WO2012023648A1 - Diagnostic composition for non-small-cell lung cancer comprising a preparation for measuring the hoxa11 gene methylation level, and a diagnostic method for non-small-cell lung cancer using the same - Google Patents

Abstract

The present invention relates to a diagnostic composition for non-small-cell lung cancer comprising a preparation for measuring the HOXA11 gene methylation level, and to a diagnostic method using the same. More specifically, the invention relates to a composition for diagnosing lung cancer depending on whether or not there is any HOXA11 gene methylation, and to a method for diagnosing lung cancer by measuring the methylation level. Because the HOXA11 gene methylation of the present invention appears specifically in lung cancer cells, the preparation for measuring the methylation level of the present invention can be used to advantage in lung cancer diagnosis.

Description

A composition for diagnosing non-small cell lung cancer comprising an agent for measuring the methylation level of the HOA110 gene and a method for diagnosing non-small cell lung cancer using the same

The present invention relates to a composition for diagnosing non-small cell lung cancer, comprising an agent for measuring methylation level of HOXA11 gene, and a diagnostic method using the same. More specifically, the present invention relates to a composition for diagnosing non-small cell lung cancer according to the methylation of the HOXA11 gene and a method for diagnosing non-small cell lung cancer by measuring the methylation level.

Lung cancer is the most common cause of cancer-related deaths worldwide. Although significant advances have been made in the detection and treatment of lung cancer over the past two decades, the prognosis for patients with disease is still poor, between 8% and 15%, combined with five-year survival rates for all stages of lung cancer. Poor prognosis for lung cancer patients is the result of some relapses, which occurs in approximately 20-50% of patients who have undergone therapeutic surgical resection with proper lymph node dissection. Even patients with stage 1 lung cancer have a five-year survival rate of less than 50% because the cancer has spread near or elsewhere in the lymph nodes, even before it is discovered.

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 into squamous cell carcinoma, adenocarcinoma, large cell carcinoma, It is classified as a tissue type such as adenosquamous carcinoma. Since small cell carcinoma has a poorer prognosis than non-small cell carcinoma, chemotherapy is usually used as the main treatment. Non-small cell carcinoma is usually used in stages 1-2 and chemotherapy in advanced cases.

Squamous cell carcinoma accounts for about 35% to 50% of all lung cancers and occurs mainly in the proximal bronchus. It is very closely related to smoking and is common in men. It is characterized by histologically forming keratin, forming a cellular bridge, and relatively slow growth rate and metastasis rate compared to other forms of lung cancer. Adenocarcinoma, on the other hand, accounts for about 15-35% of all lung cancers and is relatively more common in women than in the West. The site of development is a cancer that occurs mainly in the peripheral bronchus, the periphery of the lung, histologically forming gland and producing mucin, an animal viscous substance, especially rubber protein in mucus. Histologically, it is isolated in the lung parenchyma, and metastasis rate is relatively high, so the prognosis is poorer than that of squamous cell carcinoma.

According to these different types of tissues, there are various clinical manifestations such as prone site, progression form, speed, and symptoms (Brambilla et al., Eur Respir J.18 (6): 1059-68, 2001).

Non-small cell carcinoma, which accounts for about 80% of the lung cancers, is determined based on the size of the cancer mass, whether or not the surrounding tissues are infiltrated, the degree of lymph gland invasion, and the metastasis to distant organs. 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 together 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.

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 there is no mutation in a particular gene, the expression of the gene can be abnormal and cause disease. A typical example is methylation where a methyl group is attached to a gene transcription regulatory region, eg, a cytosine base region 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). Abnormal methylation of CpG islands, which are normally unmethylated in the promoter region of tumor suppressor genes, is associated with malignant transformation and transcriptional silencing of genes that play an important role in tumor progression (Baylin SB, Herman JG, GraV JR, Vertino PM, Issa JP.Alterations in DNA methylation: a fundamental aspect of neoplasia.Adv Cancer Res 1998; 72: 141-6, Jones PA, Baylin SB.The epigenomics of cancer.Cell 2007; 128 : 683-92). 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, and there is a need for continuous development of markers showing specific DNA methylation tendencies.

In the past, hypermethylation of homeobox genes in lung cancer has been reported in several groups. Shiraishi et al (Shiraishi M, Sekiguchi A, Oates AJ, Terry MJ, Miyamoto Y. HOX gene clusters are hotspots of de novo methylation in CpG islands of human lung adenocarcinomas.Oncogene 2002; 21: 3659-62) Hypermethylation of CpG islands was found in the promoter regions of the HOXA4, HOXA7, HOSD11, and HOXD13 genes in human lung adenocarcinoma). And Rauch et al (Rauch T, Wang Z, Zhang X, et al. Homeobox gene methylation in lung cancer studied by genome-wide analysis with a microarray-based methylated CpG island recovery assay.Proc Natl Acad Sci USA 2007; 104: 55 We also identified frequent hypermethylation of CpG islands in the promoter regions of HOXA5 and HOXA9 in early squamous cell carcinomas. However, such hypermethylation is still insufficient to be used for the diagnosis of lung cancer, and the development of more accurate markers is urgently needed.

Accordingly, the present inventors have completed the present invention by revealing a lung cancer-specific methylation tendency in the HOXA11 gene and finding lung cancer specific by measuring the methylation level.

