RU2330285C1 - Method of non-small cell lung carcinoma diagnostics and related analysis set - Google Patents

Abstract

FIELD: medicine; oncology; molecular biology.

SUBSTANCE: to diagnose non-small cell lung carcinoma potentially cancer-affected human tissue and adjacent histologically normal tissue are tested for gene WIF1 transcription level. Lowered transcription level detected in potentially cancer-affected human tissue compared to that of adjacent histologically normal tissue is diagnostic indicator of non-small cell lung carcinoma. For realisation of polymerase chain reaction within detection of gene WIF1 transcription level, primers set of sequence SEQ ID NO: 1 and 2 is used.

EFFECT: invention application provides high-reliable diagnostics of non-small cell lung carcinoma, including squamous cell carcinoma, as well as at earliest stage 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 non-small cell lung cancer, including squamous cell cancer.

State of the art

Lung cancer remains one of the leading causes of cancer death in the world. Mortality from lung cancer exceeds mortality from cancer of the breast, colon and prostate combined [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]. In Russia, mortality rates from lung cancer in men are 68.2 cases per 100 thousand of the population, in women - 6.8 cases [Zaridze D.G. 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 lung cancer (RL): small cell (MRL) and non-small cell (NSCLC), comprising 15-20 and 75-80%, respectively. NSCLC includes squamous cell lung cancer (PRL), lung adenocarcinoma (AKL), and large cell cancer. Among the various forms of lung cancer, in terms of the frequency of occurrence of PRL, it ranks first, and AKL is 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 unrealistic. 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 only through the development of early diagnostic methods.

The main methods for diagnosing lung cancer 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 - a cancer marker of colon cancer, but can be used in the evaluation of lung cancer; NSE - neuron-specific enolase - in some cases used in assessing the status of patients with lung cancer; tissue polypeptide antigen (TPA) is a fragment of cytokeratin, a more specific marker of lung cancer. These markers are used to control treatment, to identify relapses, but not to diagnose early stage RL. For the diagnosis of NSCLC, SCC markers are widely used - squamous cell carcinoma antigen and a fragment of cytokeratin-19 (CYFRA 21.1) [Pastor A., Menendez R., Cremades MJ, Pastor V., Llopis R., 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], but they are also 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 level of transcription or translation of genes, methylation of promoter regions of the gene, and others. Unlike protein molecular genetic markers have significantly greater sensitivity. Lung cancer is accompanied by a change in the functional activity of many genes. Among the promising potential marker genes involved in the carcinogenesis of various forms of NSCLC are the WIF1 gene [Mazieres J., He B., You L., Xu Z., Lee AY, Mikami I., Reguart N., Resell R, McCormick F ., Jablons DM 2004. Wnt inhibitory factor-1 is silenced by promoter hypermethylation in human lung cancer. Cancer Res. 64: 4717-4720; Wissmann C., Wild PJ, Kaiser S., Roepcke S., Stoehr R., Woenckhaus M., Kristiansen G., Hsieh JC, Hofstaedter F., Hartmann A., Knuechel R., Rosenthal A, Pilarsky C. 2003. WIF1, a component of the Wnt pathway, is down-regulated in prostate, breast, lung, and bladder cancer. J. Pathol. 201: 204-212.].

Wnt factor inhibitor 1 - WIF1 is an extracellular inhibitor of the Wnt signaling pathway, which is able to bind Wnt molecules, thereby preventing their interaction with receptor complexes and further activation of the signaling pathway [Hsieh JC, Kodjabachian L., Rebbert ML, Rattner A., Smallwood PM, Samos CH, Nusse R, Dawid IB, Nathans J. 1999. A new secreted protein that binds to Wnt proteins and inhibits their activities. Nature. 398: 431-436].

Abnormal activation of the Wnt signaling pathway is often observed in various types of tumors and contributes to the formation and progression of the tumor phenotype, in particular, in the case of lung tumors [Mazieres J., He B., You L., Xu Z., Jablons D.M. 2005. Wnt signaling in lung cancer. Cancer Lett. 222: 1-10; He B., You L., Uematsu K., Xu Z., Lee A.Y., Matsangou M., McCormick F., Jablons D.M. 2004. A monoclonal antibody against Wnt-1 induces apoptosis in human cancer cells. Neoplasia. 6: 7-14; You L., He B., Xu Z., Uematsu K., Mazieres J., Mikami I., Reguart N., Moody T.W., Kitajewski J., McCormick F., Jablons D.M. 2004. Inhibition of Wnt-2-mediated signaling induces programmed cell death in non-small-cell lung cancer cells. Oncogene. 23: 6170-6174].

