JP5044837B2 - Method for detecting esophageal cancer - Google Patents

Description

  The present invention relates to a method for detecting cancer. More specifically, the method for detecting a cancer of the present invention comprises human Cellular Retinoic Acid Binding Protein for early diagnosis of esophageal cancer, and for accurately predicting malignancy and lymph node metastasis of esophageal cancer. This invention is intended to detect inactivation of the 1 (human CRABP1) gene.

  Esophageal carcinoma is an epithelial-derived tumor (cancer) that occurs in the esophagus. Histological classification includes squamous cell carcinoma (Esophageal Squamous-cell Carcinoma) and adenocarcinoma. In the former, mucosal epithelial cells of the esophagus become cancerous, accounting for 90% or more of the total. Japanese account for 93% of all esophageal cancers. In the latter, cells of Barrett's esophagus become cancerous, and when combined, they account for more than 95% of all esophageal cancers. Diagnosis methods include esophageal imaging, endoscopy, ultrasound endoscopy, CT (computer tomography), PET (positron emission tomography), SCC (squamous cell carcinoma associated antigen) and CEA (cancer) Although methods based on tumor markers such as fetal antigen) are known, the accuracy is not necessarily high, and it is difficult to diagnose early esophageal cancer including malignancy. Esophageal cancer is known as a cancer with an extremely poor prognosis and poor response to surgery or radiation / chemotherapy due to the difficulty of early diagnosis. This is because esophageal cancer has many lymph node metastases, and unlike other digestive organs, the esophagus has no serosa (outer membrane), so the cancer is relatively easy to invade surrounding tissues. Is mentioned.

Very recently, it has been reported that reduced expression of Low Density Lipoprotein Receptor-Related Protein 1B (LRP1B) or deletion of the genomic gene can be used for diagnosis of esophageal cancer (Non-patent Document 1).
Sonoda I, Imoto I, Inoue J, Shibata T, Shimada Y, Chin K, Imamura M, Amagasa T, Gray JW, Hirohashi S, and Inazawa J: Frequent Silencing of Low Density Lipoprotein Receptor-Related Protein1B (LRP1B) Expression by Genetic and Epigenetic Mechanisms in Esophaseal Squamous Cell Carcinoma, Cancer Research 64, 3741-3747, 2004

  At present, the technique of Non-Patent Document 1 is publicly known as a genetic test method for esophageal cancer. Furthermore, new genetic testing methods have been found, and establishment of a comprehensive diagnosis method for esophageal cancer using them has been demanded. Under such circumstances, an object of the present invention is to provide a new method for detecting the gene level of esophageal cancer, which can determine even the malignancy of cancer and the possibility of lymph node metastasis.

  In general, in many cases, cancer cells have undergone genomic gene deletion and amplification, suppression of expression due to methylation of CpG islands, suppression of gene expression due to deacetylation of histone proteins, gene point mutations, etc. Has been. It is extremely important to examine chromosomal DNA and gene expression in esophageal cancer cells in detail, search for a group of genes associated with malignancy and early diagnosis of esophageal cancer, and find genes that are decisive for diagnosis. Furthermore, it is considered that a diagnostic method for esophageal cancer can be established by finding a characteristic gene change in esophageal cancer cells and analyzing the correlation between the gene change and the clinicopathological findings of cancer.

  Now, the present inventor can use inactivation of human Cellular Retinoic Acid Binding Protein 1 (human CRABP1) gene as an indicator of early esophageal cancer (esophageal epithelial cell carcinoma), and The present inventors have found that early detection of esophageal cancer, as well as early detection of esophageal cancer and determination of the possibility of lymph node metastasis, have been completed.

  That is, the present invention provides a method for detecting esophageal cancer (hereinafter also referred to as the present detection method), which detects canceration of the esophageal epithelial cells by detecting inactivation of the human CRABP1 gene in esophageal epithelial cells. It is an invention to do.

The present inventors comprehensively analyzed the methylation enhancement in the esophageal cancer cell line on the genome using the BAMCA method. BAMCA method is an abbreviation of B acterial A rtificial chromosome array-based M ethylated C pG island A mplification, is an assay method which can detect methylation in a specific gene. An example of a typical aspect of the BAMCA method is as follows. That is, DNA is prepared from esophageal cancer cells and normal esophageal cells, and each DNA is digested with the restriction enzyme SmaI (methylation sensitive restriction enzyme). In this case, when the SmaI site is methylated, since the methylated site is not digested with SmaI, it is digested with XmaI (methylation-insensitive restriction enzyme) in the next step. XmaI cleavage end-specific adapter ligation is performed, and an XmaI fragment derived from esophageal cancer cells is labeled with the fluorescent dye Cy-3 by PCR and amplified. On the other hand, the XmaI fragment derived from normal esophageal cells is labeled with the fluorescent dye Cy-5. Mix both, perform a number of BAC (B acterial A rtificial C hromosome ) competitive hybridization on high density genome array equipped with DNA (Comparative Genomic Hybridization). As a result, when both proximal SmaI sites are methylated in esophageal cancer cell DNA, an XmaI fragment corresponding to the methylated region is generated, and the ratio of Cy3 / Cy5 increases. For example, using this value of 1.5 or more as an index, a BAC clone containing a sequence that matches a cancer-specific methylated DNA fragment can be identified. This series of BAMCA process is shown in FIG.

  As a result, RP11-10K12 was found as a highly methylated BAC DNA, and as a result of gene analysis contained therein, the human Cellular Retinoic Acid Binding Protein 1 (CRABP1) gene was the target gene for methylation. It was found that esophageal cancer is frequently methylated.

