RU2324186C1 - Method and tool for diagnostics of pulmonary epidermoid carcinoma - Google Patents

Abstract

FIELD: medicine; oncology.

SUBSTANCE: method applies the AKR1B10 gene transcription as a marker for diagnostics of pulmonary epidermoid carcinoma, and the decreased transcription in human tissue that is presumably affected by cancer, as compared to transcription in healthy tissue, indicates the pulmonary epidermoid carcinoma.

EFFECT: reliability of diagnostics of pulmonary epidermoid carcinoma is increased.

7 cl, 6 ex, 2 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, 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, the mortality rate from lung cancer in men is 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), large cell cancer, etc. 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, it 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 different forms of NSCLC are the AKR1B10 gene encoding aldose reductase B 10 from the family of 1 aldo ketoreductases (AKR) [Fukumoto S., Yamauchi N., Moriguchi H., Hippo Y., Watanabe A ., Shibahara J., Taniguchi H., Ishikawa S., Ito H., Yamamoto S., Iwanari H., Hironaka M., Ishikawa Y., Niki T., Sohara Y., Kodama T., Nishimura M., Fukayama M., Dosaka-Akita H., Aburatani H. 2005. Overexpression of the aldo-keto reductase family protein AKR1B10 is highly correlated with smokers' non-small cell lung carcinomas. Clin. Cancer Res. 11, 1776-1785; Penning T.M. 2005. AKR1B10: a new diagnostic marker of non-small cell lung carcinoma in smokers. Clin. Cancer Res. 11, 1687-1690]. Protein AKR1B10 (36 kDa) belongs to the family of enzymes with aldoketoreductase activity. All members of the AKR family are hydroxy reductases, dependent on NAD (P) H, and catalyze the reduction of many compounds, including aromatic and aliphatic aldehydes and ketones [Cao D., Fan S.T., Chung S.S. 1998. Identification and characterization of a novel human aldose reductase-like gene. J. Biol. Chem. 273, 11429-11435; Hyndman D., Bauman D. R., Heredia V. V., Penning T. M. 2003. The aldo-keto reductase superfamily homepage. Chem. Biol. Interact 143-144, 621-631]. The AKR1B10 gene (13762 bp) is located on chromosome 7q33, and is not transcribed in most normal human tissues. Transcription and translation of this gene was found only in the tissues of the small and large intestines and at a low level in the liver and adrenal glands, which is consistent with the assumed role of AKR in the metabolism of steroids and inactivation of chemically active aldehydes of digested food in the digestive tract. Elevated AKR1B10 protein was found in malignant tumors of the liver, but its role in the pathogenesis is unclear [Hyndman D.J., Flynn T.G. 1998. Sequence and expression levels in human tissues of a new member of the aldo-keto reductase family. Biochim. Biophys. Acta. 1399.198-202; Martin H.J., Breyer-Pfaff U., Wsol V., Venz S., Block S., Maser E. 2006. Purification and characterization of AKR1B10 from human liver: role in carbonyl reduction of xenobiotics. Drug Metab. Dispos. 34, 464-470; Scuric Z., Stain S.C., Anderson W.F., Hwang J.J. 1998. New member of aldose reductase family proteins overexpressed in human hepatocellular carcinoma. Hepatology. 27, 943-950].

For the AKR1B10 gene, using mainly immunohistochemical staining and immunoblotting methods, a change in the amount of the corresponding protein in PRL was revealed [Fukumoto et. al. 2005, supra]. It should be noted that the transcriptional activity of this gene in normal tumors remains poorly understood, although from a diagnostic point of view, the high stability of the resulting transcript plays a significant role in choosing a candidate for the role of a diagnostic marker.

A change in 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.

It is clear from the foregoing that in this area there is an urgent need to search for a new diagnostic marker of PRL, which allows to diagnose the disease reliably and at the very early stages, and to develop a simple, sensitive, reliable method applicable to it in clinical or outpatient medical institutions Diagnosis of squamous cell lung cancer.

Disclosure of invention

This invention was made possible by the authors of a comparative analysis of the level of transcription of the AKR1B10 gene in tumor tissues of the lungs of various types and at different stages of their malignant transformation and the discovery of the fact that even the early stages of development of malignant transformation are accompanied by a significant increase in the level of transcription of the AKR1B10 gene.

