According to the method initial pair of tissue samples from patients are obtained, where one of the samples is obtained from tissue supposedly affected by cancer, and the second one is obtained from histologically normal (conditionally-normal) adjacent or located at a small distance from the tumour tissue; separation and purification of RNA preparations from initial pair of samples; synthesis of single-stranded cDNA on RNA matrix with application of oligonucleotide primers; fixing rate to cDNA concentration by control gene, whose transcription level is constant in norm even in case of large intestine cancer; carrying out quantitative and semi-quantitative reaction of gene ZG16 fragment amplificxation with application of cDNA obtained at stage c), as matrix and pair of gene-specific oligonucleotide primers with sequences SEQ ID NO: 1 and 2; comparison of quantity of amplified DNA ZG16 fragment for the sample obtained from the tissue supposedly affected with cancer, with the quantity of amplified DNA fragment for the sample, obtained from normal tissue, where said quantities of amplified DNA fragment represent gene ZG16 mRNA level, decrease of ZG16 mRNA quantity being a diagnostic sign of large intestine cancer. For realisation of the method claimed is a set which includes primers for carrying out polymerase chain reaction for determination of gene ZG16 mRNA level, possessing sequences SEQ ID NO^∧1 and 2.

EFFECT: method and set allow to diagnose large intestine cancer with high reliability, including early stages of tumour transformation progression.

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Description

FIELD OF THE INVENTION

The present invention relates to medicine, in particular oncology, and molecular biology and can be used for early diagnosis of colon cancer.

State of the art

Colon cancer ranks second or third in mortality from cancer in Russia and the industrialized countries of Europe and America [Jemal A., Siegel R., Ward E., Murray T., Xu J., Smigal C., Thun M.J. 2006, Cancer statistics, 2006. CA Cancer J. Clin., 56. 6-30]. More than 600,000 cases of primary colon cancer (RTK) are registered annually in the world, in Russia over the past 5 years - more than 50,000 primary cases annually [Bulletin of the Russian Cancer Research Center named after NN Blokhina RAMS 2006, Statistics of malignant neoplasms in Russia and the CIS countries in 2004, t.17, No. 3 (adj. 1), p.47]. The diagnosis in half of the cases of RTK is established at a far advanced stage of the tumor process, which makes radical cure unrealistic. The success of treatment is determined by the possibility of early detection of tumors.

Clinical practice in many countries uses commercially available immunological tests to predict some of the most common types of tumors (for example, antibodies to the ERBB2 receptor and the recently approved FDA MammaPrint test for predicting breast cancer). At the same time, to predict RTK in clinical practice, only one immunological test is used to increase the level of embryonic cancer antigen (ERA / CEA) in the bloodstream. The test has low predictive value and is mainly used to determine the risk of tumor re-growth after surgery. In addition, an increased level of ERA in the blood may be associated with smoking, alcohol consumption and some non-cancer diseases (bronchitis, pneumonia, etc.). To diagnose colon cancer, several ineffective types of tests are currently used:

1) The FOBT test for the presence of hemoglobin in feces allows diagnosis of RTK in 30% of cases. The advantages of this test are relative cheapness and ease of use, and the most important disadvantages are very low sensitivity and nonspecificity (stomach ulcer, duodenal ulcer, hemorrhoids also give a positive answer). This test is performed, for example, by BioMerica (EZ Detect kit) and other companies;

2) a test for the presence of DNA in the stool (PreGen-Plus, Exact Sciences) is more sensitive than hemoglobin tests, and the result of the analysis does not depend on the patient’s diet. Nevertheless, it is a non-specific and, in addition, a very expensive diagnosticum;

3) tests based on the detection of modified glycolipids and glycoproteins by monoclonal antibodies (CA 19-9 antigen, modified Lewis blood group antigen, TAG-72 glycoprotein, etc.). Such tests are ineffective due to very high differences in the level of antigen synthesis in primary colon tumors;

4) a serum test based on the detection of an increased level of synthesis of the enzyme guanylyl cyclase C (GCC) or the mRNA encoding it (GCC-B1 ™ Blood TestGCC-B1 ™). The test is effective only in the late stages of the disease with massive penetration of tumor cells into the bloodstream (in particular as a result of repeated growth of the tumor after surgery or metastasis), when treatment is futile.

The need to identify new tumor markers is determined by: 1) the lack of diagnostic kits on Russian-made RTKs; 2) the exceptional high cost and lack of reliability of diagnostic kits produced abroad (one analysis costs an average of $ 500-600); 3) an increase in the number of diseases of RTK in Russia, including cases detected in the late stages of the disease, when treatment is ineffective; 4) the duration and high cost of treatment in a hospital; 5) the need to increase the average life expectancy of the able-bodied population of the Russian Federation to improve the overall demographic situation.

Most malignant colon tumors, as a rule, arise as a result of the conversion of benign tumors (adenomas) to malignant (adenocarcinomas) [Kashida H., Kudo S. Early colorectal cancer: concept, diagnosis, and management. Int. J. Oncol. 2006, 11, 1-8]. Carcinogenesis is characterized by phenotypically different stages: hyperplasia, adenomas, adenocarcinomas, metastases. This stepwise process is accompanied by activation of transcription of a number of oncogenes, as well as a decrease or complete loss of transcriptional activity of the so-called tumor suppressor genes. Changes in the activity of these two groups of genes can serve as a diagnostic sign of malignant tumors, including RTK. This change can be determined using reverse transcription followed by a standard polymerase chain reaction (standard, semi-quantitative RT-PCR method) or real-time PCR (PCR-RV) - the most advanced way to quantify gene activity, determined by the amount of mRNA transcribed from DNA of these genes per tumor cell.