Human CRABP1 is a protein of 16 kDa molecular weight and has the property of binding to retinoic acid with high affinity. Human CRABP1 is known to be a protein that binds to retinoic acid, protects retinoic acid and has a role to transport from the cytoplasm to the nucleus. Retinoic acid in the nucleus binds to RAR (R etinoic A cid R eceptor ) and RXR (R etinoid X R eceptor) , it plays an important role regarding signaling. Human CRABP2 is a protein having high homology with human CRABP1, and both proteins have a homology of 75% at the amino acid level. The nucleotide sequence of the human CRABP1 gene and the amino acid sequence encoded by the sequence are already known [GenBank number: NM_004378 “Homo sapiens cellular retinoic acid binding protein 1 (CRABP1)” (gene): SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2].

[This detection method]
As described above, this detection method is a method characterized by detecting inactivation of the human CRABP1 gene in esophageal epithelial cells.

  The esophageal epithelial cell that is the target for detecting inactivation of the human CRABP1 gene is preferably a biopsy tissue cell of the specimen provider. This sample tissue cell may be a normal human esophageal cell or the cancer tissue of an esophageal cancer patient, but in reality, 1) As a result of endoscopy, canceration of the esophagus is suspected. The main target is the affected tissue when a lesion is found, or 2) an esophageal cancer tissue that has been confirmed to be esophageal cancer but its malignancy and progression must be determined. Can be.

  If inactivation of the human CRABP1 gene is observed in this `` detection of a lesion that is suspected to be cancerous in the esophagus as a result of endoscopy, etc. '' The lesion tissue has progressed toward canceration or is already in the state of canceration, and it has been found that the malignancy is becoming high, and immediate full-scale treatment (removal of the lesioned part by surgery, etc. The need for conventional chemotherapy). In addition, when inactivation of the human CRABP1 gene is observed in “an esophageal cancer tissue that has been confirmed to be esophageal cancer but its malignancy and progression must be determined”, the cancer Tissue malignancy is getting higher and lymph node metastasis is suspected. Therefore, it is indicated that there is a need for immediate full-scale treatment (removal of a lesion site by surgery, lymph node dissection, full-scale chemotherapy, etc.). The esophageal cell tissue collected as a specimen can be subjected to a necessary treatment, for example, DNA or RNA prepared from the collected tissue and subjected to this detection method.

  As described above, this detection method can identify canceration of the cells (particularly, initiation of canceration) by detecting inactivation of the human CRABP1 gene in esophageal cells. The main cause of inactivation of the human CRABP1 gene is methylation of the CpG island of the gene, and other causes include deletion of the gene and a marked decrease in expression. Regarding the inactivation by methylation of CpG island, it has been reported that transcription inactivation occurs when the CpG rich promoter and exon region are methylated closely (Bird, AP & Wolffe, AP: Methylation-induced repression-belts). , braces, and chromatin, Cell 99, 451-454, 1999). In cancer cells, CpG islands are densely methylated more frequently than other regions, and hypermethylation in the promoter region is deeply involved in inactivation of tumor suppressor genes in human cancer (Ehrlich, M., Jiang, G., Fiala, E., Dome, JS, Yu, MC, Long, TI, Youn, B., Sohn, OS, Widschwendter, M., Tomlinson, GE, Chintagumpala, M., Champagne, M., Parham, D., Liang, G., Malik, K. & Laird, PW: Hypomethylation and hypermethylation of DNA in Wilms tumors, Oncogene, 21, 6694-6702, 2002).

  In the present invention, in order to improve the sensitivity and accuracy of the BAMCA method and to comprehensively analyze all the chromosomes, a Comparative Genomic Hybridization (CGH) is performed on the MCG Whole Genome Array-4500, which is a high-density genome array equipped with 4523 types of BAC DNA. To identify the chromosomal region methylated in esophageal cancer cells. Furthermore, as a result of investigating genes localized in the region, the human CRABP1 genomic gene was successfully identified. Furthermore, it was found that the CpG-1 region in the CpG island of the human CRABP1 genomic gene is methylated, thereby suppressing the promoter activity and reducing the expression of the human CRABP1 gene. When the expression of human CRABP1 gene was examined using 43 types of esophageal squamous cell carcinoma cell lines, the expression of human CRABP1 gene was very low or not expressed in 34 types of cell lines. This is presumed to be due to the fact that CpG islands are highly methylated. From these results, it was concluded that inactivation of the human CRABP1 gene was mainly performed by methylation of the CpG island of the genomic gene.

  A typical example of gene inactivation without gene deletion is inactivation due to a decrease in the degree of histone protein acetylation. In human CRABP1 gene in esophageal cancer cells, Such a phenomenon has not been detected.

  Confirmation of gene inactivation without gene deletion can be performed by detecting the presence or absence of human CRABP1 protein in the specimen, ie, increase or decrease in the amount of the gene (gene May be accompanied by a deletion of. A typical method is an immunostaining method. That is, human CRABP1 protein in a specimen (typically a specimen of esophageal cell tissue) is converted into an antibody specific to the protein (monoclonal antibody or polyclonal antibody: known human CRABP1 gene or human CRABP1 protein <usually recombinant Can be prepared by a conventional method using a protein> and is also commercially available) (direct antibody binding method using an antibody against a labeled human CRABP1 protein), or preferably It is possible to detect indirectly (double antibody method or triple antibody method using labeled second or third antibody). Such immunostaining can be performed according to a conventional method.