The expression of WIF1 is often inhibited in tumors of various origins. A decrease in the level of the WIF1 transcript is often observed in tumors of the breast, nasopharynx, digestive tract, bladder, lungs [Urakami S., Shiina H., Enokida H., Kawakami T., Kawamoto K., Hirata H., Tanaka Y., Kikuno N., Nakagawa M., Igawa M., Dahiya R. 2006a. Combination analysis of hypermethylated Wnt-antagonist family genes as a novel epigenetic biomarker panel for bladder cancer detection. Clin Cancer Res. 12: 2109-2116; Urakami S., Shiina H., Enokida H., Kawakami T., Tokizane T., Ogishima T., Tanaka Y., Li LC, Ribeiro-Filho LA, Terashima M., Kikuno N., Adachi H., Yoneda T ., Kishi H., Shigeno K., Konety BR, Igawa M., Dahiya R, 2006b. Epigenetic inactivation of Wnt inhibitory factor-1 plays an important role in bladder cancer through aberrant canonical Wnt / beta-catenin signaling pathway. Clin Cancer Res. 12: 383-391; Taniguchi H., Yamamoto H., Hirata T., Miyamoto N., Oki M., Nosho K., Adachi Y., Endo T., Imai K., Shinomura Y. 2005. Frequent epigenetic inactivation of Wnt inhibitory factor-1 in human gastrointestinal cancers. Oncogene. 24: 7946-7952; Lin Y.C., You L., Xu Z., He B., Mikami I., Thung E., Chou J., Kuchenbecker K., Kim J., Raz D., Yang C.T., Chen J.K., Jablons D.M. 2006. Wnt signaling activation and WIF-1 silencing in nasopharyngeal cancer cell lines. Biochem Biophys Res Commun. 341: 635-640; Mazieres J., He B., You L., Xu Z., Lee A.Y., Mikami I., Reguart N., Resell R., McCormick F., Jablons D.M. 2004. Wnt inhibitory factor-1 is silenced by promoter hypermethylation in human lung cancer. Cancer Res. 64: 4717-4720; Wissmann et al., 2003, supra].

As an inhibitor of the Wnt signaling pathway, the abnormal activation of which can make a significant contribution to carcinogenesis, the WIF1 gene can be considered as a potential tumor suppressor gene [Urakami et al., 2006b, supra; Taniguchi et al., 2005, supra; He et al., 2004, supra]. The loss of activity of such genes results from point mutations, allelic deletions, homozygous deletions, or hypermethylation of CpG islets of genes, especially in the promoter regions [Belinsky SA, Klinge DM, Dekker JD, Smith MW, Bocklage TJ, Gilliland FD, Crowell RE, Karp DD , Stidley CA, Picchi MA 2005. Gene promoter methylation in plasma and sputum increases with lung cancer risk. Clin. Cancer Res. 11, 6505-6511], For the WIF1 gene, it was revealed that the cause of the change in its expression in tumors is hypermethylation of the CpG islands of the gene promoter [Ai L., Tao Q., Zhong S., Fields CR, Kim WJ, Lee MW, Cui Y., Brown KD, Robertson KD 2006. Inactivation of Wnt inhibitory factor-1 (WIF1) expression by epigenetic silencing is a common event in breast cancer. Carcinogenesis. 27: 1341-1348; Batra S, Shi Y, Kuchenbecker K.M., He B, Reguart N, Mikami I., You L., Xu Z., Lin Y.C., Clement G., Jablons D.M. 2006. Wnt inhibitory factor-1, a Wnt antagonist, is silenced by promoter hypermethylation in malignant pleural mesothelioma. Biochem Biophys Res Commun. 342: 1228-1232; Urakami et al., 2006a, supra; Urakami et al., 2006b, supra; Taniguchi et al., 2005, supra; Mazieres et al., 2004, supra]. For the WIF1 gene, using mainly the immunohistochemical staining method, there was a lack of correlation between its expression status and the clinical and pathological characteristics of NSCLC [Wissmann et al., 2003, supra].

Loss of transcriptional activity in NSCLC is indicated for several genes. In work (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. Clin. Oncol 132. 28-34) revealed a decrease in the 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 PRL, the most common form of radar, were analyzed. This analogue is closest in technical essence to the claimed invention and adopted as a prototype.