RTK is accompanied by a change in the functional activity of many genes. We carried out a bioinformatic search for potential RTK biomarkers based on a comprehensive analysis of the databases of cloned libraries of expressed nucleotide sequences (Expressed Sequence Tags - EST) obtained from RNA of normal and tumor PTK cells. In this case, the most promising as potential biomarkers of genes were identified, the mRNA of which, determined by the number of EST clones, most significantly differs in the norm and tumor.

EST klonoteki are widely used for various purposes, it is one of the basic elements in the study of genomic transcripts [Nagaraj S.H., Gasser R. B., Ranganathan S. A. hitchhiker's guide to expressed sequence tag (EST) analysis. Brief Bioinform. 2007 Jan; 8 (1): 6-21. Epub 2006 May 23]. EST is a short, typically 200 to 800 bp, cDNA obtained from a cDNA clonoteca synthesized from a normal or tumor tissue mRNA matrix. During systematic sequencing of cDNA libraries, only a part of the nucleotide sequence of each mRNA from the 3'- or 5'-end is determined (depending on the method of obtaining EST). The number of EST clones corresponding to a particular gene in each of the clonotes in the vast majority of cases is directly proportional to the transcription of this gene. This premise is the basis of this program. Comparing the normalized number of EST clones for the gene in the normal and tumor clonotes, one can judge not only about the level of its transcription in the tumor compared to the norm, but also about the stability and frequency of occurrence of such a change in different tumor samples from which the clonotopes were obtained .

As a result of the bioinformatic search for potential RTK biomarkers, we identified genes with the most significant changes in the mRNA level in tumors compared to the norm, among which the ZG16 gene (zymogen granule protein 16) was selected as one of the most promising genes - potential tumor markers. This gene encodes a secretory protein (167 aa) stored in zymogen granules and belonging to the family of lectins containing the Jacalin domain. Zymogen granules are rounded formations with a diameter of 0.5-1.5 microns in the cells of various human glands containing zymogens, or proenzymes. Protein ZG16 can act as a linker molecule between the submembrane matrix in zymogen granules (zgm) and aggregated secretory proteins during the formation of granules in tgn (trans-network of the Golgi apparatus, where the secreted products are separated and sorted) [Dartsch, H., R. Kleene, HFKern: In vitro condensation-sorting of enzyme proteins isolated from rat pancreatic acinar cells. Eur. J. Cell Biol. 75, 211-222 (1998); Kleene R, Dartsch H, Kern HF. The secretory lectin ZG16p mediates sorting of enzyme proteins to the zymogen granule membrane in pancreatic acinar cells. 1999 Feb; 78 (2): 79-90]. Protein ZG16 is involved in the secretion of glycoproteins.

The ZG16 gene is located on chromosome 16p13.3, contains 4 exons, and the transcript length is 1.883 bps. Normally, gene expression is detected in the liver and intestines. A significant decrease in gene expression was shown in the most common form of liver cancer - hepatocellular carcinoma (Zhou YB, Cao JB, Yang HM, Zhu H, Xu ZG, Wang KS, Zhang X, Wang ZQ, Han ZG hZG16, a novel human secreted protein expressed in liver, was down-regulated in hepatocellular carcinoma. Biochem Biophys Res Commun. 2007 Apr 13; 355 (3): 679-86. Epub 2007 Feb 12). This analogue is closest in technical essence to the claimed invention and adopted as a prototype.

However, changes in the expression of the ZG16 gene in other cancers, including PTK, have not been studied. It should be noted that the determination of changes in the transcriptional activity of this gene in colon tumors is of great interest for the diagnosis of RTK, since from a diagnostic point of view, the high stability of the resulting transcript plays an important role in choosing a candidate for the role of a diagnostic marker. It is clear from the foregoing that in this area there is an urgent need to search for a new diagnostic marker of RTK, which allows reliable and early diagnosis of the disease, and to develop on its basis a simple, sensitive, reliable, diagnostic method applicable in clinical or outpatient medical institutions RTK.

Disclosure of invention

This invention was made possible as a result of a comparative analysis of the level of ZG16 gene expression in tumor tissues of the colon of various types and at different stages of their malignant transformation and the discovery of the fact that already the early stages of the development of malignant transformation are accompanied by a significant decrease in the level of ZG16 gene mRNA.

The present invention in its aspect relates to a new marker for the diagnosis of colon cancer, which is a change in the level of mRNA of the ZG16 gene. A decreased level in a human tissue presumably affected by cancer compared with its level in healthy tissue serves as a diagnostic sign of RTK.