  Deletion of the human CRABP1 gene is less frequent than CpG island methylation. As typical methods that can directly detect gene deletion, CGH (Comparative Genomic Hybridization) method and FISH method [Fluorescence in situ hybridization (FISH): Yasui, K., Imoto, I., Fukuda, Y., Pimkahaokham, A., Yang, ZQ, Naruto, T., Shimada, Y., Nakamura, Y., And Inazawa: Identification of target genes within an amplicon at 14q12-q13 in esophageal squamous cell carcinomas. Genes Chromosomes Cancer, 32, 112-118, 2001]. In this embodiment, BAC (Bacterial Artificial Chromosome) DNA, YAC (Yeast Artificial Chromosome) DNA having human CRABP1 gene, or PAC (Phage Artificial Chromosome) DNA (hereinafter also referred to as BAC DNA etc.) is labeled and FISH is performed. The deletion part of the human CRABP1 gene can be detected. It is preferable and practical that such a method is performed using a genomic DNA fixing substrate. Since normally obtained BAC DNA and the like are in a small amount for the production and practical use of a large number of genomic DNA fixing substrates, it is necessary to obtain the DNA as a gene amplification product (this gene amplification step is referred to as “infinite storage or inexhaustible”). It is also called “recycling”). In inexhaustible storage, first, BAC DNA or the like is digested with a 4-base recognition enzyme such as RsaI, DpnI, or HaeIII, and then ligated by adding an adapter. The adapter is an oligonucleotide consisting of 10 to 30 bases, preferably 15 to 25 bases. The double strand has a complementary sequence, and after annealing, phosphorylates the 3'-end oligonucleotide that forms a blunt end. There is a need to. Next, the primer having the same sequence part as one of the oligonucleotides of the adapter can be used for amplification by PCR and inexhaustible. On the other hand, an aminated oligonucleotide of 50 to 70 bases having a sequence characteristic to each BAC DNA or the like can be spotted on an array and used on a detection substrate.

  A desired DNA fixing substrate can be produced by fixing the inexhaustible BAC DNA or the like on the substrate, preferably a solid substrate. Examples of the solid substrate include glass, plastic, membrane, and three-dimensional array, and a glass substrate such as a slide glass is preferable. The solid substrate such as glass is more preferably coated by adhesion of poly-L-lysine, aminosilane, gold / aluminum, or the like.

  It is possible to produce a desired DNA fixing substrate by fixing the inexhaustible DNA on the substrate, preferably a solid substrate, by spotting the inexhaustible DNA on the substrate and drying it. it can.

  As one of means for directly detecting the deletion of the human CRABP1 gene, Southern blotting can be mentioned. In Southern blotting, genomic DNA obtained from a specimen is digested with a restriction enzyme, electrophoresed, immobilized on a nitrocellulose membrane, and detected by this to hybridize with the human CRABP1 gene. This is a method for detecting the presence of the gene.

  According to the present invention, it is possible to accurately grasp signs of canceration, cancer progression, and malignancy in esophageal cell specimens.

[Reference Example 1] Fabrication of high-density CGH array loaded with many cancer-related genes
From the results of the BLAST search of the selected DNA and the genome database website of the National Cancer for Biotechnology and the University of California Santa Cruz Biotechnology, 4523 BAC / PAC clones present in the euchromatin region of the human genome were selected.

  BAC and PAC DNA were digested with DpnI, RsaI, and HaeIII, and then ligated with adapter synthesis oligonucleotides. Next, PCR was performed twice using a primer having an adapter sequence. This process is called inexhaustible storage, and the obtained DNA is defined as inexhaustible DNA. Inexhaustible DNA was spotted on the array with Duplicate using an ink jet type spotter (GENESHOT, NGK Insulators, Nagoya) to produce the desired high density CGH array (MCG Whole Genome Array-4500).

[Reference Example 2] Disclosure of primers used in the test The contents of the various gene amplification primers used in the test are disclosed in Table 1 below. The gene amplification step used in the following examples was performed using primers having the contents shown in Table 1 (SEQ ID NOs: 4 to 25).

[Example 1] Status of genomic DNA methylation in esophageal squamous cell carcinoma cells What chromosomal genes are highly methylated in esophageal squamous cell carcinoma cells is determined by the BAMCA method (Bacterial artificial chromosome array-based methylated CpG island amplification). ). Examples of analyzes using the BAMCA method are Misawa A., Inoue J., Sugino Y., Hosoi H., Sugimoto T., Hosoda F., Ohki M., Imoto I., Inazawa J .: Methylation-associated silencing of The nuclear receptor 1I2 gene in advanced-type neuroblastomas, identified by bacterial artificial chromosome array-based methylated CpG island amplification, Cancer Research 65, 10233-10242, 2005. A summary of the analysis using the BAMCA method and some of the results are shown in FIG. 2A.

  TE-4, TE-5, KYSE-150, and KYSE-510 were used as esophageal squamous cell carcinoma cells. Normal esophageal epithelial cell line NEK2 was used as normal esophageal epithelial cells. DNA was prepared from cancer cells and normal cells and analyzed by the BAMCA method using MCG Whole Genome Array-4500 (see FIGS. 2A: 1 and 11). Among the four types of esophageal squamous cell carcinoma cells mentioned above, 61 types of BAC clones corresponding to highly methylated chromosomal DNA having a ratio of Cy3 (cancer cells) / Cy5 (control normal cells) of 1.5 or more are included therein. It contained 105 kinds of genes (see FIG. 2A: 2). Next, for these 105 types of genes, CpG islands and restriction enzyme SmaI recognition sequences (methylation recognition sites in the BAMCA method) are examined in a region close to the transcription start site (http: //genome.ucsc. Search using edu /) and explore expression profiles in many tissues and cancers (http://www.lsbm.org/database/index.html and http://www.ncbi.nih.gov) Searched using /index.html), we selected 59 genes whose expression might be significantly reduced due to high methylation in esophageal squamous cell carcinoma cells (FIG. 2A: 21) 3). Finally, (a) the expression status of each gene, (b) the DNA methylation inhibitor 5-aza-deoxycytidine (abbreviated as 5-aza-dC) and / or the histone deacetylase inhibitor trichostatin A ( Cellular expression in BAC clone RP11-10K12 by analyzing the presence or absence of recovery of gene expression by treatment, and (c) methylation status of CpG island in each gene region in esophageal squamous cell carcinoma cells Retinoic Acid Binding Protein 1 (abbreviated as CRABP1, GenBank number: NM_004378: SEQ ID NO: 1) was found to be a methylated target gene whose gene expression was specifically suppressed in esophageal squamous cell carcinoma cells (FIG. 2A: 31). And 4, see FIG. 2B). FIG. 2B shows typical array data analyzed by the BAMCA method for the esophageal squamous cell carcinoma cell TE-4 cell line. Spots of different colors (originally three colors of green, yellow and red) were each highly methylated in TE-4 cell line compared to normal esophageal cell MEK2, each with the same degree of methylation Shows a genomic DNA fragment that is not methylated. The BAC clone RP11-10K12 indicated by the arrow has the CRABP1 genomic gene region, and all cell lines examined using the BAMCA method (TE-4, TE-5, KYSE-150, KYSE-510). The BAC clone on the array showed a high Cy3 (esophageal cancer specimen) / Cy5 (healthy person specimen) ratio.

