WO2001066719A1 - Nouveau gene clone en neuroblastome humain et nouveaux fragments de gene - Google Patents

Nouveau gene clone en neuroblastome humain et nouveaux fragments de gene Download PDF

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WO2001066719A1
WO2001066719A1 PCT/JP2001/001629 JP0101629W WO0166719A1 WO 2001066719 A1 WO2001066719 A1 WO 2001066719A1 JP 0101629 W JP0101629 W JP 0101629W WO 0166719 A1 WO0166719 A1 WO 0166719A1
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nucleic acid
dna
gene
human neuroblastoma
prognosis
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PCT/JP2001/001629
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English (en)
Japanese (ja)
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Akira Nakagawara
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Chiba-Prefecture
Hisamitsu Pharmaceutical Co., Inc.
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Publication of WO2001066719A1 publication Critical patent/WO2001066719A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • Novel genes and novel gene fragments cloned in human neuroblastoma Novel genes and novel gene fragments cloned in human neuroblastoma
  • the present invention relates to a novel gene expressed in human neuroblastoma. More specifically, the present invention relates to nucleic acids derived from a novel gene expressed in human neuroblastoma and fragments thereof.
  • Neuroblastoma is a childhood cancer that develops in sympathetic ganglion cells derived from peripheral sympathetic nervous system cells and in adrenal medulla cells. These sympathetic nervous system cells are differentiated and matured at the place where the neural crest cells in the early stage of development migrate to the ventral side and form so-called sympathetic ganglia. Some of the cells migrate further to the adrenal gland, penetrate the adrenal cortex, which is being formed, and reach the medulla where they form the medulla. Neural crest cells are also the source of other peripheral nerve cells, and differentiate into dorsal root ganglia (sensory nerves), skin pigment cells, thyroid C cells, some lung cells, enteric ganglion cells, etc. I do. Neuroblastoma spreads extensively and causes cancers such as melanoma, medullary carcinoma, small cell lung carcinoma, and Hirschsprung disease.
  • Neuroblastoma is characterized by showing various clinical features (Nakagawabara: Neuroblastoma occurrence and its molecular mechanism Pediatric Internal Medicine 30, 143, 1998). For example, neuroblastomas that occur before the age of one year have a very good prognosis, with most undergoing differentiation and cell death. Regress spontaneously. At present, many neuroblastomas that are positive for urinary mass screening of urine in 6-month-old infants belong to this spontaneously regressing pancreas. On the other hand, neuroblastomas that develop over the age of one year are highly aggressive and often kill the patient.
  • Trk A a high-affinity receptor for nerve growth factor (NGF)
  • NGF nerve growth factor
  • Trk tyrosine kinase II Ret tyrosine kinase.
  • TrkA receptor the role of the TrkA receptor is the most important, and TrkA expression is remarkably high in neuroblastomas with favorable prognosis, and the signal from this is signaled by tumor cell survival, differentiation, or cell death (apoptosis). Is strongly controlled.
  • Trk A expression is significantly suppressed. Instead, signals from Trk B or Ret promote tumor progression by promoting survival.
  • N_myc neuronal oncogene
  • the present invention has been made in view of such circumstances, and has clarified information on a novel gene that is expressed in neuroblastoma, and has also clarified information on the gene related to good or poor prognosis. It is another object of the present invention to enable a genetic diagnosis on the prognosis of neuroblastoma based on the genetic information.
  • the present inventors have examined the prognosis of human neuroblastoma and succeeded in producing cDNA libraries from clinical tissues having good and poor prognosis. Furthermore, about 2400 clones were cloned from each of these two types of cDNA libraries, and classified according to the prognosis of neuroblastoma.
  • the present inventors determined both terminal sequences of the cloned gene, and performed a homology search to select only a novel gene. As a result, it was confirmed that the cDNA library derived from the human neuroblastoma had a very high proportion of new genes as compared with those derived from other cells. Furthermore, the present inventor has found that, when comparing the novel gene groups classified as described above, expression of some genes is enhanced only in clinical tissues having a favorable prognosis of neuroblastoma.
  • the present inventors have made it possible to provide genetic information (nucleic acid sequence) for detecting and cloning a novel gene expressed in human neuroblastoma. Furthermore, based on the nucleic acid sequence information, it has become possible to make a genetic diagnosis of prognosis of neuroblastoma and to select a tumor marker that can be used for the diagnosis, thereby completing the present invention.
  • the present invention provides the nucleic acids or nucleic acid fragments described in 1 to 15 below. Furthermore, the present invention provides uses of the nucleic acids or nucleic acid fragments described in 16 to 18 below.
  • a nucleic acid consisting of one sequence selected from the group consisting of the nucleic acid sequences set forth in SEQ ID NOs: 1 to 4038 in the sequence listing, or a nucleic acid complementary thereto.
  • nucleic acid derived from a gene expressed in human neuroblastoma comprising one sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs: 1 to 4038 in the sequence listing; or Nucleic acid complementary to it.
  • nucleic acid according to the above 2 wherein one sequence is selected from the group consisting of the nucleic acid sequences set forth in SEQ ID NOs: 1914 to 3981 in the sequence listing.
  • nucleic acid according to the above item 5 wherein the nucleic acid is DNA.
  • a nucleic acid derived from a gene expressed in a human neuroblastoma having a favorable prognosis the nucleic acid being represented by SEQ ID NO: 1 to 1913 in the sequence listing or SEQ ID NO: 392 to 4038
  • a nucleic acid comprising one sequence selected from the group consisting of sequences, or a nucleic acid complementary thereto.