This method includes the following steps:

a) obtaining the initial pair of tissue samples from the patient, where one of the samples was obtained from tissue presumably affected by cancer, and the second was obtained from adjacent histologically normal (conditionally normal) tissue;

b) isolation and purification of RNA preparations from the initial pair of samples;

c) synthesis of single-stranded cDNA on an RNA template using oligonucleotide primers;

d) normalization of the concentration of ZG16 cDNA according to the control gene, the level of transcription of which is constant in normal and in colon cancer;

e) conducting a quantitative or semi-quantitative amplification reaction of the ZG16 gene fragment using the cDNA obtained in step c) as a template and a pair of gene-specific oligonucleotide primers;

f) comparing the amount of amplified ZG16 DNA fragment for a sample obtained from a tissue presumably affected by cancer with the amount of amplified DNA fragment for a sample obtained from normal tissue, where the indicated amounts of amplified DNA fragment reflect the level of transcription of the ZG16 gene, and lowering the level of ZG16 mRNA serves a diagnostic sign of colon cancer.

In one embodiment of the method of the invention, in step c), the oligonucleotide primers used for the synthesis of single-stranded cDNA are selected from oligo (dT) _n- containing primers, random hexamers or a combination thereof, as well as gene-specific primers.

In a further embodiment of the method of the present invention, oligonucleotide primers selected in such a way that they specifically hybridize with cDNA even in the presence of an impurity of genomic DNA are used to amplify the cDNA in step d).

In a separate preferred embodiment of the invention, the primer sequence is represented by SEQ ID NO: 1 and 2.

In yet another embodiment of the method of the present invention, in step e), the quantitative or semi-quantitative amplification reaction of the ZG16 gene fragment is real-time PCR or standard RT-PCR.

In one embodiment of the inventive method, in step g), a so-called “house-keeping” gene encoding beta-actin is used as a control gene, having the most constant expression level both in the normal intestinal mucosa and in the colon tumor cells. Other “housekeeping genes,” for example, a gene encoding beta-2-microglobulin (B2M), can be used as an endogenous control.

Another aspect of the present invention is a set of primers for the implementation of the polymerase chain reaction for determining the level of mRNA of the ZG16 gene having the sequence of SEQ ID NO: 1 and 2.

List of drawings

The invention will now be described in more detail with reference to certain illustrative examples and drawings.

Figure 1 shows the results of the selection of conditions for determining the level of mRNA of the ZG16 gene in samples of colon tissue. Electrophoretic separation on an agarose gel of PCR products (size 79 bp) obtained after 28, 30, 32 and 34 cycles was carried out in a 1.8% agarose gel. M is a molecular weight marker for DNA (plasmid DNA pBR222 / AluI).

Figure 2 shows the results of an RT-PCR analysis of the mRNA level of the ZG16 gene in RTK (after 30 cycles). Sample numbers are indicated above the tracks. O - tumor, H - normal (normal mucous membrane of the colon at a distance of about 10 cm from the tumor). The gene encoding beta-2-microglobulin (B2M) (25 cycles of PCR) was used as an internal control. Electrophoretic separation of PCR products was carried out on a 1.8% agarose gel.

Figure 3 shows the results of a quantitative determination of changes in the level of mRNA of the ZG16 gene during RTK using real-time PCR in 76 paired norm-tumor samples. Genes encoding beta-2-microglobulin (B2M) and beta-actin (ASTV) were used as control genes. The drawing shows the localization of tumors (distal or proximal) above and the stages of tumors (I, IIA, IIB, IIIB, IIIC, IV) below.

The implementation of the invention

This invention provides a new genetic marker for the diagnosis of colon cancer and based on the determination of the mRNA level of this marker, a simple, reliable way to diagnose PTK at different stages of the development of malignant transformation, including the initial ones. A reliably detectable difference in the level of mRNA of the ZG16 gene in normal and tumor tissues can be used to detect PTK.

Colon tissue samples for analysis

As samples for analysis, surgical and biopsy material obtained by colonoscopy with a direct biopsy of a normal and presumably cancer of the colon mucosa can be used.

Isolation of RNA from intestinal tissue samples

Methods for isolating total RNA from mammalian tissue samples are well known in the art and typically include the following steps: grinding tumor and normal tissue samples in liquid nitrogen, cell lysis, RNA isolation and purification, RNA quality control by electrophoresis in 1% agarose gel in the presence of ethidium bromide dye or in a denaturing polyacrylamide gel, as well as spectrophotometric determination of the amount of RNA. Homogenization of tissue pieces can be carried out manually, grinding with a pestle in a ceramic mortar, or using mechanical homogenizers, for example, Omni Mixer or Micro-Dismembrator U from Sartorius (Germany). Various protocols can be used to isolate RNA that are well known to those skilled in the art. Classical RNA isolation methods employ strong chaotropic agents such as guanidinochloride and guanidinoisothiocyanate, protein solubilizers, and sequential extraction with phenol and chloroform to denature and remove proteins [Sambrook J., Fritsch E.F., Maniatis T., Molecular Cloning. A laboratory manual. 2nd 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 RNases, inhibitors can be 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 can be carried out using a number of commercially available kits (Klonogen, St. Petersburg; RNeasy mini and RNeasy micro kits (Qiagen); SV Total RNA Isolation System, Promega (USA), etc.).

