KR101201157B1 - Composition for diagnosing lung cancer comprising an agent for determining level of methylation of RXRG gene and a method for diagnosing lung cancer using the same - Google Patents

Abstract

The present invention relates to a lung cancer diagnostic composition comprising an agent for measuring the methylation level of the RXRG gene promoter and a diagnostic method using the same. More specifically, the present invention relates to a composition for diagnosing lung cancer according to methylation of CpG island of the RXRG gene promoter and a method for diagnosing lung cancer by measuring the methylation level. Since methylation of the CpG island of the RXRG gene promoter of the present invention is specifically expressed in lung cancer cells, a composition comprising an agent for measuring the methylation level of the present invention can be usefully used for diagnosing lung cancer.

Description

Composition for diagnosing lung cancer comprising an agent for determining level of methylation of RXRG gene and a method for diagnosing lung cancer using the same}

The present invention relates to a lung cancer diagnostic composition comprising an agent for measuring the methylation level of the RXRG gene promoter and a diagnostic method using the same. More specifically, the present invention relates to a composition for diagnosing lung cancer according to the methylation of the CpG island of the RXRG gene promoter and a method for diagnosing lung cancer by measuring the methylation level.

Lung cancer is the biggest cause of smoking and pollution, and is mainly caused by carcinogens, and the incidence rate is increasing worldwide, especially in developed countries. According to statistics from the National Statistical Office, in 2005, 65,479 people died of cancer in Korea, with 26.7% of all deaths being cancer. Lung cancer recorded 28.4 deaths per 100,000 population (21.1%), the highest among all cancers. This ranking has not changed.

Lung cancer is classified into two types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) .Non-small cell cancer is again classified as adenocarcinoma, squamous cell carcinoma, large cell carcinoma, It is classified into a histologic type of linear squamous cell carcinoma, and various clinical forms such as prone site, progression form, speed, and symptoms vary according to these different types of tissues (Brambilla et al., Eur Respir). J. 18 (6): 1059-68, 2001).

However, lung cancer, like most other cancers, has few unique symptoms or awareness symptoms. Therefore, only 21% of patients diagnosed with lung cancer have not metastasized, and 50% have already metastasized and are inoperable. 70% of lung cancer patients die within one year, and 87% within five years. It is one of those diseases with a poor prognosis such as death. Non-small cell cancers, which account for about 80% of lung cancers, are staged and determined by the size of the cancer mass, the presence of periphery of the tissue, the degree of lymph gland involvement, and metastasis to distant organs, with the exception of surgery. It is one of the cancers that is hard to cure due to its low therapeutic effect. This poor prognosis and high mortality rate is due to the lack of effective methods for the diagnosis of lung cancer. Therefore, early diagnosis and treatment are very important in intractable lung cancer.

Until now, most of the means for testing lung cancer are physical, and biopsy, bronchoscopy, chest examination, high-voltage imaging, and sputum cytology are performed along with chest X-ray. However, this diagnostic method is not only economically burdensome but also has the disadvantage that the patient suffers during the examination. Therefore, it is necessary to develop a diagnostic agent for lung cancer that enables accurate determination of the onset and progression of lung cancer.

On the other hand, it is not yet clear how and how cancer cells are produced. However, it is thought that cancer is caused by the generation of cells that are not regulated by the modification of genes that function to control the growth of normal cells. This is a general view. Therefore, many cancer researchers have constructed DNA chips for genes that are predicted to be related to cancer, and compared the expression levels between cancer tissues and normal tissues to select cancers using genes that are specifically overexpressed in cancer tissues as tumor markers. It is a trend used for inspection. Of course, such diagnostic methods are limited in accuracy, often positive in the absence of cancer, causing confusion, but still need to be approached at the genetic level in order to diagnose and treat cancer fundamentally.

