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WO2016181979A1 - Procédé d'utilisation des niveaux d'expression de syt7, mfsd4, et etnk2 pour détecter des métastases de cancer gastrique dans le foie, kit de détection, procédé de criblage d'agents thérapeutiques ciblés moléculaires, et composition pharmaceutique - Google Patents

Procédé d'utilisation des niveaux d'expression de syt7, mfsd4, et etnk2 pour détecter des métastases de cancer gastrique dans le foie, kit de détection, procédé de criblage d'agents thérapeutiques ciblés moléculaires, et composition pharmaceutique Download PDF

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WO2016181979A1
WO2016181979A1 PCT/JP2016/063956 JP2016063956W WO2016181979A1 WO 2016181979 A1 WO2016181979 A1 WO 2016181979A1 JP 2016063956 W JP2016063956 W JP 2016063956W WO 2016181979 A1 WO2016181979 A1 WO 2016181979A1
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mfsd4
syt7
etnk2
gastric cancer
expression level
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Japanese (ja)
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光郎 神田
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国立大学法人名古屋大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a prognostic marker for gastric cancer and a method for testing using the same.
  • the present invention relates to a method for examining gastric cancer having a high risk of progressing to liver metastasis that affects the prognosis of gastric cancer, a test kit, a screening method for a molecular target therapeutic agent, and a pharmaceutical composition.
  • Gastric cancer is a common cancer in Asia, such as Japan, China, and South Korea, and South America. Looking at cancer mortality by region in Japan, the mortality rate of gastric cancer is decreasing year by year, but it is the second highest after lung cancer in men, and the third highest after colon cancer and lung cancer in women. (According to 2012 statistics.) With the spread of cancer screening, early detection has progressed and the mortality from gastric cancer has decreased. However, advanced gastric cancer still has a poor prognosis and is an important disease to be overcome in Japan, where gastric cancer prevalence is high.
  • gastric cancer with distant metastasis is treated in a lump and its treatment policy is not distinguished.
  • a multi-step process is required for free cancer cells arising from the primary lesion to engraft and proliferate to form a metastatic focus.
  • Adhesion molecules, proteolytic enzymes, growth factors, angiogenic factors, chemokines, and many other molecules Has been reported to be involved.
  • gastric cancer the three pathways of recurrent metastasis are greatly different, so the molecules involved in metastasis are also different, and the properties of metastasized cancer cells are also considered to be greatly different. Nevertheless, it is considered that the same treatment is performed by using three pathways with different metastasis formation mechanisms as distant metastases, which may contribute to the difficulty of complete cure of metastatic cancer.
  • Chemotherapy for recurrent gastric cancer and advanced gastric cancer that may recur is a therapy based on the administration of S-1, whose mechanism of action is inhibition of DNA synthesis.
  • S-1 is a drug that is generally effective for cancers with proliferating cells, and does not act specifically on metastatic gastric cancer.
  • the main cause of S-1 monotherapy significantly extending the survival period of patients with advanced gastric cancer is a decrease in the rate of recurrence of peritoneal dissemination, which is controlled in hematogenous and lymph node metastasis including liver metastasis. It's hard to say.
  • liver metastases As a form of recurrent metastasis of gastric cancer, hematogenous metastasis that metastasizes to other organs in the bloodstream is the second most common after peritoneal dissemination metastasis.
  • gastric cancer there are many liver metastases among the hematogenous metastases that metastasize to the liver. It is becoming important for follow-up of gastric cancer after surgery to detect and treat liver metastasis recurrence as soon as S-1 is not so effective against recurrence and subjective symptoms are difficult to occur.
  • Patent Documents 1 to 3 Methods for predicting and detecting recurrence of gastric cancer are also disclosed. Moreover, although there is a report regarding a marker having a high risk of liver metastasis regardless of the cancer type, research has not been made on a marker for predicting liver metastasis specific to gastric cancer (Patent Document 4).
  • a molecular target therapeutic agent refers to a drug that is made to act efficiently by capturing the properties of diseased cells such as cancer cells at the molecular level and targeting proteins or genes expressed on the surface.
