RU2390780C1 - Diagnostic technique for non-small cell carcinoma of lung and kit for implementation thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: substance of the invention involves a diagnostic technique for non-small cell carcinoma of lung of a gene NPRL2 based on real-time mRNA measurement by PCR method and a kit for implementation thereof. The reduced mPNA in a carcinoma tissue in comparison with that in a healthy tissue is a diagnostic character of non-small cell carcinoma of lung.

EFFECT: high-reliable diagnostic of non-small cell carcinoma of lung, first of all, squamous cell carcinoma, including earliest progression of neoplastic aberration.

7 cl, 7 ex, 3 tbl, 4 dwg

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 non-small cell lung cancer (NSCLC), especially squamous lung cancer (SCLC).

State of the art

Lung cancer (RL) remains one of the leading causes of cancer death in the world. Mortality from this disease exceeds mortality from breast, colon and prostate cancer combined [Jemal A., Siegel R., Ward E., Murray T., Xu J., Smigal C., Thun M.J. 2006. Cancer statistics. CA Cancer J Clin. 56, 6-30]. In Russia, lung cancer mortality rates in men are 68.2 cases per 100 thousand of the population, in women - 6.8 cases [D. Zaridze 2004. Epidemiology and etiology of malignant neoplasms, pp. 29-85 - in the book. Carcinogenesis / Ed. D.G. Zaridze. - M .: Medicine]. There are two main types of RL: small-cell (MRL) and non-small-cell (NSCLC), comprising 15-20 and 75-80%, respectively. NSCLC includes squamous cell carcinoma of the lung (SCLC), adenocarcinoma of the lung (AK) and large cell carcinoma. Among the different forms of radar cancer in terms of frequency of occurrence, PCRL ranks first, AK - second.

The diagnosis in half of the cases of NSCLC is made at a far advanced stage of the tumor process, which makes radical cure ineffective. Modern treatment methods increase the average five-year life expectancy of patients with NSCLC by no more than 15%. This indicator can be significantly improved through the development of early diagnostic methods.

The main diagnostic methods for RL are instrumental - x-ray and endoscopic. Sputum analysis for atypical cells, lung tissue biopsy, computed tomography, and fluorescence fibroscopy are also used. Immunological diagnostic methods have so far limited clinical use. The definition of tumor markers is of practical importance: CEA - cancer-embryonic antigen - an oncomarker of colorectal cancer, but can be used in the assessment of RL; NSE, a neuron-specific enolase, is in some cases used in assessing the condition of patients with RL; tissue polypeptide antigen (TPA) - a fragment of cytokeratin, a more specific marker of RL. These markers are used to control treatment, to identify relapses, but not to diagnose early stage RL. For the diagnosis of NSCLC, markers of SCCA or SCC-squamous cell carcinoma antigen (SCC) and cytokeratin fragment 19 (CYFRA 21.1 - cytokeratin fragment) [Pastor A., Menendez R., Cremades MJ, Pastor V., Llopis R. are widely used , Aznar J. 1997. Diagnostic value of SCC, CEA and CYFRA 21.1 in lung cancer: a Bayesian analysis. Eur Respir J. 10, 603-609; Buccheri G., Torchio P., Ferrigno D. 2003. Clinical Equivalence of Two Cytokeratin Markers in Non-small Cell Lung Cancer. A Study of Tissue Polypeptide Antigen and Cytokeratin 19 Fragments. Chest. 124, 622-632], which, however, are unsuitable for the early diagnosis of radar cancer.

Diagnostic signs of malignant transformation of cells can also be changes in the genome in tumor cells - point mutations in the coding and regulatory regions of genes, microsatellite instability, allelic losses, changes in the content of mRNA genes, methylation of promoter regions of genes, etc., as well as changes in the functional activity of many genes. Unlike protein, molecular genetic markers are significantly more sensitive. Among the promising potential marker genes involved in the carcinogenesis of various forms of RL are new genes, potential tumor suppressor genes (TSGs) on the short arm of human chromosome 3 [Zabarovsky E.R., Lerman M.I., Minna J.D. 2002. Tumor Suppressor Genes on Chromosome 3p Involved in the Pathogenesis of Lung and Other Cancers. Oncogene. 21, 6915-6935; Kiselev L.L., Senchenko V.N., Oparina N.Yu., Braga E.A., Zabarovsky E.R. 2005. Tumor suppressor genes located on the short arm of human chromosome 3. Molecular medicine. 3, 17-28]. Compared to others, the 3p21 cytogenetic region is enriched with protein coding genes [Bertone P., Stole V., Royce TE, Rozowsky JS, Urban AE, Zhu X., Rinn JL, Tongprasit W., Samanta M., Weissman S., Gerstein M., Snyder M. 2004. Global identification of human transcribed sequences with genome tiling arrays. Science. 24, 2242-46]. In order to identify new TSGs, a detailed mapping of hemi- and homozygous 3p deletions was performed for small cell and non-small cell RL and other human carcinomas using various methods [Braga E.A., Kiselev L.L., Zabarovsky E.R. 2004. From the identification of genomic polymorphism to diagnostic and prognostic markers of epithelial tumors. Molecular biology. 38, 145-154]. The obtained data confirmed the significant role of genes from the 3p21 region in carcinogenesis, as previously assumed [Zabarovsky E.R., Lerman M.I., Minna J.D. 2002. Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers. Oncogene. 21, 6915-35]. In the critical region of LUCA (3p21.3), which is characterized by a high deletion rate in the above tumors, a plot of homozygous deletions containing 17 genes is mapped [Zabarovsky E.R., Lerman M.I., Minna J.D. 2005. Chromosome 3 abnormalities in lung cancer. In: Lung Cancer: Principles and Practice. Third Edition. Lippincott Williams & Wilkins, Philadephia. 118-134]. These genes include the NPRL2 gene (PubMed ID: 11085536. other designations: tumor suppressor candidate 4 - TUSC4, NPR2, NPR2L, G21) with a length of 3.3 kb, containing 11 exons and encoding the main transcript with a length of 18 tp .n. with multiple spliced isoforms that are expressed in all normal tissues, including lung tissue [Lerman M.I. and Minna J.D. 2000. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium. The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. Cancer Res. 60: 6116-6133; Li J., Wang F., Haraldson K., Protopopov A., Duh F.M., Geil L., Kuzmin I., Minna J.D., Stanbridge E., Braga E., Kashuba V.I., Klein G., Lerman M.I., Zabarovsky E.R. 2004. Functional characterization of the candidate tumor suppressor gene NPRL2 located in 3p21.3C. Cancer Res. 64 (18): 6438-43]. The main product encoded by the NPRL2 gene is a soluble nuclear localization protein consisting of a protein binding domain similar to the core domain of the MutS protein and a nitrogen permease regulatory domain that transports nitrogen through cell membranes. The NPRL2 protein can be involved in the repair of unpaired bases in DNA, regulation of the cell cycle and activation of apoptosis pathways [Zabarovsky E.R., Lerman M.I., Minna J.D. 2005. Chromosome 3 abnormalities in lung cancer. In: Lung Cancer: Principles and Practice. Third Edition. Lippincott Williams & Wilkins, Philadephia. 118-134].