Numerous diseases are caused by genetic abnormalities, and the most frequent form of genetic abnormalities is a change in the genetic code sequence. These genetic changes are called mutations. When a gene is mutated, the structure and function of the protein encoded by the gene changes, abnormalities and cleavage occur, and this mutated protein causes disease. However, even if a particular gene is not mutated, an abnormality may occur in the expression of the gene and cause disease. A typical example is methylation where a methyl group is attached to a gene transcription regulatory site, eg, a cytosine base site of a CpG island. This is called epigenetic change and has the same effect as transferring to progeny cells in a manner similar to mutations, eg, loss of expression of the corresponding protein. In the case of cancer cells, expression of tumor suppressor genes is inhibited by methylation of the promoter CpG island, which is recognized as one of the important mechanisms causing cancer (Robertson, K. and Jones, P., Carcinogen, 21, 461, 2000). In fact, abnormal methylation / demethylation of CpG islands has been reported in various cancer cells such as prostate cancer, colon cancer, uterine cancer and breast cancer, and it has been found that they play an important role in the early stages of cancer formation. It is attracting attention as a marker. However, there is a lack of a sufficient number of markers for diagnosing cancer, which requires the continuous development of markers showing a tendency to cancer-specific DNA methylation.

Therefore, the present inventors have studied to develop a DNA methylation marker specific for lung cancer, and showed a tendency to lung cancer specific methylation in the CpG island in the promoter region of the retinoid X receptor gamma (RXRG) gene, and thus the measurement of the methylation level. The present invention was completed by revealing that lung cancer can be diagnosed.

An object of the present invention is to provide a composition for diagnosing lung cancer comprising an agent for measuring the methylation level in the CpG island of the RXRG gene promoter.

Another object of the present invention is to provide a method for diagnosing lung cancer by measuring the methylation level in the CpG island of the RXRG gene promoter.

As one aspect for achieving the above object, the present invention relates to a lung cancer diagnostic composition comprising an agent for measuring the methylation level of the Retinoid X receptor gamma (RXRG) gene.

In the present invention, the term "methylation" occurs in the cytosine of the CpG island (CpG island) of the promoter region of a particular gene, thereby interrupting the binding of transcription factors, thereby blocking the expression of a specific gene, For the purpose, methylation means methylation at the CpG island of the promoter region of the RXRG gene.

In the present invention, the term "measurement of methylation level" is to measure the methylation level of the promoter CpG island of the RXRG gene, such as methylation-specific PCR (methylation-specific polymerase chain reaction, hereinafter referred to as MSP) or automatic base analysis. You can check it by way. In normal lung tissue cells, the methylation status of the RXRG gene does not appear in the promoter, but in lung cancer tissue cells, the methylation in the promoter of the RXRG gene appears specifically, indicating that the expression of the RXRG gene is low. Therefore, lung cancer can be diagnosed.

As used herein, the term "CpG island" refers to a genomic region in which CpG is collected at an exceptionally high frequency, and has a C + G content of 50% or more and a CpG ratio of 3.75% or more of 0.2 to 3kb in length. Refers to the site. In the CpG, C stands for cytosine, G stands for guanine and p stands for phosphodiester bond between cytosine and guanine. There are about 45,000 CpG islands in the human genome, most of which are found at promoter sites that regulate gene expression. Indeed, the CpG islands are found in promoters of housekeeping genes, about 50% of human genes (Cross, S. and Bird, A., Curr. Opin. Gene Develop., 5: 309, 1995). . In somatic cells of normal individuals, CpG islands of the housekeeping gene promoter region are unmethylated and genes that are not expressed during development, such as imprinted genes and genes on inactive X chromosome, are methylated.

In genomic DNA of mammalian cells, in addition to A, C, G and T, there is a fifth base called 5-methylcytosine with a methyl group attached to the fifth carbon of the cytosine ring (5-mC). . 5-mC is attached only to C of CG dinucleotide (5'-mCG-3 '), called CpG. C in CpG is mostly methylated by the contact of methyl groups. Methylation of CpG inhibits the expression of alu or transposons and repeats of the genome. In addition, the CpG is a site where most epigenetic changes occur frequently in mammalian cells. This is because 5-mC of CpG is naturally deaminoated to be thymine (T).

As used herein, the term "Retinoid X receptor gamma" refers to a nuclear receptor encoded by the RXRG gene and is also known as NR2B3 (nuclear receptor subfamily 2, group B, member 3). The RXRG is a member of the retinoid X receptor (RXR) family of nuclear receptors that is involved in regulating the antiproliferative effect of retinoic acid. The receptor forms a homodimer with retinoic acid, thyroid hormones, and vitamin D receptors, and serves to enhance DNA binding and transcriptional functions to its response factors.