From the foregoing, it is clear that in this area there is an urgent need to search for a new diagnostic marker for NSCLC, which can reliably and from the very early stages diagnose the disease, and to develop a simple, sensitive, reliable method applicable to it in clinical or outpatient medical institutions for the diagnosis of non-small cell lung cancer.

Disclosure of invention

This invention was made possible as a result of a comparative analysis of the level of transcription of the WIF1 gene in tumor tissues of the lungs of various types and at different stages of their malignant transformation and the unexpected discovery of the fact that the early stages of the development of malignant transformation are accompanied by a significant decrease in the level of transcription of the WIF1 gene.

The present invention in its first aspect relates to a new marker for the diagnosis of non-small cell lung cancer, which is the level of transcription of the WIF1 gene. The reduced level in the tissue of human tissue suspected of being cancer compared with its level in healthy tissue is a diagnostic sign of non-small cell lung cancer.

In one embodiment, lung cancer, the marker of which is the level of transcription of the WIF1 gene, is squamous cell lung cancer.

In a further aspect, the present invention relates to the use of a transcription level of the WIF1 gene as a marker for the diagnosis of non-small cell lung cancer.

In one embodiment, lung cancer, for which the level of transcription of the WIF1 gene serves as a diagnostic marker, is squamous cell lung cancer.

In another aspect, the present invention relates to a method for the diagnosis of non-small cell lung cancer.

This method includes the following steps:

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) 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 WIF1 cDNA according to the control gene, the transcription level of which is constant in normal and in lung cancer;

e) conducting a quantitative or semi-quantitative amplification reaction of a WIF1 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 WIF1 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 WIF1 gene, and a decrease in the transcription of the WIF1 gene is diagnostic a sign of lung cancer.

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

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 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 WIF1 gene fragment is real-time PCR or standard 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 for the implementation of the polymerase chain reaction to determine the level of transcription of the WIF1 gene having the sequence of SEQ ID NO: 1 and 2.

Enumeration of figures.

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

Figure 1 shows the results of the selection of conditions for determining the level of transcription of the WIF1 gene in lung tissue samples. Electrophoretic separation on an agarose gel of PCR products (size 224 bp) obtained after 36 and 40 cycles was carried out on a 1.8% agarose gel. M - DNA molecular weight marker (50 bp Ladder, Fermentas, Lithuania).

Figure 2 shows the results of RT-PCR analysis of the level of transcription of the WIF1 gene in PRL with central localization of the tumor and AKL (after 36 and 40 cycles). Sample numbers and the number of amplification cycles are indicated above the tracks. T - tumor, N - conditional norm (tissue adjacent to the tumor). Samples were pre-aligned with GAPDH transcription. Electrophoretic separation of PCR products was carried out on a 1.8% agarose gel. Representative analysis results are shown.

The implementation of the invention

This invention provides a new genetic marker for the diagnosis of non-small cell lung cancer and based on the determination of the level of transcription of this marker, a simple, reliable method for the diagnosis of NSCLC at various stages of development of malignant transformation, including the initial ones. A reliably detectable difference in transcription of the WIF1 gene in normal and tumor tissues can be used to detect lung cancer.

Lung tissue 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 as samples for analysis.

Isolation of RNA from lung 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. In classical RNA isolation methods, strong chaotropic agents are used, such as guanidinochloride and guanidinoisothiocyanate, which dissolve proteins, 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 performed using a number of commercially available kits (Klonogen, St. Petersburg; RNeasy kits (Qiagen); SV Total RNA Isolation System, Promega (USA), etc.).

To exclude work with aggressive agents, to accelerate 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 template. isolated from lung tissue samples, and its conversion into double-stranded form.

The reverse transcription process, as a result of which a single-stranded DNA chain is synthesized on the RNA matrix, if necessary, with the completion of the second chain, allows us to switch from 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 therefore the amount of the lung tissue under study, 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). Thermostable Thermus thermophilus (Tth) DNA polymerase can be used, with reverse transcriptase activity in the presence of Mn ^{2+ ions} .

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. Nucleotides A, C or G are often added to the oligo (dT) sequences at the 3'end to "anchor" the primer to the boundary of the transcript and the 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.

Gene transcription analysis can be performed using single-stranded or double-stranded and amplified 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. Through the use of 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 by more than 10 ⁵ times in a short time, so it is possible to work with very small amounts of RNA (less than 1 nanogram) and, therefore, with a small amount of the studied tissue [Zhu YY, Machleder EM, 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, Evrogen, Russia (MINT kit), Clontech, USA, etc.