To exclude work with aggressive agents, to speed up and simplify the isolation of RNA, various devices are used, for example QuickGene-810 (Life Science, Japan). For the extraction of RNA, this device uses an 80 μm porous membrane, which is 12.5 times thinner than the glass filter commonly used in such devices (1000 μm). This allows you to reduce the degradation of RNA and increase its output.

Reverse transcription reaction: cDNA synthesis on an RNA matrix isolated from intestinal tissue samples.

The reverse transcription process, as a result of which a single-stranded DNA chain is synthesized on an RNA matrix, allows us to switch from more unstable RNA molecules to more stable DNA molecules and amplify using the polymerase chain reaction to the amounts necessary for detection. RT-PCR amplification allows the use of very small amounts of the original RNA (at the level of 1 nanogram), and consequently, the amount of test tissue from which RNA is isolated.

The reverse transcription reaction can be carried out using a number of 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 and others, with the help of which it is possible to obtain amplification products up to several thousand and even several tens of thousands of nucleotide pairs (kb). Thermus thermophilus thermostable DNA polymerase (Tth), which has reverse transcriptase activity in the presence of Mn ²⁺ ions, can be used.

For reverse transcription, various primers can be used, for example:

1) oligo (dT) _n- containing primers that bind to the endogenous polyA tail at the 3'-end of the mRNA (the number n is usually 12-18, but can reach a larger value). These primers are most often used to obtain full-size cDNA. To the oligo (dT) sequence, nucleotides A, C or G are often added at the 3'-end to anchor the primer to the border of the transcript and poly-A tract;

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

3) hexamers or other short oligonucleotides (10-12 nucleotides) of random composition can also be used in combination with oligo (dT) -containing primers;

4) specific oligonucleotide primers are used to transcribe the mRNA region of interest for the study. These primers are successfully used for diagnostic purposes.

Analysis of the mRNA level of the ZG16 gene using PCR.

Using the first cDNA strand as a template commercially available from a wide range of manufacturers, thermostable DNA polymerase (e.g. Taq, Pfu, Tfl, Tth, Tma, etc.) and specific primers, sites for which are located in the transcript of interest, carry out a standard , semi-quantitative PCR in which the amplified transcript fragment is detected by simple gel electrophoresis, or real-time quantitative PCR. The selection of specific primers is carried out by a method well known to specialists in this field. To select primers and annealing temperatures, it is advisable to use commercially available programs or programs that are freely available on the Internet. Among these programs are Oligo (version 6.42), PrimerSelect from the Lasergene package (www.dnastar.com). Primer Premier 5, primers3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgiC), EasyExonPrimer (Wu X., Munroe DJ 2006. EasyExonPrimer: Automated Primer Design for Exon Sequences. Appl Bioinformatics 5, 119-120), ExPrimer (Sandhu KS, Acharya KK 2005. ExPrimer: to design primers from exon-exon junctions. Bioinformatics. 21, 2091-2092), PerlPrimer, FastPCR (http://www.biocenter.helsinki .fi / bi / Programs / fastpcr.htm), PrimerQuest (http://scitools.idtdna.com/Primerquest/), and also the PrimerDesigner program developed at the Institute of Molecular Biology of the Russian Academy of Sciences. Given the complexity of the analyzed genome, the length of the primers can be selected in the range from 18 to 25 bp

PCR primers can be selected so that they specifically hybridize with cDNA even in the presence of an impurity of genomic DNA in the preparation. This can be achieved, for example, by selecting primers for different exons of the ZG16 gene. When using gene-specific primers complementary to regions of different exons, the length of the PCR product amplified with impurity genomic DNA will be significantly longer than the expected PCR product amplified with cDNA. If at least one of the selected primers overlaps the boundary between exons, impurities of genomic DNA will not affect the results of the amplification reaction.

The specificity of the selected primers is checked by alignment with mRNA and human DNA (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi).

In a preferred embodiment, primers are used:

SEQ ID NO: 1, (ZG16_F 5'-TCGTAGGTCTTCAGGTGCGCTATG-3 ') and

SEQ ID NO: 2, (ZG16_R 5'-AAGATCTCCTCCAGGTCTCCGTTG-3 ').

One skilled in the art will understand that other primer pairs can be selected that differ, for example, in length, in their localization relative to the transcript sequence of the ZG16 gene, which will provide specific and efficient amplification of the transcript fragment of the ZG16 gene even in the presence of an impurity of genomic DNA .

Quantification of mRNA levels is achieved by parallel PCR with the test transcript and the control / standard transcript. As the endogenous internal control, relative to which the amplification products of the studied ZG16 gene were normalized, “housekeeping genes” encoding beta-2-microglobulin (B2M), a low molecular weight protein of cell surface surface antigens and globular beta-actin protein (ASTV), were selected. The expression of “housekeeping genes” in all cells is usually about the same. For these two selected genes, in the case of PTK, the smallest spread of transcription levels in normal and tumor tissues is shown in comparison with other control genes [Rubie C., Kempf K., Hans J., Su T., Tilton B., Georg T., Brittner B., Ludwig B., Schilling M. Housekeeping gene variability in normal and cancerous colorectal, pancreatic, esophageal, gastric and hepatic tissues. Mol. Cell Probes. 2005, 19: 101-119].