These functions are among the key to the normal functioning of the cell and its violation is undoubtedly an important link in carcinogenesis.

To detect suppressive activity of TSG protein products, i.e. the ability to partially or completely suppress the growth of tumor cells, there are several methods [Braga EA, Kiselev LL, Zabarovsky ER 2004. From the identification of genomic polymorphism to diagnostic and prognostic markers of epithelial tumors. Molecular biology. 38, 145-154; Ito I., Ji L., Tanaka F., Saito Y., Gopalan B., Branch CD, Xu K., Atkinson EN, Bekele BN, Stephens LC, Minna JD, Roth JA, Ramesh R. 2004. Liposomal vector mediated delivery of the 3p FUS 1 gene demonstrates potent antitumor activity against human lung cancer in vivo. Cancer Gene Therapy. 11, 733-739]. The relatively new GIT functional test (Gene Inactivation Test) is based on TSG inactivation in tumor cells in vivo and in vitro. TSG is inactivated using regulated expression eukaryotic vectors, with the tested genes mutating or deletion in experimental tumors in immunodeficient SCID mice or in tumor cell lines. The loss of the ability to suppress the growth of tumor cells by mutant transgenes may serve as evidence of the gene belonging to TSG. The “suppressor" activity of the NPRL2 gene is shown in vivo and in vitro in relation to small cell and non-small cell cancer cells by various methods, including using GIT [Li J., Wang F., Haraldson K., Protopopov A., Duh FM, Geil L., Kuzmin I., Minna JD, Stanbridge E., Braga E., Kashuba VI, Klein G., Lerman MI, Zabarovsky ER 2004. Functional characterization of the candidate tumor suppressor gene NPRL2 located in 3p21.3C. Cancer Res. 64 (18): 6438-43].

Data on the quantitative assessment of the mRLNA content of the NPRL2 gene during the development of NSCLC, including SCLC, are practically absent. Thus, until recently, a detailed comparison of the mRNA content of this gene in lung tumors and normal lung tissues was not carried out.

According to the literature, a decrease in mRNA content is shown for several genes in NSCLC. In [Terauchi K., Shimada J., Uekawa N., Yaoi T., Maruyama M., Fushiki S. 2006. Cancer-associated loss of TARSH gene expression in human primary lung cancer. J. Cancer Res. din. Oncol. 132, 28-34] revealed a decrease in transcription of the TARSH gene, one of the genes associated with cell aging, in tumor cell lines and NSCLC samples compared to normal. Based on the results, the authors suggested that this gene could be used as a biomarker for the diagnosis of NSCLC. However, in this work, only 8 samples of SCRL were analyzed, the most common form of radar, and further studies are needed.

The invention, a method for diagnosing various human carcinomas, including RL, as well as lymphomas and leukemia, is based on the detection of cytogenetic changes and allelic losses for 10 tumor suppressor genes located in the critical region of LUCA chromosome 3, including NPRL2 [ Ji L., Minna J., Roth J., Lerman M. Chromosome 3p21.3 genes are tumor suppressors. 2002. International Patents No. WO 0204511, No. US 2004016006].

The invention relates to a method for diagnosing cancer of the breast, colon, bladder, based on the detection of an increase in the mRNA of multiple spliced forms of the CD44 surface glycoprotein gene in tumor tissues, corresponding metastases and biopsy samples compared to normal tissues, involves PCR with radioactively labeled primers [Tarin D., Matsumura Y. Diagnostic method. 1994. International Patent No. WO 94/02633].

An invention proposing a method for diagnosing lymphomas is based on comparing the relative content of mRNAs of the λ and κ immunoglobulin genes involved in the development of lymphomas by real-time polymerase chain reaction (PCR-RV) [Stalberg A., Kubista M. Method to measure gene expression ratio of key genes. 2002. Patent No. 20004100112 of the Russian Federation, international patent No. WO 02/099135 A1].

[Cheng M., Chen Y., Yu X., Tian Z., and Wei H. 2008. Diagnostic utility of LunXmRNA in peripheral blood and pleural fluid in patients with primary non-small cell lung cancer. BMC Cancer. 8, 156] using real-time PCR (using absolute measurements on a standard curve), the level of mRNA genes LunX, SK19, CEA, VEGF-C and hnRNP A2 / B1 in peripheral blood and pleural fluid in patients diagnosed with NSCLC, esophageal cancer was estimated , breast and healthy volunteers. In the blood, LunX mRNA was found in 75.0% (33/44) of patients with NSCLC only. In addition, the mRNA of all other genes was found both in all studied patients and in healthy people. The LunX gene mRNA content decreased in patients after treatment. It was concluded that the LunX gene mRNA level is a specific marker for lung cancer, which can be used for the diagnosis of NSCLC. This analogue is closest in technical essence to the claimed invention and adopted as a prototype.

In the above and other inventions, the reduction of the NPRL2 gene mRNA was not used, including using the PCR-PB method, for the diagnosis of lung cancer.

Various methods are used to evaluate the mRNA content of genes. The modern highly sensitive PCR-RV method differs from traditional qualitative and semi-quantitative methods in that it allows you to quickly and accurately determine the number of copies of DNA or cDNA in a wide range of concentrations [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]. The method is widely used in the world, both for scientific and clinical purposes, due to its high productivity, usually 96 reactions are carried out in one experiment and 384 reactions in the latest models of devices. Unlike conventional PCR, when the amount of products is determined at the final stage of the reaction, PCR-PB allows you to monitor the accumulation of products in the exponential phase of the reaction. In this system, along with primers, a probe (probe) labeled with a fluorescent dye is used. A probe is an oligonucleotide that hybridizes to a DNA / cDNA sequence between two flanking primers. PCR-RV consists of two main stages: 1) hybridization of the test matrix with the probe; and 2) PCR catalyzed by TaqDHK polymerase, which has 5'-exonuclease activity, splitting the probe, which leads to the appearance and increase of a fluorescent signal. Usually the reaction lasts about 2 hours (40 cycles), in new generation devices - only 40 minutes. Analysis results become available immediately after completion of the reaction. Various models of PCR-RV devices developed by well-known manufacturers - Applied Biosystems, Bio-Rad, Invitrogene, Eppendorf and others, as well as domestic devices DT-322 (DNA-Technology), ANK-32 (Institute Analytical Instrumentation RAS, http://www.syntol.ru/productank.htm).

Despite the widespread use of PCR-RV, the main method for the quantitative assessment of the mRNA content of genes today, in Russia this method has not yet received sufficient distribution both in basic research and in the clinic. The main reason limiting its use is the relatively high cost of instruments and reagents. We are encouraged by the appearance on the domestic market of inexpensive devices developed by Russian companies, as well as inexpensive domestic sets of reagents (Syntol, DNA Technologies, Isogen, etc.), comparable in efficiency to foreign sets [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. Experience in molecular oncology diagnostics. 2006. Molecular Biology. 40, 349-356].