Analysis of the level of transcription of the WIF1 gene using PCR.

Using the first or second 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 for which sites are located in the transcript of interest, conduct 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. Such programs include 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/) . Given the complexity of the analyzed genome, the length of the primers can be selected in the range from 18 to 25 bp

In order to simplify the procedure for the implementation of the method for the purposes of clinical analysis and to ensure maximum preservation of the RNA contained in the sample, it is necessary to minimize manipulations with the tissue sample, which could potentially lead to the destruction of RNA. In this regard, in one of the preferred variants of obtaining the RNA preparation in this invention do not use RNAse-free DNase. Moreover, primers for PCR are 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 WIF1 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.

In a preferred embodiment, primers are used:

WIF1_F (SEQ ID NO: 1) 5'-CCAGGCAAGAGTACTCATAG-3 'and

WIF1_R (SEQ ID NO: 2) 5'-TTGGATCTGCCATGATGCCT-3 '

One skilled in the art will understand that other pairs of primers can be selected that differ, for example, in length, in their localization relative to the transcript sequence of the WIF1 gene, which will provide specific and efficient amplification of the transcript fragment of the WIF1 gene even in the presence of an impurity of genomic DNA .

A quantitative assessment of the level of transcription is achieved by parallel PCR with the test transcript and the control / standard transcript or by preliminary equalizing the number of matrices in PCR by the number of control / standard transcript. As an endogenous internal control, relative to which the amplification products of the studied WIF1 gene were normalized, the “household gene” - GAPDH encoding glyceraldehyde-3-phosphate dehydrogenase was selected. The expression of “housekeeping genes” is the same in all cells. For the WIF1 gene in the case of NSCLC, the smallest spread of transcription levels in normal and tumor tissues is shown (DW Liu, ST Chen, HP Liu. Choice of endogenous control for gene expression in nonsmall lung cancer. 2005. Eur. Respir.J, 26, 1002- 1008).

To analyze the level of gene transcription, not only standard PCR but also real-time PCR (PCR-RV) 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 simultaneously, an economical alternative can be a system using SYBR Green dye specific for double-stranded DNA, the fluorescence intensity of which increases in real time in proportion to the increase in the number of amplicons. In this embodiment, primers without a probe can be used.

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 two to three hours (50 or 60 PCR cycles, respectively) and includes simultaneous PCR, detection of a fluorescent signal, 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. As the main method for measuring the level of gene transcription, the comparative method (relative measurement method, RQ method) is usually chosen, based on the relative measurement of the number of studied polynucleotide sequences, which allows a double comparison of the results for control and target genes, as well as for normal and tumor samples cDNA

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. Samples of lung tissue

We analyzed 29 pairs of pulmonary tissue samples (tumor is a conditional norm) of patients with NSCLC: 25 pairs of PRL samples (19 samples with central tumor localization and six with peripheral) and four pairs of AKL samples. The histologically normal lung tissue taken from the tissue adjacent to the tumor closer to the edge of the resection was taken as the conditional norm. The average age of patients, including 28 men and one woman, is 61 years old (range 34-76 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) - 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. The first stage of the disease was found in six patients, the second stage in 18 patients, the third stage in five. Signs of distant metastases were not observed in any of the patients. None of the patients were exposed to radiation therapy and chemotherapy before surgery.

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 purified by the standard method using guanidinisothiocyanate and phenol, followed by precipitation with ethanol [Sambrook et al., 1989, supra]. 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, USA) 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 [Sambrook et al., 1989, supra].

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, 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) for building the 1st 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.

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 performed using Advantage2 DNA Polymerase with 5'-AAGCAGTGGTATCAACGCAGAGT-3 'primer according to the Advantage 2 PCR kit protocol (Clontech, Heidelberg , Germany).

Conditions for PCR:

The composition of the reaction mixture (50 μl):

Advantage 2 PCR buffer (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 level of transcription of the WIF1 gene in lung tissue samples.

Protocol for determining the level of transcription

When selecting conditions for determining transcription levels, double-stranded cDNA was amplified using gene-specific primers WIF1_F (SEQ ID NO: 1) and WIF1_R (SEQ ID NO: 2), which were selected for different exons of the WIF1 gene, one of the primers overlapping the border between exons. Under these conditions, genomic DNA impurities do not affect the amplification results.