To analyze the level of gene transcription, standard PCR and real-time PCR (RT-PCR) can be used. Unlike the standard method, where only the final reaction products are recorded, real-time PCR uses fluorescently labeled oligonucleotide probes to detect DNA during its amplification; therefore, it allows the accumulation of amplified fragments in the exponential phase of the reaction, which increases the sensitivity of the method. The probe, complementary to the middle part of the amplified fragment, contains a fluorophore and a quencher at the ends. When the fluorophore and quencher are associated with an oligonucleotide probe, only slight fluorescence emission is observed. During the amplification process, due to the 5'-exonuclease activity of Taq polymerase, the fluorescent label passes into the solution, freeing itself from the quencher, and generates a fluorescent signal that amplifies in real time in proportion to the accumulation of the amplification.

The selection of probes for PCR-RV can be carried out in accordance with the standard recommendations of the manufacturer of devices for PCR-RV. If there is no need for multiplex analysis of several genes at the same time, a system using specific dyes specific for double-stranded DNA, such as SYBR Green or EvaGreen, whose fluorescence intensity increases in real time in proportion to the increase in the number of amplicons, can be an economical alternative. In this embodiment, primers without a probe can be used. EvaGreen is a third-generation saturating fluorescent dye that specifically binds to double-stranded DNA. This dye has low toxicity and inhibits the polymerase chain reaction to a much lesser extent than traditional dyes (such as SYBR Green), which allows the dye to be used in high concentrations and to obtain a higher fluorescent signal. The use of a saturating dye also allows obtaining more accurate information about the content of a double-stranded DNA fragment at a given temperature.

For PCR-RV, reagents from Applied Biosystems (USA), BioRad (USA) and others are widespread. However, the high cost of reagents limits the performance of large-scale studies. Comparison of universal branded kits from Applied Biosystems (USA) with domestic PCR kits for GenePakTM PCR Core (Isogen, Russia), kits (Syntol, Russia) and RialityTM (Biochip-IMB, Russia) showed that the latter is not inferior in all basic respects to branded sets. The RialityTM kit is selected for further research.

Varying the concentration of primers for each control and studied genes from 100 to 500 nM allows you to choose the optimal concentration at which the amplitude of the curve obtained during PCR-RV was maximum, and the reaction efficiency (E) was close to 100%.

For real-time PCR, various companies have developed amplifiers, for example: ABI Prism 7000 Sequence Detection System from Applied Biosystems (USA), Chromo4, MiniOpticon or iCycler iQ5 MJ Research (Bio-Rad), as well as domestic DT-322 devices (DNA- Technology), ANK-32 (Institute for Analytical Instrumentation RAS, http://www.syntol.ru/productank.htm), etc.

The use of PCR-RV also reduces the risk of contamination and automates the diagnostic process. The process lasts from 40 minutes to three hours (depending on the amplifier used) and includes simultaneous PCR, fluorescence signal detection, data processing and their presentation in graphical form (full kinetic curve) using special software. The results of the analysis become available immediately after the completion of the process and do not require additional purification and analysis of PCR products.

где P_n - количество молекул продукта/репортерной флуоресценции к циклу n, P₀ - исходное количество молекул, содержащих амплифицируемый фрагмент, Е - эффективность амплификации. В идеальных условиях Е=2, т.е. на каждом цикле цепной реакции происходит удвоение количества продукта. После логарифмирования обеих частей уравнения 1 преобразуют его к виду:

The exponential stage of PCR is described by the equation:

where P _n is the number of product / reporter fluorescence molecules to cycle n, P ₀ is the initial number of molecules containing the amplified fragment, E is the amplification efficiency. Under ideal conditions, E = 2, i.e. on each cycle of the chain reaction, the amount of product doubles. After the logarithm of both parts of equation 1, it is converted to:

A threshold cycle (C (T)) is a cycle n at which a certain level of reporter fluorescence is reached — threshold fluorescence P _{C (T)} = const. For n = C (T), equation 2 takes the form:

Those. the value of C (T) is directly proportional to the logarithm of the amount of substrate. Thus, PCR-RV allows you to compare the amount of substrate, provided that the reaction efficiency and a given level of threshold fluorescence are the same for each of the compared reactions.

The methods for processing PCR-RV data are based on the application of equation 3. For processing PCR-RV data, two main principles are used: the calibration graph method and direct data comparison.

1. Calibration graph method: this method involves the construction of a calibration graph in the coordinates C (T) -log P ₀ with a series of dilutions of the DNA standard, from which the substrate concentration (P ₀ ) is found in the experimental samples. The following standard options are proposed: purified RT-PCR product; recombinant DNA; recombinant RNA followed by reverse transcription; a synthetic oligonucleotide containing an amplifiable sequence.

2. The method of direct comparison of data. From equation 3 it follows that with equal efficiency of reaction E in samples 1 and 2, the relative concentration of the substrate

If we normalize these results according to amplification data with the REF control sequence and, assuming that the REF reaction in both samples proceeds with an E _ref efficiency, we obtain:

И, наконец, если обе реакции протекают со сходной эффективностью, близкой к 2, формула упрощается до вида:

Свободно распространяемая программа REST выполняет расчеты по формуле 4 с дополнительным статистическим анализом для определения средних значений С(Т).If the effectiveness of the "control" and "experimental" reactions are similar, approximately you can write:

And finally, if both reactions proceed with a similar efficiency close to 2, the formula is simplified to the form:

The freely distributed REST program performs calculations according to formula 4 with additional statistical analysis to determine the average values of C (T).