  • diseased cells such as cancer cells at the molecular level and targeting proteins or genes expressed on the surface.
  • a molecular targeted therapeutic agent for gastric cancer There are only a few molecular targeted therapeutic agents for gastric cancer, and only Trastuzumab for HER2-positive gastric cancer has received domestic approval. In addition, ramucilumab is approved for advanced gastric cancer in the United States.
  • JP 2013-102716 A JP 2005-516215 Gazette International Publication No. 2005/010180 JP 2006-119064 A
  • markers for evaluating the risk of recurrence and metastasis have been disclosed, few markers specific to liver metastasis of gastric cancer have been reported. Moreover, since the accuracy is not so high, it has not been put into practical use yet. Therefore, identification of a marker specific to liver metastasis that can accurately evaluate the risk of recurrence is desired. If a marker highly specific for liver metastasis can be found, more effective individualized treatment can be provided.
  • trastuzumab which is currently approved in Japan as a molecular targeted therapy for gastric cancer, is only effective for HER2-positive gastric cancer, which accounts for about 20% of advanced and recurrent gastric cancer.
  • lamusilmab which is approved for the treatment of advanced gastric cancer in the United States, is not very effective in prognostic extension. Therefore, the development of new molecular targeted therapeutic agents for advanced and recurrent gastric cancer is desired.
  • Another object of the present invention is to develop a method for screening a molecular target drug using the marker as an index.
  • the present invention relates to the following test methods, kits, screening methods, and pharmaceutical compositions.
  • a test method for predicting liver metastasis after gastrectomy for gastric cancer wherein the patient serum collected from the subject and / or at least one of SYT7, MFSD4, and ETNK2 in gastric cancer tissue in gastrectomy
  • the above expression level is obtained, and when at least one expression level of SYT7, MFSD4, ETNK2 in the sample is significantly different from a predetermined value, it is determined that the risk of liver metastasis is high Inspection method.
  • a test kit for diagnosing or predicting liver metastasis after gastrectomy for gastric cancer a primer for quantitative PCR for measuring the expression level of SYT7, MFSD4, or ETNK2, an anti-SYT7 antibody, A test kit comprising one or more of an anti-MFSD4 antibody, an anti-ETNK2 antibody, or a methylation detection reagent for the MFSD4 gene promoter region.
  • a method for screening a molecular target therapeutic agent for treating liver metastasis after gastrectomy for gastric cancer comprising suppressing SYT7, ETNK2 expression level, increasing MFSD4 expression level, or MFSD4 gene promoter region
  • a molecular targeted therapeutic drug screening method comprising screening a substance using at least one of methylation as an index.
  • a pharmaceutical composition for suppressing liver metastasis after gastrectomy comprising at least one siRNA of SYT7 and ETNK2.
  • the risk of liver metastasis can be predicted immediately after gastrectomy, it can be used for subsequent treatment. Specifically, a group of patients with a high risk of liver metastasis can be treated with a follow-up plan with a view to liver metastasis after gastrectomy. Furthermore, it becomes possible to screen a medicine using the expression level of SYT7, MFSD4, and ETNK2 as an index. Therefore, it is possible to develop a medicament for treating recurrence of liver cancer metastasis targeting these molecules.
  • FIG. 1A shows SYT7
  • FIG. 1B shows MFSD4
  • FIG. 1C shows the correlation between the expression level of ETNK2 and liver metastasis.
  • the expression levels of SYT7, MFSD4, and ETNK2 in the resected tissue of 200 gastric cancer patients are shown as an average value and a plot diagram.
  • B The figure which shows the analysis result of the gastric cancer cell line by methylation specific PCR.
  • the present inventor classified the cases after gastrectomy according to the course, and conducted comprehensive expression analysis using a next-generation sequencer for mRNA obtained from gastric cancer primary tissue. As a result, it was revealed that SYT7 and ETNK2 were specifically highly expressed and the expression level of MFSD4 was specifically decreased in gastric cancer with liver metastasis. When the expression level is higher or lower than a certain value, it can be diagnosed that the case has a high risk of causing liver metastasis.