The methods and protocols described in this invention mainly involve the use of domestic kits and, although performed on an ABI Prism 7000 instrument (Applied Biosystems, USA), can be adapted for domestic instruments. The combination of low-cost devices and domestic reagent kits will make this technological method available for domestic scientific and clinical laboratories.

It is clear from the foregoing that a change in the mRNA content as a molecular marker for the diagnosis of epithelial tumors, and in particular RL, has not yet found wide practical application in clinics. In this area, there is an urgent need in the search for new diagnostic markers of RL, which allow reliable and early diagnosis of the disease, as well as in the development of a simple, sensitive, reliable, applicable in the conditions of clinical or outpatient medical institutions method for diagnosing RL, in particular NSCLC.

Disclosure of invention

This invention was made possible as a result of a comparative analysis of the content of the NPRL2 gene mRNA in various tumor tissues of the lung at different stages of 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 NPRL2 gene mRNA.

The present invention in its first aspect relates to a new marker for the diagnosis of non-small cell lung cancer (NSCLC), which is a change in the content of mRLNA of the NPRL2 gene. A decrease in the gene mRNA content in the tissues of human lungs presumably affected by cancer compared with its content in normal / healthy tissues serves as a diagnostic marker for various types of NSCLC, primarily squamous cell lung cancer (SCLC).

In one embodiment, lung cancer, the marker of which is a decrease in the NPRL2 gene mRNA, is non-small cell lung cancer.

In its next aspect, the present invention relates to the use of changes in the mRLNA of the NPRL2 gene as a marker for the diagnosis of non-small cell lung cancer.

In one embodiment, lung cancer, for which a change in the NPRL2 gene mRNA content serves as a diagnostic marker, is squamous cell lung cancer.

In yet another aspect, the present invention relates to a method for diagnosing non-small cell lung cancer, especially squamous lung cancer. 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) tissues;

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

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

d) normalization of the concentration of NPRL2 cDNA according to the control gene, the mRNA content of which is relatively constant in normal and tumors;

e) conducting a quantitative or semi-quantitative amplification reaction of the NPRL2 gene fragment using the cDNA obtained in step c) as a matrix and a pair of gene-specific oligonucleotide primers and a probe (in the case of PCR-PB);

f) comparing the amount of amplified NPRL2 DNA fragment in the sample obtained from suspected lung cancer tissue with the amount of amplified DNA fragment in the sample obtained from normal tissues, where the indicated amounts of the amplified DNA fragment reflect the NPRL2 gene mRNA content, and its decrease serves as a diagnostic marker lung cancer.

In one of the embodiments of the claimed method is intended for the diagnosis of squamous cell carcinoma of the lung.

In one embodiment of the method of the invention, in step c), the oligonucleotide primers used to synthesize single or double stranded cDNA are selected from oligo (dT) -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 and a probe 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 sequence of primers and probe is represented by SEQ ID NO: 1, 2, and 3.

In yet another embodiment of the method of the present invention, in step e), the quantitative or semi-quantitative amplification reaction of the NPRL2 gene fragment is real-time PNR or RT-PCR.

In one embodiment of the inventive method, in step d), the GAPDH gene encoding a glyceraldehyde-3-phosphate dehydrogenase protein is used as a control gene.

Another aspect of the present invention is a set of primers and probe for carrying out a polymerase chain reaction to determine the mRNA content of the NPRL2 gene having the sequences of SEQ ID NO: 1, 2 and 3.

List of drawings

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

Figure 1. Electrophoretic separation in a 2% agarose gel of the PCR-PB product of the NPRL2 gene transcript (size 135 bp). 1st and 2nd lanes of the transcript of the NPRL2 gene of the expected size are normal and tumors, 3rd lane - M50, DNA molecular weight marker from 50 to 500 bp (“Isogen Laboratory”).

Figure 2. Change in the threshold cycle C _t reflecting the initial mRNA content of the control GAPDH gene in normal (N) and tumor (T). The calculated values of R (relative mRNA content, taking into account the obtained C _t , see the formula) for the GAPDH gene did not exceed 1.5-2 times (P <0.05) both in the tumor and in the norm, which indicates a slight variability of the mRNA content and the acceptability of this gene for normalizing PCR-RV data.

Figure 3. Relative content of mRLNA of the NPRL23 (R) gene in various histological types of RL (see formula). A value of R equal to 1 corresponds to the absence of changes in the mRNA content of the studied gene in the tumor compared with the norm, normalized relative to the data for the control GAPDH gene. The dashed line shows the variability of the mRNA content for the GAPDH gene. R values of 0.1 and 0.01 correspond to a decrease in mRNA content by 10 and 100 times, respectively. As can be seen, a decrease in the RBSP3 gene mRNA content was found in most of the samples studied.

Figure 4. Relative content of mRLNA of the NPRL2 (R) gene in lung tissue samples of central and peripheral localizations from healthy donors: NCL (10-14), NPL (10-14) and the NPL pool, calculated relative to the average conditional norm Ncp from 14 patients diagnosed with NSCLC ( left side of the drawing), as well as relative to the NPL pool (right side of the drawing). As can be seen from the drawing, the mRNA content of the gene in normal tissues did not vary significantly, which shows the acceptability of using conditional norms as reference samples for quantitative changes by PCR-RV, as well as additional comparison samples from healthy donors and / or their pools.

The implementation of the invention

The present invention provides a method for the diagnosis of non-small cell lung cancer (NSCLC) by real-time PCR, especially squamous cell lung cancer (SCLC), based on measuring the mRNA content of the NPRL2 gene, as well as a kit for implementing this method. This is a simple and reliable way to diagnose SCRL, as well as other types of NSCLC at different stages of the development of malignant transformation, including the initial ones. A reliably detectable difference in the NPRL2 gene mRNA content in normal and tumor tissues can be used to detect lung cancer.

Lung tissue samples for analysis

As samples for analysis, biopsy samples, punctate, including material obtained by bronchoscopy with direct biopsy (central lung cancer) and transthoracic (percutaneous) tumor puncture (peripheral cancer), can be used.

Isolation of RNA from lung tissue samples

Methods of isolating total RNA from mammalian tissue samples are well known to specialists and, as a rule, include the following stages: grinding of 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 RNase enzymes, 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 kits (Qiagen); SV Total RNA Isolation System, Promega (USA), etc.). The use of various devices, for example, QuickGene-810 (Life Science, Japan), eliminates the work with aggressive agents, accelerate and simplify the isolation of RNA. For the extraction of RNA, this device uses an 80 μm porous membrane, which is 12.5 times thinner than the glass filter (1000 μm) commonly used in such devices, which makes it possible to reduce RNA degradation and increase its yield.