In order to confirm that the restriction enzyme SmaI recognition sequence around human CRABP1 is highly methylated in the four types of esophageal squamous cell carcinoma cells used here, bisulfite sequencing analysis (Sonoda I. et. Al., Cancer Research 64 , 3741-3747, 2004) examined methylation status. As a result, it was found that all four types of squamous cell carcinoma cells of the esophagus were highly methylated in the pattern of either the front two or the back two of the three types of SmaI recognition sites (Fig. 2C). FIG. 2C shows the structure of the human CRABP1 genomic gene and the state of methylation at the SmaI site of the predicted promoter region analyzed by the BAMCA method. In FIG. 2C, three types of CpG islands (CpG-1, -2, and -3) are present in the exon 1, exon 2, and intron 2 regions of the human CRABP1 genomic gene (GenBank accession number: human CRABP1 cDNA sequence). Is NM_004378: SEQ ID NO: 1, and human CRABP1 genomic sequence is NT_010194: SEQ ID NO: 3). The results of analysis by the bisulfite sequencing method using 5 types of cell lines are shown below the CpG island. White and black fans or circles indicate unmethylated and methylated SmaI sites, respectively. In esophageal squamous cell carcinoma cells, two or more of the three types of SmaI sites were highly methylated, but NEK2, which is a normal esophageal epithelial cell, was found to be unmethylated.

  In the normal cell NEK2 used as a target, none of the three types of SmaI recognition sites are methylated. Therefore, it was found that there is a high possibility that these three types of SmaI recognition sites are highly methylated regions.

[Example 2] Expression of CRABP1 in esophageal squamous cell carcinoma cell lines In FIGS. 3A and 3B, the expression of human CRABP1 gene in esophageal squamous cell carcinoma cells by RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) method. The result of examining the situation of methylation is shown.

  FIG. 3A shows the results of human CRABP1 mRNA gene expression using RT-PCR in esophageal squamous cell carcinoma cells (KYSE and TE series) and normal esophageal epithelial cells (labeled Normal). Specifically, when 43 types of esophageal squamous cell carcinoma cell lines were examined for the expression of human CRABP1 using RT-PCR, the human CRABP1 gene was not expressed in 34 types of cell lines or at a very low level. Expression. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal standard. In the four types of esophageal squamous cell carcinoma cells used in the analysis of the BAMCA method, the KYSE-150 and KYSE-510 cell lines could not detect the expression of human CRABP1, but the TE-4 and TE-5 lines were human CRABP1. It was confirmed that the expression of was recovered.

  In FIG. 3B, after culturing the cells for 5 days in the presence of DNA methylation inhibitor 5-azadeoxycytidine (abbreviated as 5-aza-dCyd, 0 to 10 mM), human CRABP1 gene expression was expressed from cultured cells by RNA. The result of extracting and analyzing by RT-PCR method is shown. The GAPDH gene was used as an internal standard. At this time, histone deacetylase inhibitor trichostatin A (TSA, 100 ng / ml) was added and cultured for 12 hours, and the synergistic effect of both drugs was also examined. As a result, recovery of expression was observed in esophageal squamous cell carcinoma cells in which human CRABP1 gene expression was not detected. On the other hand, esophageal squamous cell carcinoma cell lines KYSE-510 and TE-14 for which human CRABP1 gene expression could not be detected were demethylated by adding the DNA methylation inhibitor 5-aza-dC, resulting in CRABP1 gene expression. However, the expression of human CRABP1 did not recover even when the histone protein deacetylation inhibitor TSA was added. Addition of TSA did not show any promoting effect on recovery of CRABP1 gene expression by 5-aza-dC (FIG. 3B).

[Example 3] Methylation of CpG island in CRABP1 gene of esophageal squamous cell carcinoma cell line
The situation of methylation of CpG island of human CRABP1 gene detected using CpGPLOT (http://www.ebi.ac.uk/emboss/cpgplot/) was analyzed. As shown in FIG. 2C and FIG. 3A, methylation of the SmaI recognition site observed in KYSE-150 and KYSE-510 cells in which the expression of the human CRABP1 gene had disappeared is one of the three types of SmaI recognition sites. In the case of TE-4 and TE-5 cells where human CRABP1 gene was expressed compared to the pattern in which the previous two sites were highly methylated, the last two of the three SmaI recognition sites The pattern was highly methylated at some points. From this, it was predicted that DNA methylation involved in suppression of human CRABP1 gene expression occurred in CpG islands around the two previous SmaI recognition sites.