The size of the PCR fragment was 224 bp The selection of PCR conditions was carried out on several cDNA samples. Amplification was carried out in 25 μl of the mixture. The reaction mixture (50 μl) containing: 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2 mm MgCl ₂ , 0.2 mm each of dNTP, 0.4 μm of each of the primers, 0.8 μg of cDNA , 2 units Taq activity of DNA polymerase (Fermentas, Lithuania) was divided into 2 parts of 25 μl for 36 and 40 PCR cycles. Conditions for PCR:

The composition of the reaction mixture (50 μl):

PCR buffer (10x) 5.0 μl MgCl ₂ (25 mm) 4.0 μl dNTP (10 mm) 1.0 μl WIF1_F primer, 10 μM 2.0 μl WIF1_R primer, 10 μM 2.0 μl cDNA (0.1 μg / μl) 8.0 μl Taq DNA polymerase, 5 u / μl 0.4 μl sterile deionized water 27.6 μl

Amplification conditions:

94 ° C, 2 min 1 cycle

94 ° C, 40 s; 63 ° C, 60 s; 72 ° C, 60 s 36 and 40 cycles

72 ° C, 3 min 1 cycle

The cDNA samples were normalized by the control GAPDH gene encoding the glyceraldehyde-3-phosphate dehydrogenase protein. To do this, we selected the number of matrices providing equal amounts of the amplification product on the GAPDH matrix in PCR with the same number of amplification rounds before saturation of the amplification product. Amplification was performed with GAPDH-for primers (5'-TTAGCACCCCTGGCCAAGG-3 ') and GAPDH-rev (5'-CTTACTCCTTGGAGGCCATG-3') in a 50 μl reaction containing 16.6 mM ammonium sulfate, 67 mM Tris-HCl, pH 8.8, 0.1% Tween 20, 2.5 mM MgCl ₂ , 0.125 mM of each deoxynucleotide triphosphate, 0.4 mM of each of the primers and 1 unit Taq DNA polymerase activity (State Research Institute of Genetics, Russia). PCR was performed under the following conditions; 94 ° C, 30 s; 58 ° C, 30 s and 72 ° C, 40 s. After 24, 27 and 30 cycles, aliquots of the reaction were selected.

PCR was performed on an amplifier OmniGene, Hybaid (UK). Amplification products were analyzed on a 1.8% agarose gel with 0.5 μg / ml ethidium bromide (see Figure 1). As a result, optimal RT-PCR conditions were selected (36–40 cycles) under which an increase in the number of PCR products with an increase in the number of cycles was obtained. All amplification reactions were repeated three times.

The fluorescence intensity of the bands after electrophoretic separation of PCR products was evaluated visually.

The amplified WIF1 gene fragments were cloned into the pCR®-Blunt vector (Invitogen, USA) 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 reagent kit. 3.1 followed by analysis of the reaction products on an ABI PRISM 3100-Avant automated DNA sequencer.

Example 6. Determination of the level of transcription of the WIF1 gene in samples of normal and tumor tissues.

Protocol for determining the level of transcription

1. Prepare a master-mix by mixing all the PCR components except the matrix. Master-mix should be prepared at the rate of 2 reactions 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 (10x) 5.0 μl MgCl ₂ (25 mm) 4.0 μl dNTP (10 mm) 1.0 μl WIF1_F primer, 10 μM 2.0 μl WIF1_R primer, 10 μM 2.0 μl cDNA 8.0 μl Taq DNA polymerase, ((Fermentas, Lithuania) 5 u / μl 0.4 μl sterile deionized water 27.6 μl

2. In 0.6 ml tubes (labeled for 36 amplification cycles) add 42 μl master-mix.

3. Add 8 μl of the matrix to the tubes, mix by pipetting several times.

4. Transfer 25 μl of the reaction mixture to 0.6 ml tubes (labeled for 36 amplification cycles).

5. In a separate tube (control), mix 21 μl of the master-mix and 4 μl of sterile deionized water.

6. Add 1 drop of mineral oil (Sigma, USA) to each tube and close the tube caps.

7. Place the tubes in the thermocycler and conduct 36 or 40 cycles of the amplification reaction.

8. After completion of the amplification reaction, withdraw 10 μl of the amplification products and mix with 2 μl of 6x Orange Loading Dye paint (Fermentas, Lithuania). 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 (50 bp Ladder, manufactured by Fermentas, Lithuania). Gel electrophoresis was performed in TBE buffer containing 0.5 mg / L ethidium bromide. The separation length is 3-5 cm of the gel. After gel electrophoresis, the amplification products were visualized and documented in ultraviolet at a wavelength of 302 nm.