The present invention, in its aspect, relates to a new marker for the diagnosis of squamous cell lung cancer, which is the level of transcription of the AKR1B10 gene. An elevated level in human tissue presumably affected by cancer compared with its level in healthy tissue is a diagnostic sign of squamous 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 AKR1B10 cDNA according to the control gene, the transcription level of which is constant in normal and with squamous cell carcinoma of the lung;

e) conducting a quantitative or semi-quantitative amplification reaction of the AKR1B10 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 DNA fragment AKR1B10 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 transcription level of the AKR1B10 gene, and the increase in transcription of the AKR1B10 gene is diagnostic a sign of 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 AKR1B10 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 AKR1B10 gene having the sequence of SEQ ID NO: 1 and 2.

List of figures

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

Figure 1 shows the results of the selection of conditions for determining the level of transcription of the AKR1B10 gene in lung tissue samples. Electrophoretic separation on an agarose gel of PCR products (size 162 bp) obtained after 25, 30, 34 and 37 cycles was carried out in a 1.8% agarose gel. M is a marker of the molecular masses of DNA (DNA of plasmid pBK222 / AluI).

Figure 2 shows the results of RT-PCR analysis of the level of transcription of the AKR1B10 gene in PRL with centralized tumor localization (after 33 cycles). Sample numbers are indicated above the tracks. T - tumor, N - conditional norm (tissue adjacent to the tumor). N1 - N3 - the norm. The GAPDH gene (28 PCR cycles) was used as an internal control. Electrophoretic separation of PCR products was carried out on a 1.8% agarose gel.

The implementation of the invention

This invention provides a new genetic marker for the diagnosis of squamous cell carcinoma of the lung and based on the determination of the level of transcription of this marker, a simple, reliable way to diagnose PRL at various stages of the development of malignant transformation, including the initial ones. A reliably detectable difference in transcription of the AKR1B10 gene in normal and tumor tissues can be used to detect squamous cell 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 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 guanidinisothiocyanate, dissolving 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 speed up and simplify the isolation of RNA, various devices are used, for example QuickGene-810 (Life Science, Japan). For the extraction of RNA, this device uses an 80 μm porous membrane, which is 12.5 times thinner than the glass filter commonly used in such devices (1000 μm). This allows you to reduce the degradation of RNA and increase its output.

Reverse transcription reaction: synthesis of cDNA 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 quantities needed 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. To the oligo (dT) sequence, nucleotides A, C or G are often added at the 3'-end to anchor the primer to the border of the transcript and poly-A tract;

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

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

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

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 an 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 AKR1B10 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. Appi Bioinformatics. 5, 119-120), ExPrimer (Sandhu KS, Acharya KK 2005. ExPrimer: to design primers from exon-exon junctions . Bioinformatics. 21, 2091-2092), PeriPrimer, 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 implementing 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 RNA destruction. 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 AKR1B10 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:

AKR1B10_F (SEQ ID NO: 1) 5'-CTTGGAAGTCTCCTCTTGGCA-3 '

AKR1B10_R (SEQ ID NO: 2) 5'-ACAGGTCCTCCCGCT TCAC-3 '

One skilled in the art will understand that other primer pairs can be selected that differ, for example, in length, in their localization relative to the AKR1B10 gene transcript sequence, which will provide specific and efficient amplification of the AKR1B10 gene transcript fragment even in the presence of an impurity of genomic DNA .

Quantification of the transcription level is achieved using parallel PCR with the test transcript and the control / standard transcript. As the endogenous internal control, relative to which the amplification products of the AKR1B10 gene under investigation were normalized, the “household gene”, GAPDH, encoding glyceraldehyde-3-phosphate dehydrogenase, was selected. The expression of “housekeeping genes” in all cells is usually about the same. The GAPDH gene for NSCLC, including PRL, shows the smallest spread in transcription levels in normal and tumor tissues (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 from 40 minutes to three hours (depending on the amplifier used) and includes simultaneous PCR, fluorescence signal detection, data processing and their presentation in graphical form (full kinetic curve) using special software. The results of the analysis become available immediately after the completion of the process and do not require additional purification and analysis of PCR products. 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. 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 the peripheral regions. 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 one in 18, the third 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 insoluble 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 EM, 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 circuit (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 AKR1B10 gene in lung tissue samples.

Protocol for determining the level of transcription

When selecting conditions for determining transcription levels for amplification of double-stranded cDNA, gene-specific primers AKR1B10_F (SEQ ID NO: 1) and AKR1B10_R (SEQ ID NO: 2) were used, which were matched to different exons of the AKR1B10 gene, one of the primers overlapping the boundary between exons. Under these conditions, genomic DNA impurities do not affect the amplification results.