  For this reason, the previous two CpG islands (CpG-1 consisting of 355 base pairs covering exon 1 from -142IVS to +67 of human CRABP1 gene and CpG- covering exon 2 from -117IVS to +25) The methylation status in 2) and the expression of the human CRABP1 gene were examined in detail.

  First, using a series of esophageal squamous cell carcinoma cell lines and NEK2 lines, we analyzed the relationship between CRABP1 expression and methylation status of CpG-1 and CpG-2 regions using the COBRA method. In FIG. 3C, COBRA (Combined Bisulfite Restriction Analysis) method in esophageal squamous cell carcinoma cells expressing human CRABP1 gene, esophageal squamous cell carcinoma cells not expressing human CRABP1 gene, and normal esophageal epithelial cells NEK2 cells. The result regarding the methylation of the CpG-1 and CpG-2 regions using is shown. In this figure, a PCR product consisting of 395 base pairs having CpG-1 and a PCR product consisting of 427 base pairs having CpG-2 are shown by horizontal thick lines. The former is digested with BstUI restriction enzyme and the latter with TaqI restriction enzyme. The vertical check line indicates the restriction enzyme digestion site, the arrow in the electropherogram indicates the unmethylated human CRABP1 genomic gene, and the arrowhead indicates the methylated allele.

  In FIG. 3C, it was shown that high methylation of the CpG-1 region was detected in the esophageal squamous cell carcinoma cell line in which human CRABP1 expression was lost, but esophageal squamous cell carcinoma cells expressing human CRABP1 were detected. Severe methylation of the CpG-1 region was not detected in the cell lines and NEK-2 cell lines. On the other hand, methylation of the CpG-2 region was observed regardless of the human CRABP1 gene expression status in the esophageal squamous cell carcinoma cell line. Therefore, it is concluded that methylation of CpG-1 region is an important site strongly related to human CRABP1 gene expression more than CpG-2 region.

  A more detailed bisulfite sequencing analysis shows that the CpG-1 region is highly methylated in the KYSE-30, KYSE-510, and KYSE-770 cell lines, which do not show CRABP1 gene expression, consistent with the COBRA results. However, it was not methylated in the TE-4 and KYSE-1260 cell lines and the NEK2 cell line in which human CRABP1 gene expression was observed (FIG. 4A).

  In FIG. 4A, the upper part is a map of CpG islands present in the region containing exon 1 of human CRABP1. CpG islands are indicated by vertical bar marks. The middle part shows cell lines that do not express human CRABP1 gene (KYSE-30, KYSE-510 and KYSE-770), human CRABP1 gene expression cell lines (TE-4 and KYSE-1260), and normal esophageal epithelial cell lines The results of bisulfite sequence analysis using genomic DNA extracted from NEK2 are shown. White and black squares indicate unmethylated and methylated CpG sites, respectively. Each column shows the methylation status in one cell line. BstUI restriction enzyme digestion sites are indicated by arrows. The lower part is classified into regions 1 to 5, and the genomic DNA fragment examined for promoter activity is indicated by a horizontal thick bar.

[Example 4] Promoter activity of CpG island (CpG-1) of human CRABP1 gene The promoter activity of CpG-1 region which is a CpG island of human CRABP1 gene was examined. The CpG-1 region consisting of 355 base pairs was divided into five types from region 1 to region 5 (FIG. 4A), and each genomic DNA fragment was cloned into pGL3-Basic reporter plasmid (Promega Madison WI). These plasmids were transfected into esophageal squamous cell carcinoma cell lines TE-4, KYSE-30, KYSE-510, KYSE-960, and firefly luciferase activity was measured 36 hours later. As a result, region 4 and region 5, particularly the latter, showed strong luciferase activity (FIG. 4B). Therefore, it was estimated that the promoter involved in human CRABP1 gene expression is localized in the CpG1 region, and in particular, methylation in region 5 plays a sufficient role in suppressing human CRABP1 gene expression. .

[Example 5] Methylation of human CRABP1 gene in primary cell lines derived from esophageal squamous cell carcinoma To examine whether abnormal methylation of CRABP1 gene occurs in clinical specimens of esophageal squamous cell carcinoma Genomic DNA derived from a clinical specimen of esophageal squamous cell carcinoma extracted from a patient was prepared, and the methylation state of the CpG-1 region was examined using the COBRA method (the method is based on FIG. 3C). As a result, clear methylation was detected in about half of the primary cancer cells examined (FIG. 4C and undescribed data: the arrowheads shown below the electropherogram of FIG. 4C indicate that the CpG-1 region was methylated). (Shows specimens of primary esophageal squamous cell carcinoma). In order to confirm this result more quantitatively, a bisulfite sequence analysis of the CpG-1 region was performed. [FIG. 4D: The method is the same as FIG. 5)]. As a result, it was confirmed that the CpG1 region was highly methylated in clinical specimens of esophageal squamous cell carcinoma (cases 1, 4, 10). Therefore, it was presumed that the general mechanism is that the expression of human CRABP1 gene is reduced by the high methylation of CpG island (CpG-1 region) of human CRABP1 gene in the process of esophageal canceration.

[Example 6] Inhibition of proliferation of esophageal squamous cell carcinoma cell line by restoring human CRABP1 gene expression In order to show that CRABP1 gene acts as a tumor suppressor gene in the process of esophageal canceration, human CRABP1 gene Using an esophageal squamous cell carcinoma cell line in which expression was lost, it was examined whether forced expression of this gene suppressed proliferation (FIGS. 5A and 5B).

  FIG. 5A shows an assay for examining colony forming ability using esophageal squamous cell carcinoma cells. Specifically, cell lines that do not express the human CRABP1 gene (KYSE-30 and KYSE-510), Myc-tag human CRABP1 expression plasmid (pCMV-Tag3-CRABP1) or empty vector (pCMV-Tag3-empty) The results are shown in which cells were transiently transfected and then selected for 3 weeks in the presence of the appropriate concentration of antibiotic G418.