The present invention will now be illustrated in detail with reference to specific examples, which are the most preferred embodiments of the present invention. It should be understood that the invention is not limited to these described embodiments. On the contrary, it is intended that it includes any alternatives, modifications, or equivalents that are acceptable given the nature and scope of the invention.

Example 1. Tissue samples of the colon.

76 pairs of colon tissue samples (tumor / normal) of patients with RTK were analyzed. The average age of patients, including 52 (68%) men and 24 (32%) women, is 61.3 years (range 35-84 years). The proportion of patients older than 50 years is 81.3%. The diagnosis in each case was established on the basis of the results of clinical, morphological, endoscopic and radiological examinations. 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 (nodulus) - 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. Stage I of the disease was found in three patients, stage II in thirteen, stage III in four, and stage IV in six. The number of patients subjected to surgery before radiation therapy and chemotherapy, 4.

Example 2. The selection of RNA from tissue samples

Total RNA was isolated from frozen samples of tumor and normal tissues, crushed in liquid nitrogen. Tissue samples were homogenized on a Micro-Dismembrator U device (Sartorius, Germany). RNA was isolated using the “Rneasy Mini kit” or “Rneasy Micro kit” kit (Qiagen, USA) according to the protocols supplied by the manufacturer, which made it possible to efficiently get rid of not only proteins, insoluble precipitates of glycoproteins, DNA, but also low molecular weight RNA. The quality of the RNA preparation was checked by electrophoresis on a 1% agarose gel in the presence of ethidium bromide. The amount of RNA was determined on a ND 1000 spectrophotometer (NanoDrop Technologies). To determine the concentration using this device, 1 μl of solution is sufficient.

Example 3. The reaction of reverse transcription.

Synthesis of the first strand of cDNA

Single-stranded cDNA was synthesized on an RNA matrix isolated as described in Example 2. To obtain cDNA, 10 pg - 5 μg of total RNA, 200-500 ng of oligo (dT) _12-18 or 50-250 ng of random hexamers, or 2 pmol of a gene-specific primer were used. Superscript ™ III reverse transcriptase (Invitrogen) -modified M-MLV reverse transcriptase with reduced RNase N.

Reaction conditions:

The composition of the reaction mixture (13 μl):

RNA (1 μg / μl) 1.0 μl oligo primer (dT) ₁₈ (50 μM) 1.0 μl dNTP (10 mm) 1.0 μl sterile deionized water 10.0 μl

The sample was heated at 65 ° C for 5 min, placed in ice and kept for 1 min.

5 μl of the mixture was added:

buffer (Invitrogen) for building the 1st chain (5x) 4.0 μl SuperScript ™ III reverse transcriptase (Invitrogen) 1.0 μl

Incubated at 50 ° C, 50 min. The reaction was stopped by heating at 70 ° C for 15 min, and the sample volume was adjusted to 20 μl.

Example 4. The normalization of the concentration of cDNA for the control gene.

Samples of cDNA were normalized by the ASTV control gene. Used primers selected for different exons of the ASTV gene, and one of the primers overlaps the boundary between exons:

ACTB_F: 5'-CCTTCCTGGGCATGGAGTC-3 'and

ACTB_R: 5'-CTTGATCTTCATTGTGCTGGGT-3 '

The size of the PCR fragment is 191 bp The selection of PCR conditions was carried out on several cDNA samples. Amplification conditions: preheating at 94 ° C for 3 min; 94 ° C 30 s, 59 ° C 30 s and 72 ° C 30 s - 26, 28 and 30 cycles; 72 ° C 5 min - 1 cycle.

PCR was performed on a MasterCycler, Eppendorf (Germany) thermocycler with a heating cap or a Tertsik thermocycler, DNA technology (Russia). Amplification products were analyzed on a 1.8% agarose gel with 0.5 μg / ml ethidium bromide. As a result, optimal RT-PCR conditions for ASTV (26-28 cycles) were selected, under which a linear relationship was obtained between the amount of PCR product and the number of cycles. All amplification reactions were repeated three times.

The fluorescence intensity of the bands after electrophoretic separation of PCR products was quantified using the GeneProfiler photo densitometry program (http: //www/scanalytics.com) and expressed as relative intensity normal / tumor (N / O) values.

Example 5. Selection of conditions for determining the level of mRNA of the ZG16 gene in intestinal tissue samples by standard semi-quantitative RT-PCR method.

Protocol for determining the level of mRNA

When selecting the conditions for determining the amount of mRNA for amplification of single-stranded cDNA, gene-specific primers ZG16_F (SEQ ID NO: 1) and ZG16_R (SEQ ID NO: 2) were used, which were selected for different exons of the ZG16 gene, one of the primers overlapping the boundary between exons. Under these conditions, genomic DNA impurities do not affect the amplification results.

The size of the PCR fragment was 79 bp The selection of PCR conditions was carried out on several cDNA samples. Amplification was carried out in 25 μl of a mixture containing 67 mm Tris-HCl, pH 8.8, 16.6 mm (NH ₄ ) ₂ SO ₄ , 0.1% Tween-20, 2.5 mm MgCl ₂ 0.2 mm each from dNTP, 0.1 μg cDNA, 0.2 μM each of the primers, 2 units SmarTaq DNA polymerase activity (Dialat Ltd., Moscow).