  • a nucleic acid derived from a gene expressed in a poorly prognostic human neuroblastoma which comprises one sequence selected from the group consisting of the nucleic acid sequences set forth in SEQ ID NOs: 1914 to 3981 in the sequence listing. Or a nucleic acid complementary thereto. 10. The nucleic acid according to 9 above, wherein the nucleic acid is DNA.
  • a nucleic acid derived from a gene whose expression is enhanced It is a nucleic acid derived from a gene whose expression is enhanced in neuroblastoma, and comprises one sequence selected from the group consisting of the nucleic acid sequences set forth in SEQ ID NOs: 3982 to 4038 in the sequence listing.
  • An isolated nucleic acid capable of hybridizing with the nucleic acid of any one of the above 1 to 12 under stringent conditions.
  • nucleic acid according to the above 14, wherein the nucleic acid is DNA.
  • a PCR primer comprising the nucleic acid according to 15 above.
  • a diagnostic method for prognosis of human neuroblastoma which comprises detecting the nucleic acid according to 11 above from a clinical tissue of human neuroblastoma.
  • a diagnostic kit for prognosis of human neuroblastoma comprising a set of PCR primers consisting of an isolated nucleic acid capable of hybridizing with the nucleic acid of the above item 12 under stringent conditions.
  • the above-mentioned preferred nucleic acids are those derived from genes whose expression is enhanced only in human neuroblastomas having a good prognosis in comparison between human neuroblastomas having a good prognosis and human neuroblastomas having a poor prognosis. And the information on the sequence of the nucleic acid allows the prognosis of human neuroblastoma to be diagnosed.
  • FIG. 1 is a view corresponding to an electrophoresis photograph showing the results of analysis of N-myc amplification in genomic DNA of human neuroblastoma clinical tissue by Southern hybridization.
  • lane 1 is a genome DNA (positive control group) from a neuroblastoma cell line with a poor prognosis.
  • Lane 2 is genomic DNA of normal human placenta (negative control group with good prognosis).
  • Lanes 3-7 are the genome of human neuroblastoma clinical tissue (Cases 1-5): DNA.
  • FIG. 2A shows an example of a gene whose expression was found to be enhanced in semi-quantitative PCR as a result of examining gene expression levels in human neuroblastomas with good and poor prognosis (human ⁇ transblastoma with good prognosis).
  • FIG. 9 is a diagram corresponding to an electrophoretic photograph showing an example of enhancement of expression in FIG.
  • lanes 1 to 8 and lanes 18 to 25 are clinical tissue samples of human neuroblastoma with good prognosis.
  • Lanes 10-17 and lanes 27-34 are clinical tissue samples of poorly prognostic human neuroblastoma.
  • Lanes 9 and 26 are size markers.
  • Figure 2B shows semi-quantitative P expression of gene expression in human neuroblastomas with good and poor prognosis.
  • FIG. 9 is a diagram corresponding to an electrophoresis photograph showing another example of a gene whose expression was found to be enhanced as a result of CR examination (an example of enhanced expression in human neuroblastoma with a poor prognosis).
  • lanes 1 to 8 and lanes 18 to 25 are clinical tissue samples of human neuroblastoma with good prognosis.
  • Lanes 10-17 and 27-34 are clinical tissue samples of human neuroblastoma with poor prognosis.
  • Lanes 9 and 26 are size markers.
  • FIG. 9 is a diagram corresponding to an electrophoresis photograph showing the expression of both human neuroblastoma and a poor prognosis.
  • lanes 1 to 8 and lanes 18 to 25 are clinical tissue samples of human neuroblastoma with good prognosis.
  • Lanes 10-17, And lanes 27-34 are clinical tissue samples of human neuroblastoma with poor prognosis.
  • Lanes 9 and 26 are size markers.
  • Figure 4A corresponds to an electrophoresis photograph showing an example of a gene whose cell cycle-specific gene expression was examined by semi-quantitative PCR and showed similar expression in all cell cycles.
  • FIG. 4A corresponds to an electrophoresis photograph showing an example of a gene whose cell cycle-specific gene expression was examined by semi-quantitative PCR and showed similar expression in all cell cycles.
  • Fig. 4B is a diagram corresponding to an electrophoresis photograph showing an example of a gene whose expression was reduced only in the G1 phase as a result of examining the expression level of a cell cycle-specific gene by semi-quantitative PCR. .
  • FIG. 4C is a diagram corresponding to an electrophoresis photograph showing an example of a gene whose expression was reduced only in the S phase as a result of examining the expression level of a cell cycle-specific gene by semi-quantitative PCR.
  • Fig. 4D is a diagram corresponding to an electrophoresis photograph showing an example of a gene in which expression was reduced only in the G2ZM phase as a result of examining the expression level of a cell cycle-specific gene by semi-quantitative PCR. is there.
  • Figure 4E is a diagram corresponding to an electrophoresis photograph showing an example of a gene whose expression was found to be enhanced only in the G1 phase as a result of examining the expression level of a cell cycle-specific gene by semi-quantitative PCR. .
  • FIG. 4F is a diagram corresponding to an electrophoresis photograph showing an example of a gene whose expression was found to be enhanced only in the S phase as a result of examining the expression level of a cell cycle-specific gene by semi-quantitative PCR.
  • Figure 4G is a diagram corresponding to an electrophoresis photograph showing an example of a gene whose expression was found to be enhanced only in the G2ZM phase as a result of examining the expression level of cell cycle-specific genes by semi-quantitative PCR. is there.
  • Figure 4H corresponds to an electrophoresis photograph showing an example of a gene whose expression was found to be enhanced in the S and G2 / M phases as a result of examining the expression levels of cell cycle-specific genes by semi-quantitative PCR.
  • Lane 2 is HeLa cells in stage 01, treated with 400 ⁇ 1 of 111; 11110 sine for 18 hours.