  • SYT7 is a membrane protein belonging to the synaptotagmin (SYT) family. It has been identified as a calcium / phospholipid-binding molecule present on synaptic vesicles, suggesting that it functions as a calcium sensor. In humans, the presence of 17 isoforms has been reported, and it is reported that it is mainly distributed in brain tissue. SYT7 is also mainly expressed in the brain and has been reported to be involved in neurotransmitter exocytosis (Non-Patent Documents 1 and 2).
  • ETNK2 (Ethanolamine kinase 2) is an enzyme involved in the first synthesis step in the CDP-ethanolamine pathway, which is one of the synthesis pathways of phosphatidylethanolamine, which is a major phospholipid of biological membranes. It is highly expressed in the liver and genital organs, and it has been suggested from knockout mouse experiments that it is involved in placental homeostasis (Non-patent Document 3).
  • MFSD4 major facilitator superfamily domaining 4
  • the risk of metastasis to the liver can be evaluated by quantifying the expression level of at least one of SYT7, MFSD4, and ETNK2.
  • SYT7, MFSD4, and ETNK2 are considered to be released from the gastric primary tissue or released from the disintegrated tumor into the blood and circulate in the blood. Therefore, by detecting the expression of SYT7, MFSD4, and ETNK2 in the serum using not only gastric cancer tissue but also serum as a sample, the risk of transition to liver metastasis can be evaluated. Moreover, methylation of MFSD4 described later can be detected not only by analyzing tissues but also by analyzing DNA obtained from serum samples.
  • SYT7, MFSD4, and ETNK2 can quantify their expression levels by quantifying mRNA and protein. Any method may be used as long as mRNA and protein can be quantitatively measured.
  • mRNA may be measured using quantitative PCR, next-generation sequencer, Northern blotting, DNA chip for gene expression analysis, or the like.
  • the quantitative PCR method is preferable because it can be measured in a short time.
  • a known method such as SYBR Green method, TaqMan probe method, RT-PCR method or the like can be used.
  • the protein may be measured using an anti-SYT7 antibody, an anti-MFSD4 antibody, or an anti-ETNK2 antibody.
  • Antibodies may be prepared by immunizing animals such as rabbits, goats, mice and the like with conventional methods.
  • the protein can be quantified by a known method such as a protein chip, ELISA method, RIA method, or Western blotting. By measuring mRNA and protein, the expression level of SYT7, MFSD4, and ETNK2 can be quantified with high sensitivity, and the risk of liver metastasis can be evaluated.
  • the promoter region of the MFSD4 gene has a CpG island and its expression is inactivated by DNA methylation. Therefore, the decrease in the expression of the MFSD4 gene may be analyzed for the degree of methylation of the promoter region.
  • known assay methods such as quantitative methylation-specific PCR, pyrosequencing, HELP assay, and chip-on-chip assay can be used.
  • the kit for examining the expression level of the SYT7, MFSD4, and ETNK2 genes can be configured to include enzymes, reagents, and the like that are optimal for quantification in addition to a PCR primer set that can quantify the expression level of each gene.
  • the primer any sequence may be used as long as it can quantitatively measure SYT7, MFSD4, and ETNK2.
  • a control primer such as a primer for amplifying GAPDH may be included.
  • kits for examining the expression levels of SYT7, MFSD4, and ETNK2 proteins include antibodies and aptamers for detecting proteins, and reagents necessary for detection in addition to molecules that bind to SYT7, MFSD4, and ETNK2 proteins. What is necessary is just composition.
  • the promoter region of the MFSD4 gene may be configured to include a reagent necessary for a known methylation detection method.
  • molecular target therapeutics can be selected by screening for compounds that suppress their expression.
  • a molecular target therapeutic agent can be selected by screening a compound that increases the expression of MFSD4.
  • a candidate for a molecular target therapeutic agent may be selected from a library composed of a low molecular compound, a natural product, or the like.
  • a library compound is added to a culture solution of a cancer cell line or gastric cancer cell line established from gastric cancer liver metastasis and a cell line that highly expresses SYT7 and ETNK2, and cell culture is performed to express SYT7 and ETNK2 What is to be suppressed may be selected.
