JP6467580B2 - Diagnostic and therapeutic agents for small cell lung cancer - Google Patents

Description

  The present invention relates to a method for detecting small cell lung cancer using the expression level of nSR100 gene as an index, a diagnostic agent used in the method, and a therapeutic agent for small cell lung cancer targeting nSR100 gene.

  Lung cancer is a leading cause of cancer-related deaths worldwide and can be broadly divided into two types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Small cell lung cancer is known to have a high relevance to smoking. For example, nicotine, a component of tobacco smoke, promotes tumor growth and angiogenesis, and inhibits apoptosis of cancer cells It has been reported. In addition, small cell lung cancer is highly malignant and rapidly grows and metastasizes, so it is very important to detect it early. In addition, small cell lung cancer is difficult to treat effectively because of its high resistance to chemotherapy. Therefore, development of a more effective treatment method is demanded.

As a detection marker gene small cell lung cancer, REST (RE 1- s ilencing t ranscription factor) gene is known (Non-Patent Document 1). The REST gene normally expresses a protein having nine zinc finger domains, but a splicing variant protein (sREST protein) having only five zinc finger domains is specifically expressed in small cell lung cancer. Therefore, small cell lung cancer can be detected by using the expression level of this sREST protein or sREST mRNA as an index.

On the other hand, NSR100 although (n eural-specific SR -related protein of 100 kDa) protein expressed from the gene is known to have the function of controlling the splicing, the expression of the brain, olfactory bulb, and eyes, etc. of the nervous Since it is limited to tissue, it is considered to be involved in splicing specific to nerve tissue (Non-patent Document 2).

CANCER RESEARCH 60, 1840-1844, April 1, 2000 Cell 138, 898-910, September 4, 2009

  An object of the present invention is to find a novel small cell lung cancer marker gene, and to provide a method for detecting small cell lung cancer and a diagnostic agent used in the method. Furthermore, it aims at providing the therapeutic agent of small cell lung cancer.

  As a result of diligent research, the present inventors have surprisingly found that the nSR100 gene, whose expression is reported to be restricted to neural tissue, is specifically expressed in small cell lung cancer cells. I found it. In connection with this, the present inventors also found that the amount of miSR-suppressed miRNA increased in the blood of SCLC patients. Furthermore, we found an association between nSR100 gene expression and malignancy of small cell lung cancer. As a result of further research based on these findings, the present invention was completed.

  That is, the present invention includes the following aspects.

Item 1.
(A) a polynucleotide having at least 15 bases continuous in the base sequence of human nSR100 mRNA and / or a polynucleotide complementary to the polynucleotide; or (b) a base sequence of human nSR100 mRNA or a base sequence complementary thereto. A polynucleotide having at least 15 bases that hybridizes under stringent conditions;
A diagnostic agent for small cell lung cancer containing any of the above.

Item 2.
Item 2. The diagnostic agent for small cell lung cancer according to Item 1, wherein the polynucleotide is a probe or a primer.

Item 3.
(A ′) a polynucleotide having at least 15 bases consecutive in the base sequence of miR-4279, miR-4419b, miR-4516, or miR-4635 and / or a polynucleotide complementary to the polynucleotide, or (b ′ A polynucleotide having at least 15 bases that hybridizes under stringent conditions to the base sequences of miR-4279, miR-4419b, miR-4516, and miR-4635, or a base sequence complementary thereto,
A diagnostic agent for small cell lung cancer containing any of the above.

Item 4.
Item 4. The diagnostic agent for small cell lung cancer according to Item 3, wherein the polynucleotide is a probe or a primer.

Item 5.
A diagnostic agent for small cell lung cancer, comprising an antibody that recognizes human nSR100 protein.

Item 6.
(1) a step of measuring the amount of human nSR100 mRNA or a nucleic acid derived therefrom in a sample derived from lung tissue collected from a subject using the diagnostic agent according to item 1 or 2, and (2) the above step (1 ) Is the amount of human nSR100 mRNA or nucleic acid derived from it (sample mRNA expression level) in human nSR100 mRNA or the amount of nucleic acid derived from it in a sample derived from a tissue not containing small cell lung cancer tissue or nerve tissue ( A step for comparison with the expression level of the control mRNA),
Have
(3) A method for detecting small cell lung cancer, wherein the test mRNA expression level is higher than the control mRNA expression level, and the subject is a determination index that the subject has small cell lung cancer.

Item 7.
(1 ") The diagnosis according to Item 3 or 4, wherein at least one amount selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635 in a blood sample collected from a subject is used And (2 ″) at least one selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635 measured in the above step (1 ″). The amount (miRNA amount to be tested) is selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635 in a blood sample collected from a subject who is not a small cell lung cancer patient (eg, a healthy subject) Comparing with at least one amount (control miRNA amount)
Have
(3 ″) A method for detecting small cell lung cancer, wherein the test miRNA amount is higher than the control miRNA amount, and the subject is an indicator for determining that the subject has small cell lung cancer.

Item 8.
(1 ′) a step of measuring the expression level of human nSR100 protein in a sample derived from lung tissue collected from a subject using the diagnostic agent according to item 3, and (2 ′) measured in the above step (1 ′). Comparing the expression level of human nSR100 protein (test protein expression level) with the expression level of human nSR100 protein (control protein expression level) in a sample derived from a tissue not containing small cell lung cancer tissue or nerve tissue,
Have
(3 ′) A method for detecting small cell lung cancer, wherein the test protein expression level is higher than the control protein expression level, and the determination index is that the subject has small cell lung cancer.

Item 9.
A therapeutic agent for small cell lung cancer, comprising a nucleic acid that suppresses human nSR100 mRNA expression, human nSR100 protein expression, or human nSR100 protein function.

Item 10.
Item 10. The therapeutic agent for small cell lung cancer according to Item 9, wherein the nucleic acid is siRNA that specifically suppresses the expression of human nSR100 mRNA.

Item 11.
Item 10. The therapeutic agent for small cell lung cancer according to Item 9, wherein the nucleic acid is at least one selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635.

Item 12.
A therapeutic agent for small cell lung cancer, comprising an antibody that recognizes human nSR100 protein.

  According to the present invention, it is possible to provide a method for detecting small cell lung cancer based on the discovery of a new small cell lung cancer marker gene (nSR100 gene), and a diagnostic agent used in the method. Furthermore, a therapeutic agent for small cell lung cancer can also be provided. According to the detection method of the present invention, small cell lung cancer can be detected easily and efficiently by using the expression product amount of nSR100 gene or the amount of miSR suppression of nSR100 gene as an index. In addition, since nSR100 protein regulates the generation of sREST mRNA (or protein), which is a known small cell lung cancer marker, it increases nSR100 mRNA (or protein) expression in small cell lung cancer cells and suppresses nSR100 gene An increase in the blood level of miRNA is expected to occur prior to an increase in the expression level of sREST mRNA (or protein). Therefore, small cell lung cancer can be detected earlier by using the nSR100 gene expression level or the nSR100 gene suppression miRNA level as an index. Furthermore, according to the present invention, the malignancy of small cell lung cancer (such as tumor-forming ability in tissues other than the lung) can also be evaluated.

The structures of REST genes (I to VI) and the expression products (REST protein and sREST protein) are shown. Each box from I to VI indicates an exon constituting the REST gene. White bars indicate the structure of the REST protein or sREST protein, and the gray box in the bar indicates the zinc finger domain. As shown in the upper part of the figure, the sREST protein is a protein having five zinc finger domains on the N-terminal side, which is generated when a stop codon appears between exon V and exon VI due to frame shift. A shows the result of expression analysis of nSR100 protein in SCLC cells, and B shows the result of expression analysis of nSR100 protein, REST protein, and sREST protein in SCLC cells. The upper part of the photographic images of A and B shows the abbreviations of the cell names used for expression analysis. Shown on the right side of B is the name of the detected protein. A shows the expression analysis results of nSR100 protein in SCLC cells (N417) adherently cultured in suspension culture or extracellular matrix, and B shows SCLC cells adherently cultured in suspension culture or extracellular matrix. The expression analysis result of sREST mRNA (upper part), REST mRNA (lower part left), and nSR100 mRNA (lower part right) is shown. In A, the left 2 lanes show the results when the primary antibody (anti-nSR100 antibody) was not reacted, the right 2 lanes show the results when the primary antibody (anti-nSR100 antibody) was reacted, w / o represents the expression analysis result in SCLC cells cultured in suspension, and w represents the expression analysis result in SCLC cells adhered and cultured on the extracellular matrix. In B, the lower part of each graph shows the type of cell used for expression analysis. + ECL indicates that cells cultured on the extracellular matrix were used, (plate) indicates that cells that had adhered to the culture dish were used, and (medium) did not adhere to the culture dish and was in the medium. It shows that the cells that were floating were used. The vertical axis of each graph of B shows the relative expression level when the expression level of GAPDH mRNA is 1. A shows the expression analysis results of REST mRNA (upper), sREST mRNA (middle), and nSR100 mRNA (lower) in NCI-N417 cells in which nSR100 gene is suppressed, and B shows the results of expression analysis of these proteins. Indicates. The lower part of each graph of A and the upper part of B show the types of cells used for expression analysis. siRNA (-) indicates that NCI-N417 cells treated with RNAi using non-specific siRNA were used, and siRNA (+) represents NCI-N417 cells treated with RNAi using siRNA against the nSR100 gene. Indicates that it was used. Shown on the right side of B is the name of the detected protein. The vertical axis of each graph of A shows the relative expression level when the expression level of GAPDH mRNA is 1. A shows the photograph of the mouse | mouth after tumor formation experiment, B shows the expression analysis result of nSR100 mRNA, sREST mRNA, and REST in the formed tumor. In A, I to III are photographs of mice 60 days after injection, and IV is a photograph of mice 46 days after injection. In B, the lower part of each graph shows the type of sample injected. N417 is a suspension culture NCI-N417 cell sample (pattern I), N417 + ECL is an adhesion culture NCI-N417 cell sample (pattern III), N417 + Matrigel suspension culture NCI-N417 cells and a mixed sample of Matrigel (pattern IV). The vertical axis of each graph of B shows the relative expression level when the expression level of GAPDH mRNA is 1. A shows the results when NCI-N417 cells are used, B shows the results when NCI-H82 cells are used, and C shows the results when NCI-H1650 cells are used. In A to C, siRNA-A indicates siRNA targeting target sequence 1 (SEQ ID NO: 10), siRNA-B indicates siRNA targeting target sequence 2 (SEQ ID NO: 11), and siRNA-C indicates target SiRNA targeted to sequence 3 (SEQ ID NO: 12), siRNA-D represents siRNA targeted to target sequence 4 (SEQ ID NO: 13), siRNA mix represents an equimolar mixture of siRNA-A to siRNA-D Show. In each figure, the vertical axis represents the blood concentration of each miRNA. “Control” on the horizontal axis shows data of healthy subjects, “SCLC” shows data on SCLC patients, “NSCLC” shows data on NSCLC patients, and “gastric cancer” shows data on gastric cancer patients. In each figure, the vertical axis represents the blood concentration of miR-4516. `` Normal '' on the horizontal axis shows data for healthy subjects, `` SCLC '' shows data for SCLC patients, `` NSCLC '' shows data for NSCLC patients, `` gastric '' shows data for gastric cancer patients, `` bladder '' Indicates data for bladder cancer patients, “prostate” indicates data for prostate cancer patients, and “kidney” indicates data for kidney cancer patients.