The results of the analysis of the level of transcription of the WIF1 gene.

The level of transcription of WIF1 was considered reduced in the tumor if, after 40 cycles of the amplification reaction in the tumor, the amount of product did not exceed the amount of the product after 36 cycles of the amplification reaction in the conventional norm. The significance of differences in transcription of WIF1 in tumors and conditional norms was assessed by Fisher's exact test. The data were considered reliable at P <0.05, where P is an indicator of the statistical significance (reliability) of the data.

We have shown that in 90% of tumors, the level of transcription is reduced in tumors compared to the conventional norm or is absent, while transcription is not detected only in 38% of adjacent tissues (P <0.0001). In eighty-three percent (83%) of the tumors, the level of transcription is absent, while transcription is absent only in 38% of the adjacent tissues (P = 0.0011) (Figure 2 and Table). In fourteen out of 29 samples, gene transcription in tumors is practically absent. In two samples, gene transcription is significantly reduced. In one sample, gene transcription is present only in the tumor. In ten samples, gene transcription was not found either in tumors or in adjacent tissues. The absence of gene transcription in tissue samples adjacent to dysplasia, but morphologically normal, shows the possibility of the spread of tumor cells into normal cells. In two samples, the level of gene transcription in tumors is approximately equal to the level in the conditional norm. No correlation was found between changes in the level of transcription of the WIF1 gene, histological differences, and stages of tumor progression (Table).

The present invention also provides that the determination of the level of transcription of the WIF1 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 level of transcription of the WIF1 gene during or at the end of the course of treatment will indicate the restoration of the activity of the tumor suppressor gene WIF1, which may indicate positive changes in treatment. A decrease in the level of transcription of the WIF1 gene will indicate a further suppression of gene activity, which may indicate a lack of treatment effectiveness.

TNM - clinical and morphological classification of tumors:

T0-T4 - categories reflecting the local distribution of the primary tumor; N0-N3 - categories reflecting a different degree of metastatic lesion of regional lymph nodes; M0-M1 - characterize the presence of distant metastases. A - lack of metastases, B - lesion of single lymph nodes, n.d. - there is no data.

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

Claims (8)

1. A method for the diagnosis of non-small cell lung cancer, comprising the following stages: a) obtaining the initial pair of tissue samples from the patient, where one of the samples is taken from tissue presumably affected by cancer, and the second is taken 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 or double-stranded cDNA on an RNA template using oligonucleotide primers; d) normalization of the concentration of WIF1 cDNA according to the control gene, the transcription level of which is constant in normal and in lung cancer; e) conducting a quantitative or semi-quantitative amplification reaction of a WIF1 cDNA 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 WIF1 cDNA fragment for a sample obtained from a tissue presumably affected by cancer with the amount of amplified cDNA fragment for a sample obtained from normal tissue, where the indicated amounts of the amplified cDNA fragment reflect the level of transcription of the WIF1 gene, and a decrease in the transcription of the WIF1 gene is diagnostic a sign of non-small cell lung cancer. 2. The method according to claim 1, in which squamous cell lung cancer is diagnosed. 3. The method according to claim 1, wherein in step c) the oligonucleotide primers used for the synthesis of single-stranded or double-stranded cDNA are selected from oligo (dT) -containing primers, random hexamers or a combination thereof, as well as gene-specific primers. 4. The method according to claim 1, in which for the amplification of cDNA in step d) oligonucleotide primers are used that are selected so that they specifically hybridize with cDNA even in the presence of an impurity of genomic DNA. 5. The method according to claim 3, in which the sequence of the primers represented by SEQ ID NO: 1 and 2. 6. The method according to claim 1, wherein in step e) a quantitative or semi-quantitative amplification reaction of a WIF1 cDNA fragment is a real-time polymerase chain reaction (PCR) or a standard reverse transcription polymerase chain reaction (RT-PCR). 7. The method according to claim 1, wherein in step d), the GAPDH gene encoding the glyceraldehyde-3-phosphate dehydrogenase protein is used as a control gene. 8. A set of primers for the implementation of the polymerase chain reaction to determine the level of transcription of the WIF1 gene in the method according to claim 1, having the sequence of SEQ ID NO: 1 and 2.

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