WO2013191242A1 - Test method and treatment means for small cell lung cancer having poor prognosis - Google Patents

Test method and treatment means for small cell lung cancer having poor prognosis Download PDF

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WO2013191242A1
WO2013191242A1 PCT/JP2013/066947 JP2013066947W WO2013191242A1 WO 2013191242 A1 WO2013191242 A1 WO 2013191242A1 JP 2013066947 W JP2013066947 W JP 2013066947W WO 2013191242 A1 WO2013191242 A1 WO 2013191242A1
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lung cancer
small cell
cell lung
hotair
poor prognosis
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石川　雄一
宏 小野
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公益財団法人がん研究会
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    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present invention relates to a test method, biomarker, and treatment means that can detect small cell lung cancer with poor prognosis among small cell lung cancer.
  • Lung cancer is one of the intractable cancers, and both morbidity and mortality begin to increase in the late 40s, and become higher as the elderly get older. There is no significant difference in the number of morbidity and death, which is related to the low survival rate of those with lung cancer, and there is a need for the development of useful laboratory diagnostics and treatments.
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • tissue types such as adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and adenosquamous cell carcinoma.
  • SCLC is classified as a neuroendocrine cancer tissue type, accounts for about 15 to 20% of lung cancer, and generally shows a unique aspect such as being easier to metastasize and increase than NSCLC.
  • chemotherapy is mainly used, and it is usually difficult to operate, so it is difficult to obtain fresh materials, and research tends to be delayed, so biomarkers that lead to SCLC testing and treatment The search for is also not progressing.
  • HOTAIR RNA called HOTAIR (hereinafter simply referred to as “HOTAIR”) transcribed antisense from the HOXC cluster, which is one of the HOX genes, is long intergenic non- It is a long non-coding RNA that does not encode a protein called coding RNA (lincRNA) and is a relatively recent cancer-related gene.
  • This HOX gene group is a group of genes that make the segment of animals, and contributes to the creation of the segment during the fetal period, but is also involved in the development and progression of cancer. HOTAIR is also thought to regulate gene expression in the HOXD cluster.
  • HOTAIR High expression of HOTAIR is confirmed in primary breast cancer tissues and metastatic breast cancer tissues, and high expression of HOTAIR in primary breast cancer tissues is considered as a strong predictor of metastasis and death (Non-patent Document 1, Patent Document 1). ).
  • Patent Document 1 Patent Document 1
  • PRC Polycomb repressive complex
  • Cancers differ in biomarkers such as genes and proteins expressed by organ characteristics and phenotypes (phenotypes) and drug sensitivities, so examination methods and treatment methods need to be examined for each cancer type.
  • Breast cancer histology is mainly adenocarcinoma (invasive)
  • colon cancer is mainly epithelial and adenocarcinoma
  • liver cancer is mainly epithelial cancer, and is different from SCLC histology
  • Small cell lung cancer includes cancer with poor prognosis and cancer with good prognosis (see, for example, Example 1 and Table 1). It has been pointed out that small cell lung cancer with a poor prognosis may have already metastasized or disseminated at the time of discovery. The treatment is difficult, and therefore, early detection and establishment of a treatment method are required. Therefore, an object of the present invention is to provide a test method and a biomarker thereof that can detect small cell lung cancer with poor prognosis among small cell lung cancer. Furthermore, an object of the present invention is to provide a therapeutic agent for small cell lung cancer with a poor prognosis. Furthermore, an object of the present invention is to provide a method for screening a drug for treating small cell lung cancer with a poor prognosis.
  • the present inventors have found that HOTAIR, which is a lincRNA, is highly expressed in small cell lung cancer with a poor prognosis. Furthermore, the present inventors have found an siRNA capable of suppressing the growth of small cell lung cancer that highly expresses HOTAIR. That is, the present invention relates to a diagnostic method for determining whether small cell lung cancer (SCLC) is a small cell lung cancer with a poor prognosis, and in a small cell lung cancer (SCLC) as a specimen. It is a test method that comprises measuring the expression level of HOTAIR, which is a lincRNA, and that the higher the expression level of HOTAIR, the poorer the prognosis.
  • SCLC small cell lung cancer
  • the present invention is a biomarker for diagnosing small cell lung cancer used for determining whether small cell lung cancer (SCLC) is a small cell lung cancer with a poor prognosis, and is a lincRNA. It is a biomarker for diagnosis and diagnosis of small prognosis of small cell lung cancer, which is composed of HOTAIR and has a poorer prognosis as the expression level of HOTAIR, which is a lincRNA, is higher. Further, the present invention is a kit for use in quantifying the biomarker for testing and diagnosis of poor prognosis of small cell lung cancer so that the cDNA (SEQ ID NO: 1) of HOTAIR, which is a lincRNA, can be quantified. A kit comprising a primer to be amplified and a polymerase and / or a probe to be paired with the cDNA.
  • the present invention provides an oligoribo corresponding to a base sequence of 30 bases or less of the base sequence of SEQ ID NO: 1 comprising the base sequence of positions 801 to 819 of the base sequence (SEQ ID NO: 1) of HOTAIR, which is a lincRNA.
  • An agent for inhibiting the growth of small cell lung cancer with a poor prognosis which comprises, as an active ingredient, a double-stranded RNA comprising a nucleotide and its complementary oligoribonucleotide.
  • the present invention relates to an oligo corresponding to a base sequence of 30 bases or less of the base sequence of SEQ ID NO: 1, comprising the base sequence of 801 to 819 of the base sequence (SEQ ID NO: 1) of cDNA for HOTAIR, which is a lincRNA.
  • a kit for inhibiting the growth of small cell lung cancer with a poor prognosis comprising a double-stranded RNA comprising ribonucleotide and its complementary oligoribonucleotide.
  • the present invention relates to an agent for inhibiting the growth of small cell lung cancer with poor prognosis, comprising screening a candidate drug for inhibiting or suppressing the expression of HOTAIR, which is a lincRNA, using human cultured small cell lung cancer cells. Screening method.
  • SCLC tissue (cancer) on the left side of the figure shows the appearance of sheet-like growth of small tumor cells that are almost naked nucleus and the nucleolus is unclear.
  • Normal tissue on the right side of the figure has alveolar structure. It shows a clear aspect. Cut along the boundary indicated by the dotted line.
  • the vertical axis in the figure indicates the expression level of HOTAIR for ACTB.
  • ACTB indicates ⁇ -actin.
  • “high-HOTAIR” indicates a high HOTAIR expression group having a HOTAIR / ACTB ratio of 1.368 or more
  • “low-HOTAIR” indicates a low HOTAIR expression group having a HOTAIR / ACTB ratio of less than 1.368.
  • SCLC small cell lung cancer
  • (B) Decrease in the expression level of HOTAIR due to RNA interference (# 1 to # 3 siHOTAIR) in the SBC-3 cell line.
  • shaft shows the expression level of HOTAIR with respect to GAPDH.
  • SiGFP is a negative control.
  • One embodiment of the present invention is a test method for determining small cell lung cancer (SCLC) with a poor prognosis based on the expression level of HOTAIR.
  • SCLC small cell lung cancer
  • “poor prognosis” means that the recurrence rate is high after SCLC surgery and postoperative chemotherapy. Death is often caused by poor prognosis of SCLC.
  • SCLC components are first collected from SCLC surgical specimens.
  • rapid diagnosis and subsequent pathological diagnosis it is necessary to confirm that the collected specimen is a small cell lung cancer.
  • the quick diagnosis is performed as follows, for example.
  • SCLC components are identified visually by slicing a frozen specimen with a cryostat and performing hematoxylin-eosin staining. SCLC is small compared to normal cells, is almost like a naked nucleus, has an obscure nucleolus, and exhibits sheet-like proliferation, so it can be roughly identified visually.
  • the pathological diagnosis is performed as follows, for example.
  • the surgical specimen is fixed in formalin for several days, and then the sample is cut out.
  • the lung is sliced at a thickness of approximately 5 mm, similar to a CT scan image, to obtain a sample. It is preferable to further divide into block sizes centering on the tumor area on each surface and the surrounding related area.
  • a specimen prepared in this manner is sliced into a paraffin-embedded specimen to prepare a preparation.
  • immunostaining using an antibody of a neuroendocrine marker for example, Chromogranin A, Synaptophysin, NCAM / CD56
  • a neuroendocrine marker for example, Chromogranin A, Synaptophysin, NCAM / CD56
  • the specimen determined to be SCLC as described above may be stored, for example, as follows until the HOTAIR expression level is examined.
  • a portion of the surgical specimen that was cut off at the time of rapid diagnosis is divided into several 3 mm square tissue sections on a serum tube that has been given the specimen number on ice for research purposes. Then, it is put into liquid nitrogen, frozen, and stored in a -80 ° C freezer.
  • the stored specimen is prepared as follows. Take the serum tube of the preserved specimen from the -80 ° C freezer, take out one piece of tissue in the tube with a sterilizing cloth, place it in a new serum tube with the same number, and immediately put it into liquid nitrogen.
  • the test method of the present invention comprises measuring the expression level of HOTAIR, which is a lincRNA, in a specimen of small cell lung cancer (SCLC), and the higher the expression level of HOTAIR, the poorer the prognosis. .
  • the poor prognosis small cell lung cancer diagnostic marker used in the present invention consists of HOTAIR, which is a lincRNA.
  • HOTAIR cDNA consists of the base sequence represented by SEQ ID NO: 1 (NCBI Accession No .: NR_003716.2).
  • the expression level of HOTAIR is preferably expressed as a relative amount with respect to the expression level of the internal standard gene.
  • internal standard genes include glyceraldehyde-3-phosphate dehydrogenase (also referred to as “GAPDH”), ⁇ -actin (also referred to as “ACTB”), IPO8, etc.
  • GAPDH Genbank Accession No .: NM_002046.3
  • GAPDH Genbank Accession No .: NM_002046.3
  • ⁇ -actin gene As an internal standard, and when examining HOTAIR in cancer cells, it is preferable to use GAPDH gene as an internal standard (BMC Mol Biol. 2008). 9: 103).
  • the test method of the present invention further includes measuring the expression level of a gene serving as an internal standard in small cell lung cancer (SCLC), and is a lincRNA HOTAIR that is an expression level of the gene serving as an internal standard.
  • SCLC small cell lung cancer
  • the small prognosis marker for small cell lung cancer of the present invention further comprises an mRNA expressed from a gene serving as an internal standard, and the expression level of mRNA expressed from the gene serving as an internal standard is that of the HOTAIR region that is a lincRNA.
  • the higher the expression level the worse the prognosis.
  • the determination of small cell lung cancer with poor prognosis may be performed using the H / R ratio of the following formula.
  • H / R HOTAIR expression level / internal standard gene expression level
  • Detection of the expression level of HOTAIR and the internal standard gene can be performed by any known gene such as RT-PCR using various methods such as SYBR Green method or TaqMan method, Northern blot method, DNA chip (Affymetrix, etc.) It can be performed using expression quantification methods.
  • a real-time quantitative PCR method which is one of the RT-PCR methods, is preferable in that a very small amount of DNA can be detected with high sensitivity.
  • HOTAIR lamRNA
  • first strand cDNA is used as a template for PCR amplification with primers specific to HOTAIR cDNA.
  • Applied Biosystems High-Capacity cDNA Reverse Transcription Kit
  • real-time quantitative PCR for example, TaqMan (registered trademark) Gene Expression Assays provided by Applied Biosystems can be used.
  • Amplified products can be separated and quantified by electrophoresis, etc., but it is accurate and easy to quantify using real-time quantitative PCR instruments such as Applied Biosystems ABI PRISM 7000 Sequence Detection System and Roche LC480 system. Is preferable.