PCR conditions

The composition of the reaction mixture (25 μl):

PCR buffer (10 ×) 2.5 μl MgCl ₂ (25 mm) 2.5 μl dNTP (10 mm) 0.5 μl primer ZG16_F, 10 μM 0.5 μl primer ZG16_R, 10 μM 0.5 μl cDNA (0.1 μg / μl) 1.0 μl SmarTaq DNA polymerase, 5 u / μl 0.3 μl sterile deionized water 17.2 μl

Amplification conditions:

94 ° C 3 min - 1 cycle

94 ° C for 30 s; 56 ° C for 30 s; 72 ° C 45 s - 28, 30, 32 and 34 cycles

72 ° C 5 min - 1 cycle

As a result, optimal RT-PCR conditions for ZG16 (30-32 cycles) were selected, under which a linear relationship was obtained between the amount of PCR product and the number of cycles (Figure 1). All amplification reactions were repeated three times.

The amplified ZG16 gene fragments were cloned into the pGEM®-T Easy vector (Promega) and sequenced. Their nucleotide sequences completely coincided with the sequences of the corresponding cDNA fragment. Sequencing was performed using the ABI PRISM® BigDye ™ Terminator v. 3.1 followed by analysis of the reaction products on an ABI PRISM 3100-Avant automated DNA sequencer.

Example 6. Determination of changes in the level of mRNA of the ZG16 gene in samples of tumor tissues compared with normal tissues by RT-PCR.

Protocol for determining the level of mRNA

1. Prepare a master-mix by mixing all the PCR components except the matrix. Master-mix should be prepared at the rate of 1 reaction with a volume of 25 μl for each sample + 1 additional reaction with a volume of 25 μl. The composition of the reaction mixture (25 μl):

PCR buffer (10 ×) 2.5 μl MgCl ₂ (25 mm) 2.5 μl dNTP (10 mm) 0.5 μl primer ZG16_F, 10 μM 0.5 μl primer ZG 16_R, 10 μM 0.5 μl cDNA 1.0 μl SmarTaq DNA polymerase, (Dialat Ltd., Moscow), 5 u / μl 0.3 μl sterile deionized water 17.2 μl

2. Add to the tubes 0.6 ml or 0.2 ml (labeled for 32 amplification cycles) add 24 μl master-mix.

3. Add 1 μl of the matrix to the tubes, mix by pipetting several times.

4. In a separate tube (control), mix 24 μl of the master-mix and 1 μl of sterile deionized water.

5. Add 1 drop of mineral oil (MP Biomedicals, LLC) to each tube and close the caps of the tubes (if PCR was performed on a Tertsik amplifier, DNA technology). In the event that PCR was performed on a MasterCycler, Eppendorf amplifier, no oil was added.

6. Place the tubes in the amplifier and carry out 30 cycles of the amplification reaction.

7. After completion of the amplification reaction, withdraw 4 μl of amplification products (30 cycles), continue the amplification reaction for another 2 cycles, withdraw 4 μl (32 cycles) and mix with 2 μl 6 × Orange Loading Dye. Samples were applied to a 1.8% agarose gel containing 0.5 mg / L ethidium bromide. A molecular weight marker of DNA was also applied to the gel, which made it possible to estimate the size of amplification products (DNA of plasmid pBR222 / AluI, manufactured by Sibenzyme, Russia, was used). Gel electrophoresis was performed in TBE buffer containing 10 mg / L ethidium bromide. The separation length is 3-5 cm of the gel. After gel electrophoresis, visualization of the amplification products and their documentation should be carried out in ultraviolet at a wavelength of 302 nm.

The results of the analysis of the mRNA level of the ZG16 gene.

The average values of changes in the mRNA levels of the studied genes and standard errors were calculated using the SPSS 13.0 computer program (SPSS Inc., USA). The reliability of the observed changes was evaluated based on the normal distribution of data. The data were considered reliable at P <0.05, where P is an indicator of the statistical significance (reliability) of the data.

It was found that in 80% (16/20) of RTK samples, the amount of ZG16 mRNA was reduced by 2 or more times (P <0.05) compared to the conventional norm (some of the results are presented in Figure 2).

Example 7. Determination of changes in the level of mRNA of the ZG16 gene in tumor tissue samples compared to normal by PCR-RV.

A quantitative analysis of changes in the ZG16 gene mRNA was performed by real-time PCR (PCR-RV) on an Applied Biosystems 7500 Fast Real-Time PCR System. The B2M and ASTV genes were selected as control genes.

The primer sequences are shown in table 1.

Table 1 Primers for PCR-RV Genes Primers F 5'-TCGTAGGTCTTCAGGTGCGCTATG-3 ' Zg16 R 5'-AAGATCTCCTCCAGGTCTCCGTTG-3 ' B2M F 5'-TATCCAGCGTACTCCAAAGA-3 ' R 5'-GACAAGTCTGAATGCTCCAC-3 ' ASTV F 5'-CCTTCCTGGGCATGGAGTC-3 ' R 5'-CTTGATCTTCATTGTGCTGGGT-3 '

Optimized primer concentration for ZG16, ASTV and B2M - 800 nM.