An object of the present invention is to provide a composition for diagnosing lung cancer comprising an agent for measuring the methylation level of the HOXA11 gene.

An object of the present invention is a primer pair comprising a nucleotide represented by SEQ ID NO: 1 and SEQ ID NO: 2 specific to the methylated sequence of the HOXA11 gene, and SEQ ID NO: 3 and specific to the unmethylated sequence of the HOXA11 gene It is to provide a primer pair comprising a nucleotide represented by SEQ ID NO: 4.

Measuring the methylation level of the HOXA11 gene from a biological sample of a patient suspected of lung cancer; And comparing the methylation level with the corresponding gene methylation level of the normal control sample.

As described above, the methylation of the HOXA11 gene of the present invention affects the expression of HOXA11 in lung cancer cells, and the methylation of the HOXA11 gene of the present invention appears specifically in lung cancer cells. The composition containing the agent to measure can be usefully used for diagnosing lung cancer.

1 shows the methylation status of HOXA11 in vitro. A. Two-way cluster analysis shows the methylation status of HOXA11 in six lung cancer cell lines (H23, H226, H460, H520 and A549) and normal cell lines (HDF). The degree of methylation was expressed as a color change in successive grades of red (0% methylation) to light yellow (100% methylation). The X axis represents the CpG region and the Y axis represents the cell line. B. The methylation status of specific CpG regions is shown in epigrams. C. Specified methylation status of specific CpG regions.

2 shows expression analysis of mRNA of HOXA11 in vitro. MRNA levels of HOXA11 were analyzed by RT-PCR. mRNA levels were significantly deregulated in normal lung cells in six lung cancer cell lines.

Figure 3 shows the expression analysis of HOXA11 mRNA in vitro. MRNA levels of HOXA11 were analyzed by quantitative real-time PCR. mRNA levels were significantly deregulated in normal lung cells in six lung cancer cell lines.

4 shows the effect of 5-aza-dC on hypermethylation and re-expression of silent HOXA11. Reexpression of silenced HOXA11 was examined by RT-PCR in six lung cancer cell lines after cells were treated with 5-aza-dC for 72 hours. Compared with other cell lines, re-expression of HOXA11 was very small in H226 and A549 cells.

5 shows the effect of 5-aza-dC on hypermethylation and re-expression of silent HOXA11. Reexpression of silenced HOXA11 was examined by quantitative real-time PCR in six lung cancer cell lines after cells were treated with 5-aza-dC for 72 hours. Compared with other cell lines, re-expression of HOXA11 was very small in H226 and A549 cells.

6 shows the effect of 5-aza-dC on hypermethylation and re-expression of silent HOXA11. The methylation status of the HOXA11 gene was also determined after treatment with 5-aza-dC for 72 hours. Heatmaps show an unusual pattern of hypermethylation, with the extent of demethylation being pronounced in H460 cells with low density methylated HOXA11 and insignificant in A549 cells with high density methylated HOXA11.

FIG. 7 shows the effect of 5-aza-dC and / or TSA on hypermethylation and re-expression of silent HOXA11. H226 and A549 cells, which showed very little re-expression of HOXA11, were treated with 10 M 5-aza-dC with 0.5 M TSA for 72 hours. TSA induced additional expression of HOXA11 with RT-PCR.

FIG. 8 shows the effect of 5-aza-dC and / or TSA on hypermethylation and re-expression of silent HOXA11. H226 and A549 cells, which showed very little re-expression of HOXA11, were treated with 10 M 5-aza-dC with 0.5 M TSA for 72 hours. TSA induced re-expression of additional HOXA11 by real-time PCR.

9 shows a promoter analysis of HOXA11. After the treatment of SssI methylase (methylase), the change in the base of cytosine (cytosine) in the CpG dinucleotide (CpG dinucleotide) was confirmed by bisulfite sequencing (bisulfite sequencing). Cytokines methylated by SssI are resistant to bisulfite mutations. U represents unmethylated bases, respectively, methylated sequences.

10 shows a promoter analysis of HOXA11. Relative luminase activity of the four HOXA11 promoter structures was measured. Luminase activity was significantly lower in structures with methylated promoter sequences (blue box) than in structures with unmethylated promoter sequences (red box).

11 shows HOXA11 hypermethylation analysis. Promoter hypermethylation of HOXA11 was analyzed in paraffin embedded tissues obtained from 354 NSCLC patients using MSP. The frequency of HOXA11 hypermethylation was closely related to the pT grade (P = 0.02) rather than the pathologic stage (P = 0.42). pT2 grade patients were divided into four groups. HOXA11 hypermethylation was more frequent in patients with tumors larger than 3 cm in size or with abnormal patterns (P = 0.004) than patients with pT1 grade. Asterisk (*) indicates no statistical difference in the frequency of HOXA11 hypermethylation compared to pT1.

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 HOXA11 gene, specifically the lung cancer is non-small cell lung cancer.

 In the present invention, the term "methylation" refers to a methyl group attached to the base changes the expression pattern of the gene. Methylation in the present invention occurs in the HOXA11 gene. Methylation of the present invention specifically includes the continuous presence of C and G bases in the nucleotide sequence of the HOXA11 gene, which occurs in the cytosine of CpG islands in which CpG is concentrated, thereby impairing the binding of transcription factors. Such that expression of a particular gene is blocked.