  • a library compound may be added to a culture solution of a cell line with a low expression level of MFSD4, cell culture may be performed, and one that increases MFSD4 expression may be selected. Since the expression levels of SYT7, MFSD4, and ETNK2 can be quantified by measuring mRNA and protein as described above, a substance that suppresses expression with high sensitivity can be selected.
  • the present inventor has found that when SYT7, MFSD4, and ETNK2 are depleted by siRNA, a change occurs in the cell migration ability. With respect to SYT7 and ETNK2, which have a correlation between increased expression and liver metastasis, it is possible to suppress liver metastasis by suppressing it with siRNA. Therefore, there is a possibility that siRNA of SYT7 and ETNK2 can be used as a therapeutic agent.
  • SYT7 expression refers to gene and / or protein expression unless otherwise specified.
  • liver metastasis recurrence group ⁇ Analysis of genes specifically expressed in the liver recurrence group (1) ⁇ Previously, advanced gastric cancer cases that had undergone gastrectomy at Nagoya University School of Medicine were divided into 4 groups: long-term recurrence group over 5 years, peritoneal dissemination recurrence group, liver metastasis recurrence group, and lymph node recurrence group. Focusing on the recurrence of liver metastasis, which accounts for about 25% of recurrent gastric cancer, and oral S-1 after gastrectomy is not effective in advanced gastric cancer. We decided to detect genes with different expression levels.
  • RNA obtained from gastric cancer primary tissue, gastric non-cancerous part, and liver metastatic lesions of 4 patients with liver metastasis recurrence group were divided into groups according to their course.
  • the group was divided into four groups, a long-term recurrence group of 5 years or more, a peritoneal dissemination recurrence group, a liver metastasis recurrence group, and a lymph node recurrence group.
  • Expression profiling by transcriptome analysis was performed on RNA obtained from gastric cancer primary tissue, gastric non-cancerous part, and liver metastatic lesions of 4 patients with liver metastasis recurrence group.
  • RNA was extracted using RNeasy kit (manufactured by QIAGEN). The extracted total RNA was subjected to sequence library adjustment according to a standard protocol using TruSeq RNA Sample Prep Kit (manufactured by Illumina).
  • next-generation sequencer Hiseq manufactured by illumina
  • paired-end sequencing was performed, and transcriptome analysis was performed.
  • Data was acquired with a read base length of 100 bases / read, a reference acquisition number of reads of 100 million read pairs (200 million reads) / lane, and a reference acquisition data amount of 20 Gb / lane.
  • HiSeq software For analysis, use HiSeq software to perform mapping processing to the specified reference sequence, calculate the expression level for each gene based on the FPKM (Fragments per kilobase of exon per million mapped sequence reads) value, and create an inter-sample comparison table Was done.
  • FPKM Frragments per kilobase of exon per million mapped sequence reads
  • the expression level of 57551 molecules in the non-cancerous part of the stomach, the primary gastric lesion, and the liver metastatic lesion in 4 cases in the liver metastasis recurrence group was comprehensively analyzed by transcriptome analysis.
  • Table 1 shows the signals of non-cancerous stomach, primary gastric lesion, and liver metastatic lesion in the group in which recurrence of liver metastasis was observed.
  • the intensity ratio (log 2 ratio) is calculated and summarized. SYT7 and MFSD4 were extracted as genes in which a significant difference was observed in the gene expression level between the non-cancerous part of the stomach and the primary gastric lesion, and no significant difference was observed in the expression between the primary gastric lesion and the liver metastatic lesion.
  • SYT7 NCBI RefSeq ID accession number: NM_00125202065.1
  • SYT7 was found to be 21-fold or more upregulated in the primary gastric lesion compared to the non-cancerous part of the stomach.
  • MFSD4 NCBI RefSeq ID accession number: NM — 181644.4
  • the expression level of the ETNK2 (NCBI RefSeq ID accession number: NM_001297760.1) gene is significantly enhanced in the liver metastasis recurrence group as compared to the long-term recurrence-free group.