The size of the PCR fragment was 162 bp The selection of the conditions for the NDP was carried out on several samples of cDNA. All primers were selected using the Primer Designer program developed at the IMB RAS. Amplification was carried out in 25 μl of a mixture containing: 67 mm Tris-HCl, pH 8.8, 16.6 mm (NH ₄ ) ₂ SO ₄ 0.1% Tween-20, 2.5 mm MgCl ₂ , 0.2 mm each of dNTP, 0.1 μg of cDNA, 0.2 μM of each of the primers, 2 units SmarTaq DNA polymerase activity (Dialat Ltd., Moscow).

Conditions for PCR:

The composition of the reaction mixture (25 μl):

PCR buffer (10x) 2.5 μl MgCl ₂ (25 mm) 2.5 μl dNTP (10 mm) 0.5 μl primer AKR1B10_F, 10 μM 0.5 μl primer AKR1B 10_R, 10 μM 0.5 μl cDNA (0.1 μg / μl) 1.0 μl SmarTaq DNA polymerase, 5 u / μl 0.3 μl sterile deionized water 17.2 μl

Amplification conditions:

94 ° C, 2 min 1 cycle

94 ° C, 30 s; 56 ° C, 30 s; 72 ° C, 45 s 25, 30, 34 and 37 cycles

72 ° C, 5 min 1 cycle

The cDNA samples were normalized by the GAPDH control gene encoding glyceraldehyde-3-phosphate dehydrogenase protein. Used primers selected for different exons of the GAPDH gene, and one of the primers overlaps the border between exons:

GAPDH_F: 5'-GGAGTCAACGGATTTGGTC-3 'and

GAPDH_R: 5'-TGGGTGGAATCATATTGGAACAT-3 '.

The size of the PCR fragment is 139 bp The selection of PCR conditions was carried out on several cDNA samples. Amplification conditions: preheating at 94 ° C for 2 min; 94 ° C, 30 s, 56 ° C, 30 s and 72 ° C, 30 s - 28 and 30 cycles; 72 ° C, 5 min - 1 cycle.

PCR was performed on a MasterCycler, Eppendorf (Germany) thermocycler with a heating cap or a Tertsik thermocycler, DNA technology (Russia). Amplification products were analyzed on a 1.8% agarose gel with 0.5 μg / ml ethidium bromide (see Figure 1). As a result, optimal RT-PCR conditions (30-33 cycles) were selected, under which a linear relationship was obtained between the number of cycles and the number of PCR products. All amplification reactions were repeated three times.

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

The amplified AKR1B10 gene fragments were cloned into the pGEM®-T Easy vector (Promega) and sequenced. Their nucleotide sequences completely coincided with the sequences of the corresponding cDNA fragment. Sequencing was performed using the ABI PRISM® BigDye ™ Terminator v 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 AKR1B10 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 1 reaction with a volume of 25 μl for each sample +1 additional reaction with a volume of 25 μl.

The composition of the reaction mixture (25 μl):

PCR buffer (10x) 2.5 μl MgCl ₂ (25 mm) 2.5 μl dNTP (10 mm) 0.5 μl primer AKR1B10_F, 10 μM 0.5 μl primer AKR1B10_R, 10 μM 0.5 μl cDNA 1.0 μl SmarTaq DNA polymerase, (Dialat Ltd., Moscow) 5 u / μl 0.3 μl sterile deionized water 17.2 μl

2. Add to the tubes 0.6 ml or 0.2 ml (labeled for 33 amplification cycles) add 24 µl of the master mix.

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

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

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

6. Place the tubes in the thermocycler and spend 33 cycles of the amplification reaction.

7. After completion of the amplification reaction, take 4 μl of amplification products (33 cycles), continue the amplification reaction for another 2 cycles, take 4 μl (35 cycles) and mix with 2 μl of 6x Orange Loading Dye. Samples were applied to a 1.8% agarose gel containing 0.5 mg / L ethidium bromide. A molecular weight marker of DNA was also applied to the gel, which made it possible to estimate the size of amplification products (DNA of plasmid pBR222 / AluI manufactured by Sibenzym, Russia was used). Gel electrophoresis was performed in TBE buffer containing 10 mg / L ethidium bromide. The separation length is 3-5 cm of the gel. After gel electrophoresis, 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 AKR1B10 gene

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

It was found that in 80% (20/25) of PRL samples (Table 2), the level of transcription of the AKR1B10 gene was significantly increased three or more times (p <0.0001) compared to the conventional norm, and in half of the samples this difference significantly exceeded tenfold ( Figure 2 and Table). In 18 out of 25 samples, transcription of the gene is normally practically absent; in the remaining seven samples, the level of transcription is insignificant. In two samples, gene transcription was not detected either in tumors or in adjacent tissue. Of the 10 samples studied, the norm in two observed transcription of the AKR1B10 gene, in the remaining eight it was not detected.