  In the upper part of FIG. 5A, after electrophoresis using a cell extract (20 μg protein), Western blotting was performed, and when human CRABP1 protein was detected using an anti-Myc antibody, a cell line transfected with the pCMV-Tag3-CRABP1 plasmid (Indicated as human CRABP1) shows that Myc-tagged CRABP1 protein was detected. On the other hand, it was shown that human CRABP1 protein was not detected in cells transfected with an empty vector (indicated as Empty). In the same middle row, G418-resistant cells were selected 3 weeks after transfection. In the cell line transfected with pCMV-Tag3-CRABP1 plasmid (labeled CRABP1), colonies were compared with cells transfected with the empty vector (labeled Empty). It has been shown that the number has decreased significantly. The lower row shows the results of quantitative analysis of colony forming ability. Specifically, colonies having a size of 2 mm or more were counted. The results of three experiments are shown as mean ± standard deviation (indicated by a small bar above the bar graph). Three experiments were performed for each.

  Further, in FIG. 5B, using the same esophageal squamous cell carcinoma cell lines KYSE-30 and KYSE-510 and a human CRABP1 expression plasmid, a cell line stably expressing human CRABP1 (indicated as CRABP1 clone) was prepared. The results of comparison of the growth with a cell line (indicated as Empty Clone) into which an empty vector has been stably introduced are shown. That is, the growth of cell lines (KYSE-30 and KYSE-510) stably expressing the human CRABP1 gene, which was transfected with pCMV-Tag3-CRABP1 plasmid or empty vector and selected with G418, was observed. It was.

  In the upper part of FIG. 5B, both the KYSE-30 and KYSE-510 strains transfected with the pCMV-Tag3-CRABP1 plasmid (20 μg protein) were analyzed by Western blot analysis using an anti-Myc-tag antibody. The cell line has been shown to express Myc-tagged CRABP1 protein. The lower panel examines the effects of stable expression of human CRABP1 on growth of the KYSE-30 and KYSE-510 strains. Specifically, using a cell expressing human CRABP1 and a cell transfected with an empty vector, the number of viable cells is measured by a water-soluble tetrazolium salt (WST) assay that measures the colorimetric method. This reagent is Cell Counting kit-8 sold by Dojin Chemical Laboratory (Kumamoto). The results of three experiments are shown as mean ± standard deviation. Statistical analysis using the Mann-Whitney U test showed a P value of 0.05 or less in all cases.

[Example 7] Promotion of proliferation in cancer cells in which CRABP1 gene expression was knocked down Human CRABP1-specific siRNA (CRABP1-siRNA) was transfected into esophageal squamous cell carcinoma cell line TE-4 to express human CRABP1 expression. Knocked down. As a result, the amount of human CRABP1 protein significantly decreased 48 to 96 hours after transfection. The cell line promoted growth compared to cells transfected with control siRNA (Luc-siRNA) (FIG. 5C: see assay description in FIG. 5B). Furthermore, in the experiments using KYSE-30 and KYSE-510 cancer cell lines derived from human CRABP1 by introducing human CRABP1 expression plasmid, the expression of human CRABP1 gene was knocked out. The growth partially recovered by going down (see FIG. 4D: description of FIG. 5C).

[Example 8] Effect of human CRABP1 gene expression on cell cycle arrest in esophageal squamous cell carcinoma To clarify the role of human CRABP1 protein on esophageal squamous cell carcinoma, human CRABP1 gene is stably expressed The cell cycle in the control cell into which the gene-transferred cell and the empty vector were introduced was analyzed using a fluorescence-activated cell sorter (FACS).

Compared with control cells, CRABP1 gene-introduced cells established from KYSE-30 and KYSE-510 cells (CRABP1 clone1 and clone2) accumulated in the G ₀ -G ₁ phase and decreased in the S ₁ phase ( FIG. 6A). Details will be described below.

In FIG. 6A, FACS analysis is performed using cells that stably express human CRABP1 (indicated as CRABP1 clone) and control cells that do not (indicated as Empty Clone), thereby analyzing the mechanism of the anti-proliferative effect of human CRABP1. Shows the results. Cells stably expressing human CRABP1 established from the KYSE-30 and KYSE-510 strains have a cell cycle arrest in the G ₀ -G ₁ phase, resulting in a decrease in S phase cells compared to the control cell line Was. This result indicates that expression of human CRABP1 arrests cells at the check point of the cell cycle of G ₁ -S phase in esophageal squamous cell carcinoma cells, that is, induces G ₀ -G ₁ arrest. However, the number of transgenic cells did not increase in the sub-G ₁ phase, and SA-β-galactosidase positive cells, an aging assay, were not observed. It was suggested that it did not happen.

Next, in FIG. 6B, the expression level of the protein related to the check point of the cell cycle in the G ₁ -S phase is examined. Specifically, electrophoresis was performed on a 20 μg protein extract prepared from cells stably expressing human CRABP1 (indicated as CRABP1) and control cells (indicated as Empty Clone), which were established from KYSE-30 and KYSE-510 strains. The expression levels of Myc-CRABP1, p21, p27, CyclinD1, and p44 / 42 protein (MAP kinase) were examined by Western blotting. The p21 and p27 proteins are inhibitors of cyclin-dependent phosphorylase. As a result, the human CRABP1 transgenic cell (KYSE-30 and KYSE-510 cells), as compared to controls, it was shown that the amount p27 suppresses a protein to S phase transition from the G ₁ phase is increased . In contrast, the amount of protein p21, which plays the same role as p27, increased in the KYSE-30 transgenic strain, but was the same as the control in the KYSE-510 transgenic strain.