  • ETNK2 was found to be approximately 5.3 times as potent in expression as the relapse-free group.
  • no significant enhancement of ETNK2 expression was observed in other metastatic forms of peritoneal dissemination and lymph node recurrence. Therefore, the ETNK2 gene expression level and protein expression level function as biomarkers for gastric cancer liver metastasis.
  • PCR primers used the following sequences, ABI STEPOnePlus Real-Time PCR System (manufactured by Applied Biosystems) was heated at 95 ° C for 10 minutes, and then amplified at 40 ° C for 40 cycles at 95 ° C for 5 seconds and 60 ° C for 60 seconds. And analyzed.
  • FIG. 1A shows the expression level of SYT7 mRNA
  • FIG. 1B shows the expression level of MFSD4 mRNA
  • FIG. 1C shows the normalized value of ETNK2 mRNA expression level of GAPDH mRNA. Box-whisker plots (minimum value, 1st case) were observed in each group of cases in which liver metastasis was not observed at the time of gastrectomy. (Quarter point, median, third quarter point, maximum value).
  • the expression levels of SYT7 and ETNK2 in the group in which liver metastasis was observed were significantly higher than those in the group other than liver metastasis.
  • the expression of MFSD4 was significantly higher in the group other than liver metastasis compared to the group in which recurrence of liver metastasis was observed.
  • the optimal cut-off value should be verified with multiple specimens, it is possible to predict the risk of gastric cancer with recurrent liver metastases by measuring the expression levels of SYT7, MFSD4, and ETNK2.
  • SYT7 and ETNK2 expression is used as a marker of liver metastasis, as shown in FIGS. 1A and 1C, among the patient groups in which liver metastasis was not observed, simultaneous liver metastasis, liver metastasis within 2 years after surgery Since the number of patients exceeding the median value of any liver metastasis group is 25% or less, the risk of gastric cancer that recurs in the liver metastasis can be predicted based on the median value of cases in the liver metastasis group.
  • the cut-off value can be predicted to vary depending on the measurement method of SYT7, MFSD4, and ETNK2, and the sample to be measured. Therefore, the cut-off value can be appropriately determined, and a patient group with a high risk of liver metastasis can be selected and followed. desirable.
  • stage II / III is divided into liver recurrence (-) and liver recurrence (+) according to the presence or absence of later liver metastasis, and stage IV is analyzed according to the presence or absence of liver metastasis, even if it has distant metastasis .
  • the average value is shown on the left side of FIG. 2, and the plot is shown on the right side.
  • the expression level of SYT7 in gastric cancer tissues was confirmed in patients with liver metastases after staged curative resection (Stage II / III, liver recurrence (+)), among those with distant metastases (Stage IV liver metastasis ( +)) was significantly higher. Conversely, in cases where there is no future or present liver metastasis, there is no difference in the expression level from normal gastric tissue, and it is considered that the presence and prediction of gastric cancer liver metastasis have high specificity.
  • the expression level of MFSD4 in gastric cancer tissues was more highly suppressed in cases where liver metastasis or future liver metastasis recurrence was observed.
  • ETNK2 The expression level of ETNK2 in gastric cancer tissues was significantly higher in cases with liver metastasis (Stage IV liver metastasis (+)), even among distant metastases. On the other hand, in other gastric cancer cases, there is no difference in the expression level from normal gastric tissue, and it is considered that the presence of gastric cancer liver metastasis has high specificity.
  • ROC curve analysis was performed on each mRNA expression level in the above 200 cases of gastric cancer patients.
  • the mRNA expression levels of SYT7, MFSD4, and ETNK2 in the primary gastric cancer lesion were all highly correlated with liver metastasis or liver metastasis within 2 years after surgery.
  • stage II / III gastric cancer Using the cut-off values obtained by ROC curve analysis of each gene, 93 patients with stage II / III gastric cancer, that is, patients who were curatively excised but at a stage with high risk of recurrence The progress was examined. As shown in FIG. 4, in cases of SYT7 expression enhancement, MFSD4 expression suppression, and ETNK2 expression enhancement in gastric cancer primary lesions, liver metastasis recurs significantly after curative resection, and independence of gastric cancer liver metastasis is also found in multivariate analysis. It was a risk factor.