Of the 25 pairs of PRL samples, 19 correspond to central localization, and six to peripheral. In the case of the AKR1B10 gene, a 10-fold or more increase in the level of transcription in tumors compared to the norm was observed in 11 of 19 samples of central PRL and only in one of six samples of peripheral PRL. Thus, an increase in the level of transcription with central PRL is observed in 84%, and with peripheral in 17% of tumor samples (p = 0.03 according to the Fisher test). It is possible that a more significant change in gene transcription is associated with a greater dependence of the development of central BPD on smoking patients, harmful working conditions and other adverse factors. In AKL, an increase in the level of transcription of the AKR1B10 gene was detected in only 25% of the samples (Table). Thus, unlike many genes associated with various forms of lung cancer [Kettunen E., Anttila S., Seppanen JX., Karjalainen A., Edgren H., Lindstrom I., Salovaara R., Nissen AM, Salo J. , Mattson K., Hollmen J., Knuutila S., Wikman H. 2004. Differentially expressed genes in non-small cell lung cancer: expression profiling of cancer-related genes in squamous cell lung cancer. Cancer Genet. Cytogenet. 149, 98-106], the AKR1B10 gene can be attributed to tumor markers characteristic of PRL.

The present invention also provides that determining the level of transcription of the AKR1B10 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 AKR1B10 gene during or at the end of the course of treatment will indicate the restoration of the activity of the tumor suppressor gene AKR1B10, which may indicate positive changes in treatment. An increase in AKR1B10 gene transcription will indicate a further suppression of gene activity, which may indicate a lack of treatment effectiveness.

Table Characterization of clinical samples and changes in the level of transcription of the AKR1B10 gene in PRL samples with central and peripheral localization of the tumor and AKL compared to the conventional norm Tumor type Sample No. Description of tumor samples Change in the level of transcription in the tumor compared to the conditional norm TNM Stage Keratinization Central PRL four T1N0M0 I - 9 80/05 98/05 IA + 21 T3N0M0 II - 26 497 300/05 6 IIB 90/05 T2N1M0 IIB - 7 T3N0M0 II + 13 fifteen 77/05 5 IIB 228/05 T3N1M0 III - 290/05 466 T3N2M0 + fourteen Peripheral PRL 475 T1N0M0 I - 2 T2N0M0 IB - 451 T3N0M0 II + 476 II - 452 II + O 301/05 T3N1M0 III + AKL 311/05 T2N1M0 II n.d. O 304/05 T3N0M0 II n.d. 406 II n.d. O 477 II n.d. Explanations for the table:

increased (> 3 times) AKR1B10 gene transcription level

transcription level did not change or increased slightly (<3 times)

lack of transcription in the tumor and in the normal norm

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 (7)

1. A method for the diagnosis of squamous cell lung cancer, comprising the following stages: a) obtaining an initial pair of tissue samples from a patient, where one of the samples was obtained from tissue presumably affected by cancer, and the second was obtained from adjacent 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 AKR1B10 cDNA according to the control gene, the transcription level of which is constant in normal and in lung cancer; d) conducting a quantitative or semi-quantitative amplification reaction of the AKR1B10 gene fragment using the cDNA obtained in stage c) as a matrix and a pair of gene-specific oligonucleotide primers with sequences SEQ ID NO: 1 and 2; f) comparing the amount of amplified DNA fragment AKR1B10 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 AKR1B10 gene transcription, and an increase in AKR1B10 gene transcription is diagnostic a sign of squamous cell lung cancer. 2. 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. 3. The method according to claim 1, in which for the amplification of cDNA in stage d) use oligonucleotide primers selected in such a way that they specifically hybridize with cDNA even in the presence of an impurity of genomic DNA in the preparation. 4. The method according to claim 3, in which the sequence of primers represented by SEQ ID NO: 1 and 2. 5. The method according to claim 1, wherein in step e), the quantitative or semi-quantitative amplification reaction of the AKR1B10 gene fragment is real-time (PCR) or standard reverse transcription polymerase chain reaction (RT-PCR). 6. The method according to claim 1, wherein in step d), the GAPDH gene encoding glyceraldehyde-3-phosphate dehydrogenase protein is used as a control gene. 7. The kit for the diagnosis of squamous cell carcinoma of the lung according to claim 1, which allows to determine the level of transcription of the AKR1B10 gene by polymerase chain reaction and includes primers with the sequences SEQ ID NO: 1 and 2 for the implementation of this reaction.

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