  • Lane 3 is HeLa cells treated with 2 mM tl ⁇ lymi dine for 20 hours and 100% in S phase.
  • Lane 4 was treated with 0.6 g Zm1 of Noco dazo 1 e for 18 hours, and 85% of the HeLa cells were in the G2ZM phase.
  • nucleic acids derived from a novel gene expressed in human neuroblastoma according to the present invention (hereinafter, referred to as “genes of the present invention”) and nucleic acid fragments related thereto (hereinafter, “nucleic acids of the present invention”) )
  • nucleic acids of the present invention and nucleic acid fragments related thereto (hereinafter, “nucleic acids of the present invention”) )
  • nucleic acid fragment of the present invention especially when it is not necessary to distinguish between nucleic acid and its fragment and describe them, they are collectively referred to as “nucleic acid of the present invention”. This will be described in detail with reference to various embodiments.
  • nucleic acid of the present invention is derived from the gene according to the present invention as described above, and constitutes the gene or is obtained from the gene by an in vivo or in vitro process.
  • nucleic acid refers to a polynucleotide that is, for example, DNA or RNA, or an active DNA or RNA derived therefrom, and preferably refers to DNA or: RNA.
  • Particularly preferred nucleic acids have sequences identical or complementary to the human cDNA sequences disclosed herein. Such DNAs and fragments thereof are hereinafter referred to as “DNA of the present invention” and “DNA fragment of the present invention”.
  • DNA of the present invention Especially when it is not necessary to distinguish between DNA and its fragments, they are collectively referred to as “DNA of the present invention”. Also called DNA.
  • the DNA is represented by a single-stranded: DNA (sense strand) sequence as shown in the nucleic acid sequence (base sequence) corresponding to the sequence number in the sequence listing, but is not only complementary to the sequence but also A single strand (antisense strand), and a double-stranded DNA containing both of them.
  • the DNA of the present invention is obtained from clinical tissue of human neuroblastoma, and has the following characteristics including the description in the examples.
  • cDNA from cells and tissues Methods for making lee are known to those skilled in the art, for example,
  • hybridizes under stringent engineering conditions refers to two nucleic acids (or fragments) that are described in Sambrook et al. Expression (Expression of Cioned Genes in E. coli) ", Molecular Cular Cloning: A Labo rat o ry Manua l (1989) Cold Spr i ng Harbo r Labo rat o ry Pre ss, New York, USA, 9.47-9.62 and 1 1.45—1.1.61, meaning that they hybridize to each other under the hybridization conditions.
  • the "stringent conditions” refers to hybridization at 6.degree. C. at 6.degree. C. and then washing at 2.degree. C. with 2.x SSC.
  • the salt concentration in the washing step should be, for example, about 2. OxSSC, 50 as low stringency. From C, it is possible to select up to about 0.2xSSC as high stringency and 50 ° C.
  • the temperature of the washing step can be increased from room temperature at low stringency conditions, about 22 ° C, to about 65 ° C under high stringency conditions.
  • isolated nucleic acid refers to a substance that is substantially free of cellular material and culture medium when produced by recombinant DNA technology and is chemically synthesized. Refers to nucleic acids or polynucleotides that are substantially free of precursor or other chemicals.
  • the nucleic acid of the present invention can also be a normally isolated nucleic acid, unless it is in a native state.
  • Neuroblastoma is a tumor of the nerve cell itself, which is known only in two types in humans Analyzing the genes that are expressed there is thought to bring enormous insights into understanding neuronal biology. In other words, it is extremely difficult and practically impossible to obtain a site-specific homogeneous tissue from the brain or peripheral nerves. Neuroblastomas, on the other hand, consist of a nearly homogenous population of neurons (albeit swollen) derived from peripheral sympathetic nerve cells, and the possibility of obtaining uniformly expressed neuronal genes is likely. high. In addition, since neuroblastoma is a cancer, it is characterized by many important genes expressed at an immature stage of neurogenesis.
  • neuroblastomas have a distinct clinical and biological distinction between those with good prognosis and those with poor prognosis.
  • Neuroblastoma cancer cells with a good prognosis have a very slow growth rate and begin to regress spontaneously at some point. From the findings so far, in this spontaneous regression, neuronal differentiation and apoptosis (neural cell death) occur, and the shunting and programmed cell death that occur at the stage of maturation of normal neurons are very similar. Is a phenomenon. Therefore, by analyzing the genes expressed in this tumor, it is extremely possible to obtain important gene information related to neural differentiation and apoptosis.
  • a poor prognosis neuroblastoma is a tumor composed of cancer cells that continue to grow malignantly. Therefore, it is highly likely that there are many important genes related to the proliferation of nerve cells and genes expressed in undifferentiated nerve cells. In other words, it is highly likely that genetic information completely different from the profile of the gene expressed in neuroblastoma with a favorable prognosis can be obtained.
  • neuroblastoma cell line (cell line) is derived from a clinical tissue with a poor prognosis, and the gene expression profile is considered to be significantly different from that of normal neurons as the tumor develops and progresses.
  • nucleic acids of the present invention include nucleic acids of genes whose expression is enhanced only in a specific cell cycle, and this indicates that It is expected that very useful genetic information on differentiation will likely be available.
  • the nucleic acid of the present invention which has various characteristics as described above and is derived from a novel gene from which useful genetic information can be obtained, can be obtained from a clinical tissue of human neuroblastoma, 038, or a part of the nucleic acid sequence.
  • the term “good prognosis” refers to human neuroblastoma in which the tumor is localized or has regressed or has become a benign sympathetic ganglion cell tumor. Based on N-myc and other tumor markers (Trk A, chromosomal abnormalities, etc.), it is judged to have low malignancy.
  • Trk A tumor marker
  • chromosomal abnormalities etc.