As used herein, the term "measurement of methylation level" measures the degree of methylation of a nucleic acid sequence. In particular, it is important to measure the methylation level of the HOXA11 gene in the present invention. Measurement of the methylation level can be used without limitation, known methods for measuring the methylation level in the art, for example, Goldengate Methylation Cancer Panel I microarray, EpiTYPER ^TM analysis, methylation-specific polymerase chain reaction (PCR), In the following, it may be examined by a method such as MSP) or automatic base analysis. In normal lung tissue cells, the methylation status of the HOXA11 gene does not appear, but in non-small cell lung cancer tissue cells, the methylation in the HOXA11 gene is specific, indicating that low levels of the HOXA11 gene are expressed. Diagnosis can be made.

The present invention relates to a lung cancer diagnostic composition comprising an agent for measuring the methylation level of the CpG island of the HOXA11 gene promoter.

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 transposons and repetitive sequences 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).

Increased efficiency of DNA methyltransferase is one of the leading factors responsible for making it easier to cause abnormal methylation of CpG islands in the promoter region of cancer suppressor genes.

As used herein, the term "HOXA11" is a gene encoding a class of transcriptional regulators called homeobox genes, and the homeobox gene consists of complexes named A, B, C and D on four separate chromosomes. (Krumlauf R. Hox genes in vertebrate development. Cell 1994; 78: 191-201). The HOXA11 gene is a class A of chromosome 7. The HOXA complex contains 12 genes (11 HOX genes and EVX1), located in the 155-kb-long genomic region on the 7p15-7p14.2 chromosome (Rauch T, Wang Z, Zhang X) , et al. Homeobox gene methylation in lung cancer studied by genome-wide analysis with a microarray-based methylated CpG island recovery assay.Proc Natl Acad Sci USA 2007; 104: 5527-32). Most HOXA promoters contain highly dense CpG islands, whose methylation is associated with control of HOXA gene expression. Homeobox is a sequence of about 180 nucleotides that encodes a homeodomain that can sequence-specifically bind DNA and is known to be present in all eukaryotes (Abate-Shen C. Deregulated homeobox gene expression in cancer: cause or consequence Nat Rev Cancer 2002; 2: 777-85). The human genome is estimated to have at least 100 homeobox genes, including the HOX family (Tupler R, Perini G, Green MR. Expressing the human genome. Nature 2001; 409: 832-33). Homeobox genes encode transcription factors that play an important role in embryo growth and adult cell differentiation. HOX genes are expressed ubiquitous and have a well characterized role in embryonic growth, which determines the identity of the embryonic region along the anteroposterior body axis (Daftary GS, Taylor HS.Endocrine regulation of HOX genes.Endocr Rev 2006; 27: 331-55.

In addition, HOX genes are known to play an important role in lung growth and expression in normal adult lungs (Golpon HA, Geraci MW, Moore MD, et al. HOX genes in human lung: altered expression in primary pulmonary hypertension and emphysema. J Pathol 2001; 158: 955-66). Altered expression of HOX genes has been linked to tumorigenesis and tumor suppression, resulting in a variety of hematological malignancies and solid tumors, some of which show up-regulation and others that show down-regulation. Shah N, Sukumar S. The Hox genes and their roles in oncogenesis.Nat Rev Cancer 2010; 10: 361-71). Dysregulated expression of HOX genes is found in a tissue-specific manner in lung cancer. For example, the expression levels of HOXA1, A5, A10, and C6 were highly unregulated in squamous cell carcinoma, and the expression levels of HOXA5 and A10 were adenomeric in adenocarcinoma (Abe M, Hamada J, Takahashi O, et al. Disordered expression of HOX genes in human non-small cell lung cancer.Oncol Rep 2006; 15: 797-802).

The present inventors investigated the hypermethylation status of the HOXA11 gene in non-small cell lung cancer and correlated the results with the clinicopathologic features of lung cancer patients. Therefore, the present inventors found that lung cancer can be diagnosed by measuring the methylation level of the HOXA11 gene. there was.

In a specific embodiment of the present invention, it was performed using the Goldengate Methylation Cancer Panel I microarray and EpiTYPER ^™ assay to investigate the methylation (FIGS. 1 to 3). As a result, hypermethylation was observed in most lung cancer cell lines. In addition, down-regulation of gene expression in lung cancer cell lines was observed using RT-PCR and quantitative real-time PCR to investigate HOXA11 expression associated with methylation. That is, in the non-small cell lung cancer tissue cells, methylation in the promoter of the HOXA11 gene was specifically observed, indicating that low-level expression of the HOXA11 gene was observed.

In addition, in order to confirm whether the expression of HOXA11 was caused by demethylation of HOXA11, 5-methyl-aza-dC treatment was performed to demethylate and analyzed for re-expression using RT-PCR, quantitative real-time PCR, and EpiTYPER ^™ assay. As a result, an increase in mRNA level was observed (FIGS. 4-8). That is, it was confirmed that expression is down-regulated by methylation in the promoter of the HOXA11 gene.