Vitamin A (retinol) and its natural and synthetic analogs, retinoids, play an important role in regulating the growth, differentiation, and death of epithelial cells, which interact with their nuclear receptors belonging to the steroid hormone receptor superfamily. Is done through. These receptors can be classified into two classes: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Both receptor classes have alpha, beta, gamma subclasses with numerous isoforms resulting from promoter use and alternative splicing. The RARs have a strong affinity for both all-trans and 9-cis isoforms of retinoic acid, while RXRs have significant specificity only for 9-cis isoforms. The RXR / RAR heterodimer is a functional unit that introduces a retinoid signal in in vivo. These heterodimers activate transcription by binding to the retinoic acid response factor found at the promoter region of the retinoic acid inducible target gene.

The present inventors investigated abnormal methylation status of the CpG island of the promoter region of the RXRA, RXRB, and RXRG genes in non-small cell lung cancer and correlated the results with the clinicopathologic features of lung cancer patients. Lung cancer could be diagnosed by measuring methylation levels in the islets.

In a specific embodiment of the present invention, using the MSP to examine the methylation of the RXRA, RXRB, RXRG gene promoter in the tissues of patients with non-small cell lung cancer, the MSP is a non-methylated-specific and methylated-specific primer, respectively Was performed (see FIG. 1). As a result, methylation of the RXRG gene was detected in 23.7% of tumor tissues, whereas methylation of the RXRA, RXRB genes was rarely detected (5.7%, 4.3%, respectively), and RXRA, RXRB in the corresponding non-tumor lung tissues. The methylation frequency of RXRG gene was 2.1%, 2.8%, and 3.6%, respectively, indicating that methylation in RXRG gene was specific in lung cancer tissues (see FIG. 2). In addition, in order to evaluate the effect of DNA methylation on the transcriptional regulation of RXRG gene, the mRNA level and methylation status showed that RXRG gene was not detected in tissues with methylated alleles, whereas non-methylated allele RXRG was detected. Non-tumoral tissues with mRNA were found to be expressed at high levels (see Figure 3). This result indicates that there is a direct correlation between RXRG promoter methylation and mRNA expression. In addition, as a result of examining whether the methylation status of the RXRG gene is related to survival, the methylation of the RXRG gene was not significantly related to the prognosis of the patients. However, when the patients were classified according to the smoking status, the RXRG methylation was no smoking experience. It was found to be related to worse survival in the patients, and it was confirmed that there was a significant difference in the overall survival rate of the smoking inexperienced patients and the smoking experienced patients (see Table 4 and FIG. 4).

As used herein, the term "nonmalignant" cell or tissue refers to a cell or tissue that is part of a diseased tissue but is not considered a tumor site.

As used herein, the term "diagnostic" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether lung cancer has developed.

The lung cancer to be diagnosed of the present invention may be small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC), preferably non-small cell lung cancer. The non-small cell lung cancer includes adenocarcinoma, squamous cell carcinoma, large cell carcinoma, or linear squamous cell carcinoma.

In the present invention, an agent for determining the methylation level of a gene is specific for a compound or methylation sensitive restriction enzyme that modifies an unmethylated cytosine base, a primer specific for the methylated allele sequence of the RXRG gene, and an unmethylated allele sequence. Can include primers.

The compound that modifies the unmethylated cytosine base may be bisulfite, but is not limited thereto, and is preferably sodium bisulfite. Methods for detecting the methylation of promoters by modifying non-methylated cytosine residues using such bisulfites are well known in the art (Herman JG et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 9821-9826; WO01 / 26536; US2003 / 0148326A1).

In addition, the methylation-sensitive restriction enzyme may be a restriction enzyme containing CG as a recognition site of the restriction enzyme as a restriction enzyme that can specifically detect methylation of CpG islands. For example, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, NotI and the like are not limited thereto. Depending on the methylation or demethylation of the restriction enzyme recognition site, the cleavage by restriction enzymes is different and can be detected by PCR or Southern blot analysis. Methylation sensitive restriction enzymes other than the restriction enzymes are well known in the art.

The methylation level at the promoter CpG island of the RXRG gene of a patient suspected of lung cancer was obtained by treating the patient's biological sample with a compound or methylation sensitive restriction enzyme that modified the cytosine base that was not methylated in the obtained DNA. Then, the treated DNA can be measured by amplifying by PCR using a primer and confirming the presence of the amplified result.