1. Definition of terms The abbreviations such as nucleotide sequences (nucleotide sequences) and nucleic acids in this specification are defined by IUPAC-IUB (IUPAc-IUB communication on Biological Nomenclature, Eur. J. Biochem., 138; 9 (1984)). , “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Patent Office) and common symbols in the field.

  In the present invention, “gene” refers to a regulatory region, a coding region, an exon, and an intron without distinction unless otherwise specified.

  In the present invention, unless otherwise specified, “DNA” includes double-stranded DNA including human genomic DNA, single-stranded DNA including cDNA and synthetic DNA (sense strand), and 1 having a sequence complementary to the sense strand. Both double-stranded DNA (antisense strand) and fragments thereof are included. In the present specification, “RNA” includes not only single-stranded RNA but also single-stranded RNA having a sequence complementary thereto, and further double-stranded RNA composed thereof, unless otherwise specified. Used for purposes. The RNA includes synthetic RNA such as total RNA, mRNA, rRNA, siRNA and the like.

  In the present invention, “nucleotide”, “oligonucleotide”, and “polynucleotide” are synonymous with nucleic acid and include both DNA and RNA. These may be double-stranded or single-stranded, and in the case of “nucleotide” having a certain sequence (or “oligonucleotide”, “polynucleotide”), it is complementary to this unless otherwise specified. “Nucleotide” (or “oligonucleotide” and “polynucleotide”) having a typical sequence is also intended to be inclusive. Furthermore, when the “nucleotide” (or “oligonucleotide” and “polynucleotide”) is RNA, the base symbol “T” shown in the sequence listing shall be read as “U”.

  In the present invention, “human nSR100 mRNA” is not particularly limited as long as it is nSR100 mRNA expressed in cells constituting the human body. Examples of human nSR100 mRNA include mRNA consisting of the base sequence shown in SEQ ID NO: 1, but as long as the above, 90% or more identity to the base sequence shown in SEQ ID NO: 1, preferably 95% or more. It may be mRNA consisting of a base sequence having identity, more preferably 98% or more, and even more preferably 99% or more.

  In the present invention, “miR-4279”, “miR-4419b”, “miR-4516”, and “miR-4635” are particularly limited as long as they are the miRNAs expressed in the cells constituting the human body. Not. These miRNAs mean precursor miRNA and / or mature miRNA, but preferably mean mature miRNA. miR-4279 includes, for example, mRNA consisting of the base sequence shown in SEQ ID NO: 14, miR-4419b includes, for example, mRNA consisting of the base sequence shown in SEQ ID NO: 15, and miR-4516 includes, for example, SEQ ID NO: MRNA having the base sequence shown in FIG. 16 is exemplified, and miR-4635 includes, for example, the mRNA having the base sequence shown in SEQ ID NO: 17, but as long as the above, the base sequences shown in these SEQ ID NOs. Alternatively, it may be an mRNA comprising a nucleotide sequence having 90% or more identity, preferably 95% or more identity, more preferably 98% or more identity, and even more preferably 99% or more identity.

  In the present invention, the “human nSR100 protein” is not particularly limited as long as it is an nSR100 protein expressed in cells constituting the human body. Examples of the human nSR protein include a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, but 90% or more identity, preferably 95% or more to the amino acid sequence shown in SEQ ID NO: 2 as far as described above. It may be a protein comprising an amino acid sequence having identity, more preferably 98% or more, and even more preferably 99% or more.

  “Nucleic acid that suppresses expression of human nSR100 mRNA” and “nucleic acid that suppresses expression of human nSR100 protein” in the present invention include not only siRNA against human nSR100 mRNA, but also miRNA, antisense poly (oligo) nucleotides, and ribozymes. And decoy are included.

  The “nucleic acid that suppresses the function of human nSR100 protein” in the present invention means a nucleic acid having an action of suppressing the function of human nSR100 protein, and includes aptamers for human nSR100 protein.

  The “antibody” referred to in the present invention includes one of the above antibodies having antigen-binding properties, such as a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or a Fab fragment or a fragment generated by a Fab expression library. Parts are included.

  Furthermore, in the present invention, the “diagnostic agent” refers to one that is used directly or indirectly to diagnose the presence or absence of small cell lung cancer. This includes (poly) (oligo) nucleotides or antibodies capable of specifically recognizing and binding to human nSR100 mRNA (or protein) whose expression is increased in lung tissue in relation to small cell lung cancer. The Based on the above properties, these (poly) (oligo) nucleotides and antibodies are used as probes for detecting the above-mentioned nSR100 mRNA (or protein) expressed in lung tissue in vivo and in vitro, and (poly) (oligo) ) Nucleotides can be effectively used as primers for amplifying the gene expressed in vivo.

2. Diagnostic agent for small cell lung cancer containing polynucleotide
2-1. Diagnostic agent using nSR100 mRNA expression level as an indicator The present invention provides (a) a polynucleotide having at least 15 bases continuous in the base sequence of human nSR100 mRNA and / or a polynucleotide complementary to the polynucleotide, or (b) A polynucleotide having at least 15 bases that hybridizes under stringent conditions to the base sequence of human nSR100 mRNA or a base sequence complementary thereto,
The present invention relates to a diagnostic agent for small cell lung cancer containing any of the above.

  In the present invention, detection (diagnosis) of small cell lung cancer is carried out by evaluating the presence or absence of the expression level (expression level) of human nSR100 mRNA in the lung tissue of a subject. In this case, the diagnostic agent of the present invention specifically recognizes human nSR100 mRNA or a nucleic acid (cDNA, etc.) derived from it as a primer for specifically recognizing and amplifying human nSR100 mRNA or a nucleic acid (cDNA, etc.) derived therefrom. It can be used as a probe for detecting automatically.

  The diagnostic agent of the present invention is a polynucleotide comprising the base sequence (full-length sequence) of human nSR100 mRNA as long as it selectively recognizes (specifically) human nSR100 mRNA or a nucleic acid derived therefrom (cDNA or the like). It may be a polynucleotide comprising its complementary sequence. Moreover, the polynucleotide which consists of a partial sequence of the said full length sequence or its complementary sequence may be sufficient. In this case, examples of the partial sequence include a polynucleotide having at least 15 consecutive base lengths arbitrarily selected from the base sequence of the full-length sequence or its complementary sequence.

  Here, “selectively (specifically)” means that human nSR100 mRNA can be specifically detected in, for example, Northern blotting, and human nSR100 mRNA or its origin in RT-PCR. This means that nucleic acid (cDNA, etc.) is specifically amplified, but is not limited thereto, and those skilled in the art can judge that the above-mentioned detection product or amplification product is derived from human nSR100 mRNA. That's fine.