In addition, to investigate whether hypermethylation inhibits transcription, the activity of the promoter that caused hypermethylation by treatment with SSI methyl transferase was measured and the promoter activity of the methylated structures was significantly lower compared to unmethylated structures (FIG. 9 and FIG. 10). And as a result of performing MSP to investigate the clinicopathological characteristics, hypermethylation was found to be higher in squamous cell carcinoma than adenocarcinoma, and hypermethylation was more frequently observed in Tp2 grade (FIG. 11).

In the present invention, histone deacetylase inhibition by TSA promotes transcriptional reactivation induced by 5-Aza-dC (FIG. 3). Methylation and histone deacetylation are known to work complementarily to maintain and establish inhibition of chromatin status and gene transcription silencing. In the present invention, six lung cancer cell lines were investigated for hypermethylation in response to small amounts of 5-Aza-dC and its effect on the re-expression of HOXA11. Specifically, gene inactivation was found in most cell lines except H226 and A549. As it is known that histone deacetylase inhibition by TSA promotes 5-Aza-dC-induced transcriptional reactivation, TSA treatment is a real-time PCR in H226 cells using RT-PCR in A549 cells. Using the method, we slightly increased the re-expression of the HOXA11 gene to detectable. This observation is in line with previous reports that DNA methylation for histone deacetyl activity may be a potent component for maintaining gene silencing associated with hypermethylation of CpG islands (Cameron EE, Bachman KE, Myohanen S). , Herman JG, Baylin SB.Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer.Nat Genet 1999; 21: 103-7.). Slight reactivation of HOXA11 in response to 5-Aza-dC and TSA in H226 cells also suggests that another type of deacetylation inhibitor (HDACI) is required for re-expression of HOXA11 in H226 cells.

As used herein, the term "diagnosis" refers to 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 preferably be non-small cell lung cancer (NSCLC), and the non-small cell lung cancer includes squamous cell carcinoma, adenocarcinoma, large cell cancer, or linear squamous cell carcinoma. More preferably, it may be squamous carcinoma.

According to a specific example of the present invention, it was observed that HOXA11 methylation occurs 1.76 times more in the pT2 grade than in the pT1 grade (Table 1). In the present invention, the pT2 grade is classified and defined by the following four elements. (1) maximum size of 3 cm or more, (2) visceral pleura invasion, (3) entire lungs, such as pneumonitis, which expands the atelectasis or vascular umbilical cord (hilum) (4) Proximity to the main bronchus is at least 2 cm away from the carina. Specifically, 118 of the 239 tumors classified as pT2 grade had a maximum size of 3 cm or more regardless of other factors, and 66 tumors showed lateral pleural infiltration regardless of tumor size. Eleven tumors were adjacent to the main bronchus regardless of tumor size, and six tumors were associated with three or more factors. In other words, the incidence of HOXA11 hypermethylation was higher in pT2 grades with tumor size of 3 cm or greater or in obstructive pattern compared to pT2 grades showing lateral pleural infiltration or adjacent to main bronchus. In addition, tumors with HOXA11 hypermethylation showed significantly higher Ki-67 levels when compared to those without HOXA11 hypermethylation (26 ± 18 vs. 19 ± 24; P = 0.02). These observations suggest that HOXA11 hypermethylation may be associated with cell proliferation or disruptive pneumopathy in NSCLC.

In the present invention, an agent for measuring the methylation level of a gene may comprise a compound or methylation sensitive restriction enzyme that modifies an unmethylated cytosine base, a primer specific for the methylated sequence of the HOXA11 gene and a primer specific for the unmethylated sequence. Can be.

The compound that modifies the unmethylated cytosine base may be bisulfite, but is not limited thereto, and is preferably sodium bisulfite. The use of such bisulfites to modify unmethylated cytosine residues to detect whether the promoter is methylated is well known in the art.

In addition, the methylation-sensitive restriction enzyme is a restriction enzyme capable of specifically detecting the methylation of CpG islands, preferably a restriction enzyme containing CG as a recognition site of the restriction enzyme. 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 of the HOXA11 gene in patients suspected of lung cancer may be obtained by obtaining genomic DNA from the patient's biological sample and treating the obtained DNA with a compound or methylation sensitive restriction enzyme that modifies unmethylated cytosine bases. 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 HOXA11 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. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers 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 pair comprises a nucleotide represented by SEQ ID NO: 1 and SEQ ID NO: 2 specific for the methylated sequence of the HOXA11 gene, and a primer specific for the unmethylated sequence of the HOXA11 gene is SEQ ID NO: 3 And it may be a primer pair comprising a nucleotide represented by SEQ ID NO: 4.

In addition to the above preparations, the composition for diagnosing or predicting lung cancer of lung cancer may further include a polymerase, agarose, a buffer solution for electrophoresis, and the like.

As another aspect, the invention provides a primer pair comprising a nucleotide represented by SEQ ID NO: 1 and SEQ ID NO: 2 specific to the methylated sequence of the HOXA11 gene, and a sequence specific to the unmethylated sequence of the HOXA11 gene It relates to a primer pair comprising a nucleotide represented by SEQ ID NO: 3 and SEQ ID NO: 4.

In another aspect, the present invention provides a method for treating a cancer, comprising measuring the methylation level of the HOXA11 gene from a biological sample of a patient suspected of lung cancer; And comparing the methylation level with the methylation level of the gene of the normal control sample. Preferably, the lung cancer is non-small cell lung cancer, more preferably squamous cell carcinoma.