  • HOTAIR lincRNA
  • a primer for PCR usually about 10 to 30 nucleotide sequences sandwiching a polynucleotide portion of at least 50 bases, preferably 100 to 1,000 bases, of a polynucleotide of HOTAIR cDNA (SEQ ID NO: 1) or an internal standard gene.
  • a fragment consisting of As the probe a fragment consisting of at least 15 consecutive nucleotide sequences of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 (HOTAIR cDNA) is usually used.
  • the probe base sequence is usually 15-30 bases, preferably 20-25 bases.
  • HOTAIR lincRNA
  • cDNA DNA array or Northern blot
  • the above primer or real-time quantitative PCR method is used. In some cases, the primer and the probe are used.
  • RNA is extracted from a surgical tumor tissue, and the expression status is confirmed by qRT-PCR using the above-mentioned primers of HOTAIR.
  • ⁇ -actin for tissues and GAPDH for cells as endogenous controls.
  • the method for detecting the expression level of HOTAIR includes, for example, the following steps.
  • SCLC small cell lung cancer
  • the steps d to e are used.
  • a step of amplification and detection of an internal standard gene may be included, and a step of comparing both expression levels may be added.
  • the poor-prognosis small cell lung cancer growth inhibitor of the present invention contains siRNA of HOTAIR that is lincRNA as an active ingredient.
  • This siRNA contains at least 19 consecutive bases in the base sequence of SEQ ID NO: 1 including the base sequences 801 to 819 of the base sequence of SEQ ID NO: 1, preferably 30 bases or less of the base sequence of SEQ ID NO: 1, Is an oligoribonucleotide corresponding to a base sequence of 27 bases or less, preferably 23 bases or less, its complementary oligoribonucleotide, or a double-stranded RNA comprising these.
  • the RNA fragment used in the present invention may be a sense RNA or a antisense RNA of the target RNA, but these are easily decomposed by RNase and are considered to be inferior in effectiveness. It is done.
  • This double-stranded RNA is usually used as a double strand by synthesizing both sense and antisense separately and then hybridizing them.
  • Oligoribonucleotides "corresponding" to these specific base sequences mean the RNA complementary to the portion corresponding to the specific base sequence of the lincRNA produced by transcription of this gene. It means that T in this specific DNA sequence is replaced with U.
  • the siRNA of the present invention may be a modified RNA that has been treated to prevent degradation by a nuclease, and may be, for example, a 2′-O-methylated or 4′-thiolated RNA aptamer.
  • an overhang sequence eg, dTdT, UU, UG, etc.
  • the small cell lung cancer growth inhibitor with poor prognosis may be a combination with other known tumor growth inhibitors and the like.
  • the agent for inhibiting small cell lung cancer growth of the present invention may be in the form of a kit containing such other drugs, or is pharmaceutically acceptable such as sterile isotonic saline, preservatives, buffering agents and the like. Media may be included.
  • siRNA may be encapsulated in a suitable carrier such as a liposome.
  • the poor prognosis small cell lung cancer growth inhibitor of the present invention is an injection kit for administering a mixture of the above-mentioned poor prognosis small cell lung cancer growth inhibitor formulated with a diluent, You may provide as kits, such as a kit for tablets for administering each formulated formulation.
  • the means for introducing siRNA into the cell is not particularly limited, and examples include calcium phosphate method, microinjection method, protoplast fusion method, electroporation, method using viral vector, etc.
  • Commercial transfection reagent based on liposome etc. Is easy to use.
  • siRNA siHOTAIR
  • it may be administered intravenously, and in the case of direct administration to the lesion, the affected area can be examined with an endoscope. In addition to administration, it may be done by inoculating the lesion at the time of surgery.
  • the small prognosis lung cancer cell growth inhibitor can be screened by the following method. Specifically, in the method of the present invention, human cultured small cell lung cancer cells are prepared, and the candidate drug and small cell lung cancer cells are contacted by culturing this cell line in the presence of the candidate drug. Inhibition or suppression of the expression of the lincRNA HOTAIR is performed to perform a primary screening of the candidate drug, and then the suppression of the proliferation and / or invasive ability of human cultured small cell lung cancer cells by the candidate drug is investigated. Perform next screening. As the SCLC cell used for screening, a cell line with a high HOTAIR RNA expression level is preferable. Examples of such an SCLC cell include the SBC-3 cell line.
  • SBC-3 cells are considered to correspond to a cell line of small lung cancer with poor prognosis because of the high expression level of HOTAIR RNA.
  • the degree of inhibition or suppression of the expression of HOTAIR which is a lincRNA, can be determined by a comparative experiment with a control to which no candidate drug is added. Expression levels are measured for total RNA or mRNA or poly A (+) RNA obtained from small cell lung cancer cell lines, or for cDNA synthesized from RNA by the reverse transcriptase-PCR (RT-PCR) method. It can be determined by a hybridization method using a radiolabeled probe (for example, Northern hybridization, Southern hybridization, DNA microarray, tissue microarray, etc.).
  • RT-PCR, PCR primers, and probes described above can be used.
  • this candidate drug Can be used as a small cell lung cancer metastasis inhibitor.
  • Example 1 The clinical specimens used in this example were prepared as follows. 35 SCLC tissue samples were obtained from multiple SCLC patients undergoing surgery at the Cancer Institute Hospital between 1995 and 2010 with written informed consent prior to surgery. It was. In addition, 16 non-cancerous tissue samples were obtained from the same patient. All specimens were immediately frozen in liquid nitrogen and stored at -80 ° C for RNA extraction. The SCLC tissue sample was collected as follows. Using lung tissue including lung cancer tissue submitted for rapid diagnosis, excise the cancerous part and non-cancerous part (area far enough from the cancerous part) (Fig. 1) and place it on a serum tube on ice. It was immediately put into liquid nitrogen and frozen.
  • the following examination was performed using the preserved tissue section at the time of rapid diagnosis of the case diagnosed as SCLC.
  • all the preserved tissue sections at the time of rapid diagnosis were sliced again with a cryostat, hematoxylin-eosin staining was performed, and it was confirmed later that the SCLC component was contained.
  • Table 1 shows the patient background and clinicopathological factors.
  • SCLC recurrence refers to a case in which new lesions were found in the lung and other organs during recurrence during the postoperative follow-up (up to about 15 years after surgery), and medical records and images were searched retrospectively.
  • the recurrent metastasis destination is described, but includes metastasis metastasized to a plurality.
  • follow-up results using medical records and follow-up survey results (about 15 years at the longest postoperatively) of Akenake Hospital's medical history room are shown.
  • HOTAIR expression was performed using quantitative real-time PCR (qRT-PCR) from the 35 SCLC samples collected and stored at the time when specimens were submitted to the pathology department for rapid diagnosis during surgery. I checked the level.
  • qRT-PCR was performed as follows. Total RNA was extracted from tissues and cells using RNeasy mini kit or RNeasy mini kit plus (Qiagen). CDNA was generated from 30 ng of total RNA using High Capacity RNA-to-cDNA (R) kit (AB, catalog number 4387406).
  • the obtained cDNA was subjected to PCR amplification for 45 cycles, followed by real-time PCR reaction using LightCycler (registered trademark) 480 SYBR Green I Master protocol (Roche, catalog number 4707516) and the following primers.
  • the expression level was examined. Each gene in each sample of a 96-well plate on the LightCycler (registered trademark) 480 real time PCR System (Roche) was tested three times.
  • the expression level of HOTAIR RNA was normalized with respect to the expression level of ⁇ -actin (ACTB) for tissue samples and xenografts.
  • ACTB ⁇ -actin
  • HOTAIR (F): 5'-GGTAGAAAAAGCAACCACGAAGC-3 '(SEQ ID NO: 4)
  • HOTAIR (R): 5'-ACATAAACCTCTGTCTGTGAGTGCC-3 '(SEQ ID NO: 5)
  • ACTB (F): 5'-AGAAAATCTGGCACCACACC-3 '(SEQ ID NO: 6)
  • ACTB (R): 5'-AGAGGCGTACAGGGATAGCA-3 '(SEQ ID NO: 7)
  • the HOTAIR / ACTB ratio was examined in 35 SCLC tissues and 15 non-cancerous tissues in the same subject group (FIG. 2).
  • a level where the HOTAIR / ACTB ratio was higher than 1.368 was regarded as high HOTAIR expression. According to this criteria, 12 out of 35 SCLC patients were classified as having high HOTAIR expression and 23 were classified as low.
  • the clinicopathological factors of these two groups are shown in Table 1.
  • the high HOTAIR expression group showed more recurrence and death than the low HOTAIR expression group.
  • Example 3 The cell line used in this example was prepared as follows. SCLC cell lines COLO-668, COR-L51, COR-L88, and DMS-79 were obtained from ECACC (The European Collection of Cell Cultures). SCLC cell lines DMS-53, LU-134A, MS-1, SBC-3, SBC-5, SBC-1, A549 and MRC-5 are either JCRB (Japanese Collection of Research Bio-resources) or RIKEN Obtained from the BioResource Center (BRC). These cell lines were cultured in Dulbecco's modified Eagle medium (DMEM, Nacalai Tesque, catalog number 08488-55, 500 ml) in a 37 ° C, 5% CO 2 incubator.
  • DMEM Dulbecco's modified Eagle medium
  • This Dulbecco's modified Eagle medium consists of 5.0 ml of 200 mM L-alanyl-L-glutamine solution (Nacalai Tesque catalog number 04260-64), 5.5 ml of antibiotic antifungal mixed stock solution (Nacalai Tesque catalog number 02892-54), And 50 ml of fetal bovine serum (Biowest catalog number S1560).
  • RNA interference experiments with HOTAIR were performed as follows. Using Lipofectamine TM RNAiMAX (Invitrogen, catalog number 13778-075), 20 nM siRNA targeting HOTAIR was introduced into the cells according to the manufacturer's instructions. The efficiency of this introduction is determined by introducing a labeled positive control (Blex-IT TM Alexa Fluor® red fluorescent oligo (Invitrogen, Catalog No. 14750-100)), and then placing the cells in a humidified environment containing 5% CO 2. And incubated at 37 ° C. for 72 hours, and then evaluated using a fluorescence microscope (Leica DMIRE2).
  • RNA interference was similarly performed for the GFP gene as a negative control. Quantitative RT-PCR was performed in the same manner as in Example 1.
  • siHOTAIR Sense: 5′-GAACGGGAGUACAGAGAGAUU-3 ′ (corresponding to positions 801 to 819 of SEQ ID NO: 10 and SEQ ID NO: 1)
  • the expression level of HOTAIR RNA is shown in FIG. 4B. # 1-3 It can be seen that the introduction of siHOTAIR decreased the expression level of HOTAIR RNA. Furthermore, SBC-3 cell line after introduction of # 1-3 siHOTAIR (below) was cultured in Dulbecco's modified Eagle medium (DMEM, Nacalai Tesque catalog number 08488-55, 500 ml) in a 37 ° C, 5% CO2 incubator. . This Dulbecco's modified Eagle medium contains 5.0 ml of 200 mM L-alanyl-L-glutamine solution (Nacalai Tesque catalog number 04260-64) and 50 ml of fetal calf serum (Biowest catalog number S1560).