Reverse transcription reaction: cDNA synthesis on an RNA template isolated from lung tissue samples, conversion to double-stranded form

The reverse transcription reaction, as a result of which a single-stranded DNA 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. Further PCR amplification allows the use of very small amounts of the original RNA (at the level of several nanograms), and, consequently, the amount of lung tissue studied, from which RNA is isolated.

The reverse transcription reaction can be 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 and etc., 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 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) hybridize with RNA in numerous sites. When reverse transcription with these primers receive short cDNA. Random hexamers are used to overcome difficulties associated with the 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 oligot-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.

Gene transcription analysis can be performed using single-stranded or double-stranded cDNA. Specific primers are most often used for the synthesis of the second chain and its amplification. Commercially available kits for cDNA synthesis, based on the use of various reverse transcriptases and various primers for seed. To obtain cDNA, the SMART method (switching mechanism at the 5 'end of RNA templates of reverse transcriptase) was also developed, which is based on the property of reverse transcriptases to add several nucleotide residues, mainly dC, to the 3'-end of the synthesized first cDNA chain. This oligo (dC) sequence serves as an annealing site for the oligonucleotide adapter having a complementary oligo (dG) sequence at the 3'-end. Reverse transcriptase perceives the primer as an extension of the RNA template and continues the synthesis of the first strand [Schmidt WM, Mueller MW 1999. CapSelect: a highly sensitive method for 5 'CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs. Nucleic Acids Res. 27, e31]. Thus, the first cDNA strand is flanked on one side by a 3'-primer sequence with oligo (dT) at the 3'-end, and on the other hand, by a sequence complementary to the adapter. These primers have the same external sequence, differing only at the 3'-end. Then the first chain is amplified in PCR with a primer corresponding to the outer part of the 3'-primer and adapter. The nucleotide sequence of the common part of these primers is selected depending on further goals, for example, the production of clonotes, the use of subtractive hybridization, etc. The result is double-stranded DNA enriched with full-sized sequences. By using an adapter with a blocked 3'-end, a significant reduction in background amplification is achieved. When using the modified SMART method, the source material is amplified in a short time by more than 10 ⁵ times, which allows working with very small amounts of RNA (less than 1 nanogram), and therefore with a small number of tissues [Zhu YY, Machleder E .M., Chenchik A., Li R., Siebert PD 2001. Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques 30, 892-897]. Kits for producing cDNA by this method are produced by various companies, for example, Eurogen, Russia (MINT kit), Clontech, USA, etc.

Selection of specific primers and probes

The selection of specific primers and probes is carried out in a manner well known to specialists in this field, for which they use the available programs, 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 /) and others. High specificity of PCR-RV is ensured by using a probe containing a fluorophore or fluorescent dye at the 5'-end (in this case, FAM - 6-carboxy-fluoroscein), and at the 3'-end - t. n a quencher (in this case, 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. 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 a preferred embodiment, primers and a probe are used:

NPRL2_F (SEQ ID NO: 1) 5'-GGACCTCACTACACAACAAATCCTG-3 ';

NPRL2_R (SEQ ID NO: 2) 5'-GTCACAACGCCGTAGTACAGCA-3 ';

NPRL2_Z (SEQ ID NO: 3) 5 '- [FAM] -ACATCCAGAAGATTTCAGCAGAG-

GCAGAT- [Dabcyl] -3 '.

Analysis of the mRLNA of the NPRL2 Gene by PCR-PB

A quantitative assessment of the mRNA content is achieved using parallel PCR-RV with the test and control samples. As an internal control, relative to which the amplification products of the studied NPRL2 gene were normalized, the “household gene” - GAPDH encoding glyceraldehyde-3-phosphate dehydrogenase was selected. According to a preferred embodiment of the quantitative assessment of the mRNA content of genes, it is necessary to select a control gene having a slight variability of the mRNA content in tumor and normal lung tissues compared to the variability of the mRNA content of the studied genes. The selection of a suitable control gene for rationing quantitative data has been the subject of many reviews [Radonic A., Thuike S., Mackay I.M., Landt O., Siegert W., Nitsche A. 2004. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 313, 856-862; Huggett J., Dheda K., Bustin S., Zumla A. 2005. Real-time RT-PCR normalization; strategies and considerations. Genes and Immunity. 6. 279-284; Wong M.L., Medrano J.F. 2005. Real-time PCR for mRNA quantification. BioTechniques. 39, 1-11]. Most often, the “housekeeping” genes are chosen as controls, 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 actually depends on the degree of accuracy selected / required. In the present invention, the traditional and frequently used control GAPDH gene is selected. According to the literature, this gene is most suitable as a control for NSCLC samples and normal lung tissue [D.W. Liu, S. T. Chen, H. P. Liu. Choice of endogenous control for gene expression in nonsmall lung cancer. 2005. Eur. Respir.J. 26, 1002-1008]. However, the variability of the mRNA content must be checked for each studied sample of samples of this type of tissue.

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.

Selection of reference samples

According to a preferred form of quantifying the mRNA content of the genes, it is important to verify the suitability of the reference samples, in this case conditional norms. A “conditional norm” is considered to be a sample of lung tissue with missing macro- and microscopic signs of tumor growth. If the conditionally normal tissue sample was “unsuitable” (that is, in cases when tumor cells were detected under microscopy or the material was difficult to interpret), additional comparison samples obtained from “healthy” donors (donors that did not suffer lung cancer during their lifetime) were used ) or averaged norms of several conditional norms.

Since not all tumor samples had suitable paired conditional norms, the relative NPRL2 mRNA content (R _cDNA ) was calculated using different comparison samples - paired conditional norms, if any, and averaged conditional norms from several patients, if there were no paired norms . As additional comparison samples, we used 5 samples from healthy donors of central and peripheral localization, for which ΔCt = Ct (NPRL2) -Ct (GAPDH) was averaged and R _cDNA was calculated further.

Reaction Effectiveness

According to the preferred form of quantifying the mRNA content of genes, it is necessary to take into account the effectiveness of the reactions that can have a significant effect on the final result. For this, a program for the mathematical processing of experimental PCR-RV data has been developed, which allows calculating the effectiveness of reactions. The program is compatible with experimental data files (Ct, ΔRn, etc.) from the Applied Biosystems software (ABI Prism SDS Software) and also allows a statistical assessment of the reliability of the measured changes and assessment of the suitability of the selected control gene.

The present invention will now be illustrated in detail with reference to specific examples representing the most preferred embodiments of the invention. The invention is not limited to the described embodiments, but rather is intended to include any alternatives, modifications, or equivalents that are acceptable given the nature and scope of the invention.