  • patients with a stage of 1 or 2 an onset age of less than 1 year, surviving for at least 5 years after surgery without recurrence, and having no evidence of N-myc in clinical tissue have a favorable prognosis
  • poor prognosis refers to the state of progression of human neuroblastoma in which tumor progression is observed, and the degree of malignancy is determined based on N-myc and other tumor markers. Is determined to be high.
  • a patient with stage 4 onset age of 1 year or older, died within 3 years after surgery, and an increase in N-myc in clinical tissues was regarded as poor prognosis.
  • stage 4 onset age of 1 year or older, died within 3 years after surgery, and an increase in N-myc in clinical tissues was regarded as poor prognosis.
  • it is not limited to such a specific example.
  • nucleic acids having the nucleic acid sequences of SEQ ID NOs: 1 to 4038 in the sequence listing nucleic acids derived from genes expressed in neuroblastomas having favorable prognosis are represented by SEQ ID NO: 1.
  • the nucleic acid derived from the gene expressed in the neuroblastoma having a poor prognosis is a nucleic acid having the nucleic acid sequence of SEQ ID NO: 1914 to 3981.
  • These two groups of nucleic acids are the nucleic acids of the present invention. Together they constitute suitable nucleic acids.
  • nucleic acid sequence of SEQ ID NO: 3982 or 4038 in the sequence listing was compared. Very significant differences were found in the corresponding genes. That is, the expression of these genes was enhanced in human neuroblastoma with a favorable prognosis. Therefore, the nucleic acid sequence of SEQ ID NOs: 3982 to 4038 can be used as a tumor marker for diagnosing the quality of neuroblastoma by detecting nucleic acids having those nucleic acid sequences in addition to the useful genetic information described above. It can also be used as information on one car. For this reason, nucleic acids having the nucleic acid sequences of SEQ ID NOs: 3982 to 4038 in the above sequence listing constitute particularly suitable nucleic acids among the nucleic acids of the present invention.
  • the nucleic acid of the present invention has utility in the following procedures for obtaining human neuroblastoma and various information related thereto.
  • the present invention it is possible to detect a gene expressed in human neuroblastoma by using the nucleic acid of the present invention or a fragment thereof as a probe for hybridization. is there. Furthermore, by using the nucleic acid of the present invention or a fragment thereof as a probe for hybridization, the distribution of the gene expression can be identified by examining the gene expression in various tumors and normal tissues.
  • the hybridization method itself is not particularly limited. Preferred methods include, for example, Northern hybridization, Southern hybridization, Colony hybridization, Dot hybridization, Fluorescence in situ hyoridazation ⁇ I SH), Insanchu Hyprideization [ins itu hy brida zation (I SH)], DNA chip method, microarray method, and the like.
  • One application example of the above-mentioned hybridization is to use the DNA of the present invention or a fragment thereof as a probe for Northern hybridization, to measure the length of mRNA in a sample to be assayed, and to perform gene expression. Can be quantitatively detected.
  • FI SH F 1 uorescence in situ hybrid dazation
  • tissue distribution of gene expression by using the DNA of the present invention or a fragment thereof as a probe for insanthu hybridization.
  • the DNA of the present invention or a fragment thereof is used as a probe for hybridization, at least 40 bases are required, and 40 or more consecutive bases in the DNA or a fragment thereof of the present invention are required. Some are preferably used. More preferably, those having 60 or more bases are used.
  • DNA probe reactions in the above-described various hybridizations are well known.
  • appropriate hybridization conditions between a DNA probe of the present invention of each length and a target DNA can be easily determined. it can.
  • the probes according to the invention are labeled so that they can be easily detected.
  • Detectable labels are detected either visually or using instruments. It can be any kind, element or compound that can be issued. Commonly used detectable labels include radioisotopes, avidin or biotin, and fluorescent materials (such as FITC or rhodamine).
  • the radioisotope is 32 P, 14 C, 125 I, 3 H, 35 S and the like.
  • the probe labeled with biotin is detected after hybridization using a labeling means such as avidin Z streptavidin, fluorescent label, enzyme, colloidal gold complex and the like.
  • the probe according to the present invention may be labeled by binding to a protein.
  • a labeling means such as avidin Z streptavidin, fluorescent label, enzyme, colloidal gold complex and the like.
  • the probe according to the present invention may be labeled by binding to a protein.
  • radioactive or fluorescent histone single-chain binding proteins are used.
  • PCR Polynucleotide Rase Chain Reactio n
  • mRNA can be extracted from a sample to be assayed, and gene expression can be measured semi-quantitatively by RT-PCR.
  • a method is performed by a method known to the parties concerned. For example, Sambrook et al., “Molecular cloning: an experimental method (Mo1ecu1arCloning: A Laboratory Manu nu a 1), Vol. 2, 2nd edition, Cold Spring Herba 1 (1989) and Introduction to Genetic Diseases (Fumimaro Takahisa: Nankodo).
  • the DNA or its fragment of the present invention When used as a primer for PCR, it needs to have a base length of 10 to 60, and among the DNA or its fragment of the present invention, there are 10 to 60 consecutive bases. Are preferably used. More preferably, those having 15 to 30 bases are used. Generally, those having a GC content of 40 to 60% in a primer sequence are preferred. Furthermore, it is desired that there is no difference in the Tm value between the two primers used for amplification. It is also desirable that the primers do not anneal at the 3 'end and do not take secondary structures within the primers. (3) Gene expression screening
  • nucleic acid of the present invention By using the nucleic acid of the present invention or a fragment thereof, it is possible to detect the expression distribution of the target gene expressed in various tissues and cells. This can be achieved, for example, by using the DNA of the present invention or a fragment thereof as a hybridization probe or PCR primer as described above.