Measuring the methylation level of the HOXA11 gene relates to a method for providing information for diagnosing lung cancer comprising measuring the mRNA level of the HOXA11 gene by RT-PCR or real-time PCR. Specifically, the expression level of HOXA11 is normalized to GAPDH, wherein the HOXA11 primer used is one pair of SEQ ID NO: 5 and SEQ ID NO: 6, and the GAPDH primer may be a pair of SEQ ID NO: 7 and SEQ ID NO: 8.

Measuring the methylation level of the HOXA11 gene includes measuring the methylation level of the CpG island of the HOXA11 gene promoter.

As used herein, the term “biological sample” includes, but is not limited to, samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, or urine, which differ in methylation levels of the HOXA11 gene due to the development of lung cancer. Preferably, the biological sample of the present invention may be tissue of the patient.

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 separation (Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980) or commercially available DNA extraction kits. In the present invention, it was extracted using MagAttract DNA Blood M48 kit (Qiagen, Hilden, Germany), QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), and DNeasy Tissue kit (Qiagen, Valencia, CA).

 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 HOXA11 gene.

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 unmethylated 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) is a restriction enzyme that can specifically detect methylation of CpG island, as described above, and preferably a restriction enzyme containing CG as a recognition site of restriction enzyme. For example, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, NotI, and the like, but are 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 HOXA11 gene is a primer consisting of a pair of sequences of SEQ ID NO: 1 and SEQ ID NO: 2, the primer specific for the unmethylated allele sequence of the HOXA11 gene is SEQ ID NO: It may be a primer consisting of a pair of 3 and SEQ ID NO: 4.

 Measuring the methylation level of the HOXA11 gene 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 genomic sequencing method is used to amplify all sites of the CpG island of the HOXA11 gene by PCR and analyze the sequencing of the amplified sites for methylation. You can judge.

 In addition, even when the restriction enzyme is used, if a PCR result is found in a method known in the art, for example, mock DNA and a PCR result is shown in the mock DNA, it is determined that the promoter is methylated, and 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 HOXA11 gene promoter is methylated and diagnose the non-small cell lung cancer.

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: Cell Culture

Six human lung cancer cell lines (H23, H226, H460, H520, H1650, A549) and one normal cell line (HDF, human skin fibroblasts) were obtained from the American Type Culture Collection (Manassa, VA). Lung cancer cells and the normal cells, RPMI1640 (Cellgro ^ⓡ Mediatech, Herndon) with DMEM raise respectively from (WelGENE, KOREA), 10% heat-deactivated (heat-inactivated) fetal calf serum (Hyclone, Logan, UT) and 1% antibiotic Antimycotic (Gibco, New York, NY). And incubated at 37˚C in 5% carbon dioxide (CO ₂ ) state.

Example 2: TSA and 5-Aza-dC Treatment of Cells

Cells were incubated with 10 μM of 5-Aza-2'-deoxycytidine (5-Aza-dC; Sigma Aldrich, St. Louis, MO) for a total of 72 hours, or 0.5 μM trichostatin A (TSA) for the last 24 hours of 72 hours. ) Were put together and incubated. Cells were harvested and washed with ice-cold PBS. Then, genomic DNA and mRNA were separated, the methylation status was analyzed by EpiTYPER ^™ assay, and the expression level was analyzed by RT-PCR and quantitative real-time PCR.

Example 3: Tissue Sample Preparation

 97 rapid freezing tumors and their corresponding normal tissues and 354 paraffin-embedded tumor tissues were treated between August 1994 and November 2005 at the Department of Thoracic and Cardiovascular Surgery, Samsung. Medical Center, Seoul, Korea) from 451 NSCLC patients with experience of therapeutic resection. Written informed consent for the use of tumor samples was received from all participants. Information on patient demographics was obtained through interviewer-run surveys. The average duration of follow-up after therapeutic resection was 4.9 years. Staging was determined through the guidelines of the American Joint Committee on Cancer (AJCC).

Example 4 Genomic DNA Extraction and Sodium Bisulfite Mutation

 Genomic DNA obtained from cultured cells, paraffin-embedded tissues, and fast-freezing tumors and their corresponding normal tissues were determined by MagAttract DNA Blood M48 kit (Qiagen, Hilden, Germany), QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), and DNeasy Tissue kit (Qiagen, Valencia, CA) using the instructions according to the instructions. One microgram of genomic DNA was mutated by sodium bisulfite using the EZ DNA Methylation-Gold Kit (ZYMO Research, Orange, Calif.) According to the instructions.

Example 5 Genome Whole Methylation Analysis Using Microarrays

 To find candidate genes involved in the development of NSCLC, the methylation status of 97 rapid freeze tumors and their corresponding normal tissues was determined according to conventional methods (Bibikova M, Lin Z, Zhou L, et al. High-throughput DNA methylation). profiling using universal bead arrays.Genome Res 2006; 16: 383-93), and Illumina's Golden Gate Methylation Cancer Panel I.