  • DMEM Dulbecco's modified Eagle medium
  • This Dulbecco's modified Eagle medium contains 5.0 ml of 200 mM L-alanyl-L-glutamine solution (Nacalai Tesque catalog number 04260-64) and 50 m

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Abstract

[Problem] In small cell lung cancer (SCLC), there are a type having poor prognosis and a type having good prognosis. It has been demanded to detect small cell lung cancer at an earlier stage and establish a treatment method for small cell lung cancer. The present invention provides: a test method which can detect small cell lung cancer having poor prognosis; a means for treating small cell lung cancer having poor prognosis; and a method for screening for a drug for treating small cell lung cancer having poor prognosis. [Solution] It is found that HOTAIR (for which cDNA comprises the nucleotide sequence represented by SEQ ID NO: 1), which is lincRNA, is highly expressed in small cell lung cancer having poor prognosis. The present invention is a diagnosis method for determining whether or not small cell lung cancer is one having poor prognosis, wherein the prognosis of the small cell lung cancer is determined as being poorer when the amount of HOTAIR, which is lincRNA, expressed in a sample of the small cell lung cancer is higher. Further, siRNA which can inhibit the proliferation of small cell lung cancer in which HOTAIR is highly expressed is discovered.

Description

予後不良の肺小細胞がんの検査方法及び治療手段Examination method and treatment method for small cell lung cancer with poor prognosis
　本発明は、肺小細胞がんのうち、予後不良の肺小細胞がんを検出できる検査方法及びバイオマーカー、並びに治療手段に関する。 The present invention relates to a test method, biomarker, and treatment means that can detect small cell lung cancer with poor prognosis among small cell lung cancer.
　肺がんは、難治性のがんのひとつで、罹患率と死亡率は、ともに40歳代後半から増加し始め、高齢ほど高くなる。罹患数と死亡数に大きな差はなく、これは、肺がん罹患者の生存率が低いことと関連しており、有用な検査診断法と治療法の開発が求められている。
　肺がんを組織学的に分類すると、小細胞がん（Small cell lung cancer：SCLC）と非小細胞がん（Non-small cell lung cancer: NSCLC）の２つに大別される。大多数を占めるNSCLCは、さらに腺がん、扁平上皮がん、大細胞がん、腺扁平上皮がんなどの組織型に分類される。SCLCは、神経内分泌がんの組織型に分類され、肺がんの約15～20％を占め、一般にNSCLCに比べて転移し易く、増大し易い等の特異な様相を示す。SCLCに対しては、化学療法が主に行われており、通常は手術の対象となりにくいことから、新鮮材料の入手が難しく、研究が遅れがちであるため、SCLCの検査や治療につながるバイオマーカーの探索も進んでいない。 Lung cancer is one of the intractable cancers, and both morbidity and mortality begin to increase in the late 40s, and become higher as the elderly get older. There is no significant difference in the number of morbidity and death, which is related to the low survival rate of those with lung cancer, and there is a need for the development of useful laboratory diagnostics and treatments.
When lung cancer is classified histologically, it is roughly classified into two types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC, which accounts for the majority, is further classified into tissue types such as adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and adenosquamous cell carcinoma. SCLC is classified as a neuroendocrine cancer tissue type, accounts for about 15 to 20% of lung cancer, and generally shows a unique aspect such as being easier to metastasize and increase than NSCLC. For SCLC, chemotherapy is mainly used, and it is usually difficult to operate, so it is difficult to obtain fresh materials, and research tends to be delayed, so biomarkers that lead to SCLC testing and treatment The search for is also not progressing.
　近年、数多くのがん関連遺伝子が報告されているが、HOX遺伝子群の一つであるHOXCクラスターからantisenseに転写されるHOTAIRと呼ばれるRNA（以下単に「HOTAIR」という。）は、long intergenic non-coding RNA (lincRNA)と呼ばれるタンパクをコードしていない長鎖非コードRNAで、比較的最近見出されたがん関連遺伝子である。このHOX遺伝子群は、動物の体節をつくる遺伝子群であり、胎児期には体節をつくるのに寄与しているが、がんの発生や進展にも関わっている。また、HOTAIRはHOXDクラスターの遺伝子発現を制御していると考えられている。
　原発性乳がん組織や転移性乳がん組織においてHOTAIRの高発現が確認され、原発性乳がん組織におけるHOTAIRの高発現が転移及び死亡の強力な予測因子として考えられている（非特許文献１、特許文献１）。これらの文献において、乳がんセルラインを用いてHOTAIRを強発現させると、PRC（Polycomb repressive complex）依存的に浸潤能や転移能を増進させ、siHOTAIRを用いると逆にこれらが抑制されることが報告されている。
　また、HOTAIRは、大腸がんの予後不良や肝がんの転移に関与していることが報告されている（非特許文献２，３）。
　なお、がんは臓器特性やフェノタイプ（表現型）によって発現する遺伝子やタンパク質などのバイオマーカーや薬剤感受性が異なるため、検査法や治療法はがん種ごとに検討される必要がある。乳がんの組織型は主に腺がん（浸潤性）、大腸がんは主に上皮性がんと腺がん、肝がんは主に上皮性がんであり、SCLCの組織型とは異なるため、これらの検査法や治療法はSCLCには適用することができないと考えられている。 In recent years, many cancer-related genes have been reported, but RNA called HOTAIR (hereinafter simply referred to as “HOTAIR”) transcribed antisense from the HOXC cluster, which is one of the HOX genes, is long intergenic non- It is a long non-coding RNA that does not encode a protein called coding RNA (lincRNA) and is a relatively recent cancer-related gene. This HOX gene group is a group of genes that make the segment of animals, and contributes to the creation of the segment during the fetal period, but is also involved in the development and progression of cancer. HOTAIR is also thought to regulate gene expression in the HOXD cluster.
High expression of HOTAIR is confirmed in primary breast cancer tissues and metastatic breast cancer tissues, and high expression of HOTAIR in primary breast cancer tissues is considered as a strong predictor of metastasis and death (Non-patent Document 1, Patent Document 1). ). In these documents, it is reported that when HOTAIR is strongly expressed using a breast cancer cell line, invasion ability and metastasis ability are enhanced depending on PRC (Polycomb repressive complex), and these are suppressed when siHOTAIR is used. Has been.
In addition, HOTAIR has been reported to be involved in poor prognosis of colorectal cancer and metastasis of liver cancer (Non-patent Documents 2 and 3).
Cancers differ in biomarkers such as genes and proteins expressed by organ characteristics and phenotypes (phenotypes) and drug sensitivities, so examination methods and treatment methods need to be examined for each cancer type. Breast cancer histology is mainly adenocarcinoma (invasive), colon cancer is mainly epithelial and adenocarcinoma, liver cancer is mainly epithelial cancer, and is different from SCLC histology These tests and treatments are not considered applicable to SCLC.
US 2012/0004278US 2012/0004278
　肺小細胞がん(SCLC)には、予後不良のがんと予後良好のがんがある（例えば、実施例１、表１を参照のこと）。予後不良の肺小細胞がんは、発見時には既に転移、全身播種している可能性があることが指摘されている。その治療は困難であり、そのため、早期の発見と治療方法の確立が求められている。
　従って、本発明は、肺小細胞がんのうち、予後不良の肺小細胞がんを検出できる検査法とそのバイオマーカーを提供することを目的とする。
　更に、本発明は、予後不良の肺小細胞がんの治療剤を提供することを目的とする。
　更に、本発明は、予後不良の肺小細胞がんを治療するための薬剤をスクリーニングする方法を提供することを目的とする。 Small cell lung cancer (SCLC) includes cancer with poor prognosis and cancer with good prognosis (see, for example, Example 1 and Table 1). It has been pointed out that small cell lung cancer with a poor prognosis may have already metastasized or disseminated at the time of discovery. The treatment is difficult, and therefore, early detection and establishment of a treatment method are required.
Therefore, an object of the present invention is to provide a test method and a biomarker thereof that can detect small cell lung cancer with poor prognosis among small cell lung cancer.
Furthermore, an object of the present invention is to provide a therapeutic agent for small cell lung cancer with a poor prognosis.
Furthermore, an object of the present invention is to provide a method for screening a drug for treating small cell lung cancer with a poor prognosis.
　本発明者らは、予後不良の肺小細胞がんにlincRNAであるHOTAIRが高発現していることを見出した。更に、HOTAIRを高発現している肺小細胞がんの増殖を抑制することのできるsiRNAを見出した。
　即ち、本発明は、肺小細胞がん（SCLC）が予後不良の肺小細胞がんであるか否かを判断するための診断方法であって、検体である肺小細胞がん（SCLC）における、lincRNAであるHOTAIRの発現量を測定することから成り、HOTAIRの発現量が高いほど予後不良であるとする検査方法である。
　また、本発明は、肺小細胞がん（SCLC）が予後不良の肺小細胞がんであるか否かを判断するために用いる肺小細胞がん検査診断用バイオマーカーであって、lincRNAであるHOTAIRから成り、lincRNAであるHOTAIRの発現量が、高いほど予後不良であるとする、予後不良肺小細胞がん検査診断用バイオマーカーである。
　また、本発明は、この予後不良肺小細胞がん検査診断用バイオマーカーを定量するために使用するためのキットであって、lincRNAであるHOTAIRのｃＤＮＡ（配列番号１）を定量可能なように増幅するプライマー及びポリメラーゼ、及び/又は該ｃＤＮＡに対合させるプローブから成るキットである。 The present inventors have found that HOTAIR, which is a lincRNA, is highly expressed in small cell lung cancer with a poor prognosis. Furthermore, the present inventors have found an siRNA capable of suppressing the growth of small cell lung cancer that highly expresses HOTAIR.
That is, the present invention relates to a diagnostic method for determining whether small cell lung cancer (SCLC) is a small cell lung cancer with a poor prognosis, and in a small cell lung cancer (SCLC) as a specimen. It is a test method that comprises measuring the expression level of HOTAIR, which is a lincRNA, and that the higher the expression level of HOTAIR, the poorer the prognosis.
In addition, the present invention is a biomarker for diagnosing small cell lung cancer used for determining whether small cell lung cancer (SCLC) is a small cell lung cancer with a poor prognosis, and is a lincRNA. It is a biomarker for diagnosis and diagnosis of small prognosis of small cell lung cancer, which is composed of HOTAIR and has a poorer prognosis as the expression level of HOTAIR, which is a lincRNA, is higher.
Further, the present invention is a kit for use in quantifying the biomarker for testing and diagnosis of poor prognosis of small cell lung cancer so that the cDNA (SEQ ID NO: 1) of HOTAIR, which is a lincRNA, can be quantified. A kit comprising a primer to be amplified and a polymerase and / or a probe to be paired with the cDNA.
　更に、本発明は、lincRNAであるHOTAIRのcDNAの塩基配列（配列番号１）の801～819番目の塩基配列を含む配列番号１の塩基配列の連続する３０塩基以下の塩基配列に相当するオリゴリボヌクレオチド及びその相補的オリゴリボヌクレオチドから成る２本鎖ＲＮＡを有効成分として含む予後不良肺小細胞がん増殖抑制剤である。
　更に、本発明は、lincRNAであるHOTAIRのcDNAの塩基配列（配列番号１）の801～819番目の塩基配列を含む、配列番号１の塩基配列の連続する３０塩基以下の塩基配列に相当するオリゴリボヌクレオチド及びその相補的オリゴリボヌクレオチドから成る２本鎖ＲＮＡを含む予後不良肺小細胞がん増殖抑制用キットである。
　更に、本発明は、ヒト培養肺小細胞がん細胞を用いて、lincRNAであるHOTAIRの発現の阻害又は抑制について、候補薬剤をスクリーニングすることから成る、予後不良肺小細胞がん増殖抑制剤のスクリーニング方法である。 Furthermore, the present invention provides an oligoribo corresponding to a base sequence of 30 bases or less of the base sequence of SEQ ID NO: 1 comprising the base sequence of positions 801 to 819 of the base sequence (SEQ ID NO: 1) of HOTAIR, which is a lincRNA. An agent for inhibiting the growth of small cell lung cancer with a poor prognosis, which comprises, as an active ingredient, a double-stranded RNA comprising a nucleotide and its complementary oligoribonucleotide.