Thus, the formulations of the present invention may comprise primers specific for the methylated allele sequence of the RXRG gene and primers specific for the unmethylated allele sequence. In the present invention, the term “primer” refers to a short nucleic acid sequence that can form base pairs with complementary templates with nucleic acid sequences having short free 3-terminal hydroxyl groups and that serves as a starting point for template strand copying. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. In addition, primers, as sense and antisense nucleic acids having 7 to 50 nucleotide sequences, may incorporate additional features that do not change the basic properties of the primers serving as the starting point for DNA synthesis.

Primers of the present invention can be preferably designed according to the sequence of the CpG island to be analyzed for methylation, each primer pair and methylation capable of specifically amplifying cytosine that is methylated and not modified by bisulfite May be a primer pair capable of specifically amplifying cytosine modified by bisulfite. Preferably, the primer specific for the methylated sequence of the RXRG gene is a primer consisting of a pair of sequences of SEQ ID NO: 1 and SEQ ID NO: 2, and the primer specific for the unmethylated sequence of the RXRG gene is SEQ ID NO: 3 and SEQ ID NO: It may be a primer consisting of a pair of sequences of 4.

The lung cancer diagnostic composition may further include a polymerase agarose, a buffer solution for electrophoresis, etc., in addition to the formulation.

As another aspect of the present invention, the present invention comprises the steps of measuring the CpG island methylation level of the Retinoid X receptor gamma (RXRG) gene promoter from biological samples of patients suspected of lung cancer; And comparing the methylation level with the CpG island methylation level of the corresponding gene promoter of the normal control sample. Preferably, the lung cancer is non-small cell lung cancer.

As used herein, the term “biological sample” includes, but is not limited to, tissues, cells, whole blood, serum, plasma, saliva, sputum, or urine, which differ in methylation levels of the RXRG gene due to the development of lung cancer. Preferably, the biological sample of the present invention may be tissue.

First, genomic DNA is obtained from a patient suspected of lung cancer to measure methylation level. The genomic DNA is obtained by phenol / chloroform extraction, SDS extraction (Tai et al., Plant Mol. Biol. Reporter) commonly used in the art. , 8: 297-303, 1990), CTAB isolation (Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980) or commercially available DNA extraction kits.

Determination of the methylation level of the gene comprises the steps of: a) treating the obtained genomic DNA with a compound or methylation sensitive restriction enzyme that modifies an unmethylated cytosine base; And b) amplifying the treated DNA by PCR using a primer capable of amplifying the CpG island of the RXRG gene promoter.

The compound for modifying the unmethylated cytosine base in step a) may be bisulfite, preferably sodium bisulfite. Methods for detecting the methylation of promoters by modifying non-methylated cytosine residues using such bisulfites are well known in the art (Herman JG et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 9821-9826; WO01 / 26536; US2003 / 0148326A1).

In addition, the methylation-sensitive restriction enzyme in step a) may be a restriction enzyme containing CG as a recognition site of the restriction enzyme as a restriction enzyme that can specifically detect methylation of CpG island, as described above. For example, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, NotI and the like, but is not limited thereto.

Amplification in step b) may be performed by a conventional PCR method. In this case, the primer used may be preferably designed according to the sequence of the CpG island to be analyzed for methylation, as described above, and may specifically amplify cytosine that has been methylated and not modified by bisulfite. Primer pairs and primer pairs capable of specifically amplifying cytosine modified by bisulfite due to unmethylation. Preferably, the primer specific for the methylated allele sequence of the RXRG gene is a primer consisting of a pair of sequences of SEQ ID NO: 1 and SEQ ID NO: 2, and the primer specific for the unmethylated allele sequence of the RXRG gene is SEQ ID NO: It may be a primer consisting of a pair of 3 and SEQ ID NO: 4.

Measuring the CpG island methylation level of the Retinoid X receptor gamma (RXRG) gene promoter may further comprise c) confirming the presence of the result amplified in step b). The presence of the result amplified in step c) may be performed according to a method known in the art, for example, electrophoresis, according to whether a band of a desired position is detected. For example, in the case of using a compound that modifies an unmethylated cytosine residue, a primer pair capable of specifically amplifying two kinds of primer pairs used in the step a), ie, cytosine which was methylated and not modified by bisulfite. And methylation based on the presence or absence of the PCR result amplified by primer pairs which are not methylated and thus can specifically amplify cytosine modified by bisulfite. Preferably, the sample genomic DNA is treated with sodium bisulfite, the bisulfite genome sequencing method of amplifying all sites of the CpG island of the RXRG gene by PCR and analyzing the sequencing of the amplified sites for methylation or not. You can judge.