  • the DNA of the present invention or a fragment thereof is directly attached on the chip or array.
  • a method of spotting such DNA or the like on a substrate using a high-precision dispenser is known (for example, see US Pat. No. 5,807,522).
  • mRNA extracted from cells of the test tissue is labeled with a fluorescent substance or the like and hybridized, and it is possible to analyze in which tissue cells the gene is highly expressed.
  • the DNA to be attached to the chip or array may be a PCR reaction product using the DNA of the present invention or a fragment thereof as a probe.
  • the DNA fragments of the invention can be synthesized directly on a substrate to form a DNA chip or array (see, eg, US Pat. No. 5,424,186).
  • the DNA of the present invention or a fragment thereof can be used as an index for various gene screenings. For example, when screening for oncogenes, it is possible to preferentially analyze genes of interest that are strongly expressed in cancer cells and weakly expressed in normal cells.
  • Genes targeted for such cloning include genes whose expression levels differ between neuroblastomas with a good prognosis and neuroblastomas with a poor prognosis, and genes in other tissues and cancer cells. Expression is expressed by a gene that is expressed differently from the expression mode, a gene that is expressed in a cell cycle-dependent manner, a gene that is induced by neural differentiation, an oncogene or a tumor suppressor gene. Controlled genes and the like.
  • Cloning is performed by incorporating the DNA of the present invention or a fragment thereof into an appropriate plasmid or bacteriophage, producing an expression vector, and introducing this into a host cell for transformation in accordance with ordinary gene recombination techniques. (Introduction) and culturing the transformant.
  • Such individual operations are described in, for example, “Synbrook et al., Molecular Cloning: An Experimental Method (Molaboratory: A Laboratory Manu a 1), Vol. 3, Second Edition, Cold Spring One Bar (1989) "and other well-known literature.
  • the gene corresponding to the nucleic acid sequence described in SEQ ID NO: 3982 to 4038 in the sequence listing is a human neuroblastoma having a favorable prognosis. Expression was enhanced.
  • the prognosis of human neuroblastoma can be diagnosed by detecting a nucleic acid having the nucleic acid sequence of any of SEQ ID NOs: 3982 to 4038 as a tumor marker. That is, it can be performed by examining the presence or absence of the enhancement of the expression of the gene in a sample containing cells from a tissue (clinical tissue) collected from a subject.
  • Examples of the method for detecting a gene (or RNA) include the above-mentioned Northern blot hybridization method, in situ hybridization method, and RT-PCR method.
  • the amount of nucleic acid hybridized with a probe comprising the DNA of the present invention (particularly, having one of the nucleotide sequences of SEQ ID NOs: 3982 to 4038) in a sample is increased. If so, it can be diagnosed that the prognosis is good.
  • the RT-PCR method is used, mRNA is extracted from a sample, reversely transcribed into DNA, amplified with a primer prepared based on the DNA, and the gene expression is measured semi-quantitatively. If the gene expression is increased in this way, it can be diagnosed that the prognosis is good.
  • a diagnostic kit containing one set of the primer as an essential component.
  • the diagnostic kit contains a PCR buffer, Contains well-known components such as purified solution and enzymes.
  • an antisense oligonucleotide to the nucleic acid of the present invention.
  • the antisense oligonucleotide is capable of hybridizing to the nucleic acid of the present invention, and includes antisense DNA and antisense: NA.
  • Antisense DNA inhibits transcription of DNA to mRNA; antisense; RNA inhibits translation of mRNA.
  • Such an antisense oligonucleotide can be synthesized using an automatic synthesizer if it is a natural type, or by a PCR method using the DNA of the present invention as a ⁇ type.
  • the antisense oligonucleotide also includes an antisense oligonucleotide derivative having improved binding ability to target mRNA, tissue selectivity, cell permeability, nuclease resistance, and intracellular stability.
  • an antisense oligonucleotide derivative having improved binding ability to target mRNA, tissue selectivity, cell permeability, nuclease resistance, and intracellular stability.
  • Such a derivative can be synthesized using a known antisense technique.
  • the antisense oligonucleotide having a sequence complementary to the sequence of the gene for the neuroblastoma or the translation initiation codon of the mRNA, the ribosome binding site, the cabling site, and the splice site according to the present invention is capable of synthesizing the RNA. Can be prevented, and the effect of suppressing gene expression is particularly high. Therefore, the present invention preferably includes such an antisense oligonucleotide.
  • a group of genes expressed in human neuroblastoma found by the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these Examples. .
  • Clinical tissue of human neuroblastoma was quasi-sterilely frozen immediately after surgical removal, and then
  • the prognosis of the sample obtained in 1 above was determined based on the following criteria.
  • ⁇ Age of onset is less than 1 year ⁇ Age of onset is 1 year or older
  • RNA Extraction Kit manufactured by QIGEN
  • total RNA was extracted.
  • the extracted total RNA was purified using an oligo dT cellulose column (manufactured by Co 11 aborative) as a pool of mRNA having a po yA structure.
  • the total amount of pools of de Kiyadzupu mRNA prepared in the above 5 was dissolved in distilled sterile water containing 1 1 1 0. 1% DE PC, 5 of 4 ⁇ 1, 10x1 one oligo RNA (5 5 ⁇ 10 ⁇ 1 i gat ion buffer (T ris— HC 1 (5
  • DNA (—) oligocap mRNA pool prepared in the above 7 was reverse-transcribed using Super Script II (kit manufactured by Life Tech Oriental) to obtain a pool of 1st strand c DNA.