Furthermore, the present invention relates to an oligo corresponding to a base sequence of 30 bases or less of the base sequence of SEQ ID NO: 1, comprising the base sequence of 801 to 819 of the base sequence (SEQ ID NO: 1) of cDNA for HOTAIR, which is a lincRNA. A kit for inhibiting the growth of small cell lung cancer with a poor prognosis, comprising a double-stranded RNA comprising ribonucleotide and its complementary oligoribonucleotide.
Furthermore, the present invention relates to an agent for inhibiting the growth of small cell lung cancer with poor prognosis, comprising screening a candidate drug for inhibiting or suppressing the expression of HOTAIR, which is a lincRNA, using human cultured small cell lung cancer cells. Screening method.
小細胞肺がん（SCLC）組織を示す写真である。図の左側のSCLC組織(cancer)は、ほぼ裸核に近く核小体が不明瞭な小型腫瘍細胞のシート状増殖の様相を示し、図の右側の正常組織(normal)は、肺胞構造が明瞭な様相を示す。点線で示す境界に沿って切り取る。It is a photograph which shows a small cell lung cancer (SCLC) structure | tissue. SCLC tissue (cancer) on the left side of the figure shows the appearance of sheet-like growth of small tumor cells that are almost naked nucleus and the nucleolus is unclear. Normal tissue on the right side of the figure has alveolar structure. It shows a clear aspect. Cut along the boundary indicated by the dotted line. 35人のSCLC患者を、SCLC組織におけるHOTAIR発現レベルに基づいて並べた図である。図中の縦軸はACTBに対するHOTAIRの発現レベルを示す。ACTBはβ-アクチンを示す。図中の横線は高HOTAIR発現グループ（n=12）と低HOTAIR発現グループ（n=23）の境界線（HOTAIR/ACTB比=1.368）を示す。It is the figure which arranged 35 SCLC patients based on the HOTAIR expression level in SCLC tissue. The vertical axis in the figure indicates the expression level of HOTAIR for ACTB. ACTB indicates β-actin. The horizontal line in the figure indicates the boundary line (HOTAIR / ACTB ratio = 1.368) between the high HOTAIR expression group (n = 12) and the low HOTAIR expression group (n = 23). HOTAIRの発現レベルと臨床的特徴を示す図である。図中、"high-HOTAIR"はHOTAIR/ACTB比が1.368以上の高HOTAIR発現グループ、"low-HOTAIR"はHOTAIR/ACTB比が1.368未満の低HOTAIR発現グループを示す。(A) NAC(-) AC(+)-SCLC患者の肺小細胞がん（SCLC）の無再発率を示す。SCLCが再発するに従って数値（無再発率）は下がる。(B) NAC(-) AC(+)-SCLC患者の肺小細胞がん（SCLC）に対する生存率を示す。SCLCのみの理由により死亡した場合に生存していないとし、SCLC以外の理由により死亡した場合には生存したものとみなす。It is a figure which shows the expression level and clinical characteristic of HOTAIR. In the figure, “high-HOTAIR” indicates a high HOTAIR expression group having a HOTAIR / ACTB ratio of 1.368 or more, and “low-HOTAIR” indicates a low HOTAIR expression group having a HOTAIR / ACTB ratio of less than 1.368. (A) Shows the recurrence-free rate of small cell lung cancer (SCLC) in NAC (-) AC (+)-SCLC patients. As SCLC relapses, the value (no recurrence rate) decreases. (B) The survival rate for small cell lung cancer (SCLC) in patients with 生存 NAC (-) AC (+)-SCLC. If a person dies for reasons other than SCLC, he / she is not alive. If he / she dies for reasons other than SCLC, he / she is considered alive. (A) qRT-PCRにより調べたSCLC細胞株のHOTAIR発現レベルを示す。HOTAIRの発現量はGAPDHの発現量に対して標準化した。縦軸はGAPDHに対するHOTAIRの発現量を示す。(B) SBC-3細胞株におけるRNA干渉（#1～#3のsiHOTAIR）によるHOTAIRの発現量の減少を示す。縦軸はGAPDHに対するHOTAIRの発現量を示す。また、siGFPはネガティブコントロールである。(C) RNA干渉によるSBC-3細胞株の増殖の抑制を示す。横軸は培養開始後の経過時間を示し、縦軸は細胞数を示す。(A) shows the HOTAIR expression level of SCLC cell lines examined by qRT-PCR. The expression level of HOTAIR was normalized to the expression level of GAPDH. A vertical axis | shaft shows the expression level of HOTAIR with respect to GAPDH. (B) Decrease in the expression level of HOTAIR due to RNA interference (# 1 to # 3 siHOTAIR) in the SBC-3 cell line. A vertical axis | shaft shows the expression level of HOTAIR with respect to GAPDH. SiGFP is a negative control. (C) Suppression of SBC-3 cell line growth by RNA interference. The horizontal axis shows the elapsed time after the start of culture, and the vertical axis shows the number of cells.
　本発明の一態様は、予後不良の肺小細胞がん（SCLC）を、HOTAIRの発現レベルに基づいて判定する検査方法である。
　本発明において「予後不良」とは、SCLCの手術後及び術後化学療法施行後に再発率が高いことをいう。SCLCの予後不良により死に至ることが多い。 One embodiment of the present invention is a test method for determining small cell lung cancer (SCLC) with a poor prognosis based on the expression level of HOTAIR.
In the present invention, “poor prognosis” means that the recurrence rate is high after SCLC surgery and postoperative chemotherapy. Death is often caused by poor prognosis of SCLC.
　HOTAIRの発現レベルを調べるために、まずSCLCの手術検体からSCLC成分を採取する。
　迅速診断とその後の病理診断にて、採取した検体が、肺小細胞がんであることを確認する必要がある。診断方法としては、迅速診断だけでもよいが、迅速診断と病理診断の両方を行うことが好ましい。
　迅速診断は、例えば、以下のようにして行う。凍結検体でクライオスタットを用いてスライスし、ヘマトキシリン‐エオジン染色を行うことによりSCLC成分を目視にて識別する。SCLCは、正常細胞に比べて小型でほぼ裸核に近く、核小体も不明瞭であり、シート状増殖を示すという特徴があるため、目視にておおよそ識別可能である。
　病理診断は、例えば、以下のようにして行う。手術検体をホルマリンで数日間固定し、その後サンプルを切り出す。例えば、肺をCT scan画像と同様におよそ5mmの厚さでスライスして、サンプルを得る。それぞれの面における腫瘍領域と周辺の関連領域を中心に更にブロックサイズに切り分けることが好ましい。こうして作成された検体をパラフィン包埋したものを薄切し、プレパラートを作成する。これらの内、SCLC腫瘍成分を多く含む領域で神経内分泌マーカー(例えば、Chromogranin A, Synaptophysin, NCAM/CD56)の抗体を用いた免疫染色を行い、神経内分泌性を確認する。 To examine the expression level of HOTAIR, SCLC components are first collected from SCLC surgical specimens.
In the rapid diagnosis and subsequent pathological diagnosis, it is necessary to confirm that the collected specimen is a small cell lung cancer. As a diagnostic method, only rapid diagnosis may be used, but it is preferable to perform both rapid diagnosis and pathological diagnosis.
The quick diagnosis is performed as follows, for example. SCLC components are identified visually by slicing a frozen specimen with a cryostat and performing hematoxylin-eosin staining. SCLC is small compared to normal cells, is almost like a naked nucleus, has an obscure nucleolus, and exhibits sheet-like proliferation, so it can be roughly identified visually.
The pathological diagnosis is performed as follows, for example. The surgical specimen is fixed in formalin for several days, and then the sample is cut out. For example, the lung is sliced at a thickness of approximately 5 mm, similar to a CT scan image, to obtain a sample. It is preferable to further divide into block sizes centering on the tumor area on each surface and the surrounding related area. A specimen prepared in this manner is sliced into a paraffin-embedded specimen to prepare a preparation. Among these, immunostaining using an antibody of a neuroendocrine marker (for example, Chromogranin A, Synaptophysin, NCAM / CD56) is performed in a region containing many SCLC tumor components to confirm neuroendocrine properties.
　上記のようにSCLCと判断した検体を、HOTAIRの発現レベルを調べるまで、例えば、次のように保存してもよい。迅速診断時に切り分けた手術検体の一部を研究用としてon iceで検体番号を付与したセラムチューブに3mm角大の組織切片として数個ずつ取り分ける。その後、液体窒素に投入し凍結して、-80℃冷凍庫に保存する。
　HOTAIRの発現レベルを調べる際には、この保存しておいた検体を、例えば、次のようにして調製する。この保存検体のセラムチューブを-80℃冷凍庫より出し、チューブ内の組織片を１つ滅菌セッシで取り出し、同じ番号を付与した新たなセラムチューブ内に入れ、直ちに液体窒素に投入する。これを実験室に運び、1症例ずつクライオスタット内に予め用意していたステージの上に置き、蒸留水を検体周囲にまき、アイシングスプレーを用いて凍結させる。これを所定の場所に設置し薄切する。薄切検体はプレパラートに2つほど接着させ、直ちに固定液内（ホルマリン＋メタノール1:1溶液）へ投入する。このプレパラートをヘマトキシリン‐エオジン染色し、腫瘍領域を特定する。一方で、ステージ上の残りの検体は周囲の凍結蒸留水を可能な限り除去し再びセラムチューブにもどし、直ちに液体窒素へ投入する。その後に、組織からのRNA抽出の際にはこのプレパラートに対応する腫瘍成分をon iceで滅菌したメスで切り分け使用する。 The specimen determined to be SCLC as described above may be stored, for example, as follows until the HOTAIR expression level is examined. A portion of the surgical specimen that was cut off at the time of rapid diagnosis is divided into several 3 mm square tissue sections on a serum tube that has been given the specimen number on ice for research purposes. Then, it is put into liquid nitrogen, frozen, and stored in a -80 ° C freezer.
When examining the expression level of HOTAIR, for example, the stored specimen is prepared as follows. Take the serum tube of the preserved specimen from the -80 ° C freezer, take out one piece of tissue in the tube with a sterilizing cloth, place it in a new serum tube with the same number, and immediately put it into liquid nitrogen. This is transported to the laboratory, placed one by one on a stage prepared in advance in the cryostat, sprinkled with distilled water around the specimen, and frozen using an icing spray. This is installed in a predetermined place and sliced. Adhere approximately 2 specimens to the slide and immediately put them into the fixative (formalin + methanol 1: 1 solution). This preparation is stained with hematoxylin-eosin to identify the tumor area. On the other hand, the remaining specimens on the stage are removed as much as possible from the surrounding frozen distilled water, returned to the serum tube, and immediately put into liquid nitrogen. Thereafter, when extracting RNA from the tissue, the tumor component corresponding to this preparation is cut and used with a scalpel sterilized on ice.
　本発明の検査方法は、検体である肺小細胞がん（SCLC）における、lincRNAであるHOTAIRの発現量を測定することから成り、HOTAIRの発現量が高いほど予後不良であるとするものである。
　本発明で用いられる予後不良肺小細胞がん診断マーカーは、lincRNAであるHOTAIRから成る。
　HOTAIRのcDNAは配列番号１で表される塩基配列から成る（NCBI Accession No.：NR_003716.2）。 The test method of the present invention comprises measuring the expression level of HOTAIR, which is a lincRNA, in a specimen of small cell lung cancer (SCLC), and the higher the expression level of HOTAIR, the poorer the prognosis. .