In addition, even when restriction enzymes are used, if a PCR result is found in a method known in the art, for example, in a mock DNA where PCR results are displayed, and the restriction enzyme is determined to be methylated, the restriction is determined. If there is no PCR result in the DNA treated with the enzyme, it can be determined whether or not the methylation according to the promoter is determined to be unmethylated, which is obvious to those skilled in the art. The mock DNA in the above means the sample DNA which is separated from the sample and is not treated at all.

Therefore, by using the method of providing information for diagnosing non-small cell lung cancer of the present invention, it is possible to effectively determine whether the RXRG gene promoter is methylated and diagnose the non-small cell lung cancer.

As described above, methylation of CpG island of the RXRG gene promoter of the present invention affects expression of RXRG in lung cancer cells, and methylation of CpG island of the RXRG gene promoter of the present invention appears specifically in lung cancer cells. Therefore, a composition comprising an agent for measuring the methylation level of the present invention can be usefully used for diagnosing lung cancer.

Figure 1 shows the methylation of RXRA, RXRB, RXRG gene promoter in the tissues of patients with non-small cell lung cancer using MSP (methylation specific PCR). MSP was performed using non-methylated-specific primers (U) and methylated-specific primers (M), respectively, with Pos representing positive controls, Neg representing negative controls, T representing tumor lung tissue, and N representing non-tumor Nonmalignant lung tissue.
Figure 2 shows the methylation pattern of the RXR gene in non-small cell lung cancer tissue (T) and non-tumor lung tissue (N).
Figure 3 shows the results of RT-PCR and MSP analysis of the RXRG gene in non-small cell lung cancer tissue (T) and non-tumor lung tissue (N). (A) shows the expression results of RXRG mRNA obtained by performing RT-PCR on a sample tissue sample. When the amplification product was passed through a 2% agarose gel, the product amplified for RXRG was found at 244 bp. , β-actin was used as internal loading control. (B) shows the results of analyzing the methylation status of the RXRG gene in the same tissue using the MSP method. Lane UD is the result of amplification products obtained using non-methylation (UM) specific primers. Results for amplification products obtained using methylation (M) specific primers.
4 shows overall survival of 139 non-small cell lung cancer patients according to RXRG methylation status: (A) all patients; (B) smoking-naive patients; (C) Smoking-experienced patient. P = 0.003, test for homogeneity.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Materials and methods

Example  1: patient and tissue sample

Non-tumor lung tissue samples corresponding to tumor tissue were obtained from 139 patients with non-small cell lung cancer (NSCLC) who underwent resection between January 2002 and July 2006 at Kyungpook National University Hospital (Daegu, Korea). The patients had not received chemotherapy and radiation prior to surgery and informed consent for tissue collection was obtained from each patient prior to surgery. The study of the present invention has been approved by the Institutional Review Board of Kyungpook National University Medical School Hospital.

Both tumor and gross normal lung tissue were obtained at the time of surgery, rapidly frozen in liquid nitrogen and stored at -80 ° C until the DNA of the genome was ready. Optimal tumors containing only 80% or more of the tumor components were used for DNA extraction and methylation analysis. Visually, normal lung tissue was confirmed normal by hematoxylin-eosin staining. Genomic DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN, VALencia, CA).

Example  2: methylation analysis

The methylation status of the RXR gene is processed in genomic DNA with sodium bisulfite, followed by polymerase chain reaction PCR (PCR) specific for methylation using primers specific for methylation and unmethylation alleles of each gene. Methylation-specific polymerase chain reaction (hereinafter also referred to as MSP). The primer sequences and unwinding temperatures are summarized in Table 1 below. All PCR amplifications were performed using reagents provided by the GeneAmp DNA Amplification Kit with AmpliTaq Gold, such as polymerases in the PTC-100 system (PE Applied Biosysytem [ABI], Foster City, Calif.). Cpgenome Universal methylated and unmethylated DNA (Chemicon, Temecula, Calif.) were used as positive controls for methylated and unmethylated genes, respectively. Negative control samples without DNA were included in each PCR set. PCR results were separated on 2% agarose gel, stained with ethidium bromide and visualized under UV. Each MSP was repeated at least once to confirm the results.