Example 1. Samples of lung tissue

Tissue samples of various histological types of RL (T), morphologically normal tissues adjacent to tumors (the so-called conditional norms (N), as well as normal lung tissue of central (NCL) and peripheral localization (NPL) from suddenly died donors were collected and characterized independently by two different groups of employees of the NII Blokhin RAMS Scientific Research Institute of Pediatric Surgery, Russian Academy of Medical Sciences.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 at bronchoscopy, transthoracic punctures) and the surgical material. Samples of lung tissue (tumor, conditionally normal tissue) were obtained immediately after removal of the tumor. Each sample was divided into approximately 3 equal parts and stored in liquid nitrogen. The total collection of tumor samples to be studied was 52 (31 + 21) samples of various histological types of lung cancer, of which 38 were squamous cell carcinomas, 11 were lung adenocarcinomas (3 were bronchioalveolar lung adenocarcinomas, 1 was small cell lung cancer, 1 was large cell lung cancer, 1 - carcinoid), as well as 50 samples of the conditional norm. In addition, we used lung tissue obtained postmortally from 10 people who did not have a history of cancer (normal): five samples taken in the central regions of the lungs and five in peripheral regions. “Morphologically normal” is considered to be a tissue in which microscopic examination did not detect signs of cell atypia, the tissue could be represented by cells of the ciliated epithelium, cubic epithelium, alveolar macrophages, lymphoid elements. The average age of patients, including 45 men, 6 women, for 1 patient - there is no data on gender and age, is about 60 years (range 31-76 years). 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) - T ₀ -T ₄ are categories reflecting an increase in the size and / or local distribution of the primary tumor, N (nodules) - N ₀ -N ₃ - categories reflecting a varying degree of metastasis of the regional lymph nodes; M (metastasis) - M ₀ -M ₁ - characterizes distant metastases. All possible combinations of TNM are combined into larger groups - stages that reflect the course of the tumor process.

Example 2. The selection of RNA from tissue samples

The following methods have been used by various research groups.

Method 1.

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 purified by the standard method using guanidinisothiocyanate and phenol, followed by precipitation with ethanol [Maniatis T., Fritsch E., Sambrook D. 1984. Molecular cloning. Moscow, "Mir" 186-196]. To remove impurities of glycoproteins that are rich in lung tissue, additional RNA precipitation was used with saline [Chomczynski P., Mackey K. 1995. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. Biotechniques 19, 942-945]. After salt precipitation, all RNA preparations were purified using the RNeasy Mini kit (Qiagen, Germany) according to the protocol provided by the manufacturer. Such a three-stage RNA purification procedure allowed us to effectively get rid of not only soluble glycoprotein precipitates, 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 spectrophotometrically on a Nanodrop spectrophotometer (Nanodrop, USA).

Method 2.

RNA from normal and tumor tissues was isolated using the Trizol RNA Prep reagent kit (Isogen Laboratory, Moscow). The main stages included: 1) 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; 2) adding a mixture of chloroform and isoamyl alcohol in the ratio of their volumes 49: 1 and centrifugation for 5 minutes at 14000 rpm (4 ° C); 3) repeated deproteinization of the aqueous phase containing RNA, phenol and centrifugation for 5 minutes at 14000 rpm (4 ° C); 4) precipitation of RNA with an equal volume of isopropanol from the aqueous phase formed after centrifugation. Then, the RNA pellet after repeated centrifugation was washed with aqueous 75% ethanol, dried and dissolved in the stabilizing agent ExtraGene E (Isogen Laboratory). RNA concentration was 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).

Method 3.

RNA from normal and tumor human tissues was isolated using the RNeasy Mini Kit reagent kit (Qiagen, Germany) according to the protocol. The main steps included: 1) destruction of tissue frozen in liquid nitrogen using a micro-desmembrator (Sartorius Germany) and homogenization of the destroyed sample in RLT lysis buffer per 600 μl per 30 μg of tissue. 2) centrifugation for 3 minutes at 14000 rpm (4 ° C); 2) adding an equal volume of aqueous 70% ethyl alcohol to the supernatant; 3) application to the column; 4) washing the column after sorption of RNA once with RW1 buffer with a volume of 700 μl and twice with RPE buffer with a volume of 500 μl; 5) RNA elution with RNase-free water. RNA concentration was determined spectrophotometrically (Nanodrop, USA) at a wavelength of 260 nm. The quality of the isolated RNA was checked by electrophoresis in 1% agarose in the presence of ethidium bromide and spectrophotometrically (Nanodrop, USA).

The nucleotide composition of the amplicons was confirmed by sequencing on a 3730 DNA Analyzer automated sequencer (Applied Biosystems, USA). Sequencing was performed separately from the 5 ′ and 3 ′ end primers using the DYEnamic ET Terminator Cycler Sequencing Kit reagents (Amersham, UK). All primers and probes were specific for PCR-PB; amplicons had the expected nucleotide sequences and size.

Example 3. The reaction of reverse transcription

The synthesis of the first cDNA strand was performed by two different research groups.

Method 1.

Single-stranded cDNA was synthesized on an RNA matrix isolated as described in Example 2. To obtain cDNA, a modified SMART method was used [Zhu Y. Y., Machleder E.M., Chenchik A., Li R., Siebert P.D. 2001. Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques 30, 892-897], 2 ng -1 μg of total RNA, primers:

SMART (5'-AAGCAGTGGTATCAACGCAGAGTACGCrGrGrG-3 ') and

CDS (5'-AGCAGTGGTATCAACGCAGAGTAC (T) ₃₀ N _-1 N-3 '),

and PowerScript reverse transcriptase (Clontech, USA).

Reaction conditions:

The composition of the reaction mixture (10 μl):

RNA (1 μg / μl) 1.0 μl SMART primer (6 μM) 2.0 μl CDS primer (10 μM) 1.0 μl sterile deionized water 1.0 μl

The sample was heated at 72 ° C for 3 min and placed on ice. 5 μl of the mixture was added:

buffer (Clontech) to build 1 chain (5x) 2.0 μl dNTP (10 mm) 1.0 μl DTT (20 mM) 1.0 μl PowerScript reverse transcriptase (Clontech) 1.0 μl

Incubated at 42 ° C, 60 min. The reaction was stopped by heating at 65 ° C for 5 min, 2 μl of 60 mM EDTA was added, and the sample volume was adjusted to 20 μl.

Method 2.

For the reverse transcription reaction, 1 μg of RNA obtained by one of the two methods described above using Trizol RNA Prep reagent kits (Isogen Laboratory) or RNeasy Mini Kit reagents (Qiagen, Germany) pretreated with RNase-free DNase I was taken (Invitrogene), 100 ng hexanucleotide primers, 1 mM dNTP, lx reaction buffer (Fermentas, Lithuania) containing 250 mM Tris-HCl (pH 8.3, 25 ° С), 250 mM KCl, 20 mM MgCl2, 50 mM DTT , and 200 units of reverse transcriptase M-MuLV (Fermentas, Lithuania). The reaction was carried out in a volume of 20 μl at the following temperature conditions: 25 ° С - 10 min, 42 ° С - 60 min, 50 ° С - 10 min, 70 ° С - 10 min.