Example 6 Quantitative Analysis of HOXA11 Hypermethylation Using EpiTYPER

For validation of microarray data, six lung cancer cell lines were quantitatively analyzed for methylation at 90 CpG on a promoter of HOXA11 using EpiTYPER ^™ assay (Sequenom, San Diego, Calif.). Primers were designed using EpiDesigner software. After PCR amplification, amplified fragments (amplicons) transferred in vitro were treated with shrimp alkaline phosphatase, cut with RNaseA, and placed in MALDI-TOF Mass Spectrometry to determine the methylation status. The result is EpiTYPER ^TM ver. The analysis was performed using 1.0 software.

Example 7: Methylation-specific Polymerase Chain Reaction (MSP) Assay

 The methylation status of the HOXA11 gene was determined in the genome with sodium bisulfite, followed by polymerase chain reaction PCR specific for methylation using primers specific for methylation and unmethylated alleles of each gene.

 PCR conditions and compositions of the cell lines are shown in Table 1 below, and PCR conditions and compositions of the tissue samples are shown in Table 2 below.

Table 1 PCR conditions Initial denaturation 94 ℃ 15 mins Denaturation 94 ℃ 30 sec Anealling 55 ℃ 30 sec Extension 72 ℃ 1 min Final extension 72 ℃ 7 minutes Step 2 ~ 4: 35 cycles PCR composition Hotstar 10X Buffer (Qiagen) final 1 X 10 mM dNTP Mix (Intron) final 0.1 mM Primer final 100 nM each Hotstar Taq enzyme 0.05 U / 15ul reaction Bisulfite treated gDNA 10 ng

TABLE 2 PCR conditions Initial denaturation 94 ℃ 10 minutes Denaturation 94 ℃ 30 sec Anealling 60 ℃ 30 sec Extension 72 ℃ 30 sec Final extension 72 ℃ 7 minutes Step 2 ~ 4: 35 cycles PCR composition 2X HS prime Taq premix (GENET BIO) final 1 X Bisulfite treated gDNA 10 ng Primer final 1uM each

The methylation status of the HOXA11 gene in tissues embedded in 354 formalin-fixed paraffins was determined using MSP, with two sets of primers. Described by Herman et al (Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB.Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA 1996; 93: 9821-6) As shown, one of the two primers is for the unmethylated promoter and the other is for the methylated promoter. Sequences related to amplification of methylated HOXA11 were 5'-GTTTAGGGTAGGGGGTTTTC-3 '(sense; SEQ ID NO: 1) and 5'-CAATCTTTCCCGACTACGAC-3' (antisense; SEQ ID NO: 2), and related to amplification of unmethylated HOXA11 Primer sequences were 5′-GGTTTAGGGTAGGGGGTTTTT-3 ′ (sense; SEQ ID NO: 3) and 5′-CCCAATCTTTCCCAACTACAAC-3 ′ (antisense; SEQ ID NO: 4). All PCR reactions were performed at least twice.

Example 8: Analysis of HOXA11 Expression

MRNA levels of HOXA11 were analyzed using RT-PCR and quantitative real-time PCR. Total RNA was extracted from cells cultured using TRIzol® Reagent (Invitrogen, Carlsbad, CA), and cDNA was used using SuperScript ^TM III First-Strand Synthesis System used with RT-PCR (Invitrogen) kit. Synthesis was according to instructions. RT-PCR was tested using a one step RT-PCR kit (Qiagen, Valencia, CA) according to the instructions for use in a tube containing 0.5 μg of total RNA and HOXA11 specific primers at a final concentration of 0.6 μM. PCR results were assessed with GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA). The expression level of HOXA11 was normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer pairs used are as follows. HOXA11, 5'-CAGCAGAGGAGAAAGAGCGG-3 '(sense; SEQ ID NO: 5) and 5'-TGCAGGCGCTTCTCTTTGTTA-3'(antisense; SEQ ID NO: 6); GAPDH, 5-'TGCACCACCAACTGCTTA-3 '(sense; SEQ ID NO: 7) and 5'-GGATGCAGGGATGATGTTC-3'(antisense; SEQ ID NO: 8). Quantitative real-time PCR was performed with the SYBR Green PCR Master Mix (QIAGEN, Germany) on the LightCycler®480 Real-Time PCR System (Roche, Germany). Quantitative PCRs were run three times for each sample primer set and the average of the three experiments was used as relative quantitative value.

Example 9 In Vitro Methylation Analysis by Luciferase Assay

  PCR of four regions on the promoter of HOXA11 (-779--1, -576--1, -309--1, and -191--1) with KPNI (5 ') and HindIII (3') sequences The primers were amplified (Table 3) and replicated with pGL3-based luciferase constructs (Promega, Madison, Wis.).

TABLE 3 Primer ID Sequence HOXA11-promoter Forward 1 (-779) CCC GGTACC GAAGGCACTAAAGCGCTTCG (SEQ ID NO: 9) ( KPNⅠ ) HOXA11-promoter Forward 2 (-779) CCC GGTACC ACTCCAGGAGAACAGACTCC (SEQ ID NO: 10) HOXA11-promoter Forward 3 (-779) CCC GGTACC TCGGTGAGAACACCGAGTGA (SEQ ID NO: 11) HOXA11-promoter Forward 4 (-779) CCC GGTACC GGACTAGCTAGCAGCTTGTC (SEQ ID NO: 12) HOXA11-promoter Reverse CCC AAGCTT TATTGGGCTACCTTGGGCTC (SEQ ID NO: 13) ( Hind III )

In each structure, 20 μg plasmid DNA was cut with KPNI and Hind III and the inserts were separated using gel electrophoresis. The isolated inserts were methylated in vitro by CpG-specific SssI methylase (New England Biolabs, Ipswich, Mass.) And cloned back into pGL3-based structures. H23 cells were transfected into the structure using Lipofectamine 2000 (Invitrogen). A pGL3-based vector containing firefly luciferase was transfected with 10 ng of the pRL-CMV vector containing Renilla luminase as an internal regulator for transfer efficiency. Luminase activity was observed 24 hours after transfection.