In Figure 6C, using the cells expressing human CRABP1 stably established from KYSE-30 and KYSE-510 strain (CRABP1 a display) and control cells (Empty and display), the cell cycle G ₁ -S phase The analysis result by RT-PCR of the genes (p21, p27, and CyclinD1 genes) associated with checkpoints is shown. Analysis by RT-PCR did not increase p21 and p27 gene expression in human CRABP1 gene-introduced cells. This result indicates that the increase in p21 and p27 protein levels in human CRABP1 gene-introduced cells is due to increased protein stability and not a result of transcription and mRNA stability.

  Furthermore, in FIG. 6D, to check the influence of endogenous human CRABP1 gene expression, esophageal squamous cell carcinoma cell TE-4 was transfected with CRABP1-siRNA and control siRNA [luciferase-siRNA (Luc-siRNA)]. 72 hours later, the amounts of p21 and p27 were examined by Western blotting. As a result, knocking down the human CRABP1 gene did not change the level of the p21 protein, but decreased the p27 protein (FIG. 6D).

[Example 9] Examination of human CRABP1 gene somatic mutations in esophageal squamous cell carcinoma As the inactivation factor of human CRABP1 is considered to be a possibility of human CRABP1 gene somatic mutations, 43 cell lines and 30 types Of esophageal squamous cell carcinoma clinical specimens. As a result, only in the KYSE-110 strain, a novel gene mutation of the human CRABP1 gene was found in the A1C mutation of exon 1 (A in the start codon ATG was replaced with C). This mutation deletes the start codon and deletes the first 9 amino acid residues of human CRABP1. However, the same mutation was not detected in clinical specimens of esophageal cancer derived from the establishment of the KYSE-110 strain, which was considered to be a mutation acquired in the process of cell line establishment. Therefore, it is considered that the probability of somatic mutation of the human CRABP1 gene is low.

[Example 10] Relationship between human CRABP1 protein expression and pathology in esophageal squamous cell carcinoma clinical specimens In order to examine the reduction of human CRABP1 expression in esophageal squamous cell carcinoma and the clinicopathological significance that it provides, 113 Expression of human CRABP1 protein in various clinical specimens of esophageal squamous cell carcinoma was examined by immunostaining using an antibody that specifically binds to human CRABP1 (FIGS. 7A to 7C show immunostaining under a microscope (× 200)). A statue).

  Human CRABP1 protein staining was performed by an indirect fluorescent antibody method using formalin-fixed, paraffin-embedded esophageal cancer tissue sections. That is, esophageal cancer sections on silane-coated glass slides were deparaffinized and dehydrated by changing the ethanol concentration stepwise. Next, the antigen protein on the slide was treated with a microwave oven at 95 ° C. for 10 minutes in a citrate buffer (pH 6.0), and then cooled to room temperature. Tissue-derived peroxidase was blocked using 5% hydrogen peroxide. In addition, nonspecific staining was blocked with 2% porcine serum. The slides were incubated overnight at 4 ° C. with an anti-human CRABP1 mouse monoclonal antibody (using Sigma clone C-1 diluted 100-fold). In order to detect human CRABP1 protein in esophageal cancer tissue, the slide was further reacted at room temperature for 2 hours with a dextran polymer reagent containing a secondary antibody and peroxidase (Dako Envision Plus). 0.2% Diaminobenzidine tetrahydrochloride and hydrogen peroxide were added to develop human CRABP1. Furthermore, counterstaining was performed using Mayer ’s hematoxylin.

  Esophageal squamous cell carcinoma cells (TE-4) fixed with formalin expressing human CRABP1 mRNA had intracellular human CRABP1 staining detected in 50% or more of the cells, which was used as a positive control. Tissue sections incubated with porcine serum were used as negative controls. The percentage of total cells that stained positive for human CRABP1 protein was determined and evaluated in each experiment. A case where the expression of human CRABP1 protein was detected in 10% or more of cancer cells was determined as positive, a case where the detection was less than 10%, or a case where the detection was not detected was determined as negative. Final evaluation was performed by microscopic observation of two people. If there was a significant difference between the two, they discussed and presented a unified view. The clinical information of the patient who provided the tissue was concealed and the result of the tissue staining was obtained.

  In the non-cancerous part around the normal esophageal epithelial tissue, staining of CRABP1 started from the spinous cell layer, and the staining became stronger according to the direction of the superficial layer (FIG. 7A). On the other hand, even in the case of squamous cell carcinoma of the esophagus, human CRABP1 staining was positive in a well differentiated region (FIG. 7B: human CRABP1 protein staining was found in 10% or more of cancer cells. Esophageal squamous cell carcinoma clinical specimen), and was not detected in low-differentiated cancer sites (negative, FIG. 7C: human CRABP1 protein staining does not express CRABP1 gene in 10% or less of cancer cells). Human CRABP1 immunostaining was detected mainly in the cytoplasm of normal esophageal cells and esophageal squamous cell carcinoma cells. Of the 113 clinical specimens of squamous cell carcinoma of the esophagus that were examined, 39 (34.5%) were positive for CRABP1 in more than 10% of cancer cells, and 74 (65.5%) had an immune response of 10 % Or negative (Table 2). Table 2 also shows the relationship between human CRABP1 expression and clinical pathology in 113 cases of esophageal squamous cell carcinoma.

  As shown in Table 2, cases in which the detection of human CRABP1 was negative were markedly observed in cancers with a lower degree of differentiation than well differentiated cancers. That is, the pT cancer category was more pT1 than pT2 / 3, the pM category cancer was pM1 / 1a / 1b than pM0, and the stage was more negative at stage I than stage II or III.