  That is, the diagnostic agent of the present invention comprises a polynucleotide having at least 15 bases continuous in the base sequence of human nSR100 mRNA and / or a polynucleotide complementary to the polynucleotide. Here, a complementary polynucleotide (complementary strand, reverse strand) is a full-length sequence of a polynucleotide consisting of the base sequence of human nSR100 mRNA, or a partial sequence thereof having a base sequence of at least 15 bases continuous in the base sequence (Hereinafter, these are also referred to as “positive strands” for the sake of convenience), it means a polynucleotide having a base-complementary relationship based on a base pair relationship such as A: T and G: C. . However, such a complementary strand is not limited to the case where it forms a completely complementary sequence with the target positive strand base sequence, but has a complementary relationship that allows it to hybridize with the target positive strand under stringent conditions. You may have. Note that stringent conditions here bind the complex or probe as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, as washing conditions after hybridization, conditions of about “1 × SSC, 0.1% SDS, 37 ° C.” can be mentioned. The complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions. Although there is no particular limitation, the more stringent hybridization conditions are about “0.5 × SSC, 0.1% SDS, 42 ° C.”, and the more severe hybridization conditions are “0.1 × SSC, 0.1% SDS, 65 ° C.”. Can do. Specifically, as such a complementary strand, a strand consisting of a base sequence that is completely complementary to the base sequence of the target positive strand, and at least 90%, preferably 95%, more preferably Examples thereof include a chain composed of a base sequence having 98% or more identity, more preferably 99% or more identity.

  The diagnostic agent of the present invention can be designed using, for example, the vector NTI (manufactured by Infomax) based on the base sequence of human nSR100 mRNA represented by SEQ ID NO: 1. Specifically, a primer or probe candidate sequence obtained by applying the nucleotide sequence of human nSR100 mRNA to the vector NTI software, or a sequence containing at least the sequence as a part can be used as a primer or probe.

  As long as the diagnostic agent of the present invention has a length of at least 15 consecutive bases as described above, specifically, the length can be appropriately selected and set according to the use of the diagnostic agent. it can.

  The diagnostic agent of the present invention can be used as a primer or a probe according to a conventional method in a known method for specifically detecting a specific gene, such as a Northern blot method, an RT-PCR method, an in situ hybridization method, etc. . By the use, it is possible to evaluate the presence or level of expression of human nSR100 mRNA in the lung tissue of the subject or the expression level (expression level).

  Depending on the type of detection method used, the subject's lung tissue and its surrounding tissues, or a part of the lung tissue are collected with a biopsy, etc., and total RNA prepared according to a conventional method is used as the sample to be measured. Alternatively, a nucleic acid (such as cDNA) derived from the RNA may be used.

  When the diagnostic agent of the present invention is used as a primer in the detection (gene diagnosis) of small cell lung cancer, a polynucleotide having at least 15 consecutive nucleotides in the nucleotide sequence of human nSR100 mRNA and / or a polynucleotide complementary thereto Examples thereof include those having a base length of usually 15 bases to 100 bases, preferably 15 bases to 50 bases, and more preferably 15 bases to 35 bases. When used as a detection probe, those having a base length of usually 15 bases to the entire sequence, preferably 15 bases to 1000 bases, more preferably 100 bases to 1000 bases can be exemplified.

  The diagnostic agent (probe or primer) of the present invention includes a suitable label for detecting human nSR100 mRNA or a nucleic acid (cDNA, etc.) derived therefrom, such as a fluorescent dye, enzyme, protein, radioisotope, chemiluminescent substance. And those with biotin added thereto.

  As the fluorescent dye used in the present invention, those generally labeled with nucleotides and used for detection and quantification of nucleic acids can be suitably used. For example, HEX (4, 7, 2 ′, 4 ′, 5 ′, 7 '-hexachloro-6-carboxylfluorescein (green fluorescent dye), fluorescein, NED (trade name, Applied Biosystems, yellow fluorescent dye), or 6-FAM (trade name, Applied Biosystems, yellow) Green fluorescent dye), rhodamine or a derivative thereof (for example, tetramethylrhodamine (TMR)), but is not limited thereto. As a method for labeling nucleotides with a fluorescent dye, an appropriate one of known labeling methods can be used [see Nature Biotechnology, 14, 303-308 (1996)]. Commercially available fluorescent labeling kits can also be used (for example, oligonucleotide ECL 3'-oligo labeling system manufactured by Amersham Pharmacia).

  In addition, the probe (oligo or polynucleotide) can be immobilized on an arbitrary solid phase and used. For this reason, the diagnostic agent of the present invention is a probe in which the probe (oligo or polynucleotide) is immobilized (for example, a DNA chip, cDNA microarray, oligo DNA array, membrane filter, etc. on which the probe is immobilized. )).

  The solid phase used for immobilization is not particularly limited as long as it can immobilize oligos or polynucleotides. Examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries or other substrates. be able to. The immobilization of the oligo or polynucleotide to the solid phase is a method of placing a pre-synthesized oligo or polynucleotide on the solid phase, or a method of synthesizing the target oligo or polynucleotide on the solid phase. Also good. The immobilization method is well known in the art depending on the type of immobilization probe, for example, using a commercially available spotter (such as Amersham) in the case of a DNA microarray [for example, photolithographic technique (Affymetrix) In situ synthesis of oligonucleotides by inkjet technology (Rosetta Inpharmatics).

  According to the DNA chip or the like on which the diagnostic agent (probe) of the present invention is immobilized, the probe formed by hybridization with labeled DNA or RNA prepared based on RNA collected from a biological tissue. The presence or expression level (expression level) of human nSR100 mRNA in lung tissue can be detected by detecting the complex of the labeled DNA or RNA with the label of the labeled DNA or RNA as an index. it can.

  The DNA chip or the like only needs to contain one or more kinds of the diagnostic agent (probe) of the present invention that can bind to human nSR100 mRNA or a nucleic acid derived therefrom (cDNA or the like). By using a DNA chip or the like containing a plurality of diagnostic agents (probes), it is possible to simultaneously evaluate the presence or absence of expression of a plurality of genes or the expression level of one biological sample.

  The diagnostic agent of the present invention is useful for detecting small cell lung cancer in a subject. Specifically, the diagnosis of small cell lung cancer using the diagnostic agent is performed by measuring the amount of human nSR100 mRNA or a nucleic acid derived from it in a sample derived from the lung tissue of the subject (test mRNA expression level), the small cell lung cancer tissue and This can be done by determining the difference in the amount of human nSR100 mRNA or nucleic acid derived therefrom (control mRNA expression level) in a tissue-derived sample that does not contain nerve tissue. In this case, specifically, if the test mRNA expression level is increased by 50% or more compared to the control mRNA expression level, preferably increased by 100% or more, more preferably increased by 200% or more, the subject has small cell lung cancer. Is likely to be.

2-2. Diagnostic agent using nSR100 gene-suppressed miRNA amount as an index The present invention is (a ′) at least 15 bases continuous in the base sequence of miR-4279, miR-4419b, miR-4516, or miR-4635 (nSR100 gene-suppressed miRNA) And / or a polynucleotide complementary to the polynucleotide, or (b ′) a string to the base sequence of miR-4279, miR-4419b, miR-4516, and miR-4635 or a base sequence complementary thereto A polynucleotide having at least 15 bases that hybridizes under mild conditions;
The present invention relates to a diagnostic agent for small cell lung cancer containing any of the above.

  In the present invention, detection (diagnosis) of small cell lung cancer is performed by evaluating the amount of nSR100 gene-suppressed miRNA in a blood sample of a subject. In this case, the diagnostic agent of the present invention is a primer for specifically recognizing and amplifying nSR100 gene-suppressed miRNA or a nucleic acid derived from it (cDNA or the like), or a nSR100 gene-suppressed miRNA or a nucleic acid derived from it (cDNA or the like). Can be used as a probe for specifically detecting.

  As long as the diagnostic agent of the present invention can selectively (specifically) recognize nSR100 gene-suppressing miRNA or a nucleic acid derived from it (cDNA or the like), the diagnostic agent of nSR100 gene-suppressing miRNA is a polynucleotide comprising the base sequence (full-length sequence) of nSR100 gene-suppressing miRNA It may be a nucleotide or a polynucleotide consisting of its complementary sequence. Moreover, the polynucleotide which consists of a partial sequence of the said full length sequence or its complementary sequence may be sufficient. In this case, examples of the partial sequence include a polynucleotide having at least 15 consecutive base lengths arbitrarily selected from the base sequence of the full-length sequence or its complementary sequence.

  Here, “selectively (specifically)” means that, for example, in Northern blotting, nSR100 gene-suppressed miRNA can be specifically detected, and in RT-PCR method, nSR100 gene-suppressed miRNA or it can be detected. This means that the derived nucleic acid (cDNA or the like) is specifically amplified, but is not limited thereto, and those skilled in the art can determine that the detected product or amplified product is derived from nSR100 gene-suppressed miRNA. Anything is acceptable.

  That is, the diagnostic agent of the present invention is characterized by comprising a polynucleotide having at least 15 bases continuous in the base sequence of the nSR100 gene suppression miRNA and / or a polynucleotide complementary to the polynucleotide. Here, the complementary polynucleotide (complementary strand, reverse strand) is the full-length sequence of a polynucleotide consisting of the base sequence of the nSR100 gene-suppressing miRNA, or a portion thereof having a base sequence of at least 15 bases in length in the base sequence This refers to a polynucleotide having a base complementary relationship to a sequence (for convenience, these are also referred to as “positive strands”) based on base pair relationships such as A: T and G: C. is there. However, such a complementary strand is not limited to the case where it forms a completely complementary sequence with the target positive strand base sequence, but has a complementary relationship that allows it to hybridize with the target positive strand under stringent conditions. You may have. Note that stringent conditions here bind the complex or probe as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, as washing conditions after hybridization, conditions of about “1 × SSC, 0.1% SDS, 37 ° C.” can be mentioned. The complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions. Although there is no particular limitation, the more stringent hybridization conditions are about “0.5 × SSC, 0.1% SDS, 42 ° C.”, and the more severe hybridization conditions are “0.1 × SSC, 0.1% SDS, 65 ° C.”. Can do. Specifically, as such a complementary strand, a strand consisting of a base sequence that is completely complementary to the base sequence of the target positive strand, and at least 90%, preferably 95%, more preferably Examples thereof include a chain composed of a base sequence having 98% or more identity, more preferably 99% or more identity.