  • DNA (-) Origo cap mRNA pool is dissolved in 21 ⁇ 1 sterile distilled water, 10 ⁇ 1 10 XF irst Str and buffer (kit accessory), 8 ⁇ 1 dNTP mix (5 mM, kit included) Product), 6 ⁇ 1 01 1 (0.1 M, kit accessory), 2.5 l oligo-dT adapter primer (5 pmo5, -G TTTTT—3), 2.01: RNa sin (40 unit / 1), 21 Superscript IIRTase (kit accessory) added. This mixture was reacted at 42 ° C. for 3 hours to perform a reverse transcription reaction. Then, all RNAs were degraded by phenol-cloth form treatment, alkali treatment, and neutralization treatment, and purified by ethanol precipitation.
  • the pool of 1st st rand c DNA prepared in 8 above was subjected to PCR amplification using Gene Amp (a kit manufactured by PerkinElmer Inc.). 1 st st rand. Dissolve the pool of 8 in 52.4 1 sterile distilled water, and add 30 303.3 XR eaction buffer (kit accessory), 8 ⁇ 1 dNTP mix (2.5 mM, kit accessory), 4 . 4/1 of magnesium acetate (25 mM, kit accessory), 1. 6 ⁇ 1 primer one F (10 pmo 5 5 -AGC ATCGAGTCGGCCTTGTTG- 3,), primer one R (1 1.
  • the pool of 2nd st rand c DNA prepared in 9 above was dissolved in 871 sterile distilled water, and 10XNEB buffer (manufactured by NEB), 100XBS A (Bac serum albumin, manufactured by NEB), 2 ⁇ 1 Sfil (restriction enzyme, 20 units / l, NEB) was added. This mixture was reacted at 50 ° C for a
  • the SfiI-treated cDNA pool prepared in the above 10 was electrophoresed on a 1% agarose gel, and the fraction of 2 kb or more was purified using GeneClIIII (manufactured by Bio101).
  • the purified cDNA pool was dissolved in 100 ⁇ 1 sterile distilled water and left at 37 ° C for 6 hours. Thereafter, the mixture was purified by phenol / cloth form treatment and ethanol precipitation to obtain a long-chain cDNA pool.
  • the cDNA library prepared in 12 of Example 1 was transformed into Escherichia coli (TOP-10: manufactured by Invitrogen).
  • the cDNA library was dissolved in 10 ⁇ 1 sterile distilled water and mixed with TOP-10. Then, the mixture was incubated for 30 minutes on water, 1 minute at 40 ° C, and 5 minutes on ice. 500 zl of SOB medium was added, followed by shaking culture at 37 C for 60 minutes. An appropriate amount was seeded on an agar medium containing ampicillin, and cultured at 37 ° C for 24 hours to obtain an E. coli clone. 2.
  • E. coli clone Preparation of glycerol stock
  • the glycerol stock of 101 prepared in Example 2-2 was transferred to a 15 ml centrifugal tube, 3 ml of LB medium and 50 g / ml of ampicillin were added, and the mixture was shaken at 37 ° C to culture E. coli. . Thereafter, plasmid DNA was extracted and purified from Escherichia coli using a QI A rep Spin Miniprep Kit (manufactured by QIAGEN).
  • the sequences of both ends of the plasmid DNA prepared in 1 above were determined using a DNA Sequencing-Kit (kit manufactured by ABI). A mixture of 600 ng of plasmid DNA, 8 zl of premix (supplied with the kit), and 3.2 pmo 1 of primer was prepared using sterilized distilled water to make a total of 20 ⁇ 1.
  • the primer used was 5, one type for the terminal sequence (CTTCTGCTCTAAA AGCTGCG), and three types for the terminal sequence (CGACCTGCAG). C, 10 seconds-50 ° C, 5 seconds ⁇ 60 ° (25 cycles were repeated with one cycle consisting of 4 minutes. Thereafter, purification was performed by ethanol precipitation. Electrophoresis was performed on polyacrylamide gel under denaturing conditions. The sequence was determined using ⁇ 377 (manufactured by ⁇ ).
  • Example 3 the nucleotide sequence information of the sample obtained by analyzing the both terminal sequences was subjected to a homology search of the nucleotide sequence via the Internet. To search BLAST of NCBI (National Center of Biotechnology, USA) was used. Good prognosis. Poor. As a result of analyzing about 8000 genes in total, there was no homozygosity for about half of the genes. The nucleotide sequences of these genes are shown in SEQ ID NOs: 1 to 4038 in the sequence listing.
  • Example 5 Comparison of gene expression levels in human neuroblastoma with good and poor prognosis by semi-quantitative PCR.
  • a PCR primer was synthesized from a part of the novel gene group obtained in Example 4, and the expression level was compared and quantified in clinical tissues with good or poor prognosis of human neuroblastoma.
  • MRNA was extracted from the clinical tissue of the human neuroblastoma by the method described in Examples 1-3, and a PCR reaction was performed using rTaq (Takara Shuzo). Specifically, 5/1 sterile distilled water, 2 ⁇ l of mRNA, 1/1 of 10 X rTaq buffer, 1/1 of 2 mM dNTPs, each set of 0.51 synthetic primer, 0. 5 1; rTaq mixed.
  • Example 6 Measurement of expression level of cell cycle-dependent gene by semi-quantitative PCR A PCR primer was synthesized from a part of the novel gene obtained in Example 4, and the cells were obtained using HeLa cells. The expression level of cycle-specific genes was compared and quantified. The used HeLa cells were treated as follows.