  Because the presence or absence of lymph node metastasis is important information for selecting treatment in patients with esophageal squamous cell carcinoma, we investigated whether CRABP1 immunostaining is useful for predicting distant lymph node metastasis. Patients with pM1 esophageal squamous cell carcinoma (16 out of 18 accounts for 88.9%) were negative for CRABP1 immunostaining regardless of cancer progression [Table 3: with metastases (levels pM1, pM1a, and pM1b) And disclosed background data for each patient].

  Furthermore, patients with pT2 / T3 squamous cell carcinoma of the esophagus need to correctly predict cancer micrometastasis in order to select the treatment correctly, but patients with negative human CRABP1 immunostaining are significantly positive for lymph node metastasis (Table 4: Disclosed relationship between pM2 patient pM category and human CRABP1 expression). These results confirm that more advanced esophageal squamous cell carcinoma patients are negative for human CRABP1 immunostaining.

  As described above, from the matters disclosed in the Examples, a part of a tissue having a possibility of cancer is collected from the esophagus of a patient suspected of esophageal cancer, and DNA is prepared. It was shown that the presence or absence of methylation of the human CRABP1 genomic gene can be analyzed using the COBRA method or methylation-specific PCR method using the DNA of healthy subjects as a control. Alternatively, human CRABP1 protein mRNA is immunized by Northern blotting, RT-PCR, microarray analysis of human CRABP1 gene mRNA expression, and human CRABP1 protein immunostaining. It was possible to examine whether or not it was detected by staining, and as a result, it was shown that prediction and diagnosis of the malignancy of esophageal cancer and the possibility of lymph node metastasis can be performed. Furthermore, it was revealed that the presence or absence of metastasis to distant organs can be predicted and diagnosed by detecting the presence or absence of methylated human CRABP1 gene in blood using a methylation-specific PCR method.

It is drawing which showed the outline of the procedure of BAMCA method. It is the figure which showed the summary of the analysis using a BAMCA method, and a part of result. It is the figure which showed the data of the typical array analyzed by the BAMCA method about the esophageal squamous cell carcinoma cell TE-4 cell line. It is a drawing showing the structure of the human CRABP1 genomic gene and the state of methylation at the SmaI site of its predicted promoter region analyzed by the BAMCA method. It is a drawing showing the results of human CRABP1 mRNA gene expression using RT-PCR in esophageal squamous cell carcinoma cells and normal esophageal epithelial cells. It is the figure which showed the result analyzed by RT-PCR method after culture | cultivating a cell in presence of a methylation inhibitor. Results for CpG region methylation using COBRA method in esophageal squamous cell carcinoma cells expressing human CRABP1 gene, esophageal squamous cell carcinoma cells not expressing the same gene, and normal esophageal epithelial cells were shown It is a drawing. It is one of the drawings which examined methylation and promoter activity of CpG-1 area | region which is a CpG island of human CRABP1 gene. It is the other figure which examined the promoter activity of CpG-1 area | region which is a CpG island of human CRABP1 gene. It is one of the drawings which examined the methylation of the human CRABP1 gene in the primary cell line derived from esophageal squamous cell carcinoma. It is the other figure which examined the methylation of the human CRABP1 gene in the primary cell line induced from esophageal squamous cell carcinoma. It is one of the drawings which examined the suppression of the proliferation of an esophageal squamous cell carcinoma cell line by restoring human CRABP1 gene expression. The other of the drawings which examined suppression of the proliferation of an esophageal squamous cell carcinoma cell line by restoring | restoring human CRABP1 gene expression. It is one of the drawings which examined the promotion of the proliferation in the cancer cell which knocked down human CRABP1 gene expression. It is the other of the drawings which examined the promotion of the proliferation in the cancer cell which knocked down human CRABP1 gene expression. It is the figure which showed the result of having analyzed the mechanism of the antiproliferative effect of human CRABP1 by performing FACS analysis using the cell which stably expresses human CRABP1, and the control cell which is not so. Is a drawing showing the results of examining the expression level of proteins involved in cell cycle checkpoints G ₁ -S phase. And cells stably expressing human CRABP1, using the control cell, is a diagram showing an analysis result of the RT-PCR of genes associated with cell cycle checkpoints G ₁ -S phase. It is a figure which shows the result of having examined the influence of endogenous human CRABP1 gene expression. It is a drawing showing the results of examination by immunostaining in a normal cell line regarding the relationship between the expression of human CRABP1 protein and clinical pathology in clinical specimens of esophageal squamous cell carcinoma. It is a figure which shows the result of having examined the expression of human CRABP1 protein in a clinical specimen of esophageal squamous cell carcinoma and clinical pathology by immunostaining in a well-differentiated cancer line (low malignancy). It is a drawing showing the results of examination by immunostaining in a poorly differentiated cancer strain (high malignancy) regarding the relationship between human CRABP1 protein expression and clinical pathology in clinical specimens of esophageal squamous cell carcinoma.

Claims (4)

A method for detecting esophageal cancer, wherein the canceration of the esophageal epithelial cell is detected by detecting inactivation of the human CRABP1 gene in the esophageal epithelial cell. In the detection method, inactivation of the human CRABP1 gene is mediated by genomic DNA of the gene, mRNA expressed using the gene as a template, cDNA using the mRNA as a template, or human CRABP1 protein in esophageal epithelial cells. The method for detecting esophageal cancer according to claim 1, wherein the esophageal cancer is detected. The said detection method WHEREIN: The said gene in a sample is detected using a DNA chip method, a Southern blot method, a Northern blot method, real-time RT-PCR method, FISH method, CGH method, or an immuno-staining method. Or the method for detecting esophageal cancer according to 2. The method for detecting esophageal cancer according to any one of claims 1 to 3, wherein inactivation of the human CRABP1 gene is inactivation resulting from methylation of a CpG island of the gene.