  The diagnostic agent of the present invention can be designed using known program software, for example, based on the base sequence of the nSR100 gene-suppressed miRNA shown in SEQ ID NO: 14, 15, 16, or 17.

  As long as the diagnostic agent of the present invention has a length of at least 15 consecutive bases as described above, specifically, the length can be appropriately selected and set according to the use of the diagnostic agent. it can.

  The diagnostic agent of the present invention can be used as a primer or a probe according to a conventional method in a known method for specifically detecting a specific gene, such as a Northern blot method, an RT-PCR method, an in situ hybridization method, etc. . By using this, it is possible to evaluate the amount of nSR100 gene-suppressed miRNA in the blood sample of the subject.

  As a sample to be measured, depending on the type of detection method to be used, a blood sample of a subject may be collected by blood sampling or the like, and total RNA prepared according to a conventional method may be used, or derived from the RNA. Nucleic acids (cDNA, etc.) may be used.

  When the diagnostic agent of the present invention is used as a primer in detection of small cell lung cancer (genetic diagnosis), a polynucleotide having at least 15 consecutive nucleotides in the base sequence of the nSR100 gene-suppressing miRNA and / or a complementary polynucleotide thereto. Examples of nucleotides include those usually having a length of 15 to 21 bases, preferably 16 to 21 bases. When used as a detection probe, in the base sequence of the nSR100 gene suppression miRNA, as a polynucleotide having at least 15 consecutive bases and / or a polynucleotide complementary thereto, usually 15 to 21 bases, preferably 16 bases to One having 21 bases can be exemplified.

  The diagnostic agent (probe or primer) of the present invention includes an appropriate label for detecting nSR100 gene-suppressed miRNA or a nucleic acid (cDNA, etc.) derived therefrom, such as fluorescent dye, enzyme, protein, radioisotope, chemiluminescence Includes substances, biotin and the like added.

  As the fluorescent dye used in the present invention, those generally labeled with nucleotides and used for detection and quantification of nucleic acids can be suitably used. For example, HEX (4, 7, 2 ′, 4 ′, 5 ′, 7 '-hexachloro-6-carboxylfluorescein (green fluorescent dye), fluorescein, NED (trade name, Applied Biosystems, yellow fluorescent dye), or 6-FAM (trade name, Applied Biosystems, yellow) Green fluorescent dye), rhodamine or a derivative thereof (for example, tetramethylrhodamine (TMR)), but is not limited thereto. As a method for labeling nucleotides with a fluorescent dye, an appropriate one of known labeling methods can be used [see Nature Biotechnology, 14, 303-308 (1996)]. Commercially available fluorescent labeling kits can also be used (for example, oligonucleotide ECL 3'-oligo labeling system manufactured by Amersham Pharmacia).

  In addition, the probe (oligo or polynucleotide) can be immobilized on an arbitrary solid phase and used. For this reason, the diagnostic agent of the present invention is a probe in which the probe (oligo or polynucleotide) is immobilized (for example, a DNA chip, cDNA microarray, oligo DNA array, membrane filter, etc. on which the probe is immobilized. )).

  The solid phase used for immobilization is not particularly limited as long as it can immobilize oligos or polynucleotides. Examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries or other substrates. be able to. The immobilization of the oligo or polynucleotide to the solid phase is a method of placing a pre-synthesized oligo or polynucleotide on the solid phase, or a method of synthesizing the target oligo or polynucleotide on the solid phase. Also good. The immobilization method is well known in the art depending on the type of immobilization probe, for example, using a commercially available spotter (such as Amersham) in the case of a DNA microarray [for example, photolithographic technique (Affymetrix) In situ synthesis of oligonucleotides by inkjet technology (Rosetta Inpharmatics).

  According to the DNA chip or the like on which the diagnostic agent (probe) of the present invention is immobilized, the probe formed by hybridization with labeled DNA or RNA prepared based on RNA collected from a biological tissue. The amount of nSR100 gene-suppressed miRNA in the blood can be evaluated by detecting the complex of the labeled DNA or RNA with the label of the labeled DNA or RNA as an index.

  The DNA chip or the like only needs to contain one or more kinds of the diagnostic agent (probe) of the present invention that can bind to the nSR100 gene-suppressing miRNA or a nucleic acid derived therefrom (cDNA or the like). By using a DNA chip or the like containing a plurality of diagnostic agents (probes), it is possible to simultaneously evaluate the presence or absence of expression of a plurality of genes or the expression level of one biological sample.

  The diagnostic agent of the present invention is useful for detecting small cell lung cancer in a subject. Specifically, the diagnosis of small cell lung cancer using the diagnostic agent is performed by measuring the amount of nSR100 gene-suppressed miRNA or nucleic acid derived therefrom (amount of test miRNA) in a subject's blood sample and a subject who is not a patient with small cell lung cancer ( For example, it can be carried out by determining the difference in the amount of nSR100 gene-suppressed miRNA or nucleic acid derived therefrom (control miRNA amount) in a blood sample collected from a healthy person. In this case, specifically, if the test miRNA level is increased by 50% or more, preferably 100% or more, more preferably 200% or more, compared to the control miRNA level, the subject has small cell lung cancer. The possibility increases.

3. Diagnostic Agent for Small Cell Lung Cancer Containing Antibody The present invention provides an antibody capable of specifically recognizing human nSR100 protein as a diagnostic agent. Specific examples of the antibody include an antibody capable of specifically recognizing a protein consisting of the amino acid sequence represented by SEQ ID NO: 2.

  The present invention is based on the knowledge that human nSR100 protein expression is specifically increased in lung tissue of patients with small cell lung cancer, and by detecting the presence or absence of human nSR100 protein expression and the degree thereof in a subject. This is based on the idea that whether or not the subject has small cell lung cancer and the degree thereof can be specifically detected.

  Therefore, the above-mentioned antibody is a tool (diagnostic agent) that can diagnose whether or not the subject has small cell lung cancer by detecting the presence or absence or the extent of the increased expression of the human nSR100 protein in the subject. ).

  The form of the antibody of the present invention is not particularly limited, and may be a polyclonal antibody using human nSR100 protein as an immunizing antigen or a monoclonal antibody thereof. Furthermore, an antibody having antigen-binding ability to a polypeptide consisting of at least continuous amino acids of the human nSR100 protein, usually 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids, is also included in the antibodies of the present invention. .

  Methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced according to these conventional methods (Current protocols in Molecular Biology, Chapter 11.12 to 11.13 (2000)). Specifically, when the antibody of the present invention is a polyclonal antibody, an oligopeptide having a partial amino acid sequence of the human nSR100 protein using a human nSR100 protein expressed and purified in Escherichia coli or the like according to a conventional method or according to a conventional method Can be synthesized to immunize non-human animals such as rabbits, and obtained from the sera of the immunized animals according to a conventional method. On the other hand, in the case of a monoclonal antibody, a human nSR100 protein expressed and purified in Escherichia coli or the like according to a conventional method, or a non-human animal such as a mouse immunized with an oligopeptide having a partial amino acid sequence of the protein, It can be obtained from hybridoma cells prepared by cell fusion with myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11).

  The human nSR100 protein used as an immunizing antigen for antibody production is based on the gene sequence information (for example, SEQ ID NO: 1) provided by the present invention, DNA cloning, construction of each plasmid, transfection into a host, It can be obtained by culturing the transformant and recovering the protein from the culture. These operations are based on methods known to those skilled in the art or methods described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). Can be done.

  Specifically, a recombinant DNA (expression vector) capable of expressing a gene encoding human nSR100 protein in a desired host cell is prepared, introduced into the host cell, transformed, and the transformant is cultured. By collecting the target protein from the obtained culture, a protein as an immunizing antigen for producing the antibody of the present invention can be obtained. The partial peptide of human nSR100 protein can also be produced by a general chemical synthesis method (peptide synthesis) according to the amino acid sequence information (SEQ ID NO: 2) provided by the present invention.

  The antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of human nSR100 protein. The oligo (poly) peptide used for the production of such an antibody does not need to have a functional biological activity, but preferably has an immunogenic property similar to that of human nSR100 protein. An oligo (poly) peptide preferably having this immunogenic property and consisting of at least 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of human nSR100 protein can be exemplified.

  Production of antibodies against such oligo (poly) peptides can also be carried out by enhancing the immunological reaction using various adjuvants depending on the host. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfaces such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Active substances, human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum are included.

  Since the antibody of the present invention has a property of specifically binding to human nSR100 protein, human nSR100 protein expressed in the lung tissue of a subject can be specifically detected by using the antibody. That is, the antibody is useful as a probe for detecting the presence and level of human nSR100 protein in a sample derived from lung tissue of a subject.