  • the liver metastasis can be predicted using not only the gastrectomy tissue sample but also the blood sample, the diagnosis can be performed with the blood sample before and after the operation, which is very beneficial for the patient. Therefore, an analysis was performed to determine whether there was a change in the expression level of SYT7 using patient serum.
  • the amount of SYT7 in patient serum was measured using an ELISA kit (ELISA Kit for Synaptotagmin VII (SYT7), manufactured by Cloud-Clone).
  • the subjects were 16 healthy patients, 10 stage I gastric cancer patients, 9 stage II gastric cancer patients, 10 stage III gastric cancer patients, and 39 stage IV gastric cancer patients. The results are shown in FIG.
  • the serum SYT7 value can be a diagnosis of the degree of progression of gastric cancer and a biomarker for predicting metastasis recurrence.
  • liver metastases can be predicted by measuring the protein expression level using not only gastrectomy tissue but also a blood sample.
  • EMT Epithelial to Mesenchymal Transition
  • QIAGEN Quality of Service
  • SYT7 was significantly positively correlated with the expression of TGFB3, an epithelial-mesenchymal transition (EMT) -related molecule.
  • EMT epithelial-mesenchymal transition
  • the expression level of MFSD4 has a significant inverse correlation with the expression of BMP2, which is an EMT-related molecule that is deeply involved in cancer metastasis and progression, and conversely, it has a positive correlation with NUDT13 and OCLN, which are suppressed by EMT. Admitted.
  • ETNK2 expression level showed a significant positive correlation with AHNAK, ITGAV, FN1, MMP3, TGFB1, and EGFR.
  • AHNAK and ITGAV are known as EMT promoters
  • FN1 and MMP3 are known as extracellular matrix proteins
  • TGFB1 and EGFR are known as cell growth factors.
  • MKN1 which is a gastric cancer cell line with high expression of SYT8 and SYT13
  • selective expression inhibition (knockdown) experiments of each gene were conducted to evaluate the migration ability of gastric cancer cells.
  • siRNA 40 nM each of siRNA was introduced into MKN1 cells using Accel siRNA transfection methods (manufactured by Dharmacon), and after 72 hours of culturing in serum-free DMEM medium, the migration ability was evaluated.
  • SYT7 siRNA, MFSD4 siRNA, ETNK2 siRNA, and control siRNA (Accel Green Non-targeting) were all obtained from Dharmacon.
  • MKN1 cells to be 2 ⁇ 10 4, 12-well plates were seeded to form ibidi Culture insert method wound gap at a predetermined width (ibid Inc.), were cultured in serum-free medium. The insert was removed 24 hours after sowing, and the width was measured at intervals of 200 ⁇ m every 6 hours. The measurement was carried out using a microscope with a magnification of 40 times, and each well was measured at 10 locations to determine the average and standard deviation.
  • FIG. 7 shows changes over time in the microscopic image and the wound width.
  • an asterisk (*) indicates that there is a significant difference compared to a control not subjected to siRNA introduction treatment. It was clarified that knockdown of SYT7 and ETNK2 significantly decreased the migration ability of gastric cancer cell line MKN1, and significantly increased by knockdown of MFSD4.
  • the invasion ability of cells was evaluated by Matrigel invasion assay for cells cultured in a serum-free medium for 72 hours after introduction of siRNA.
  • the assay was performed using BioCoat Matrigel inversion Chambers (BD Siosciences) according to the protocol. Specifically, siRNA-introduced MKN1 cells were seeded at 2.5 ⁇ 10 4 per well, cultured for 24 hours in serum-free DMEM medium, and the cells on the bottom of the membrane were fixed. The number of cells was counted by observing under a microscope. Microscopic observation was performed at a magnification of 200 times, and the average and standard deviation of five randomly selected fields were obtained.
  • the microscopic image is shown on the left side of FIG. 8, and the number of cells having invasive ability is shown on the right side of FIG.
  • the expression of MFSD4 was inhibited, it was revealed that gastric cancer cells significantly suppressed invasive ability.