  • MRNA was extracted from the above four types of HeLa cells by the method shown in Examples 13 and 13, and a PCR reaction was performed using rTaq (manufactured by Takara Shuzo). Specifically, 5 ⁇ 1 sterile distilled water, 2 ⁇ l mRNA, 11 ⁇ 10 X rTaq buffer, 1 ⁇ 1 2 mM dNTPs, 0.51 each of synthetic primer set of 0.51 0.51 rT aq was mixed. After denaturing this mixture at 95 ° C for 2 minutes, repeat 35 cycles of 95 ° C, 15 seconds, 55 ° C, 15 seconds, 72 ° C, and 20 seconds as one cycle, and further at 72 ° C for 6 minutes. After standing, the PCR reaction was performed. The reaction was electrophoresed on a 1% agarose gel. Fig. 4 shows an example of the results of the electrophoresis. Analysis was performed on 66 new genes. Table 1 shows the analysis results.
  • the nucleic acid of the present invention is a nucleic acid derived from a novel gene expressed in neuroblastoma, and thus reveals information on the gene.
  • the DNA of the present invention or a fragment thereof can be used as a probe or a primer in various hybridization or PCR methods, and the expression of the gene in other tissues and cells can be detected, and its structure and function can be analyzed. .
  • the nucleic acid of the present invention includes nucleic acids of genes that have been expressed in neuroblastomas with good prognosis and neuroblastoma with poor prognosis, respectively. And the degree of progress can be diagnosed by the expression level of the gene.
  • the nucleic acid of the present invention is a gene whose expression is enhanced in human neuroblastoma having a good prognosis when human neuroblastoma having a good prognosis is compared with human neuroblastoma having a poor prognosis. Therefore, the prognostic genetic diagnosis of neuroblastoma can be performed based on such genetic information.
  • the gene is N-my c Since the offspring are poor prognostic factors, they are regarded as good prognostic factors like the TrkA gene, and serve as an index (tumor marker) of neuroblastoma malignancy and sensitivity to anticancer drugs. obtain.

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Abstract

L'invention concerne des acides nucléiques originaires d'un nouveau gène exprimé dans un neuroblastome humain comprenant une séquence choisie parmi le groupe de séquences d'acides nucléiques représentées par SEQ ID NOS : 1 à 4038 dans la liste des séquences ou des acides nucléiques complémentaires à ceux-là, des fragments de ces acides nucléiques, l'utilisation de ces derniers en tant que sonde ou amorce, et un procédé de diagnostic du pronostic du neuroblastome humain en utilisant l'un d'eux.
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WO2002026997A2 (fr) * 2000-09-26 2002-04-04 Ceptyr, Inc. Dsp-16, phosphatase a double specificite
WO2002031126A2 (fr) * 2000-10-11 2002-04-18 Bayer Aktiengesellschaft Regulation d'une peptidyl-prolyl cis-trans isomerase de type cyclophiline humaine
WO2002097093A1 (fr) * 2001-05-30 2002-12-05 Chiba-Prefecture Acides nucleiques isoles dans le neuroblastome
WO2002103017A1 (fr) * 2001-05-31 2002-12-27 Chiba-Prefecture Acides nucleiques isoles dans le neuroblastome
WO2005014818A1 (fr) 2003-08-08 2005-02-17 Perseus Proteomics Inc. Gene surexprime dans le cancer
EP1546172A2 (fr) * 2002-05-31 2005-06-29 Schering Aktiengesellschaft Sequences de commande du gene de corine humain
EP1666490A2 (fr) * 2000-07-25 2006-06-07 Genentech, Inc. Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci
US7115727B2 (en) 2002-08-16 2006-10-03 Agensys, Inc. Nucleic acids and corresponding proteins entitled 282P1G3 useful in treatment and detection of cancer
WO2005012875A3 (fr) * 2003-07-29 2007-03-15 Bristol Myers Squibb Co Biomarqueurs de modulation des kinases dependantes des cyclines
WO2007048978A2 (fr) * 2005-10-28 2007-05-03 Biomerieux Sa Procede de detection du cancer
WO2007066698A1 (fr) 2005-12-06 2007-06-14 Kyowa Hakko Kogyo Co., Ltd. Anticorps anti-perp génétiquement recombiné
US7291719B2 (en) 2000-07-25 2007-11-06 Genentech, Inc. PRO4332 antibodies
WO2008015942A1 (fr) * 2006-07-31 2008-02-07 Hisamitsu Pharmaceutical Co., Inc. Agent thérapeutique contre le neuroblastome, procédé de criblage à la recherche de l'agent thérapeutique et procédé de détermination du pronostic d'un neuroblastome
JPWO2006064864A1 (ja) * 2004-12-17 2008-06-12 第一化学薬品株式会社 癌状態の判定方法及びその方法に用いる遺伝子産物検出試薬
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WO2009110383A1 (fr) 2008-03-04 2009-09-11 久光製薬株式会社 Procédé de criblage d'un agent thérapeutique pour le cancer
US7601532B2 (en) 2003-09-25 2009-10-13 Hisamitsu Pharmaceutical Co., Inc. Microarray for predicting the prognosis of neuroblastoma and method for predicting the prognosis of neuroblastoma
US7736654B2 (en) 2001-04-10 2010-06-15 Agensys, Inc. Nucleic acids and corresponding proteins useful in the detection and treatment of various cancers
WO2015035305A1 (fr) * 2013-09-09 2015-03-12 Somalogic, Inc. Aptamères de pdgf et vegf présentant une stabilité améliorée et leur utilisation dans le traitement de maladies et de troubles médiés par pdgf et vegf