The poor prognosis small cell lung cancer diagnostic marker used in the present invention consists of HOTAIR, which is a lincRNA.
HOTAIR cDNA consists of the base sequence represented by SEQ ID NO: 1 (NCBI Accession No .: NR_003716.2).
　本発明において予後不良肺小細胞がんの判定を行う場合、HOTAIRの発現量は、内部標準遺伝子の発現量に対する相対量として表すことが好ましい。
　内部標準となる遺伝子としては、グリセルアルデヒド-3-リン酸デヒドロゲナーゼ（「GAPDH」ともいう。）、β-アクチン(「ACTB」ともいう。)、IPO8等が挙げられるが、組織検体を扱う場合にβ-アクチン（配列番号２、Genbank Accession No.:NM_001101.3）を、一方、細胞検体を扱う場合にはGAPDH（配列番号３、Genbank Accession No.:NM_002046.3）が好ましい。また、がん組織におけるHOTAIRを調べる場合には、内部標準としてβ-アクチン遺伝子を用い、がん細胞におけるHOTAIRを調べる場合には、内部標準としてGAPDH遺伝子を用いることが好ましい（BMC Mol Biol. 2008,9:103)。 In the present invention, when determining small lung cancer with poor prognosis, the expression level of HOTAIR is preferably expressed as a relative amount with respect to the expression level of the internal standard gene.
Examples of internal standard genes include glyceraldehyde-3-phosphate dehydrogenase (also referred to as “GAPDH”), β-actin (also referred to as “ACTB”), IPO8, etc. Β-actin (SEQ ID NO: 2, Genbank Accession No .: NM_001101.3) is preferred, while GAPDH (SEQ ID NO: 3, Genbank Accession No .: NM_002046.3) is preferred when handling cell samples. Further, when examining HOTAIR in cancer tissue, it is preferable to use β-actin gene as an internal standard, and when examining HOTAIR in cancer cells, it is preferable to use GAPDH gene as an internal standard (BMC Mol Biol. 2008). 9: 103).
　従って、本発明の検査方法は、更に、肺小細胞がん（SCLC）における、内部標準となる遺伝子の発現量を測定することを含み、内部標準となる遺伝子の発現量に対する、lincRNAであるHOTAIRの発現量が、高いほど予後不良であるとしてもよい。
　また、本発明の予後不良肺小細胞がん診断マーカーは、更に、内部標準となる遺伝子から発現するmRNAを含み、内部標準となる遺伝子から発現するmRNAの発現量に対する、lincRNAであるHOTAIR領域の発現量が、高いほど予後不良であるとしてもよい。
　予後不良肺小細胞がんの判定を下式のＨ／Ｒ比を用いて行ってもよい。このＨ／Ｒ比が基準より高いと予後不良、基準より低いと良性と判断でき、また、このＨ／Ｒ比が基準より高いほど肺小細胞がんの再発度が高いと判定できる。基準となるＨ／Ｒ比は、本研究の症例におけるROC curveを用いた検討では、例えば、1.368であった。
　　Ｈ／Ｒ＝HOTAIR発現量／内部標準遺伝子発現量 Therefore, the test method of the present invention further includes measuring the expression level of a gene serving as an internal standard in small cell lung cancer (SCLC), and is a lincRNA HOTAIR that is an expression level of the gene serving as an internal standard. The higher the expression level, the worse the prognosis.
Further, the small prognosis marker for small cell lung cancer of the present invention further comprises an mRNA expressed from a gene serving as an internal standard, and the expression level of mRNA expressed from the gene serving as an internal standard is that of the HOTAIR region that is a lincRNA. The higher the expression level, the worse the prognosis.
The determination of small cell lung cancer with poor prognosis may be performed using the H / R ratio of the following formula. When this H / R ratio is higher than the standard, it can be determined that the prognosis is poor, and when the H / R ratio is lower than the standard, it can be determined that the benignity. In the examination using the ROC curve in the case of this study, the standard H / R ratio was 1.368, for example.
H / R = HOTAIR expression level / internal standard gene expression level
　HOTAIRと内部標準遺伝子の発現量の検出は、例えば、SYBR Green法やTaqMan法など各種方法を用いたRT-PCR法、Northern blot法、DNAチップ（アフィメトリクス社製等）など、任意の公知の遺伝子発現定量法を用いて行うことができる。 Detection of the expression level of HOTAIR and the internal standard gene can be performed by any known gene such as RT-PCR using various methods such as SYBR Green method or TaqMan method, Northern blot method, DNA chip (Affymetrix, etc.) It can be performed using expression quantification methods.
　RT-PCR法の１つであるリアルタイム定量PCR法（qRT-PCR）は微量なDNAを高感度かつ定量的に検出できるという点で好ましい。RT-PCRでは、HOTAIR(lincRNA)を逆転写してfirst strand cDNAを作成し、これを鋳型にHOTAIR cDNAに特異的なプライマーによりPCR増幅する。逆転写反応は、例えば、High-Capacity cDNA Reverse Transcription Kit（Applied Biosystems社）などを用いることができる。リアルタイム定量PCR法（qRT-PCR）は、例えば、Applied Biosystems社が提供するTaqMan(登録商標) Gene Expression Assaysを利用することができる。増幅産物は、電気泳動等により分離し、定量することもできるが、Applied Biosystems社ABI PRISM 7000 Sequence Detection SystemやRoche社のLC480 systemなどのリアルタイム定量PCR機器を利用して定量することが正確かつ簡便で好ましい。
　リアルタイム定量PCR以外に、様々な測定法（DNAアレイ、ノーザンブロット、ATAC-PCR法など）を用いて、HOTAIR(lincRNA)を定量することができる。 A real-time quantitative PCR method (qRT-PCR), which is one of the RT-PCR methods, is preferable in that a very small amount of DNA can be detected with high sensitivity. In RT-PCR, HOTAIR (lincRNA) is reverse transcribed to produce first strand cDNA, which is used as a template for PCR amplification with primers specific to HOTAIR cDNA. For the reverse transcription reaction, for example, High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) can be used. For real-time quantitative PCR (qRT-PCR), for example, TaqMan (registered trademark) Gene Expression Assays provided by Applied Biosystems can be used. Amplified products can be separated and quantified by electrophoresis, etc., but it is accurate and easy to quantify using real-time quantitative PCR instruments such as Applied Biosystems ABI PRISM 7000 Sequence Detection System and Roche LC480 system. Is preferable.
In addition to real-time quantitative PCR, HOTAIR (lincRNA) can be quantified using various measurement methods (DNA array, Northern blot, ATAC-PCR method, etc.).
　PCR用プライマーとしては、通常、HOTAIR cDNA（配列番号１）や内部標準となる遺伝子のポリヌクレオチドの少なくとも50塩基、好ましくは100～1,000塩基のポリヌクレオチド部分を挟む約１０～３０個程度の塩基配列からなる断片が用いられる。
　プロ―ブとしては、通常、配列番号１の塩基配列から成るポリヌクレオチド(HOTAIR cDNA)の連続した少なくとも１５個の塩基配列からなる断片が用いられる。プロ―ブの塩基配列は通常１５～３０塩基、好ましくは２０～２５塩基である。
　HOTAIR(lincRNA)又はそのcDNAの発現量を測定するために、DNAアレイやノーザンブロットを用いる場合には、上記プローブ、ATAC-PCR法などを用いる場合には、上記プライマー、リアルタイム定量PCR法を用いる場合には、上記プライマーと上記プローブが用いられている。 As a primer for PCR, usually about 10 to 30 nucleotide sequences sandwiching a polynucleotide portion of at least 50 bases, preferably 100 to 1,000 bases, of a polynucleotide of HOTAIR cDNA (SEQ ID NO: 1) or an internal standard gene. A fragment consisting of
As the probe, a fragment consisting of at least 15 consecutive nucleotide sequences of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 (HOTAIR cDNA) is usually used. The probe base sequence is usually 15-30 bases, preferably 20-25 bases.
In order to measure the expression level of HOTAIR (lincRNA) or its cDNA, when using a DNA array or Northern blot, when using the above probe or ATAC-PCR method, the above primer or real-time quantitative PCR method is used. In some cases, the primer and the probe are used.
　HOTAIRを検出する好ましい方法は、手術腫瘍組織からtotal RNAを抽出し、HOTAIRの上記プライマーを用いてqRT-PCR法で発現状況を確認する。その際、内因性コントロールとして、組織ではβ-アクチン、細胞ではGAPDHを用いることが好ましい。
　HOTAIRの発現量の検出方法は、例えば、以下の工程から成る。
　ａ）肺小細胞がん（SCLC）患者の手術検体から、SCLC組織を採取する工程、
　ｂ）得られたSCLC組織から、RNAを抽出する工程、
　ｃ）抽出されたRNAを逆転写し、cDNAを得る工程、
　ｄ）得られたcDNAから少なくともHOTAIRの一部を増幅する工程、及び
　ｅ）増幅された少なくともHOTAIRの一部を検出する工程
　内部標準遺伝子の発現量を利用する場合には、ｄ～ｅ工程に内部標準遺伝子の増幅及び検出する工程を含み、更に、両発現量を比較する工程を加えればよい。 As a preferred method for detecting HOTAIR, total RNA is extracted from a surgical tumor tissue, and the expression status is confirmed by qRT-PCR using the above-mentioned primers of HOTAIR. In this case, it is preferable to use β-actin for tissues and GAPDH for cells as endogenous controls.
The method for detecting the expression level of HOTAIR includes, for example, the following steps.
a) collecting SCLC tissue from a surgical specimen of a patient with small cell lung cancer (SCLC);
b) extracting RNA from the obtained SCLC tissue,
c) reverse transcription of the extracted RNA to obtain cDNA;
d) a step of amplifying at least a part of HOTAIR from the obtained cDNA, and e) a step of detecting at least a part of the amplified HOTAIR. When using the expression level of the internal standard gene, the steps d to e are used. A step of amplification and detection of an internal standard gene may be included, and a step of comparing both expression levels may be added.
　本発明の予後不良肺小細胞がん増殖抑制剤は、lincRNAであるHOTAIRのsiRNAを有効成分として含む。
　このsiRNAは、配列番号１の塩基配列の801～819番目の塩基配列を含む配列番号１の塩基配列中の連続する１９塩基を少なくとも含む、配列番号１の塩基配列の連続する３０塩基以下、好ましくは２７塩基以下、好ましくは２３塩基以下の塩基配列に相当するオリゴリボヌクレオチド、その相補的オリゴリボヌクレオチド、又はこれらから成る２本鎖ＲＮＡである。 The poor-prognosis small cell lung cancer growth inhibitor of the present invention contains siRNA of HOTAIR that is lincRNA as an active ingredient.
This siRNA contains at least 19 consecutive bases in the base sequence of SEQ ID NO: 1 including the base sequences 801 to 819 of the base sequence of SEQ ID NO: 1, preferably 30 bases or less of the base sequence of SEQ ID NO: 1, Is an oligoribonucleotide corresponding to a base sequence of 27 bases or less, preferably 23 bases or less, its complementary oligoribonucleotide, or a double-stranded RNA comprising these.