  • * mark in the right graph of FIG. 8 shows that there was a significant difference between each sample.
  • Non-Patent Documents 5 and 6 DNA methylation is the addition of a methyl group to the C position of the CpG sequence of DNA.
  • the CpG island present at the promoter site of the gene is methylated, resulting in gene inactivation resulting in a decrease in gene expression. It was analyzed whether the decrease in MFSD4 gene expression in the liver cancer metastasis recurrence group was due to DNA methylation.
  • FIG. 9A shows a CpG island in the promoter region of the MFSD4 gene.
  • the sequence upstream of MFSD4 (NC — 000001.11. Reference GRCh38.p2) was analyzed.
  • the MFSD4 promoter region is indicated by a thin line.
  • CpG sequences that can undergo methylation are shown as vertical lines on thin lines.
  • CpG islands are portions that are gathered at a frequency that is far from the numerical value calculated with the normal probability.
  • a region indicated by a thick line is a region where 55% or more of GC is included in the array corresponding to the definition of the CpG island.
  • DNA was extracted from the cells, and bisulfite treatment was performed using EpiTect Bisulfite Kits (manufactured by QIAGEN) according to the protocol.
  • the PCR primer used the following sequence and analyzed the sequence upstream of MFSD4. As shown in FIG. 9A, the PCR primer is set in the CpG island upstream from the transcription start site.
  • Platinum (registered trademark) Taq DNA Polymerase manufactured by Invitrogen
  • Veriti registered trademark
  • Thermal Cycler manufactured by Applied Biosystems
  • MFSD4 methylation detection primer Forward: CGGTTTTCGGTTGGTTTCG (SEQ ID NO: 7) Reverse: CGCCCTCTACGACGGAATAAAAC (SEQ ID NO: 8)
  • the primer was changed and the bisulfite sequencing method was used for the analysis.
  • An outline of the position of the primer is shown in FIG. It is set further upstream than the position of the primer shown in FIG.
  • the primer sequences used are as follows.
  • MFSD4 methylation detection primer Forward: AGTAGTTTTGTTTTTGTTGGGTAG (SEQ ID NO: 9) Reverse: CCAAAAATCCACTTCAATCTCTAAA (SEQ ID NO: 10)
  • Each cell was cultured for 96 hours in a medium supplemented with 10 ⁇ M 5-aza-dC (Sigma Aldrich), demethylated, and analyzed for the expression level of MFSD4.
  • the black bar is the result for a normal cell
  • the white bar is the result for a cell line that has been subjected to demethylation treatment with 5-aza-dC (FIG. 10 (B) graph).
  • the cell line whose expression level was suppressed to a higher degree than the control FHs74 cell line was frequently accompanied by DNA methylation, and their expression recovered by demethylation treatment. From these results, it is clear that suppression of MFSD4 expression is based on DNA methylation as a main regulator.
  • MFSD4 Since analysis with a gastric cancer cell line is considered to occur in gastric cancer tissue, MFSD4 in which a decrease in the expression level is observed can be analyzed by analyzing the methylation of the MFSD4 promoter region in the gastric cancer tissue. You may be inspected for risk.
  • MFSD4 expression level was analyzed using patient serum. DNA was extracted from patient sera, bisulfite-treated, and analyzed by quantitative methylation-specific PCR. In order to quantify methylation, the following primers were used.
  • Quantitative PCR primer Forward: GTATTGTTTATTCGGTCGTAGGC (SEQ ID NO: 11) Reverse: ATAACGCCAAACTAACGAACTCG (SEQ ID NO: 12) probe: AAACCCCGCCCCTTAAAACCGA (SEQ ID NO: 13)
  • Quantitative primers and probes were combined with DNA extracted from patient serum and mixed, and PCR reaction was performed to specifically detect MFSD4 methylation.
  • the PCR reaction was analyzed using ABI STEPOnePlus Real-Time PCR System (Applied Biosystems) at 95 ° C for 10 minutes, followed by amplification under 40 cycles of PCR conditions at 95 ° C for 5 seconds and 60 ° C for 60 seconds.