Example 10 Statistical Analysis

  Wilcoxon rank sum test (or t-test) and Fisher's exact test (or the Chi-squared test) were used for the univariate analysis of continuous and assertive variables, respectively. Multivariate logistic regression analysis was performed to determine the relationship between HOXA11 hypermethylation and covariates found statistically significant in univariate analysis, and to calculate odds ratios (ORs). . All statistics were two-sided with a Type I error rate of 5%. The effect of HOXA11 methylation at relapse or death was measured by Kaplan-Meier survival curves, and noticeable differences in prognosis between the two groups were measured by log-rank test.

result

Promoter Hypermethylation and Inhibition of HOXA11 in Lung Cancer Cell Lines

 To find epigenetic markers associated with lung cancer development, we analyzed methylation status using GoldenGate Methylation Cancer Panel I (Illumina) microarrays in rapid freezing tumors from 97 NSCLC patients and corresponding normal tissues. As a result, 63 of them (67%) found HOXA11 hypermethylation.

Methylation status of HOXA11 was confirmed using EpiTYPER ^™ Assay in six lung cancer cell lines (NCI-H23, NCI-H226, NCI-H460, NCI-H520, NCI-H1650 and A549) and one normal cell line (HDF). The promoter sequence of HOXA11 was searched using the Transcriptional Regulatory Element Database program (http://rulai.cshl.edu/cgibin/TRED/tred.cgi?process=searchOrthForm) and 1.8 from the start of transcription. The methylation status of 90 CpGs in the promoter region including kb upstream was quantitatively analyzed using EpiTYPER ^™ .

Although partially unmethylated in several CpG regions of H23, H520 and H1650, most of them were highly methylated in six lung cancer cell lines (FIG. 1A). H460 cells were found to be less methylated than other cell lines in the proximal region (dotted box), the starting point for transcription. Two epigrams and spectra (FIGS. 1B and 1C) showed a low rate of HOXA11 hypermethylation in H460 cells compared to other cell lines. HOXA11 expression was analyzed using RT-PCR (FIG. 2) and quantitative real-time PCR, which was deeply associated with this methylation state. MRNA levels in six lung cancer cell lines were down regulated compared to HDF normal cells, except for weak expression in H460 (FIGS. 2 and 3). These results suggest that HOXA11 hypermethylation may be responsible for the transcriptional deregulation of HOXA11.

Re-expression of 5-Aza-dC derived methylated and silenced HOXA11

To confirm that the expression of HOXA11 is due to the demethylation of HOXA11, to verify that the expression of HOXA11 is important in inhibiting methylation and inducing demethylation, the demethylation of the HOXA11 promoter plays an important role in the expression of HOXA11. We treated the lung cancer cell line with 10 μM 5-aza-dC for 72 hours, followed by RT-PCR (FIG. 4), quantitative real-time PCR (FIG. 5) for demethylation and re-expression of silent HOXA11. , And EpiTYPER ^™ assay (FIG. 6). Treatment of 5-aza-dC on H123, H460, H520 and H1650 cells resulted in a significant increase in mRNA levels (FIGS. 4 and 5). Demethylation was found in all cell lines after treatment, and FIG. 6 shows a typical aspect of demethylation. Demethylation was more evident in H460 with CpGs methylated at a lower density than A549 cells with densely methylated CpGs in the cells. Reexpression of silenced HOXA11 in response to 5-Aza-dC was insignificant in H226 and A549 cells (FIG. 4). Since histone deacetylase inhibitors are known to assist in the re-expression of hypermethylated hereds, these cells were initially treated with 5-aza-dC for 48 hours and then with 0.5 μM TSA. Incubation was further performed with -aza-dC for 24 hours at the mRNA level by RT-PCR (FIG. 7) and real-time PCR (FIG. 8) using 5-aza-dC and TSA on H226 cells and A549 cells. Reactivation of silenced HOXA11 was induced.

Reporter assay of the HOXA11 promoter

  To determine whether HOXA11 hypermethylation affects gene transcription, promoter activity was measured by a Dual Luciferase assay. Promoter sequences were obtained from TESS (http://www.cbil.upenn.edu/cgi-bin/tess/tess), which sequences included binding regions for TFII, SP1, and GATA. Four structures (-779--1, -576--1, -309--1, -191--1) for each methylated and unmethylated promoter were made, and their promoter activity was determined by Analyzes by measuring luminescence (RLU). The methylated promoter was obtained by hypermethylation of HOXA11 using SSI methyltransferase (New England Biolabs, Inc., Beverly, Mass.) In the presence of S-adenosylmethionine. Sequences modified by treatment of SSI methyltransferase were confirmed by sodium bisulfite sequencing (FIG. 9). However, the promoter activity of the methylated structures appeared to be significantly lower compared to the corresponding unmethylated structures (FIG. 10). The results demonstrate that promoter hypermethylation of HOXA11 is closely related to downregulation of HOXA11.