　本発明で用いるRNA断片としては、標的RNAのセンス又はアンチセンスのものでもよいが、これらはRNaseで容易に分解され、また効果が劣ると考えられるため、これらから成る２本鎖RNAが好ましく用いられる。この２本鎖RNAは通常センスとアンチセンスの２本を別々に合成し、それをハイブリダイズさせて２本鎖にして用いられる。
　これらの特定の塩基配列に「相当する」オリゴリボヌクレオチドとは、この遺伝子が転写されて生成するlincRNAの、特定の塩基配列に相当する部分に相補的なRNAという意味であり、具体的にはこの特定のＤＮＡ配列のＴをＵに置き換えたものという意味である。 The RNA fragment used in the present invention may be a sense RNA or a antisense RNA of the target RNA, but these are easily decomposed by RNase and are considered to be inferior in effectiveness. It is done. This double-stranded RNA is usually used as a double strand by synthesizing both sense and antisense separately and then hybridizing them.
Oligoribonucleotides "corresponding" to these specific base sequences mean the RNA complementary to the portion corresponding to the specific base sequence of the lincRNA produced by transcription of this gene. It means that T in this specific DNA sequence is replaced with U.
　本願発明のsiRNAは、ヌクレアーゼによる分解を防ぐ処理を施した修飾RNAであってもよく、例えば、2'-O-メチル化や4'-チオ化したRNAアプタマーなどであってもよい。また、抑制能を良好にするため、siRNAの3'末端にoverhang配列（例えば、dTdT、UU、UG等）を付加してもよい(EMBO J. 20: 6877-6888.）。
　また予後不良肺小細胞がん増殖抑制剤は、他の公知の腫瘍増殖抑制剤等との合剤であってもよい。本発明の予後不良肺小細胞がん増殖抑制剤は、このような他の薬剤を含むキットの形態であってもよいし、滅菌等張塩水、防腐剤、緩衝剤などの薬学的に許容される媒体を含有してもよい。
　また、siRNAをリポソームなど適当なキャリヤーに封入して投与してもよい。
　また、本発明の予後不良肺小細胞がん増殖抑制剤は、上記予後不良肺小細胞がん増殖抑制剤を製剤化したものを希釈剤などと混合して投与するための注射用キットや、製剤化した個々の製剤を投与するための錠剤用キットなどのキットとして提供してもよい。 The siRNA of the present invention may be a modified RNA that has been treated to prevent degradation by a nuclease, and may be, for example, a 2′-O-methylated or 4′-thiolated RNA aptamer. In order to improve the suppression ability, an overhang sequence (eg, dTdT, UU, UG, etc.) may be added to the 3 ′ end of siRNA (EMBO J. 20: 6877-6888.).
Further, the small cell lung cancer growth inhibitor with poor prognosis may be a combination with other known tumor growth inhibitors and the like. The agent for inhibiting small cell lung cancer growth of the present invention may be in the form of a kit containing such other drugs, or is pharmaceutically acceptable such as sterile isotonic saline, preservatives, buffering agents and the like. Media may be included.
In addition, siRNA may be encapsulated in a suitable carrier such as a liposome.
Moreover, the poor prognosis small cell lung cancer growth inhibitor of the present invention is an injection kit for administering a mixture of the above-mentioned poor prognosis small cell lung cancer growth inhibitor formulated with a diluent, You may provide as kits, such as a kit for tablets for administering each formulated formulation.
　siRNAを細胞に導入する手段については、特に制限はなく、リン酸カルシウム法、マイクロインジェクション法、プロトプラスト融合法、エレクトロポレーション、ウイルスベクターを用いる方法などが挙げられるが、リポソーム等に基づく市販のトランスフェクション試薬を用いるのが簡便である。
　高純度・高品質のsiRNA(siHOTAIR)を直接体内へ投与する場合や全身投与の場合などには、静脈内投与で行ってもよく、また病巣に対する直接投与の場合には、内視鏡で患部に投与するほか、手術時に病巣に接種することで行ってもよい。 The means for introducing siRNA into the cell is not particularly limited, and examples include calcium phosphate method, microinjection method, protoplast fusion method, electroporation, method using viral vector, etc. Commercial transfection reagent based on liposome etc. Is easy to use.
When high-purity and high-quality siRNA (siHOTAIR) is administered directly into the body or systemically, it may be administered intravenously, and in the case of direct administration to the lesion, the affected area can be examined with an endoscope. In addition to administration, it may be done by inoculating the lesion at the time of surgery.
　予後不良肺小細胞がん増殖抑制剤は以下の方法によりスクリーニングすることができる。具体的には、本発明の方法は、ヒト培養肺小細胞がん細胞を準備し、この細胞株を候補薬剤の存在下で培養すること等により候補薬剤と肺小細胞がん細胞とを接触させ、lincRNAであるHOTAIRの発現の阻害又は抑制を調べて候補薬剤の一次スクリーニングを行い、その後、候補薬剤によるヒト培養肺小細胞がん細胞の増殖能及び/又は浸潤能の抑制を調べて二次スクリーニングを行う。スクリーニングに用いるSCLC細胞としては、HOTAIR RNAの発現量の高い細胞株が好ましく、このようなSCLC細胞として、例えば、SBC-3細胞株が挙げられる。SBC-3細胞は、HOTAIR RNAの発現量が高いため、予後不良肺小細胞がんのセルラインに相当すると考えられる。
　この方法において、lincRNAであるHOTAIRの発現の阻害又は抑制の程度は、候補薬剤を添加しない対照との比較実験によって判定できる。発現レベルは、肺小細胞がん細胞株から得たtotal RNA又はmRNA又はポリA(+)RNAについて、又は逆転写酵素-PCR (RT-PCR)法によってRNAから合成されたcDNAについて、蛍光又は放射性標識したプローブを用いるハイブリダイゼーション法（例えばノーザンハイブリダイゼーション、サザンハイブリダイゼーション、DNAマイクロアレイ、組織マイクロアレイなど）によって決定することができる。
　RT-PCR、PCR用プライマー、プロ―ブとしては上記のものを使用できる。
　このスクリーニングにおいて、HOTAIRの発現が、候補薬剤無添加の対照と比べて、有意に阻害又は抑制され、さらに、肺小細胞がん細胞の増殖能、浸潤能が抑制された場合に、この候補薬剤を肺小細胞がん転移抑制剤とすることができる。 The small prognosis lung cancer cell growth inhibitor can be screened by the following method. Specifically, in the method of the present invention, human cultured small cell lung cancer cells are prepared, and the candidate drug and small cell lung cancer cells are contacted by culturing this cell line in the presence of the candidate drug. Inhibition or suppression of the expression of the lincRNA HOTAIR is performed to perform a primary screening of the candidate drug, and then the suppression of the proliferation and / or invasive ability of human cultured small cell lung cancer cells by the candidate drug is investigated. Perform next screening. As the SCLC cell used for screening, a cell line with a high HOTAIR RNA expression level is preferable. Examples of such an SCLC cell include the SBC-3 cell line. SBC-3 cells are considered to correspond to a cell line of small lung cancer with poor prognosis because of the high expression level of HOTAIR RNA.
In this method, the degree of inhibition or suppression of the expression of HOTAIR, which is a lincRNA, can be determined by a comparative experiment with a control to which no candidate drug is added. Expression levels are measured for total RNA or mRNA or poly A (+) RNA obtained from small cell lung cancer cell lines, or for cDNA synthesized from RNA by the reverse transcriptase-PCR (RT-PCR) method. It can be determined by a hybridization method using a radiolabeled probe (for example, Northern hybridization, Southern hybridization, DNA microarray, tissue microarray, etc.).
The RT-PCR, PCR primers, and probes described above can be used.
In this screening, when the expression of HOTAIR is significantly inhibited or suppressed compared to the control without addition of the candidate drug, and further, the proliferation ability and invasive ability of small cell lung cancer cells are suppressed, this candidate drug Can be used as a small cell lung cancer metastasis inhibitor.
　以下、実施例にて本発明を例証するが本発明を限定することを意図するものではない。
実施例１
　この実施例で用いた臨床検体を以下のように調整した。
　1995年と2010年の間にがん研究会のがん研究所病院で手術を受けた複数のSCLC患者から、手術前に書面によるインフォームドコンセントを得た上で、35のSCLC組織サンプルを得た。また同じ患者から、16の非がん組織サンプルを得た。すべての検体を直ちに液体窒素中で凍結し、-80℃で保存し、RNA抽出を行った。
　SCLC組織サンプルの採取は以下のようにして行った。迅速診断のために提出された肺がん組織を含む肺組織を用いて、がん部及び非がん部（がん部より充分に離れた領域）を切除し（図１）、氷上でセラムチューブに入れ、ただちに液体窒素に投入、凍結した。その後の病理診断においてSCLCと診断した症例の迅速診断時保存組織切片を用いて以下の検討を行った。なお、RNA抽出前に、すべての迅速診断時保存組織切片を、クライオスタットで再度スライスし、ヘマトキシリン‐エオジン染色を行っておき、後でSCLC成分が含まれることを確認した。 The following examples illustrate the invention but are not intended to limit the invention.
Example 1
The clinical specimens used in this example were prepared as follows.
35 SCLC tissue samples were obtained from multiple SCLC patients undergoing surgery at the Cancer Institute Hospital between 1995 and 2010 with written informed consent prior to surgery. It was. In addition, 16 non-cancerous tissue samples were obtained from the same patient. All specimens were immediately frozen in liquid nitrogen and stored at -80 ° C for RNA extraction.
The SCLC tissue sample was collected as follows. Using lung tissue including lung cancer tissue submitted for rapid diagnosis, excise the cancerous part and non-cancerous part (area far enough from the cancerous part) (Fig. 1) and place it on a serum tube on ice. It was immediately put into liquid nitrogen and frozen. In the subsequent pathological diagnosis, the following examination was performed using the preserved tissue section at the time of rapid diagnosis of the case diagnosed as SCLC. In addition, before RNA extraction, all the preserved tissue sections at the time of rapid diagnosis were sliced again with a cryostat, hematoxylin-eosin staining was performed, and it was confirmed later that the SCLC component was contained.
　患者の背景や臨床病理学的因子を表１に示す。
　SCLCの再発は、術後経過観察中（術後最長で約15年）の再発精査にて肺および他臓器に新たな病変を認めたものをいい、レトロスペクティブに診療録や画像を検索した。表中、再発した転移先を記載したが、複数に転移したものを含む。
　生存又は死亡については、診療録やがん研有明病院病歴室の後追い調査結果（術後最長で約15年）を用いて追跡調査したものを示す。 Table 1 shows the patient background and clinicopathological factors.
SCLC recurrence refers to a case in which new lesions were found in the lung and other organs during recurrence during the postoperative follow-up (up to about 15 years after surgery), and medical records and images were searched retrospectively. In the table, the recurrent metastasis destination is described, but includes metastasis metastasized to a plurality.
For survival or death, follow-up results using medical records and follow-up survey results (about 15 years at the longest postoperatively) of Akenake Hospital's medical history room are shown.