  • FIG. 11 shows the analysis results of 10 healthy patients, 6 stage I gastric cancer patients, 10 stage II / III gastric cancer patients, and 12 stage IV gastric cancer patients.
  • Serum methylation of MFSD4 was not detected in healthy patients and stage I gastric cancer patients.
  • stage II / III gastric cancer patients 2 out of 10 cases were detected (20%), and in stage IV gastric cancer patients, methylation was detected in serum samples in 5 out of 12 cases. Therefore.
  • MFSD4 DNA methylation in serum can also function as a diagnostic biomarker of gastric cancer progression.
  • DNA methylation of the promoter region may be analyzed.
  • using a gene or protein whose expression level decreases as a marker may cause a problem in terms of accuracy, but it can be detected with high sensitivity by detecting DNA methylation.
  • the expression level of SYT7, MFSD4, and ETNK2 in a sample obtained at the time of surgery can be used as a marker for predicting the risk of liver metastasis.
  • the patient can be treated under a finer treatment policy.
  • siRNA of SYT7 and ETNK2 can also function as a pharmaceutical composition.

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Abstract

La présente invention concerne un procédé de détection permettant de prédire des métastases de cancer gastrique dans le foie, un kit de détection, et un procédé de criblage d'un médicament qui agit sélectivement sur des métastases hépatiques. En ciblant SYT7, MFSD4, et ETNK2, dont l'expression change de manière spécifique lors de métastases du cancer gastrique dans le foie, les métastases hépatiques postopératoires peuvent être prédites ou un agent thérapeutique ciblé moléculaire peut être criblé.
PCT/JP2016/063956 2015-05-13 2016-05-11 Procédé d'utilisation des niveaux d'expression de syt7, mfsd4, et etnk2 pour détecter des métastases de cancer gastrique dans le foie, kit de détection, procédé de criblage d'agents thérapeutiques ciblés moléculaires, et composition pharmaceutique WO2016181979A1 (fr)

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CN111172274A (zh) * 2020-01-22 2020-05-19 清华大学 Synaptotagmin-7在双向情感障碍诊断和治疗中的用途
JP2021019564A (ja) * 2019-07-30 2021-02-18 国立大学法人大阪大学 細胞系譜生成方法、プログラム、及び細胞系譜生成装置
JP2022505327A (ja) * 2018-10-19 2022-01-14 コリア リサーチ インスティチュート オブ バイオサイエンス アンド バイオテクノロジー Ｓｙｔ１１抑制剤を有効成分として含む胃癌治療用組成物

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2022505327A (ja) * 2018-10-19 2022-01-14 コリア リサーチ インスティチュート オブ バイオサイエンス アンド バイオテクノロジー Ｓｙｔ１１抑制剤を有効成分として含む胃癌治療用組成物
JP7645178B2 (ja) 2018-10-19 2025-03-13 コリア リサーチ インスティチュート オブ バイオサイエンス アンド バイオテクノロジー Ｓｙｔ１１抑制剤を有効成分として含む胃癌治療用組成物
JP2021019564A (ja) * 2019-07-30 2021-02-18 国立大学法人大阪大学 細胞系譜生成方法、プログラム、及び細胞系譜生成装置
JP7355325B2 (ja) 2019-07-30 2023-10-03 国立大学法人大阪大学 細胞系譜生成方法、プログラム、及び細胞系譜生成装置
CN111172274A (zh) * 2020-01-22 2020-05-19 清华大学 Synaptotagmin-7在双向情感障碍诊断和治疗中的用途
CN111172274B (zh) * 2020-01-22 2021-10-15 清华大学 Synaptotagmin-7在双向情感障碍诊断和治疗中的用途
EP4067504A4 (fr) * 2020-01-22 2023-01-18 Tsinghua University Utilisation de synaptotagmine-7 dans le diagnostic et le traitement d'un trouble bipolaire
JP2023506637A (ja) * 2020-01-22 2023-02-17 清華大学 双極性障害の診断及び治療におけるＳｙｎａｐｔｏｔａｇｍｉｎ－７の使用

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