Example 4. The synthesis of the second strand of cDNA

For the synthesis of the second cDNA strand and amplification, 1/10 of the volume of the reaction mixture obtained in Example 3 was taken. Synthesis was carried out using Advantage2 DNA Polymerase with 5'-AAGCAGTGGTATCAACGCAGAGT-3 'primer according to the Advantage 2 PCR kit protocol (Clontech) , Germany).

Conditions for PCR:

The composition of the reaction mixture (50 μl):

NDP Buffer Advantage 2 (Clontech) (10x) 5.0 μl dNTP (2.5 mm) 5.0 μl 10 μM primer 1.0 μl cDNA (single stranded) 1.0 μl Advantage 2 DNA Polymerase (Clontech) 1.0 μl sterile deionized water 37.0 μl

Amplification conditions:

95 ° C, 1.5 min 1 cycle

95 ° C, 20 s; 65 ° C, 20 s; 72 ° C, 3 min 14-17-20-23 cycles

For each sample, the number of cycles was selected, allowing to obtain the same amount of amplified material.

Example 5. Selection of conditions for determining the content of mRNA of the NPRL2 gene in lung tissue samples

The same specific primers NPRL2_F (SEQ ID NO: 1) and NPRL2_R (SEQ ID NO: 2), which were matched to different exons of the NPRL2 gene, were used for RT-PCR and PCR-PB, with one of the primers overlapping the border between exons.

For PCR-PB, in addition to the primers, an NPRL2_R probe (SEQ ID NO: 3) was used. Amplicon size - 135 bp

The sequence of the selected primers and probes for the control gene:

Direct GAPDH-F 5'-CGGAGTCAACGGATTTGGTC-3 '

Reverse GAPDH-R 5'-TGGGTGGAATCATATTGGAACAT-3 '....

GAPDH-Z Probe - 5'-CCCTTCATTGACCTCAACTACATGGTTTACAT-3 '

Amplicon size - 141 bp

For PCR-RV, optimal concentrations of primers and probes of the studied and control genes were selected. The primer concentrations were varied in the range of 250-400 nM with a constant concentration of probes equal to 100 nM, then the concentration of the probes was varied with the selected optimal concentration of primers in the range of 150-250 nM. For NPRL2, the optimal concentrations of primers were 350 nM and the probe 150 nM, for GAPDH the concentration of primers was 300 nM, and the probe was 150 nM.

Example 6. Quantification of the content of mRNA of the NPRL2 gene

For quantitative measurements, an ABI PRISM ® 7000 Sequence Detection System, Applied Biosystems, USA was used.

Protocol for determining the mRNA content by PCR-RV

1. The reaction mixtures were prepared for the NPRL2 and GAPDH genes, carefully mixing all the components of the reaction, except the template (cDNA), primers and probe in optimal concentrations, based on 1 reaction with a volume of 25 μl for each sample + 1 additional reaction of 25 μl.

The composition of the reaction mixture for the GAPDH gene:

Reagent The concentration of the stock solutions (mm) Volume per reaction (μl) The final concentration in the reaction (mm) PCR buffer 10X 2.5 1X dNTPs 2.5 2.5 0.250 MgCl ₂ 2.5 four four Direct primer 1.0 · 10 ^-5 0.75 3 · 10 ^-4 Reverse primer 1.0 · 10 ^-5 0.75 3 · 10 ^-4 Probe 5 · 10 ^-6 0.5 1.5 · 10 ^-4 cDNA 5 one Taq DNA polymerase 5 u / μl 0.5 0.1 u / μl H ₂ O 8.5

The composition of the reaction mixture for the NPRL2 gene:

Reagent The concentration of the stock solutions (mm) Volume per reaction (μl) The final concentration in the reaction (mm) PCR buffer 10X 2.5 1X dNTPs 2.5 2.5 250 μM MgCl ₂ 25 four 4 mM Direct primer 1.0 · 10 ^-5 0.875 5.0 · 10 ^-4 ; Reverse primer 1.0 · 10 ^-5 0.875 5.0 · 10 ^-4 Probe 5 · 10 ^-6 0.5 3.0 · 10 ^-4 cDNA 5 Taq DNA polymerase 5 u / μl 0.5 0.1 u / μl H ₂ O 8.25

2. In a 96-well plate, 20 μl of the prepared matrixless reaction mixtures were added.

3. 5 μl of the matrix were added, and the plate was tightly covered with a film.

4. The tablet was placed in the instrument compartment, the names of the cells were set, the temperature regime for 40 cycles: 95 ° С - 10 min - denaturation and enzyme activation, 95 ° С - 15 sec - denaturation, 60 ° С - 1 min - probe annealing and polymerization .

5. The reactions were carried out in the mode of relative quantitative measurements (RQ software (Relative Quantification, Applied Biosystems, USA).

6. After completion of the amplification reaction, data was saved on a CD.

7. The analysis of the reaction products was carried out using gel electrophoresis in TBE buffer containing 10 mg / l of ethidium bromide. For this, 1–4 μl of amplification products were taken (40 cycles), 1.8% agarose gel containing 0.5 mg / L ethidium bromide samples and a molecular weight marker for DNA were used to evaluate the size of the amplification products (M50, Isogen).

8. The nucleotide composition of the amplicons was confirmed by sequencing on a 3730 DNA Analyzer automated sequencer (Applied Biosystems, USA). Sequencing was performed separately from the 5 ′ and 3 ′ end primers using the DYEnamic ET Terminator Cycler Sequencing Kit reagents (Amersham, UK).

9. Conducted mathematical processing of PCR-RV data and presented the results in graphical and / or tabular form.

Example 7. Mathematical processing of PCR-RV data

PCR-RV data was transferred as a text file to Microsoft Excel and mathematical data processing was performed. The relative content of mRNA - R _cDNA (hereinafter R), representing the ratio of the number of copies of the studied cDNA sequence to the number of copies of the control cDNA sequence in the tumor (T) compared with the norm (N), is associated with the main parameters of PCR-RV threshold cycle Ct and reaction efficiency E by the general expression (Bulletin 2, Applied Biosystems):

The range of extreme values of R was calculated taking into account the calculated deviations e for the Ct values:

где

i=1, 2, …, n, где n - число повторов реакции (в нашем случае n=3). При суммировании величин отклонения вычисляют по формуле:

Where

i = 1, 2, ..., n, where n is the number of repetitions of the reaction (in our case, n = 3). When summing the values of the deviation is calculated by the formula:

j=1, 2, …, k, где k - число суммируемых величин.

j = 1, 2, ..., k, where k is the number of summed quantities.

The effectiveness of PCR-RV (E) for the GAPDH and NPRL2 genes was calculated using an original program developed in the laboratory (Gene Transcription Analysis Program, registration certificate No. 20088612585, 2008, Rospatent, RF). In this program, the reaction efficiency is calculated by the slope of the straight line, which approximates the kinetic curve in its linear section using the least squares method. In further data processing, the obtained efficiencies were taken into account, which amounted to NPRL2 gene

E _tumor = E _norm = (78 ± 8)%, for the GAPDH gene E _tumor = E _norm = (91 ± 6)%. The reliability of the observed changes was evaluated based on the normal distribution of data.