  Specifically, the lung tissue of the subject and its surrounding tissues, a part of the lung tissue is collected with a biopsy, etc., and using a tissue extract or protein prepared according to a conventional method, for example, immunoblotting, ELISA, etc. In known detection methods such as those described above, human nSR100 protein can be detected by using the antibody of the present invention as a probe according to a conventional method.

  When detecting small cell lung cancer, the amount of human nSR100 protein (test protein expression level) in the sample derived from the lung tissue of the subject and the amount of human nSR100 protein in the sample derived from the tissue not including small cell lung cancer tissue and nerve tissue (control) This can be done by determining the difference in protein expression level. In this case, specifically, if the test protein expression level is increased by 50% or more, preferably by 100% or more, more preferably by 200% or more compared to the control protein expression level, the test subject has small cell lung cancer. Is likely to be.

4). Diagnostic Agent Kit The present invention also provides a diagnostic kit for use in the detection and diagnosis of small cell lung cancer comprising the above diagnostic agent. The kit contains at least one oligo or polynucleotide (which may be labeled or immobilized on a solid phase) or the antibody used as the probe or primer. In addition to the probe or primer described above, the kit includes other reagents and instruments necessary for carrying out the method of the present invention, such as a hybridization reagent, a probe label, a label detection agent, and a buffer as necessary. May be included as appropriate.

5). Detection method (diagnosis method) of small cell lung cancer
One embodiment of the detection method of the present invention includes the following steps (I) to (III):
(I) a step of measuring the amount of human nSR100 mRNA or a nucleic acid derived therefrom in a sample derived from lung tissue collected from a subject, or the amount of human nSR100 protein,
(II) The amount of human nSR100 mRNA or nucleic acid derived from it measured in the above step (I) or the amount of human nSR100 protein (test mRNA / protein expression level) does not include small cell lung cancer tissue and nerve tissue Steps in comparison with the amount of human nSR100 mRNA or nucleic acid derived from it in a tissue-derived sample, or the amount of human nSR100 protein (control mRNA / protein expression level), and (III) the test compared to the control mRNA / protein expression level A step in which a high mRNA / protein expression level is used as an indicator for determining that a subject has small cell lung cancer.

  If the test mRNA / protein expression level is higher than the control mRNA / protein expression level obtained by such a method, it can be determined that the subject has small cell lung cancer.

  Examples of the sample used here include a sample prepared from lung tissue collected from a subject. Specifically, an RNA-containing sample prepared from the tissue, a sample containing a nucleic acid (cDNA or the like) further prepared therefrom, or a sample containing a protein prepared from the tissue can be mentioned. A sample containing such RNA, nucleic acid or protein can be prepared from a lung tissue of a subject using a biopsy or the like, and prepared therefrom according to a conventional method.

  Whether or not a subject has small cell lung cancer is determined based on the fact that the test mRNA / protein expression level is higher than the control mRNA / protein expression level. Specifically, for example, the test mRNA / protein expression level is increased by 50% or more, preferably by 100% or more, more preferably by 200% or more compared to the control mRNA / protein expression level. be able to.

One embodiment of the detection method of the present invention includes the following steps (I ′) to (III ′):
(I ′) an amount of at least one selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635 (nSR100 gene-suppressed miRNA) in a blood sample collected from a subject, A step of measuring using the diagnostic agent according to 3 or 4,
(II ′) at least one amount selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635 (amount of test miRNA) measured in the above step (1 ″), At least one amount selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635 in a blood sample collected from a subject who is not a small cell lung cancer patient (eg, a healthy subject) (control miRNA And (III ′) a step in which the test miRNA amount is higher than the control miRNA amount is used as an indicator for determining that the subject has small cell lung cancer.

  If the amount of test miRNA is higher than the amount of control miRNA obtained in such a method, the subject can be judged to have small cell lung cancer.

  Examples of the sample used here include a sample prepared from blood collected from a subject. Specifically, an RNA-containing sample prepared from blood or a sample containing a nucleic acid (cDNA or the like) further prepared therefrom can be mentioned. A sample containing such RNA or nucleic acid can be prepared from a subject's blood by blood sampling or the like and prepared therefrom according to a conventional method.

  Whether or not a subject has small cell lung cancer is determined based on the test miRNA level being higher than the control miRNA level. Specifically, for example, the amount of test miRNA can be increased by 50% or more, preferably 100% or more, more preferably 200% or more compared to the control miRNA amount.

  The detection method of the present invention is specifically performed as follows according to the type of measurement object.

5-1. When the measurement target is human nSR100 mRNA or a nucleic acid derived therefrom, the detection method (diagnostic method) is carried out by measuring the amount of human nSR100 mRNA or nucleic acid derived therefrom in the sample. Specifically, Northern blot method, RT-PCR method, DNA chip analysis method, in situ hybridization analysis method, etc., using the diagnostic agent (polynucleotide) of the present invention containing the aforementioned polynucleotide as a primer or probe It can implement by performing the well-known method of these.

When the Northern blot method is used, the presence or amount of human nSR100 mRNA or a nucleic acid derived from it in a sample can be measured by using the diagnostic agent of the present invention as a probe. Specifically, the diagnostic agent of the invention (complementary strand) radioisotopes (such as ³² P, ³³ P: RI) or a fluorescent substance labeled with a, it, of the subject and transferred to a nylon membrane or the like according to a conventional method After hybridization with the sample-derived mRNA, a signal derived from the diagnostic agent label (RI or a fluorescent substance) such as a duplex between the diagnostic agent and the subject sample mRNA Examples thereof include a method of detecting and measuring with a radiation detector (BAS-1800II, manufactured by Fuji Film) or a fluorescence detector. In addition, using the AlkPhos Direct Labeling and Detection System (manufactured by Amersham Pharmacia Biotech), a diagnostic agent is labeled according to the protocol, hybridized with mRNA derived from the subject's sample, and then a signal derived from the label of the diagnostic agent. A method of detecting and measuring with a multi-bioimager STORM860 (manufactured by Amersham Pharmacia Biotech) can also be used.

  When the RT-PCR method is used, the presence or amount of human nSR100 mRNA or a nucleic acid derived from it in a sample can be measured by using the above diagnostic agent of the present invention as a primer. Specifically, a pair of primers prepared from the diagnostic agent of the present invention (the above cDNA (−)) is prepared so that a target region can be amplified using a template prepared from RNA derived from a biological tissue of a subject according to a conventional method. The method can be exemplified by a method in which a normal strand that binds to the strand (strand) and a reverse strand that binds to the strand +) are hybridized with this and subjected to PCR according to a conventional method, and the resulting amplified double-stranded DNA is detected. . The detection of the amplified double-stranded DNA was performed by a method for detecting the labeled double-stranded DNA produced by performing the PCR using a primer previously labeled with RI or a fluorescent substance. For example, a method can be used in which double-stranded DNA is transferred to a nylon membrane or the like according to a conventional method, and a labeled diagnostic agent is used as a probe to hybridize with the probe to detect it. The produced labeled double-stranded DNA product can be measured with an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems). In addition, prepare an RT-PCR reaction solution according to the protocol using SYBR Green RT-PCR Reagents (Applied Biosystems) and react with ABI PRISM 7700 Sequence Detection System (Applied Biosystems) to detect the reaction product. You can also

  When using DNA chip analysis, prepare a DNA chip to which the above-mentioned diagnostic agent of the present invention is attached as a DNA probe (single-stranded or double-stranded), and extract RNA from biological tissue of a subject by a conventional method. An example is a method in which a double strand of DNA and cRNA formed by hybridization with the prepared cRNA is bound to a labeled probe prepared from the diagnostic agent of the present invention and detected.

5-2. When the measurement target is human nSR100 protein, the detection method (diagnostic method) is carried out by measuring the amount of human nSR100 protein in the sample. Specifically, it can be carried out by performing a known method such as an immunoblot method using the diagnostic agent (antibody) of the present invention containing the antibody described above.

The immunoblot method uses a diagnostic antibody of the present invention as a primary antibody, followed by a labeled antibody (primary antibody) labeled with a radioisotope such as ¹²⁵ I, a fluorescent substance, an enzyme such as horseradish peroxidase (HRP) as a secondary antibody. Using a radiometer (BAS-1800II: manufactured by Fuji Film Co., Ltd.), a fluorescence detector, etc., detect the signal derived from the labeling substance such as a radioisotope of the obtained labeled compound or a fluorescent substance. And can be implemented by measuring. In addition, after using the diagnostic agent of the present invention as a primary antibody, the ECL Plus Western Blotting Detection System (manufactured by Amersham Pharmacia Biotech) was detected according to the protocol, and the multibioimager STORM860 (manufactured by Amersham Pharmacia Biotech) was used. It can also be measured.

5-3. When the measurement target is nSR100 gene-suppressed miRNA or a nucleic acid derived therefrom, the detection method (diagnostic method) is carried out by measuring the amount of nSR100 gene-suppressed miRNA or nucleic acid derived therefrom in the sample. Specifically, Northern blot method, RT-PCR method, DNA chip analysis method, in situ hybridization analysis method, etc., using the diagnostic agent (polynucleotide) of the present invention containing the aforementioned polynucleotide as a primer or probe It can implement by performing the well-known method of these.