　また、手術中に迅速診断のために病理部へ検体が提出された時点で、採取、保存しておいた上記の35のSCLCサンプルから、定量的リアルタイムPCR(qRT-PCR)を用いてHOTAIR発現レベルを調べた。qRT-PCRは以下のようにして行った。
　RNeasyミニキット又はRNeasyミニキットプラス（Qiagen社）を用いて、組織及び細胞からtotal RNAを抽出した。High Capacity RNA-to-cDNA(R) kit (AB社製、カタログ番号4387406)を用いて、30ngの全RNAからcDNAを生成した。得られたcDNAを45サイクルのPCR増幅を行った後、LightCycler(登録商標)480 SYBR Green I Master protocol (Roche社, カタログ番号4707516)と、下記プライマーを用いてリアルタイムPCR反応を行い、各遺伝子の発現量を調べた。LightCycler(登録商標)480 real time PCR System (Roche社)上の96ウェルプレートの各サンプルの各遺伝子についてそれぞれ３回試験を行った。組織試料及び異種移植片についてHOTAIR RNAの発現量をβ-アクチン(ACTB)の発現量に対して標準化した。
プライマー：
HOTAIR（F）：5'-GGTAGAAAAAGCAACCACGAAGC-3' （配列番号４）
HOTAIR（R）：5'-ACATAAACCTCTGTCTGTGAGTGCC-3' （配列番号５）
ACTB（F）：5'-AGAAAATCTGGCACCACACC-3' （配列番号６）
ACTB（R）：5'-AGAGGCGTACAGGGATAGCA-3' （配列番号７） In addition, HOTAIR expression was performed using quantitative real-time PCR (qRT-PCR) from the 35 SCLC samples collected and stored at the time when specimens were submitted to the pathology department for rapid diagnosis during surgery. I checked the level. qRT-PCR was performed as follows.
Total RNA was extracted from tissues and cells using RNeasy mini kit or RNeasy mini kit plus (Qiagen). CDNA was generated from 30 ng of total RNA using High Capacity RNA-to-cDNA (R) kit (AB, catalog number 4387406). The obtained cDNA was subjected to PCR amplification for 45 cycles, followed by real-time PCR reaction using LightCycler (registered trademark) 480 SYBR Green I Master protocol (Roche, catalog number 4707516) and the following primers. The expression level was examined. Each gene in each sample of a 96-well plate on the LightCycler (registered trademark) 480 real time PCR System (Roche) was tested three times. The expression level of HOTAIR RNA was normalized with respect to the expression level of β-actin (ACTB) for tissue samples and xenografts.
Primer:
HOTAIR (F): 5'-GGTAGAAAAAGCAACCACGAAGC-3 '(SEQ ID NO: 4)
HOTAIR (R): 5'-ACATAAACCTCTGTCTGTGAGTGCC-3 '(SEQ ID NO: 5)
ACTB (F): 5'-AGAAAATCTGGCACCACACC-3 '(SEQ ID NO: 6)
ACTB (R): 5'-AGAGGCGTACAGGGATAGCA-3 '(SEQ ID NO: 7)
　上記のように、定量RT-PCRを用いて、各新鮮凍結サンプルからRNAを評価した。同じ被験者群の35のSCLC組織と15の非がん組織におけるHOTAIR/ACTB比を調べた（図２）。HOTAIR/ACTB比が1.368より上のレベルを高HOTAIR発現とした。この基準によれば、SCLC患者35人のうち12人はHOTAIRの発現が高いと分類され、23人は低いと分類された。
　これら２つのグループの臨床病理学的因子を表１に示す。高HOTAIR発現グループは、低HOTAIR発現グループよりも、より多くの再発と死亡を示した。 RNA was evaluated from each fresh frozen sample using quantitative RT-PCR as described above. The HOTAIR / ACTB ratio was examined in 35 SCLC tissues and 15 non-cancerous tissues in the same subject group (FIG. 2). A level where the HOTAIR / ACTB ratio was higher than 1.368 was regarded as high HOTAIR expression. According to this criteria, 12 out of 35 SCLC patients were classified as having high HOTAIR expression and 23 were classified as low.
The clinicopathological factors of these two groups are shown in Table 1. The high HOTAIR expression group showed more recurrence and death than the low HOTAIR expression group.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
実施例２
　上記35のSCLCサンプルのうち、病理診断で他の組織型を含まない純粋なSCLC(pure-SCLC)と診断された症例に限定し、且つ、腫瘍の縮小効果を狙って行われる化学療法（Neo-adjuvant chemotherapy (NAC):ネオアジュバント化学療法）の影響を排除するために、手術前にこの化学療法を行っておらず、手術で腫瘍を取り除いた後に補助療法としての術後化学療法（Adjuvant chemotherapy(AC):アジュバント化学療法）を行った患者（NAC(-)AC(+)-SCLC）の18のサンプルについて、高HOTAIR発現グループ(n=8)と低HOTAIR発現グループ(n=10)に分けて、SCLCに起因する患者の死亡及びSCLCの再発について調べた。結果を図３Ａ及びＢに示す。
　高HOTAIRグループは、低HOTAIRグループよりも、SCLCの再発が顕著に高く（図３Ａ）、SCLCに起因する患者の死亡も顕著に高い（図３Ｂ）。 Example 2
Of the 35 SCLC samples mentioned above, chemotherapy is limited to cases diagnosed as pure SCLC (pure-SCLC) that does not include other tissue types in pathological diagnosis, and is aimed at reducing the tumor size (Neo -Adjuvant chemotherapy (NAC): In order to eliminate the influence of neoadjuvant chemotherapy, this chemotherapy is not performed before surgery, and postoperative chemotherapy (Adjuvant chemotherapy as adjuvant therapy after removing the tumor by surgery) (AC): 18 samples of patients who received adjuvant chemotherapy (NAC (-) AC (+)-SCLC) were divided into high HOTAIR expression group (n = 8) and low HOTAIR expression group (n = 10). Separately, patient death due to SCLC and recurrence of SCLC were examined. The results are shown in FIGS. 3A and B.
The high HOTAIR group has significantly higher SCLC recurrence than the low HOTAIR group (FIG. 3A) and the patient death due to SCLC is also significantly higher (FIG. 3B).
実施例３
　この実施例で用いた細胞株を以下のように調整した。
　SCLC細胞株COLO-668、COR-L51、COR-L88、DMS-79はECACC（The European Collection of Cell Cultures）から得た。SCLC細胞株DMS-53、LU-134A、MS-1、SBC-3、SBC-5、SBC-1、A549とMRC-5は、JCRB（Japanese Collection of Research Bio-resources）又は独立行政法人理化学研究所バイオリソースセンター（BRC）から得た。これらの細胞株を、37℃、5％CO2インキュベーター中で、ダルベッコ改変イーグル培地（DMEM、ナカライテスク社 カタログ番号08488-55、500ml）で培養した。このダルベッコ改変イーグル培地は、200mMのL-アラニル-L-グルタミン溶液（ナカライテスク社 カタログ番号04260-64）5.0 ml、抗生物質抗真菌混合ストック溶液（ナカライテスク社 カタログ番号02892-54）5.5 ml、及びウシ胎児血清（Biowest社 カタログ番号S1560）50mlを含む。 Example 3
The cell line used in this example was prepared as follows.
SCLC cell lines COLO-668, COR-L51, COR-L88, and DMS-79 were obtained from ECACC (The European Collection of Cell Cultures). SCLC cell lines DMS-53, LU-134A, MS-1, SBC-3, SBC-5, SBC-1, A549 and MRC-5 are either JCRB (Japanese Collection of Research Bio-resources) or RIKEN Obtained from the BioResource Center (BRC). These cell lines were cultured in Dulbecco's modified Eagle medium (DMEM, Nacalai Tesque, catalog number 08488-55, 500 ml) in a 37 ° C, 5% CO 2 incubator. This Dulbecco's modified Eagle medium consists of 5.0 ml of 200 mM L-alanyl-L-glutamine solution (Nacalai Tesque catalog number 04260-64), 5.5 ml of antibiotic antifungal mixed stock solution (Nacalai Tesque catalog number 02892-54), And 50 ml of fetal bovine serum (Biowest catalog number S1560).
　実施例１と同様に、これら細胞株におけるHOTAIR発現レベルを調べた。HOTAIRの発現量をGAPDHの発現量に対して標準化した。GAPDH用プライマーとして下記のプライマーを用いた。結果を図４Ａに示す。
GAPDH（F）：5'-CCGGGAAACTGTGGCGTGATGG-3' （配列番号８）
GAPDH（R）：5'-AGGTGGAGGAGTGGGTGTCGCTGTT-3' （配列番号９） Similar to Example 1, the HOTAIR expression level in these cell lines was examined. The expression level of HOTAIR was normalized to the expression level of GAPDH. The following primers were used as GAPDH primers. The results are shown in FIG. 4A.
GAPDH (F): 5'-CCGGGAAACTGTGGCGTGATGG-3 '(SEQ ID NO: 8)
GAPDH (R): 5'-AGGTGGAGGAGTGGGTGTCGCTGTT-3 '(SEQ ID NO: 9)
　HOTAIRの発現量が高かったSBC-3細胞株について、HOTAIRのRNA干渉実験を以下のように行った。LipofectamineTM RNAiMAX（Invitrogen社製、カタログ番号13778-075）を使用して、製造元の指示に従って、細胞に、HOTAIRを標的とする20nMのsiRNAを導入した。この導入の効率は、標識ポジティブコントロール（BLOCK-ITTMのAlexa Fluor(R)赤色蛍光オリゴ（Invitrogen社製、カタログ番号14750-100））を導入後、細胞を5％CO2を含む加湿環境下で37℃で72時間インキュベーションした後、蛍光顕微鏡（ライカDMIRE2）を用いて評価した。その結果、siRNAを20nM（最終濃度）及びLipofectamine RNAiMAXを1.5μlを用いたが、これはSBC-3を24ウェルベースで分析するために最適な条件と考えられる（形質導入効率：100％、ノックダウン効率：50％）。この条件でSBC-3に＃1-3 siHOTAIR（下記）を導入し、72時間後に、このsiRNAを導入された細胞からto-tal RNAを抽出して、定量RT-PCR解析を行った。比較のため、ネガティブコントロールであるGFP遺伝子について同様にRNA干渉を行った。定量RT-PCRは実施例１と同様にして行った。
　#1 siHOTAIR：
センス：5'-GAACGGGAGUACAGAGAGAUU-3'、（配列番号１０、配列番号１の801～819番目に相当する。）
アンチセンス：3'-UUCUUGCCCUCAUGUCUCUCU-5'
　#2 siHOTAIR：
センス：5'-CCACAUGAACGCCCAGAGAUU-3'（配列番号１１、配列番号１の840～858番目に相当する。）
アンチセンス：3'-UUGGUGUACUUGCGGGUCUCU-5'
　#3 siHOTAIR：
センス：5'-UAACAAGACCAGAGAGCUGUU-3'（配列番号１２、配列番号１の1005～1023番目に相当する。）
アンチセンス：3'-UUAUUGUUCUGGUCUCUCGAC-5'
　siGFP：
センス：5'-CUACAACAGCCACAACGUCdTdT-3' （配列番号１３）
アンチセンス：3'-TTGAUGUUGUCGGUGUUGCAG-5 ' For the SBC-3 cell line with high HOTAIR expression level, RNA interference experiments with HOTAIR were performed as follows. Using Lipofectamine ™ RNAiMAX (Invitrogen, catalog number 13778-075), 20 nM siRNA targeting HOTAIR was introduced into the cells according to the manufacturer's instructions. The efficiency of this introduction is determined by introducing a labeled positive control (Blex-IT ™ Alexa Fluor® red fluorescent oligo (Invitrogen, Catalog No. 14750-100)), and then placing the cells in a humidified environment containing 5% CO 2. And incubated at 37 ° C. for 72 hours, and then evaluated using a fluorescence microscope (Leica DMIRE2). As a result, 20 nM (final concentration) of siRNA and 1.5 μl of Lipofectamine RNAiMAX were used. This is considered to be the optimal condition for analyzing SBC-3 on a 24-well basis (transduction efficiency: 100%, knocking) Down efficiency: 50%). # 1-3 siHOTAIR (described below) was introduced into SBC-3 under these conditions, and 72 hours later, to-tal RNA was extracted from the cells into which this siRNA had been introduced, and quantitative RT-PCR analysis was performed. For comparison, RNA interference was similarly performed for the GFP gene as a negative control. Quantitative RT-PCR was performed in the same manner as in Example 1.