Data were considered reliable at P <0.05, where P is an indicator of the statistical significance of the data.

NPRL2 Gene mRNA Assessment Results

The NPRL2 gene mRNA content was estimated on cDNA samples of two groups obtained by researchers using various methods at different institutes (group 1 - samples with clinical numbers from 1, 2 ... 497, IBCh RAS, group 2 - samples with numbers 1006, ... 1086, IMB RAS ), while both foreign and domestic reagent kits were used. The characteristics of all NSCLC tissue samples from which RNA and cDNA were obtained are shown in Table 1.

Table 1 Characterization of samples of various histological types of lung cancer Histological type of cancer Localization Clinical stage Lymph node metastases Clinical number of samples Group 1 PCRL central cancer Stage I No metastases 9, 80/05, 98/05 PCRL peripheral Stage I No metastases 2, 4, 475 PCRL central Stage II No metastases 6, 21, 26, 497, 300/05, 5, 7, 13, 15, 77/05 PCRL peripheral Stage II No metastases 451, 452, 476 PCRL central Stage II With metastases 90/05 PCRL central Stage III With metastases 228/05, 14, 466, 290/05 PCRL peripheral Stage III With metastases 1, 301/05 AK, BAAK peripheral Stage II No metastases
With metastases 304/05, 406, 477; 311/05 MRL peripheral Stage III With metastases 229/05 Group 2 PCRL central Stage I No metastases 1007, 1032 PCRL there is no data Stage I No metastases 1022, 1073 PCRL central Stage II With metastases 1023 PCRL peripheral Stage II With metastases 1085 PCRL there is no data Stage II With metastases 1026, 1031 PCRL central Stage III With metastases 1025, 1021; 1082 PCRL peripheral Stage III With metastases 1086 AK, BAAK peripheral Stage I No metastases 1006, 1010, 1044, 1053 AK peripheral Stage III No metastases With metastases 1043
1078 AK central Stage III With metastases 1059 KRL peripheral Stage I No metastases 1030 TO central Stage I No metastases 1011 Notes: SCRL - squamous cell carcinoma, AK - pulmonary adenocarcinoma, BAAK - bronchioalveolar adenocarcinoma of the lung, MRL - small cell lung cancer, SCLC large cell lung cancer, K - carcinoid

Comparison of the NPRL2 gene mRNA content in tumor samples relative to different comparison samples - norms (conditional norm, average conditional norm and average norm from healthy donors who did not have cancer) showed close R values with overlapping intervals calculated taking into account deviations, regardless of the use of different methods RNA isolation and cDNA production. In addition, close values of the relative mRNA (R) content were obtained on cDNA samples of the same tumors obtained in two different ways.

The frequency of decreases in NPRL2 mRNA gene in two groups of samples.

In each group of samples, a decrease in the NPRL2 gene mRNA content in the tumor was revealed compared with the norm in most samples — about 80% (Table 2).

table 2 Change in the content of mRLNA (R) of the NPRL2 gene in samples of different histological types of lung cancer compared with the norm No.
p / p Clinical Sample Number Histological type TNM classification stage Clinical stage R Drop interval (times) one 2 PCRL T ₂ N ₀ M ₀ IB 4-5,3 2 four PCRL T ₁ N ₀ M ₀ IA 13-22 3 9 PCRL T ₁ N ₀ M ₀ IA 7-11 four 80/05 PCRL T ₁ N ₀ M ₀ IA absent 5 98/05 PCRL T ₁ N ₀ M ₀ IA 5-6 6 475 PCRL T ₁ N ₀ M ₀ IA 8-9 7 1006 AK T ₁ N ₀ M ₀ IA 2-3 8 1007 PCRL T ₂ N ₀ M ₀ IB 3-4 9 1010 AK T ₁ N ₀ M ₀ IA 1.4-2.4 10 1011 TO N ₀ M ₀ IB 2.3-3.6 ↑ eleven 1022 PCRL T ₁ N ₀ M ₀ IA 7-11 12 1030 KRL T ₂ N ₀ M ₀ IB preservation 13 1032 PCRL T ₂ N ₀ M ₀ IB 2-3 fourteen 1044 BAAK T ₁ N ₀ M ₀ IA 2.1-3.9 ↑ fifteen 1053 AK T ₁ N ₀ M ₀ IA 2-2.5 16 1073 PCRL T ₂ N ₀ M ₀ IB 4-6 17 5 PCRL T ₃ N ₀ M ₀ IIB absent eighteen 6 PCRL T ₃ N ₀ M ₀ IIB 5-7 19 7 PCRL T ₃ N ₀ M ₀ IIB absent twenty 13 PCRL T ₃ N ₀ M ₀ IIB 4-5 21 fifteen PCRL T ₃ N ₀ M ₀ IIB preservation 22 21 PCRL T ₃ N ₀ M ₀ IIB preservation 23 26 PCRL T ₃ N ₀ M ₀ IIB absent 24 77/05 PCRL T ₃ N ₀ M ₀ IIB preservation 25 90/05 PCRL T ₂ N ₁ M ₀ IIB preservation 26 300/05 PCRL T ₃ N ₀ M ₀ IIB 3-4 27 304/05 AK T ₃ N ₀ M ₀ IIB 1.6-2 28 311/05 BAAK T ₂ N ₁ M ₀ IIB preservation 29th 406 AK T ₃ N ₀ M ₀ IIB 7-9 thirty 451 PCRL T ₃ N ₀ M ₀ IIB 14-20 31 452 PCRL T ₃ N ₀ M ₀ IIB 2-3 32 476 PCRL T ₃ N ₀ M ₀ IIB 1.6-2.5 33 477 BAAK T ₃ N ₀ M ₀ IIB preservation 34 497 PCRL T ₃ N ₀ M ₀ IIB 13-17 35 1023 PCRL T ₂ N ₁ M ₀ IIB 3-4 36 1026 PCRL T ₂ N ₁ M ₀ IIB preservation 37 1031 PCRL T ₂ N ₁ M ₀ IIB 4-6 38 1085 PCRL T ₂ N ₁ M ₀ IIB 2-3 39 one PCRL T ₃ N ₁ M ₀ IIIA 14-20 40 fourteen PCRL T ₃ N ₂ M ₀ IIIA preservation 41 228/05 PCRL T ₃ N ₁ M ₀ IIIA absent 42 229/05 MRL T ₃ N ₁ M ₀ IIIA 6-11 43 290/05 PCRL T ₃ N ₁ M ₀ IIIA 10-16 44 301/05 PCRL T ₃ N ₁ M ₀ IIIA 3-6 45 466 PCRL T ₂ N ₂ M ₀ IIIA 2-3 46 1021 PCRL T ₃ N ₁ M ₀ IIIA 7.5-13 47 1025 PCRL T ₃ N ₂ M ₀ IIIA 5-7 48 1043 AK T ₄ N ₀ M ₀ IIIB 2.5-4 49 1059 AK T ₃ N ₂ M ₀ IIIA 2.1-2.3 fifty 1078 AK T ₂ N ₂ M ₀ IIIA 2-4 51 1082 PCRL T ₃ N ₂ M ₀ IIIA 17-25 52 1086 PCRL T ₁ N ₂ M ₁ IIIA 2-3 Notes: 1) group 1 consisted of samples with numbers 1, 2 ... up to 497, group 2 - samples with numbers 1006, 1007, ....... up to 1086. 2) For the second group, a history of smoking is known for 16 patients, of which only 5 of them did not smoke in the past and present, and all the rest (69%) are malicious smokers with a smoking history of 15 to 50 years