When using the Northern blot method, the presence or amount of the nSR100 gene-suppressed miRNA or nucleic acid derived therefrom can be measured in the sample by using the diagnostic agent of the present invention as a probe. Specifically, the diagnostic agent of the invention (complementary strand) radioisotopes (such as ³² P, ³³ P: RI) or a fluorescent substance labeled with a, it, of the subject and transferred to a nylon membrane or the like according to a conventional method After hybridization with the miRNA derived from the sample, a signal derived from the diagnostic agent label (RI or a labeling substance such as a fluorescent substance) is used as the duplex between the diagnostic agent and the miRNA derived from the subject sample. Examples thereof include a method of detecting and measuring with a radiation detector (BAS-1800II, manufactured by Fuji Film) or a fluorescence detector. In addition, using the AlkPhos Direct Labeling and Detection System (manufactured by Amersham Pharmacia Biotech), a diagnostic agent is labeled according to the protocol, hybridized with mRNA derived from the subject's sample, and then a signal derived from the label of the diagnostic agent. A method of detecting and measuring with a multi-bioimager STORM860 (manufactured by Amersham Pharmacia Biotech) can also be used.

  When the RT-PCR method is used, the presence or amount of nSR100 gene-suppressed miRNA or a nucleic acid derived therefrom can be measured in the sample by using the above diagnostic agent of the present invention as a primer. Specifically, a pair of primers prepared from the diagnostic agent of the present invention (the above cDNA (−)) is prepared so that a target region can be amplified using a template prepared from RNA derived from a biological tissue of a subject according to a conventional method. The method can be exemplified by a method in which a normal strand that binds to the strand (strand) and a reverse strand that binds to the strand +) are hybridized with this and subjected to PCR according to a conventional method, and the resulting amplified double-stranded DNA is detected. . The detection of the amplified double-stranded DNA was performed by a method for detecting the labeled double-stranded DNA produced by performing the PCR using a primer previously labeled with RI or a fluorescent substance. For example, a method can be used in which double-stranded DNA is transferred to a nylon membrane or the like according to a conventional method, and a labeled diagnostic agent is used as a probe to hybridize with the probe to detect it. The produced labeled double-stranded DNA product can be measured with an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems). In addition, prepare an RT-PCR reaction solution according to the protocol using SYBR Green RT-PCR Reagents (Applied Biosystems) and react with ABI PRISM 7700 Sequence Detection System (Applied Biosystems) to detect the reaction product. You can also

  When using DNA chip analysis, prepare a DNA chip to which the above-mentioned diagnostic agent of the present invention is attached as a DNA probe (single-stranded or double-stranded), and extract RNA from biological tissue of a subject by a conventional method. An example is a method in which a double strand of DNA and cRNA formed by hybridization with the prepared cRNA is bound to a labeled probe prepared from the diagnostic agent of the present invention and detected.

6). The present invention relates to a therapeutic agent or preventive agent for small cell lung cancer, which comprises a nucleic acid that suppresses human nSR100 mRNA expression, human nSR100 protein expression, or human nSR100 protein function, or an antibody that recognizes human nSR100 protein. It relates to therapeutic or prophylactic drugs.

  The siRNA targeted by the present invention is double-stranded, and is formed by hybridizing a sense strand that is complementary to the target sequence of human nSR100 mRNA and an antisense strand that is complementary to the sense strand. ing. The duplex may be a molecule having a closed structure at one end, for example, an siRNA (shRNA) having a hairpin structure. Such siRNA is not particularly limited as long as it suppresses the expression of human nSR100 mRNA (or protein). As such siRNA, those designed using a known siRNA design program may be used, or commercially available siRNA with a guaranteed suppression function may be used. Preferred target sequences for siRNA include SEQ ID NOs: 10, 11, 12, 13 and the like, preferably among these, SEQ ID NOs: 11, 12, 13 and the like, more preferably SEQ ID NOs: 12, 13 and the like. It is done.

  Moreover, not only siRNA that suppresses expression of human nSR100 mRNA (or protein), but also nucleic acid that suppresses human nSR100 gene can be used as an active ingredient of the therapeutic agent of the present invention. Examples of such nucleic acid include miRNA (preferably miR-4279, miR-4419b, miR-4516, or miR-4635), antisense poly (oligo) nucleotide, ribozyme, aptamer, and decoy for human nSR100 gene. Here, the antisense poly (oligo) nucleotide has a base sequence complementary to or substantially complementary to the base sequence of human nSR100 mRNA, and hybridizes with human nSR100 mRNA. , Which inhibits the synthesis or function of the RNA or regulates / controls the expression of human nSR100 mRNA through the interaction with the RNA. The antisense polynucleotide may be any antisense poly (oligo) nucleotide as long as it has the above-described action, and includes antisense RNA, antisense DNA, and the like.

  The antisense poly (oligo) nucleotide is usually composed of about 10 to 1000 bases, preferably about 15 to 500 bases, and more preferably about 16 to 30 bases. In order to prevent degradation by a hydrolase such as nuclease, the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are changed to chemically modified phosphate residues such as phosphorothioate, methylphosphonate and phosphorodithionate. May be substituted. These antisense poly (oligo) nucleotides can be produced using a known DNA synthesizer or the like.

  Furthermore, those having an action of suppressing the function of human nSR100 protein can also be used as an active ingredient of the therapeutic agent for small cell lung cancer of the present invention. Examples of such an action include antibodies against human nSR100 protein, particularly neutralizing antibodies.

  Here, the neutralizing antibody means an antibody having the property of inhibiting the function or activity originally possessed by the antigen by binding to the antigen. The neutralizing antibody against human nSR100 protein refers to an antibody having a property of inhibiting the function or activity of human nSR100 protein by binding to human nSR100 protein.

  The therapeutic or preventive agent for small cell lung cancer of the present invention is an active ingredient (siRNA, antisense poly (oligo) nucleotide) that suppresses the expression of the above human nSR100 mRNA (or protein) or suppresses the function of the human nSR100 protein. , Antibodies, etc.), and optional carriers and additives such as pharmaceutically acceptable carriers and additives.

  Examples of pharmaceutically acceptable carriers and additives include excipients such as sucrose and starch; binders such as cellulose and methylcellulose; disintegrants such as starch and carboxymethylcellulose; lubricants such as magnesium stearate and aerosil Agents: Fragrances such as citric acid and menthol; Preservatives such as sodium benzoate and sodium bisulfite; Stabilizers such as citric acid and sodium citrate; Suspending agents such as methylcellulose and polyvinylpyrrolide; Surfactants and the like Dispersants; diluents such as water and saline; base waxes and the like, but are not limited thereto.

  Preparations suitable for oral administration include liquids, capsules, sachets, tablets, suspensions, emulsions and the like. Formulations suitable for parenteral administration (eg, subcutaneous injection, intramuscular injection, local infusion, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained. In addition, parenteral administration preparations include aqueous and non-aqueous sterile suspensions, including suspensions, solubilizers, thickeners, stabilizers, preservatives, and the like. It may be included.

  The dose of the preventive or therapeutic agent of the present invention varies depending on the body weight and age of the subject to be administered, the severity of the disease, etc., and cannot be set unconditionally. As examples, several mg to several tens mg / kg body weight can be mentioned, which can be administered once to several times a day.

  In the case where the above active ingredient is encoded by DNA, it may be considered to incorporate the DNA into a gene therapy vector and perform gene therapy. Furthermore, when the active ingredient is an antisense polynucleotide, gene therapy can be performed as it is or by incorporating it into a gene therapy vector. Also in these cases, the dosage and administration method of the gene therapy composition vary depending on the patient's weight, age, symptoms, etc., and can be appropriately selected by those skilled in the art.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Example 1. Expression analysis of nSR100 gene in SCLC cells The expression of nSR100 protein in small cell lung cancer (SCLC) cells was examined by immunoblotting. At the same time, the expression of the REST protein (lower part of FIG. 1) and the known SCLC marker sREST protein (REST protein splicing variant: upper part of FIG. 1) was also examined. Specifically, it was performed as follows.

<1.1. Preparation of sample for immunoblotting>
As human SCLC cells, NCI-N417 cells (American Type Culture Collection: ATCC) and NCI-H82 cells (ATCC) were prepared. On the other hand, NCI-H1650 cells (ATCC), which are human non-small cell lung cancer (NSCLC) cells, were prepared as control cells. These cells were cultured in RPMI1640 medium (containing 10% FBS, 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin) in a humid environment in the presence of 5% CO _2. Suspension culture was performed at ℃. The cultured cells were collected and the protein was extracted according to a conventional method. The obtained protein extract was used as a sample.

<1.2. Immunoblot>
A sample containing 100 μg of protein was developed by electrophoresis using a 10% SDS gel according to a conventional method, and the protein in the gel after the electrophoresis was transferred to a PVDF membrane. The PVDF membrane after transfer was blocked in a TBS-T solution (20 mM TBS, pH 7.2, 0.1% Tween20 (registered trademark)) containing 1% skim milk for 1 hour at room temperature. Anti-nSR100 antibody (Santa Cruz, sc-139291) diluted to 1/500 with TBS-T solution containing 1% skim milk, and then diluted to 1/1000 with TBS-T solution containing 1% skim milk The anti-REST antibody (Santa Cruz, H-290) or anti-REST antibody (Millipore, 07-579) diluted to 1/5000 with a TBS-T solution containing 1% skim milk was reacted at room temperature for 1 hour. After that, the PVDF membrane was washed with TBS-T solution and diluted to 1/15000 or 1/20000 with TBS-T solution containing 1% skim milk (IRDye700 or IRDye800 labeled antibody (Li-COR)) For 1 hour at room temperature. Detection was performed using Odyssey fluorescence detection system (LI-COR).