Example  3: full RNA  Isolation and Reverse Transcriptase PCR

For reverse transcriptase-polymerase chain reaction (RT-PCR), total RNA was cultured using TRIzol (Invitrogen, Mt Waverly, VIC, Australia) according to the product instructions for lung cancer and corresponding non-malignant lung tissue specimens. Extracted from the NSCLC cell line. Structural integrity of total RNA was confirmed by electrophoresis on 1.2% agarrose-formaldehyde gel. The DNA of the remaining genome was compiled with RNase-free DNase (Invitrogen). The first strand cDNA was reverse transcribed from 2 μg of total RNA in 20 μL total volume using oligo (dT) and SuperScript preamplification kit (Invitrogen). The resulting cDNA was amplified using primers specific for the RXRG gene with ABI AmpliTaq Gold. Primer sequences are as follows: forward, 5'-CCCTTGAGGCCTACACCAAGC-3 '(SEQ ID NO: 5) and reverse, 5'-CACACCTGCCCAGGGGTCATC-3' (SEQ ID NO: 6). PCR was performed on PTC-100 (MJ Research, Watertown, Mass.) As follows: 35 cycles of 95 ° C. for 12 minutes, then 45 seconds at 95 ° C., 45 seconds at 58 ° C., and 72 ° C. 45 seconds at, and finally 5 minutes at 72 ° C. The amplified result was separated from 2% agarose gel and visualized using ethidium bromide and photographed.

Example  4: Statistical Analysis

The relationship between methylation and clinicopathological features was analyzed using the Chi-square test or Fisher's exact test for categorical variables. P- value <0.05 was considered statistically significant. A logistic regression test was performed to assess the relationship between methylation and covariates of age, sex, smoking history, tumor histology, and pathological status. Overall survival was measured from the day of surgery to death for any reason or until the last follow-up. Survival evaluation was calculated using the Kaplan-Meier method. Differences in overall survival of patients with or without RXR methylation were compared using the log-rank test.

Harzard ratios and 95% confidence intervals (CIs) were associated with age (≤ 63 vs.> 63 years), gender (male vs female), smoking status (naive vs. experienced smokers), and pathological stage (I vs. II-IIIA). Multivariate Cox proportional hazards models were evaluated. Homogeneity tests were performed to compare the differences between Harzard ratios of different groups. All analyzes were performed using a statistical analysis system for Windows (SAS Institute, Cary, NC), version 9.1.

result

NSCLCs From tissue samples RXR  Gene MSP Wow RT - PCR  analysis

The methylation status of the RXRA, RXRB, and RXRG genes was determined using MPS in surgically cut NSCLC samples and their corresponding non-tumor lung tissues in 139 patients. Complete CpG islands (CGIs) were found in the 1.0-kb 5 'flanking region of the gene containing the first exon (ENSG 186350, 204231, 143171; http://www.ensembl.org/), MethPrimer ( http: //www.urogene.org/methprimer/ ) The computer program allowed us to design MSP primer pairs near the start of transcription.

Each particular primer set exhibited a single band of expected size; Representative examples of MSP analysis are shown in FIG. 1A. In all genes, unmethylated bands were found in both malignant and non-tumor tissues, thus confirming the integrity of the DNA in this sample. The tumor sample was a naked eye sample containing both tumor and non-tumor tissue, showing the expected results. Bisulfite-sequencing of the sample PCR products confirmed their methylation status and showed that all cytosines are converted to thymine at positions other than CpG (data not shown). This ruled out the possibility that successful amplification could be due to incomplete bisulfite conversion. Methylation of the RXRG gene was detected in 23.7% in tumors, whereas methylation of the RXRA and RXRB genes was rarely detected (5.7% and 4.3%, respectively). The methylation frequencies of the RXRA, RXRB and RXRG genes in the corresponding non-tumor lung tissues were 2.1%, 2.8% and 3.6%, respectively (FIG. 2). At least one of the three genes was methylated (31.7%) of 139 tumors, and two of these genes were methylated (2.1%) in three tumors.