The results of PCR-RV for the entire sample of the studied samples are presented in figure 3. As can be seen from figure 3 and figure 4, changes in the content of mRNA (R) gene in tumor samples are much more significant compared with samples of different norms. The level of decrease and the frequency of reduction of mRNA of the NPRL2 gene by stages and types of RL As can be seen from table 2 and figure 3, and the content of mRNA of the NPRL2 gene is reduced in PCRL samples mainly from 2 to 10 times. However, in some samples, more significant decreases were observed with an increase in the prevalence of tumor disease; in 5 samples, the mRNA content, unlike the norm, was not detected in the tumor. An analysis of the data showed that the decrease in the mRLNA of the NPRL2 gene occurs in 79% (41/52, P <0.05) of NSCLC samples, in 84% (32/38, P <0.05) of SCRL samples and 64% (9/14, P < 0.05) samples of various types of lung cancer other than SCLC (table 3). It should be noted that at stage I of SCRL, the NPRL2 gene mRNA decreases in 100% of samples (10/10, P <0.05), at stages II and III - in 72% (13/18, P <0.05) and 90% ( 9/10, P <0.05) samples, respectively. Correlation between different characteristics and a decrease in the mRLNA content of the NPRL2 gene. Table 3 shows the frequency of decrease in the mRLNA content of the NPRL2 gene for samples (NSCLC, SCRL, and other species other than SCLC) in the presence and absence of metastases to regional lymph nodes (category N).

Table 3 Summary data on the frequency of decreases in the mRLNA of the NPRL2 gene in samples of SCRL and other types of lung cancer Stages The frequency of decreases in mRNA content,% in samples of different types of NSCLC in PCRL samples in radar samples, except for PCRL I 81 (13/16)
P <0.05 100 (10/10)
P <0.05 50 (3/6) II 68 (15/22)
P <0.05 72 (13/18)
P <0.05 50 (2/4) III 93 (13/14)
P <0.05 90 (9/10)
P <0.05 100 (4/4) I + II + III
without metastases 79 (26/33)
P <0.05 87 (20/23)
P <0.05 60 (6/10)
P <0.05 II + III
with metastases 79 (15/19)
P <0.05 80 (12/15)
P <0.05 75 (3/4) Total: The total number of decreases in 79% (41/52) of samples P <0.05 The total number of decreases in 84% (32/38) P <0.05 The total number of declines in 64% (9/14) P <0.05 Note: The first digit is%, the ratio of the number of samples with a reduced mRNA content to the total number of samples is indicated in brackets

In all three cases, in the presence and absence of metastases, the reduction frequencies are almost the same. The level and frequency of the decrease in mRNA content was practically independent of the location of the primary tumor (central or peripheral), since the calculations of the relatively average norm (the so-called NCL and NPL pools) of 5 samples from lung tissue from healthy donors of one or another localization were also given by close R.

There was no correlation between the level and frequency of the decrease in the mRNA content of the gene and such characteristics as the age and gender of the patient, the location and size of the primary tumor, the clinical stage, the duration and intensity of smoking.

Thus, the main result is that already in the early stages of NSCLC, a decrease in the NPRL2 gene mRNA content occurs in most samples (about 80%), primarily PCRL, for which the reduction rate reaches 100%.

The present invention also provides that the determination of the mRLNA content of the NPRL2 gene will be useful in monitoring the effectiveness of ongoing anti-cancer therapy, and the analysis by the method of the present invention should be carried out before and after the course of treatment, and if necessary during the course of treatment. An increase in the NPRL2 gene mRNA during or at the end of the course of treatment may indicate the restoration of the activity of the tumor suppressor gene NPRL2, which may indicate positive changes in treatment. A further decrease in the NPRL2 gene mRNA content may indicate a lack of treatment effectiveness.

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 a person skilled in the art that various changes can be made, which, however, will correspond to the nature and scope of the present invention, which are determined by the attached claims.

Claims (7)

1. A method for the diagnosis of non-small cell lung cancer by real-time PCR, including 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 histologically normal (conditionally normal) tissue;
b) synthesis of single-stranded or double-stranded cDNA on an RNA template using oligonucleotide primers;
c) normalizing the concentration of NPRL2 cDNA according to the control gene, the mRNA content of which is relatively constant in normal conditions and in lung cancer;
d) quantitative amplification or fragment of the NPRL2 gene using the cDNA obtained in stage b) as a matrix, a pair of gene-specific oligonucleotide primers with sequences of SEQ ID NO: 1, 2 and a probe with the sequence of SEQ ID NO: 3;
e) comparing the amount of amplified NPRL2 DNA fragment for the sample obtained from the tissue presumably affected by cancer with the amount of amplified DNA fragment for the sample obtained from normal tissue, where the indicated amounts of the amplified DNA fragment reflect the NPRL2 gene mRNA content, and a decrease in the NPRL2 gene mRNA content serves a diagnostic sign of lung cancer. 2. The method of claim 1, wherein squamous cell lung cancer is diagnosed. 3. The method according to claim 1 or 2, wherein in step b) the oligonucleotide primers used to synthesize single or double stranded cDNA are selected from oligo (dT) -containing primers, random hexamers or a combination thereof, as well as primers specific for the NPRL2 gene. 4. The method according to claim 1 or 2, wherein in step d) the quantitative amplification reaction of the NPRL2 gene fragment is real-time PCR coupled to a reverse transcription reaction (RT-PCR). 5. The method according to claim 1 or 2, in which, in step c), the GAPDH gene encoding glyceraldehyde-3-phosphate dehydrogenase protein is used as a control gene. 6. The kit for the diagnosis of non-small cell lung cancer according to claim 1 and 2, respectively, which allows you to determine the mRNA content of the NPRL2 gene by real-time PCR and includes primers with the sequence SEQ ID NO: 1 and 2, as well as an oligonucleotide probe with the sequence SEQ ID NO: 3. 7. The method according to claim 1 or 2, characterized in that the first strands or amplifications of the first strands of cDNA are used.

Images