<1.3. Result>
FIG. 2A shows the result of examining only the expression of nSR100 protein, and FIG. 2B shows the result of examining the expression of REST and sREST in addition to nSR100 protein. From FIG. 2A, a band (near 75 kDa) specifically expressing nSR100 protein was observed in NCI-N417 cells and NCI-H82 cells, which are SCLC cells. FIG. 2B shows that the nSR100 protein and the known SCLC marker sREST protein are expressed specifically in SCLC cells. In view of the finding that expression of nSR100 gene is not observed in tissues other than nerve tissue (Non-patent Document 2), FIG. 2 suggests that nSR100 gene is useful as an SCLC marker.

Example 2 Analysis of the effects of extracellular matrix components on the expression of nSR100 and REST genes
It is known that when SCLC cells are cultured on an extracellular matrix, the tumor-forming ability of SCLC cells is enhanced (Reference 1: PNAS USA 87, 6698-6702, 1990). Therefore, it was examined how the expression of the nSR100 gene and the REST gene is affected by the enhancement of the tumorigenicity. Specifically, it was performed as follows.

<2.1. Cell culture>
NCI-N417 cells (human SCLC cells), NCI-H1650 cells (human NSCLC cells), or Hela cells are <1.1. Suspension culture was performed for 48 hours under the same conditions as in Preparation of immunoblot sample>. Separately, NCI-N417 cells or NCI-H1650 cells were cultured on a culture dish coated with an extracellular matrix component (Millipore, ECL cell attachment matrix) for 48 hours under the same conditions as in the above suspension culture. Adhesive culture was performed. The coating was performed by spreading a diluted solution obtained by diluting the ECL cell attachment matrix with PBS to 20 μg / ml on a culture dish, incubating at 37 ° C. for 1 hour, and removing excess water.

<2.2. Immunoblot>
Samples were prepared from the cells cultured in suspension and the cells cultured on the extracellular matrix in the same manner as in Example 1, and immunoblotting was performed using the samples.

<2.3. Quantitative real-time PCR>
Total RNA was extracted from cells cultured in suspension and cells cultured on an extracellular matrix using an RNA extraction kit (Qiagen, Total RNeasy mini Kit). Using the total RNA as a template, a reverse transcription reaction kit (Qiagen, Quantitect reverse transcriptase or Takara,
A reverse transcription reaction was performed using Primescript RT). Furthermore, using the sample after the reverse transcription reaction as a template, nucleic acid amplification in a PCR device (Rotor-Gene Q, Qiagen) using a real-time PCR reaction kit (Qiagen, SYBR Green PCR kit or Takara, SYBR Premix EX TaqII) Reaction was performed. The nucleic acid amplification reaction was performed at a temperature cycle of 95 ° C. for 5 minutes → [(95 ° C. for 5 seconds → 60 ° C. for 5 seconds) × 40 cycles]. The primer sets used are shown in Table 1 by detection target mRNA.

<2.4. Result>
The expression analysis results of nSR100 protein are shown in FIG. 3A, and the expression analysis results of nSR100 mRNA, REST mRNA, and sREST mRNA are shown in FIG. 3B. From FIG. 3A, it was observed that the expression level of nSR100 protein was enhanced by culturing SCLC cells on the extracellular matrix. From the lower right of FIG. 3B, it was shown that the tendency shown in FIG. 3A is also observed at the mRNA level. In addition, the same tendency was observed for sREST mRNA from the top of FIG. 3B. From these results, it was shown that the expression behavior in SCLC cells was the same for both nSR100 and sREST, which is a known SCLC marker. That is, it was strongly suggested that the nSR100 gene is useful as an SCLC marker.

Example 3 Analysis of the effect of nSR100 gene suppression on REST gene expression
It has been clarified that nSR100 acts as a regulator of alternative splicing in a cell-specific manner in neural tissue (Non-patent Document 2). Therefore, the influence of nSR100 gene suppression on the expression of REST gene splicing variants (sREST and REST) was examined. Specifically, it was performed as follows.

<3.1. Cell culture>
NCI-N417 cells (human SCLC cells) or NCI-H1650 cells (human NSCLC cells) were adherently cultured on the extracellular matrix in the same manner as in Example 2.

<3.2. RNAi>
NCI-N417 cells in logarithmic growth phase were seeded in a 6-well plate at 1 × 10 ^ 5 cells / well and cultured for 24 hours. Thereafter, siRNA (Dharmacon, SMART-pool siRNAs) or non-specific siRNA (Dharmacon, SMART-pool siRNAs) for the nSR100 gene was transfected using a commercially available transfection reagent (DharmaFECT siRNA Transfection Reagents (Thermo Scientific)). . The culture was further continued, and the cells were collected 48 hours after transfection. In addition, siRNA (Dharmacon, SMART-pool siRNAs) for the nSR100 gene is a mixture of siRNAs targeting each of the four sequences on nSR100 mRNA shown in Table 2.

<3.3. Quantitative real-time PCR>
Quantitative real-time PCR was performed in the same manner as in Example 2 from cells not treated with RNAi and cells treated with RNAi.

<3.4. Immunoblot>
Samples were prepared from cells not treated with RNAi and cells treated with RNAi in the same manner as in Example 1, and immunoblotting was performed using the samples.

<3.5. Result>
The expression analysis results of REST mRNA, sREST mRNA, and nSR100 mRNA are shown in FIG. 4A, and the expression analysis results of REST protein, sREST protein, and nSR100 protein are shown in FIG. 4B. 4A and B show that the suppression of the nSR100 gene decreases the expression of sREST mRNA and protein, and increases the expression of REST mRNA and protein. This suggests that in SCLC cells, the enhancement of nSR100 protein controls the splicing of the REST gene, and as a result, the expression of splicing variants sREST mRNA and protein is increased. Thus, the enhancement of the known SCLC marker (sREST) in SCLC cells is controlled by the nSR100 protein, which strongly suggests that the nSR100 gene is useful as an SCLC marker. In addition, the results of FIG. 4 that the expression of REST mRNA and protein is enhanced by suppression of the nSR100 gene and the finding that apoptosis of SCLC cells can be induced by forced expression of REST protein (Reference 2: Oncogene 22, 5636- 5645, 2003) suggests that suppression of the nSR100 gene can induce apoptosis of SCLC cells, that is, suppression of the nSR100 gene is effective for the treatment of SCLC.

Example 4 Relationship between tumorigenic potential of SCLC cells and expression of nSR100 gene and REST gene
The relationship between the tumorigenic ability of SCLC cells and the expression of nSR100 gene and REST gene was examined. Specifically, it was performed as follows.

<4.1. Preparation of cells for transplantation>
NCI-N417 cells (human SCLC cells) were adherently cultured on the extracellular matrix in the same manner as in Example 2. On the other hand, similarly to Example 2, suspension-cultured NCI-N417 cells were also prepared. The culture was performed for 7 days.

<4.2. Tumorigenesis experiment>
The following four patterns of samples were injected subcutaneously into 6-week-old BALB / c Slc-nu / nu athymic nude mice (female).
(I) 0.1 ml suspension culture NCI-N417 cell solution (5 × 10 ^ 5 cells / serum-free RPMI medium)
(II) 0.1 ml serum-free RPMI medium (III) 0.1 ml adhesion culture NCI-N417 cell solution (5 × 10 ^ 5 cells / serum-free RPMI medium)
(IV) Between 0.05 ml suspension culture NCI-N417 cell solution (5 × 10 ^ 5 cells / serum-free RPMI medium) and 0.05 ml Matrigel (BD biosciences, Lot No.38073) solution (0.45 mg / Dulbecco's PBS) The degree of tumor formation was visually observed 60 days after injection for the mice injected with the mixed solutions (I) to (III) and 46 days after the injection for the mice injected with (IV).

<4.3. Quantitative real-time PCR>
Above <4.2. After observing tumor formation in a tumor formation experiment>, a tumor portion was collected. From the collected sample, quantitative real-time PCR was performed in the same manner as in Example 2.

<4.4. Result>
A photograph of the mouse after the tumor formation experiment is shown in FIG. 5A, and the expression analysis results of nSR100 mRNA and sREST mRNA in the formed tumor are shown in FIG. 5B. As shown in FIG. 5A, no significant tumor formation was observed when SCLC cells cultured in suspension were injected (the middle part on the left side of the mouse of I), but SCLC cells cultured on an extracellular matrix or cultured in suspension. When a mixture of SCLC cells and Matrigel was injected, larger tumor formation was seen in mice I (lower left of III mice and lower right of IV mice). From FIG. 5B, compared to the smaller tumor formation seen in FIG. 5A (corresponding to N417, A I), the larger tumor formation (N417 + ECL and N417 + Matrigel, corresponding to A III and IV) ) Showed higher expression of nSR100 mRNA and sREST mRNA. These results showed that the ability of SCLC to form tumors was correlated with the expression of the nSR100 gene and the expression of the sREST splicing variant of the REST gene. This suggests that suppression of the nSR100 gene can suppress the ability of SCLC to form tumors.

Example 5. SCLC cell-specific apoptosis induction by suppression of nSR100 gene
Using LDH activity as an index, the presence and extent of apoptosis due to suppression of the nSR100 gene was evaluated. Specifically, it was performed as follows.