To assess the potential role of DNA methylation in the regulation of transcription of the RXRG gene, mRNA levels and methylation status were evaluated in sample tissue samples. RT-PCR and MSP analysis indicated that RXRG gene was not detected in tissues with methylated alleles, whereas high levels were expressed in non-tumor tissues with unmethylated allele RXRG mRNA (FIG. 3). ). These results indicate that there is a direct correlation between RXRG promoter methylation and mRNA expression.

Correlation Between Methylation Status and Clinicopathological Features

We compared the clinicopathological features to determine the methylation status of the RXRA, RXRB, and RXRG genes according to the patient's profile. RXRG methylation was significantly more frequent in Stage I tumors than Stage II-IV tumors ( P = 0.02). However, as shown in Table 2 below, in adenocarcinoma, methylation of the RXR gene did not show any significant correlation with other clinicopathological factors such as age, sex, smoking status, histological type, or EGFR mutation status.

For survival RXRG  Impact of Gene Methylation

62 of the 139 patients died (44.6%). Five-year survival rate was estimated at 50.0%. As shown in Table 3, among the pathologic factors, age and pathological stage were significantly related to overall survival: adjusted hazard ratio HR _adj for age> 63 years = 1.75, 95% CI = 1.05e2.94, P = 0.03; HR _adj for stage II-IIIA = 3.02, 95% CI = 1.81-5.03, P <0.0001.

The relationship between survival results and methylation status of the RXR gene is shown in FIG. 4 and Table 4 below. Methylation of the RXRG gene was not significantly associated with the prognosis of the patient (FIG. 4A). However, by classifying patients by smoking status, it was found that RXRG methylation had an effect on showing a significant difference in overall survival between smoking inexperienced and smoking experienced patients ( P = 0.003, [test for homogeneity]). . Oddly, RXRG Methylation was associated with significantly worsened survival in no-smokers (HRadj = 12.62, 95% CI = 2.15-74.17, P = 0.005) (FIG. 4B), while RXRG methylation, although not statistically significant, was associated with smoking. There was a better overall survival rate in experienced subjects (HRadj = 0.68, 95% CI = 0.33-1.41, P = 0.30) (Figure 4c). This indicated that the presence of methylation in the RXRG gene had a direct effect contrary to survival outcomes according to smoking status ( P = 0.01, test for homogeneity) (Table 3).

Attach an electronic file to a sequence list

Claims (12)

Non-small cell lung cancer diagnostic composition comprising an agent for measuring the methylation level of the Retinoid X receptor gamma (RXRG) gene.
The method of claim 1, wherein the agent measuring the methylation level of the gene is a compound or methylation sensitive restriction enzyme that modifies an unmethylated cytosine base, a primer specific for the methylated sequence of the RXRG gene, and a non-methylated sequence. A composition comprising a primer.
delete According to claim 2, wherein the primers specific for the methylated sequence of the RXRG gene is a pair of primers consisting of SEQ ID NO: 1 and SEQ ID NO: 2, the primer specific for the unmethylated sequence of the RXRG gene is SEQ ID NO: A primer pair consisting of 3 and SEQ ID NO of SEQ ID NO: 4.
The composition of claim 2, wherein the compound that modifies the unmethylated cytosine base is sodium bisulfite.
Measuring the CpG island methylation level of the RXRG gene promoter from a biological sample of a patient suspected of non-small cell lung cancer; And comparing the methylation level with the CpG island methylation level of the corresponding gene promoter in the normal control sample.
The method of claim 6, wherein measuring the methylation level of the gene is
a) treating the obtained genomic DNA with a compound or methylation sensitive restriction enzyme that modifies the unmethylated cytosine base; And
b) amplifying the treated DNA by PCR using a primer capable of amplifying the CpG island of the RXRG gene promoter.
The method of claim 7, wherein the primer is a methylation specific primer pair consisting of SEQ ID NO: 1 and SEQ ID NO: 2 and a nonmethylation specific primer pair consisting of SEQ ID NO: 3 and SEQ ID NO: 4.
8. The method of claim 7, wherein the compound that modifies the unmethylated cytosine base is sodium bisulfite.
The method of claim 6, wherein the method of measuring methylation level is a methylation-specific polymerase chain reaction.
delete The method of claim 6, wherein said biological sample is tissue of a patient.

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