Reactions were carried out in a volume of 25 μl in standard 96-well optical dies (MicroAmp ™ Fast Optical 96-Well Reaction Plate). The reaction mixture included EvaGreen ™ intercalating dye (Biotium, Inc. http://biotium.com), primers, template (cDNA), passive dye ROX, Riality ™ thermostable reaction mixture [Manzenyuk O.Yu., Malakho S.G. ., Pekhov V.M., Kosorukova I.S., Poltaraus A.B. Characterization of universal domestic sets of reagents for real-time PCR. Molecular biology. 2006. 40 (2), C.349-356] containing Taq polymerase, dNTP, MgCl ₂ , PCR buffer. Each plate contained three negative controls (samples without DNA). Each sample was applied in triplicate. Reaction temperature profile: 95 ° С 10 min; 40 cycles: 95 ° C 15 s, 60 ° C 1 min.

PCR products were tested for specificity by melting curve method and electrophoretic separation in a 1.8% agarose gel. These fragments were sequenced, their nucleotide sequences coincided with fragments of annotated sequences.

Relative quantitative analysis was performed using AEGIS software [Dmitriev et al. Engelhardt Institute of Molecular Biology].

Quantification of mRNA AKR1B10 gene in colon cancer

It was found that in 84% (64/76) of the RTK samples, the amount of ZG16 mRNA was reduced by 2 or more times (P <0.05) compared to the norm, and in 68% (52/76) of the samples this difference significantly exceeded tenfold (Figure 3).

The level of mRNA reduction for ZG16 on average changes by 39.3 times (from 2 to 3175 times), while in 68% of cases (52/76) this decrease exceeds 10 times. A decrease in the mRNA content is already observed in the initial stages of the disease.

The results of the quantification are presented in table 2.

Thus, a very high frequency (84%) and a degree of decrease in the mRNA of the ZG16 gene at RTK (in 68% (52/76) of samples, this difference exceeds tenfold) were detected (Figure 3). Correlations between the degree of decrease in mRNA content and age, sex, tumor stage, and the presence of metastases were not detected. A decrease in the mRNA level in tumor samples occurs already at the first stage, which indicates the inactivation of the gene in the early stages of tumor development. The obtained data of a quantitative assessment of the mRNA level of the ZG16 gene during PTK indicate their inactivation in intestinal tumors, which indicates the functional importance of this gene in carcinogenesis.

The presented results for the ZG16 gene were obtained for the first time.

The present invention also provides that determining the level of mRNA of the ZG16 gene will be useful in monitoring the effectiveness of ongoing anti-cancer therapy, and analysis by the method of the present invention should be carried out before and after the course of treatment, and also, if necessary, during the course of treatment. An increase in the amount of mRNA of the ZG16 gene during or at the end of the course of treatment will indicate the restoration of the activity of the ZG16 gene, which may indicate positive changes in treatment.

The above detailed description of the invention and its specific embodiments given in the examples with reference to the drawings is intended solely to more fully clarify the essence of the claimed invention, but not to limit it. It will be clear to one skilled in the art that various changes may be made, which nevertheless will correspond to the nature and scope of the present invention, which are determined by the appended claims.

Claims (5)

1. A method for the diagnosis of colon cancer, comprising the following stages:
a) obtaining an initial pair of tissue samples from a patient, where one of the samples was obtained from tissue presumably affected by cancer, and the second was obtained from adjacent or located at a small distance from the tumor histologically normal (conditionally normal) tissue;
b) isolation and purification of RNA preparations from the initial pair of samples;
c) synthesis of single-stranded cDNA on an RNA template using oligonucleotide primers;
d) normalization of the cDNA concentration according to the control gene, the transcription level of which is constant in normal and with colon cancer;
e) conducting a quantitative or semi-quantitative amplification reaction of the ZG16 gene fragment using the cDNA obtained in step c) as a matrix and a pair of gene-specific oligonucleotide primers with the sequences SEQ ID NO: 1 and 2;
f) comparing the amount of amplified ZG16 DNA fragment for a sample obtained from a tissue presumably affected by cancer with the amount of amplified DNA fragment for a sample obtained from normal tissue, where the indicated amounts of amplified DNA fragment reflect the level of ZG16 mRNA, and a decrease in the amount of ZG16 mRNA serves a diagnostic sign of colon cancer. 2. The method according to claim 1, wherein in step c) the oligonucleotide primers used for the synthesis of single-stranded cDNA are selected from oligo (dT) -containing primers, random hexamers or a combination thereof, as well as gene-specific primers. 3. The method according to claim 1, wherein in step e), the quantitative or semi-quantitative amplification reaction of the ZG16 gene fragment is real-time PCR or standard RT-PCR. 4. The method according to claim 1, in which, in step d), the ASTV gene encoding the globular beta-actin protein or the B2M gene encoding the low molecular weight protein of cell surface surface antigens, beta-2-microglobulin is used as a control gene. 5. The kit for the diagnosis of colon cancer according to claim 1, including primers for the implementation of the polymerase chain reaction for determining the level of mRNA of the ZG16 gene having the sequences of SEQ ID NO: 1 and 2.

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