<5.1. Cell culture>
NCI-N417 cells (human SCLC cells) or NCI-H82 cells (human SCLC cells) were adherently cultured on the extracellular matrix in the same manner as in Example 2. Further, NCI-H1650 cells (human NSCLC cells) were cultured for adhesion on a culture dish.

<5.2. RNAi>
NCI-N417 cells, NCI-H82 cells, or NCI-H1650 cells in the logarithmic growth phase were seeded in a 96-well plate at 1 × 10 4 cells / well and cultured for 24 hours. Subsequently, siRNA (Dharmacon, SMART-pool siRNAs) for the nSR100 gene was transfected to 2.5 pmol per well using a commercially available transfection reagent (DharmaFECT siRNA Transfection Reagents (Thermo Scientific)) for 72 hours. The culture was continued. SiRNA-A (siRNA targeting target sequence 1 (SEQ ID NO: 10)), siRNA-B (siRNA targeting target sequence 2 (SEQ ID NO: 11)), siRNA-C (target sequence) 3 (siRNA targeting SEQ ID NO: 12), siRNA-D (siRNA targeting target sequence 4 (SEQ ID NO: 13)), siRNA mix (equimolar mixture of siRNA-A to siRNA-D) 5 in total The type was used. On the other hand, instead of siRNA, camptothecin, an anticancer agent, was added so that the concentration in the medium was 0.63, 1.25, 2.5, or 3 μg / ml, and the culture was continued for 72 hours.

<5.3. LDH activity measurement>
An LDH activity measurement kit (Takara LDH Cytotoxicity Detection kit, MK401) was used according to the method described in the package insert.

<5.4. Result>
The results when NCI-N417 cells are used are shown in FIG. 6A, the results when NCI-H82 cells are used are shown in FIG. 6B, and the results when NCI-H1650 cells are used are shown in FIG. 6C. 6A and B, when the nSR100 gene was suppressed by siRNA in SCLC cells, LDH activity comparable to or higher than that of camptothecin known as an anticancer agent was detected. In particular, when siRNA-mix, siRNA-C, or siRNA-D was used as siRNA, more remarkable LDH activity was detected. On the other hand, from FIG. 6C, even when the nSR100 gene was suppressed by siRNA in NSCLC cells, only LDH activity lower than that of camptothecin was detected. From the above results, it was suggested that suppression of the nSR100 gene can induce apoptosis specifically for SCLC cells, that is, suppression of the nSR100 gene is useful for the treatment of SCLC.

Example 6 Analysis of nSR100 gene-suppressed miRNA levels in the blood of SCLC patients
As a result of analyzing the expression of various miRNAs in SCLC cells, miRNAs (hsa-miR-4279 (SEQ ID NO: 14), hsa-miR-4419b (SEQ ID NO: 15), hsa-miR-, which are expected to suppress the expression of the nSR100 gene. It was found that the expression level of 4516 (SEQ ID NO: 16) and hsa-miR-4635 (SEQ ID NO: 17) was decreased in SCLC cells compared to normal cells. From this, it was considered that miRNAs that suppress the expression of the nSR100 gene are excreted outside the SCLC cells in SCLC patients, and as a result, the expression level of the nSR100 gene in the SCLC cells is increased. If this hypothesis is correct, SCLC diagnosis is possible by measuring the amount of miRNA excreted outside the SCLC cells. Therefore, in order to confirm this hypothesis, the amount of miRNA in the blood of SCLC patients was analyzed. Specifically, it was performed as follows.

<6.1. Preparation of serum>
Blood was collected from healthy subjects, SCLC patients, NCLC patients, and gastric cancer patients, and serum was prepared according to a conventional method.

<6.2. Quantitative real-time PCR>
Total RNA was extracted from 200 μl of serum using an RNA extraction kit (Qiagen, miRNeasy Serum / Plasma Kit). In order to suppress errors during RNA extraction of each sample, miR-39 was spiked in and used as a standard control. Using 2 μl of the total RNA extract (14 μl), cDNA was prepared using a reverse transcription reaction kit (Qiagen, miScript II RT Kit). Furthermore, a nucleic acid amplification reaction was performed in a PCR apparatus (Rotor-Gene Q, Qiagen) using the sample after the reverse transcription reaction as a template and using a real-time PCR reaction kit (Qiagen miRNA SYBR Green PCR kit). The nucleic acid amplification reaction was performed at a temperature cycle of 95 ° C. for 15 minutes → [(94 ° C. for 15 seconds → 55 ° C. for 30 seconds → 70 ° C. for 30 seconds) × 40 cycles]. In addition, as a primer, each miRNA (hsa-miR-4279 (sequence number 14), hsa-miR-4419b (sequence number 15), hsa-miR-4516 (sequence number 16), and hsa-miR-4635 (sequence) A primer commercially available from Qiagen was used as the detection primer of No. 17)).

<6.3. Result>
The results are shown in FIG. In each graph of FIG. 7, each black circle represents the data of each subject. From FIG. 7, the amount of miRNA (miR-4279, miR-4419b, miR-4516, and miR-4635) that suppresses the expression of the nSR100 gene was increased in the blood of SCLC patients. This supported the hypothesis that in SCLC patients, miRNAs that suppress nSR100 gene expression were excreted outside the SCLC cells, resulting in increased expression levels of the nSR100 gene in SCLC cells. It was also shown that SCLC diagnosis is possible by measuring the amount of miRNA in the blood.

Example 7 Expression analysis of nSR100 gene regulatory factor in blood of various cancer patients From the results of Example 6 (FIG. 7), miR-4516, which had the highest blood volume, was treated with various cancer patients (SCLC, NSCLC, gastric cancer, bladder cancer, The amount in the blood of prostate cancer, kidney cancer, and healthy subjects was measured in the same manner as in Example 6.

  The results are shown in FIG. In FIG. 8, each bar shows the data of each subject. FIG. 8 shows that the amount of miR-4516 in blood tends to increase in SCLC patients among various cancer patients. From this, it was shown that SCLC diagnosis can be performed with high accuracy by measuring the amount of miR-4516 in the blood.

Claims (8)

(A) a polynucleotide having at least 15 bases continuous in the base sequence of human nSR100 mRNA and / or a polynucleotide complementary to the polynucleotide; or (b) a base sequence of human nSR100 mRNA or a base sequence complementary thereto. It hybridizes under stringent conditions, and has a nucleotide sequence or complementary nucleotide sequence and 95% identity to that of human NSR100 mRNA, polynucleotide having at least 15 bases,
Contain any of,
When the polynucleotide is a primer, the base length of the polynucleotide is 15-50,
A diagnostic agent for malignancy of small cell lung cancer. The diagnostic agent for malignancy of small cell lung cancer according to claim 1, wherein the polynucleotide is a probe or a primer. (A ′) a polynucleotide having at least 15 bases consecutive in the base sequence of miR-4279, miR-4419b, miR-4516, or miR-4635 and / or a polynucleotide complementary to the polynucleotide, or (b ′ ) miR-4279, miR-4419b , miR-4516, and miR-4635 in the nucleotide sequence or it hybridizes under stringent conditions to the complementary base sequence, and miR-4279, miR-4419b, miR-4516 And a polynucleotide having at least 15 bases having at least 95% identity with the base sequence of miR-4635 or a base sequence complementary thereto ,
Contain any of,
When the polynucleotide is a primer, the polynucleotide has a base length of 15 to 21,
A diagnostic agent for small cell lung cancer. The diagnostic agent for small cell lung cancer according to claim 3, wherein the polynucleotide is a probe or a primer. A diagnostic agent for malignancy of small cell lung cancer, comprising an antibody that recognizes human nSR100 protein. (1) a step of measuring the amount of human nSR100 mRNA or human nSR100 cDNA in a sample derived from a small cell lung cancer collected from a subject using the diagnostic agent according to claim 1 or 2, and (2) the above step ( Contrast the amount of human nSR100 mRNA or human nSR100 cDNA (test mRNA expression level) measured in 1) with the amount of human nSR100 mRNA or human nSR100 cDNA (control mRNA expression level) in other small cell lung cancer samples. Having a process of
(3) A method for evaluating the malignancy of a small cell lung cancer, wherein the test mRNA expression level is higher than the control mRNA expression level, and the determination index is that the malignancy of the subject's small cell lung cancer is higher. (1 ") The amount of at least one selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635 in a blood sample collected from a subject according to claim 3 or 4 A step of measuring using a diagnostic agent, and (2 ″) at least one selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635 measured in the above step (1 ″) Is selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635 in blood samples collected from subjects who are not small cell lung cancer patients (eg, healthy subjects) Comparing with at least one amount (control miRNA amount)
(3 ″) A method for detecting small cell lung cancer, wherein the test miRNA amount is higher than the control miRNA amount, and the subject is an indicator for determining that the subject has small cell lung cancer. (1 ′) a step of measuring the expression level of human nSR100 protein in a sample derived from a small cell lung cancer collected from a subject using the diagnostic agent according to claim 3, and (2 ′) in the step (1 ′) Comparing the measured expression level of human nSR100 protein (test protein expression level) with the expression level of human nSR100 protein in other small cell lung cancer-derived samples (control protein expression level),
(3 ′) A method for evaluating the malignancy of a small cell lung cancer, wherein the test protein expression level is higher than the control protein expression level, and the determination index is that the malignancy of the subject's small cell lung cancer is higher.

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