Clinical Significance of HOXA11 Hypermethylation in Early NSCLCs

   To understand the clinicopathological significance of HOXA11 hypermethylation, we re-analyzed the methylation status of the HOXA11 gene using methylation-specific PCR (MSP) in tissues embedded with 354 formalin-fixed paraffins (FIG. 11). HOXA11 hypermethylation was found in 269, 76% of the 354 patients studied. HOXA11 hypermethylation was not associated with age, sex, smoking, differentiation, and tumor recurrence in patients.

 However, HOXA11 hypermethylation was found to be higher in squamous cell carcinoma than in adenocarcinoma. Specifically, HOXA11 hypermethylation was seen in 149, 81% of 185 squamous cell carcinomas, and in 96, 70% of 137 adenocarcinomas.

 Moreover, HOXA11 hypermethylation was more closely associated with grade of tumor than with stage of tumor (FIG. 11). HOXA11 hypermethylation was diagnosed as 45 of 63% of 71 patients with pT1 grade, 185 of 77% of 239 persons with pT2 grade, 19 of 90% of 21 persons with pT3 grade, and pT4 grade. It was found in 21 patients (85% of 23 patients) (P = 0.02). In addition, 239 tumors were separated into four groups based on the pT2 grading criteria and then compared the frequency of HOXA11 hypermethylation between pT2 grading tumors. HOXA11 hypermethylation was more frequent in patients with tumors with a maximum size of 3 cm or more (P = 0.02) or patients with obstruction pattern (P = 0.04) than patients with a pT1 grade (FIG. 11). That is, HOXA11 methylation was found in 38, 88% of 43 tumors with disturbance patterns, and 153 of 77% of 198 tumors were larger than 3 cm in diameter.

 Multivariate logistic regression analysis was performed to control the potential confounding effects of variability factors such as age and gender, and to calculate ORs (Table 4).

Table 4 HR ratio 95% confidence interval P-value Histology Adenocarcinoma 1.00 Squamous cell carcinoma 1.48 1.08-2.02 0.01 pT1 1.00 pT2 1.76 1.21-2.56 0.03 pT3 1.12 0.60-2.09 0.72 pT4 1.27 0.68-2.34 0.46

Age, gender control

 HOXA11 hypermethylation was 1.48 times higher in squamous cell carcinoma than adenocarcinoma (95% CI = 1.08-2.02; P = 0.01). Moreover, HOXA11 hypermethylation was observed 1.76 times more frequently in the pT2 grade than in the pT1 grade (95% CI = 1.21-2.56; P = 0.003). Finally, the relationship between HOXA11 hypermethylation and patient survival was examined. Since the tumor stage of the disease in NSCLC is closely related to the survival rate of the patient, the data were separated according to the tumor stage.

Claims (13)

Lung cancer diagnostic composition comprising an agent for measuring the methylation level of the HOXA11 gene. The method of claim 1, wherein the agent for measuring the methylation level of the gene comprises a compound that modifies an unmethylated cytosine base or a methylation sensitive enzyme, a primer specific for the methylated sequence of the HOXA11 gene, and a primer specific for the unmethylated sequence. Composition comprising. The composition of claim 1, wherein the lung cancer is non-small cell lung cancer. The composition of claim 3, wherein the non-small cell lung cancer is squamous cell carcinoma. The primer of claim 2, wherein the primer specific for the methylated sequence of the HOXA11 gene is a pair of primers including a nucleotide represented by SEQ ID NO: 1 and SEQ ID NO: 2, and specific for the unmethylated sequence of the HOXA11 gene. A primer is a pair of primers comprising a nucleotide represented by SEQ ID NO: 3 and SEQ ID NO: 4. The composition of claim 2, wherein the compound that modifies the unmethylated cytosine base is sodium bisulfite. A pair of primers comprising a nucleotide represented by SEQ ID NO: 1 and SEQ ID NO: 2 specific for the methylated sequence of the HOXA11 gene, and the sequence of SEQ ID NO: 3 and SEQ ID NO: 4 specific for the unmethylated sequence of the HOXA11 gene Primer pairs comprising nucleotides represented by numbers. Measuring the methylation level of the HOXA11 gene from a biological sample of a patient suspected of lung cancer; And comparing the methylation level with the methylation level of the gene of the normal control sample. The method of claim 8, wherein measuring the methylation level of the gene 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 HOXA11 gene. The method of claim 9, wherein the primer is a methylation-specific primer pair comprising a nucleotide represented by SEQ ID NO: 1 and SEQ ID NO: 2 and a ratio comprising a nucleotide represented by SEQ ID NO: 3 and SEQ ID NO: 4 A methylation specific primer pair. The method of claim 9, wherein the compound that modifies the unmethylated cytosine base is sodium bisulfite, and the method of measuring the methylation level is methylation-specific polymerase chain reastion. The method of claim 8, wherein the lung cancer is non-small cell lung cancer. The method of claim 12, wherein the non-small cell lung cancer is squamous cell carcinoma.

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