US9410156B2 (en) 2012-03-28 2016-08-09 Somalogic, Inc. Aptamers to PDGF and VEGF and their use in treating PDGF and VEGF mediated conditions

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US7335731B2 (en) 2000-07-25 2008-02-26 Genentech, Inc. PRO4332 polypeptides
US7291719B2 (en) 2000-07-25 2007-11-06 Genentech, Inc. PRO4332 antibodies
EP1666490A3 (fr) * 2000-07-25 2006-11-02 Genentech, Inc. Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci
EP1666490A2 (fr) * 2000-07-25 2006-06-07 Genentech, Inc. Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci
WO2002026997A3 (fr) * 2000-09-26 2003-01-09 Ceptyr Inc Dsp-16, phosphatase a double specificite
WO2002026997A2 (fr) * 2000-09-26 2002-04-04 Ceptyr, Inc. Dsp-16, phosphatase a double specificite
WO2002031126A2 (fr) * 2000-10-11 2002-04-18 Bayer Aktiengesellschaft Regulation d'une peptidyl-prolyl cis-trans isomerase de type cyclophiline humaine
WO2002031126A3 (fr) * 2000-10-11 2002-12-05 Bayer Ag Regulation d'une peptidyl-prolyl cis-trans isomerase de type cyclophiline humaine
US7736654B2 (en) 2001-04-10 2010-06-15 Agensys, Inc. Nucleic acids and corresponding proteins useful in the detection and treatment of various cancers
WO2002097093A1 (fr) * 2001-05-30 2002-12-05 Chiba-Prefecture Acides nucleiques isoles dans le neuroblastome
US7335755B2 (en) 2001-05-30 2008-02-26 Hisamitsu Pharmaceutical Co., Inc. Nucleic acids isolated in neuroblastoma
WO2002103017A1 (fr) * 2001-05-31 2002-12-27 Chiba-Prefecture Acides nucleiques isoles dans le neuroblastome
EP1546172A2 (fr) * 2002-05-31 2005-06-29 Schering Aktiengesellschaft Sequences de commande du gene de corine humain
EP1546172A4 (fr) * 2002-05-31 2005-11-30 Schering Ag Sequences de commande du gene de corine humain
US7115727B2 (en) 2002-08-16 2006-10-03 Agensys, Inc. Nucleic acids and corresponding proteins entitled 282P1G3 useful in treatment and detection of cancer
US7612172B2 (en) 2002-08-16 2009-11-03 Agensys, Inc. Nucleic acids and corresponding proteins entitled 282P1G3 useful in treatment and detection of cancer
US7429451B2 (en) 2002-10-30 2008-09-30 Hisamitsu Pharmaceutical Co., Inc. Nucleic acids isolated from stage 4 neuroblastoma
WO2005012875A3 (fr) * 2003-07-29 2007-03-15 Bristol Myers Squibb Co Biomarqueurs de modulation des kinases dependantes des cyclines
EP2311468A1 (fr) 2003-08-08 2011-04-20 Perseus Proteomics Inc. Gène surexprimé dans le cancer
WO2005014818A1 (fr) 2003-08-08 2005-02-17 Perseus Proteomics Inc. Gene surexprime dans le cancer
US7601532B2 (en) 2003-09-25 2009-10-13 Hisamitsu Pharmaceutical Co., Inc. Microarray for predicting the prognosis of neuroblastoma and method for predicting the prognosis of neuroblastoma
US8895243B2 (en) 2004-12-17 2014-11-25 Sekisui Medical Co., Ltd. Method of assessing cancerous conditions and reagent for detecting gene product to be used in the method
JP5089172B2 (ja) * 2004-12-17 2012-12-05 積水メディカル株式会社 がん組織の検出方法及びその方法に用いるがん組織の検出試薬
JPWO2006064864A1 (ja) * 2004-12-17 2008-06-12 第一化学薬品株式会社 癌状態の判定方法及びその方法に用いる遺伝子産物検出試薬
WO2007048978A2 (fr) * 2005-10-28 2007-05-03 Biomerieux Sa Procede de detection du cancer
WO2007048978A3 (fr) * 2005-10-28 2007-09-07 Biomerieux Sa Procede de detection du cancer
WO2007066698A1 (fr) 2005-12-06 2007-06-14 Kyowa Hakko Kogyo Co., Ltd. Anticorps anti-perp génétiquement recombiné
WO2008015942A1 (fr) * 2006-07-31 2008-02-07 Hisamitsu Pharmaceutical Co., Inc. Agent thérapeutique contre le neuroblastome, procédé de criblage à la recherche de l'agent thérapeutique et procédé de détermination du pronostic d'un neuroblastome
WO2009110383A1 (fr) 2008-03-04 2009-09-11 久光製薬株式会社 Procédé de criblage d'un agent thérapeutique pour le cancer
US9410156B2 (en) 2012-03-28 2016-08-09 Somalogic, Inc. Aptamers to PDGF and VEGF and their use in treating PDGF and VEGF mediated conditions
US9701967B2 (en) 2012-03-28 2017-07-11 Somalogic, Inc. Aptamers to PDGF and VEGF and their use in treating PDGF and VEGF mediated conditions
US10221421B2 (en) 2012-03-28 2019-03-05 Somalogic, Inc. Post-selec modification methods
US11208663B2 (en) 2012-03-28 2021-12-28 Somalogic, Inc. Post-selex modification methods
WO2015035305A1 (fr) * 2013-09-09 2015-03-12 Somalogic, Inc. Aptamères de pdgf et vegf présentant une stabilité améliorée et leur utilisation dans le traitement de maladies et de troubles médiés par pdgf et vegf
US9695424B2 (en) 2013-09-09 2017-07-04 Somalogic, Inc. PDGF and VEGF aptamers having improved stability and their use in treating PDGF and VEGF mediated diseases and disorders
US9994857B2 (en) 2013-09-09 2018-06-12 Somalogic, Inc. PDGF and VEGF aptamers having improved stability and their use in treating PDGF and VEGF mediated diseases and disorders
US10544419B2 (en) 2013-09-09 2020-01-28 Somalogic Inc. PDGF and VEGF aptamers having improved stability and their use in treating PDGF and VEGF mediated diseases and disorders

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