# 1 siHOTAIR:
Sense: 5′-GAACGGGAGUACAGAGAGAUU-3 ′ (corresponding to positions 801 to 819 of SEQ ID NO: 10 and SEQ ID NO: 1)
Antisense: 3'-UUCUUGCCCUCAUGUCUCUCU-5 '
# 2 siHOTAIR:
Sense: 5′-CCACAUGAACGCCCAGAGAUU-3 ′ (SEQ ID NO: 11, corresponding to positions 840 to 858 of SEQ ID NO: 1)
Antisense: 3'-UUGGUGUACUUGCGGGUCUCU-5 '
# 3 siHOTAIR:
Sense: 5'-UAACAAGACCAGAGAGCUGUU-3 '(SEQ ID NO: 12, corresponding to positions 1005 to 1023 of SEQ ID NO: 1)
Antisense: 3'-UUAUUGUUCUGGUCUCUCGAC-5 '
siGFP:
Sense: 5'-CUACAACAGCCACAACGUCdTdT-3 '(SEQ ID NO: 13)
Antisense: 3'-TTGAUGUUGUCGGUGUUGCAG-5 '
　HOTAIR RNAの発現量を図４Ｂに示す。＃1-3 siHOTAIR導入により、HOTAIR RNAの発現量が減少したことが分かる。
　更に、＃1-3 siHOTAIR（下記）を導入後のSBC-3細胞株を37℃、5％CO2インキュベーター中で、ダルベッコ改変イーグル培地（DMEM、ナカライテスクカタログ番号08488-55、500ml）で培養した。このダルベッコ改変イーグル培地は、200mMのL-アラニル-L-グルタミン溶液（ナカライテスク カタログ番号04260-64）5.0 ml及びウシ胎児血清（Biowest社 カタログ番号S1560）50mlを含む。培養開始後、0,24,48,72,96時間後のそれぞれの時点における細胞数を自動セルカウンター（BioRad社、TC-10）でカウントした。結果を図４Ｃに示す。#1 siHOTAIRの導入により、SBC-3細胞株の増殖が抑制されたことが分かる。 The expression level of HOTAIR RNA is shown in FIG. 4B. # 1-3 It can be seen that the introduction of siHOTAIR decreased the expression level of HOTAIR RNA.
Furthermore, SBC-3 cell line after introduction of # 1-3 siHOTAIR (below) was cultured in Dulbecco's modified Eagle medium (DMEM, Nacalai Tesque catalog number 08488-55, 500 ml) in a 37 ° C, 5% CO2 incubator. . This Dulbecco's modified Eagle medium contains 5.0 ml of 200 mM L-alanyl-L-glutamine solution (Nacalai Tesque catalog number 04260-64) and 50 ml of fetal calf serum (Biowest catalog number S1560). The number of cells at each time point after 0, 24, 48, 72, and 96 hours after the start of culture was counted with an automatic cell counter (BioRad, TC-10). The results are shown in FIG. 4C. It can be seen that the introduction of # 1 siHOTAIR suppressed the growth of the SBC-3 cell line.

Claims (11)

  1. 肺小細胞がん（SCLC）が予後不良の肺小細胞がんであるか否かを判断するための診断方法であって、検体である肺小細胞がん（SCLC）における、lincRNAであるHOTAIR（そのcDNAは配列番号１で表される塩基配列を有する。）の発現量を測定することから成り、HOTAIRの発現量が高いほど予後不良であるとする検査方法。 This is a diagnostic method for determining whether small cell lung cancer (SCLC) is a small cell lung cancer with a poor prognosis. In the small cell lung cancer (SCLC) specimen, the lincRNA HOTAIR ( The cDNA has a base sequence represented by SEQ ID NO: 1.), and the higher the HOTAIR expression level, the poorer the prognosis.
  2. 更に、肺小細胞がん（SCLC）における、内部標準となる遺伝子の発現量を測定することを含み、内部標準となる遺伝子の発現量に対する、lincRNAであるHOTAIRの発現量が、高いほど予後不良であるとする請求項１に記載の検査方法。 Furthermore, it includes measuring the expression level of the internal standard gene in small cell lung cancer (SCLC). The higher the expression level of HOTAIR, the lincRNA, compared to the internal standard gene expression level, the poorer the prognosis. The inspection method according to claim 1, wherein
  3. 前記内部標準となる遺伝子が、β-アクチン又はGAPDHである請求項２に記載の検査方法。 The test method according to claim 2, wherein the gene serving as the internal standard is β-actin or GAPDH.
  4. 肺小細胞がん（SCLC）が予後不良の肺小細胞がんであるか否かを判断するために用いる肺小細胞がん検査診断用バイオマーカーであって、lincRNAであるHOTAIR（そのcDNAは配列番号１で表される塩基配列を有する。）から成り、lincRNAであるHOTAIRの発現量が、高いほど予後不良であるとする、予後不良肺小細胞がん検査診断用バイオマーカー。 HOTAIR, a lincRNA, is a biomarker for diagnostic diagnosis of small cell lung cancer used to determine whether small cell lung cancer (SCLC) is a small cell lung cancer with a poor prognosis. A biomarker for diagnostic diagnosis of small cell lung cancer with a poor prognosis, wherein the higher the expression level of HOTAIR, which is a lincRNA, the poorer the prognosis is.
  5. 更に、内部標準となる遺伝子から発現するmRNAを含み、内部標準となる遺伝子から発現するmRNAの発現量に対する、lincRNAであるHOTAIR領域の発現量が、高いほど予後不良であるとする、請求項４に記載の予後不良肺小細胞がん検査診断用バイオマーカー。 Furthermore, it contains mRNA expressed from a gene serving as an internal standard, and the higher the expression level of the HOTAIR region that is a lincRNA relative to the expression level of mRNA expressed from a gene serving as an internal standard, the poorer the prognosis. Biomarker for diagnosis and diagnosis of small cell lung cancer described in 1.
  6. 請求項４に記載の予後不良肺小細胞がん検査診断用バイオマーカーを定量するために使用するためのキットであって、lincRNAであるHOTAIRのｃＤＮＡ（配列番号１）を定量可能なように増幅するプライマー及びポリメラーゼ、及び/又は該ｃＤＮＡに対合させるプローブから成るキット。 A kit for use in quantifying the biomarker for diagnosis and diagnosis of poor prognosis of small cell lung cancer according to claim 4, wherein the cDNA for HOTAIR, which is a lincRNA (SEQ ID NO: 1), is amplified so that it can be quantified. A kit comprising a primer and a polymerase, and / or a probe to be paired with the cDNA.
  7. lincRNAであるHOTAIRのcDNAの塩基配列（配列番号１）の801～819番目の塩基配列を含む配列番号１の塩基配列の連続する３０塩基以下の塩基配列に相当するオリゴリボヌクレオチド及びその相補的オリゴリボヌクレオチドから成る２本鎖ＲＮＡを有効成分として含む予後不良肺小細胞がん増殖抑制剤。 Oligoribonucleotide corresponding to a base sequence of 30 bases or less of the base sequence of SEQ ID NO: 1 comprising the base sequence of 801 to 819 of the base sequence of cDNA of HOTAIR (SEQ ID NO: 1) as lincRNA and its complementary oligo An agent for inhibiting the growth of small cell lung cancer having a poor prognosis, comprising a double-stranded RNA comprising ribonucleotides as an active ingredient.
  8. lincRNAであるHOTAIRのcDNAの塩基配列（配列番号１）の801～819番目の塩基配列を含む、配列番号１の塩基配列の連続する３０塩基以下の塩基配列に相当するオリゴリボヌクレオチド及びその相補的オリゴリボヌクレオチドから成る２本鎖ＲＮＡを含む予後不良肺小細胞がん増殖抑制用キット。 Oligoribonucleotide corresponding to a base sequence of 30 bases or less of the base sequence of SEQ ID NO: 1, including the base sequence of 801 to 819 of the base sequence (SEQ ID NO: 1) of the cDNA of HOTAIR which is lincRNA and its complementary A kit for suppressing the growth of small cell lung cancer with a poor prognosis, comprising a double-stranded RNA comprising oligoribonucleotides.
  9. ヒト培養肺小細胞がん細胞を用いて、lincRNAであるHOTAIR（そのcDNAは配列番号１で表される塩基配列を有する。）の発現の阻害又は抑制について、候補薬剤をスクリーニングすることから成る、予後不良肺小細胞がん増殖抑制剤のスクリーニング方法。 Using human cultured small cell lung cancer cells, screening candidate drugs for inhibition or suppression of the expression of LOTRNA HOTAIR (the cDNA has the nucleotide sequence represented by SEQ ID NO: 1), A screening method for an agent for inhibiting the growth of small cell lung cancer with a poor prognosis.
  10. ヒト培養肺小細胞がん細胞株を候補薬剤の存在下で培養し、lincRNAであるHOTAIR（そのcDNAは配列番号１で表される塩基配列を有する。）の発現の阻害又は抑制を調べることから成る請求項９に記載の方法。 A human cultured small cell lung cancer cell line is cultured in the presence of a candidate drug, and the inhibition or suppression of the expression of LOTRNA HOTAIR (the cDNA has the base sequence represented by SEQ ID NO: 1) is examined. 10. A method according to claim 9 comprising.
  11. 前記ヒト培養肺小細胞がん細胞株としてSBC-3細胞株を用いる請求項９又は１０に記載の方法。 The method according to claim 9 or 10, wherein an SBC-3 cell line is used as the human cultured small cell lung cancer cell line.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023074135A1 (en) * 2021-11-01 2023-05-04 国立研究開発法人国立がん研究センター Method and reagent kit for assisting determination of lung cancer prognosis

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6597316B2 (en) * 2016-01-06 2019-10-30 コニカミノルタ株式会社 Image processing apparatus and program
US20200362420A1 (en) * 2017-11-12 2020-11-19 The Regents Of The University Of California Non-coding rna for detection of cancer

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 24 February 2012 (2012-02-24), "Definition: Homo sapiens HOX transcript antisense RNA (non-protein coding) (HOTAIR), antisense RNA", retrieved from http://www.ncbi.nlm.nih.gov/nuccore/ NR_003716.2 accession no. R_ 003716.2 *
HIROSHI ONO ET AL.: "Haishosaibogan ni Okeru HOTAIR no Hatsugen wa Jutsugo Saihatsu to Kanren shiteiru", NIPPON BYORI GAKKAISHI, vol. 101, no. 1, 26 March 2012 (2012-03-26), pages 258 *
NOBUYUKI HARA ET AL.: "Tokushu/Lung cancer Tissue types and clinical characteristics", RINSHO TO KENKYU, vol. 74, no. 6, 1997, pages 1333 - 1336 *
TAKARA BIO INC.: "Tokushu 1 RNAi no Kiso to sono Oyo", BIO VIEW, 2004, pages 2 - 13 *
TOSHIHIKO IIZUKA ET AL.: "Haisengan ni Okeru Chosa noncoding RNA no Hatsugen to Idenshi Hen'i, Yogo tono Sokan", NIPPON BYORI GAKKAISHI, vol. 101, no. 1, 26 March 2012 (2012-03-26), pages 258 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023074135A1 (en) * 2021-11-01 2023-05-04 国立研究開発法人国立がん研究センター Method and reagent kit for assisting determination of lung cancer prognosis

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