WO2014126233A1 - マイクロrnaの測定方法、並びに、がん治療剤及びこれを含有するがん治療のための医薬組成物 - Google Patents
マイクロrnaの測定方法、並びに、がん治療剤及びこれを含有するがん治療のための医薬組成物 Download PDFInfo
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Definitions
- the present invention is primarily an invention relating to measurement of a biological specimen, and more specifically, an invention for distinguishing cancer malignancy by measuring microRNA in tumor tissue.
- the invention relates to an anticancer agent, and more specifically, an invention related to an anticancer agent and an anticancer pharmaceutical composition according to a specific microRNA.
- the Human Genome Project which ended in 2003, has accelerated the movement to understand cancer at the genetic level and protein molecular level, and to use it for cancer diagnosis and treatment. As a result, the early diagnosis and treatment methods for cancer have made great strides.
- NF-E2-related factor 2 NRF2
- NRF2 is a transcriptional regulator for cytoprotection against cell damage caused by chemotherapy or oxidative stress (Non-patent Documents 1 and 2). Under physiological conditions, NRF2 is ubiquitinated by the KEAP1 (Kalin 3 (CUL3) -Kelch-like ECH-related protein) ubiquitin E3 ligase complex and is constantly degraded in the proteasome, resulting in low intracellular concentrations of NRF2 protein. It is kept. Under cellular stress, KEAP1 is inactivated, NRF2 is stabilized in the nucleus, and the target gene [NRF2 is directly bound to the antioxidant response element (ARE) in the promoter in each target gene.
- ARE antioxidant response element
- Non-patent Documents 3 and 4 Leads to cell survival (Non-patent Documents 3 and 4).
- NRF2 has also been reported to be able to contribute to the growth of cancer cells by regulating metabolism (Non-patent Document 5).
- Gene mutations lead to gain of function for NRF2 or loss of function for KEAP1 in various types of human cancers, resulting in activation of NRF2, leading to cancer cell survival and proliferation mediated by NRR2 (6). ⁇ 9).
- NRF2 is stabilized by competitively interacting with KEAP1 in hepatocyte epithelial malignant tumor (HCC) by an aggregate in which p62 protein (substance for proteolysis by autophagy) is excessively accumulated ( Non-patent documents 10 to 13).
- HCC hepatocyte epithelial malignant tumor
- NRF2 has carcinogenic activity in cancer cells, and high levels of NRF2 protein are considered to be associated with a poor prognosis (Non-Patent Documents 14 to 16). From these facts, it was considered that therapeutic inhibition of the NRF2-mediated carcinogenic pathway is particularly effective for cancers in which NRF2 is stabilized and resistant to various treatments. In addition, it was thought that by finding an index that reflects the degree of stabilization of NRF2, it is possible to distinguish the malignancy of cancer that is directly linked to the prognosis of the patient.
- miRNAs are endogenous small non-coding RNAs that regulate gene expression by interfering with translation or stabilization of target transcripts through binding to the 3′-untranslated region (UTR) (17). 18). Some miRNAs can negatively regulate oncogenes, and inactivation of tumor suppressive miRNAs leads to activation of oncogenic pathways (Non-Patent Documents 19 to 22). Importantly, a single transcript can be targeted by multiple miRNAs, whereas a single miRNA can target multiple transcripts (Non-Patent Documents 23, 24).
- the present inventors made it a specific subject of the present invention to find a miRNA strongly related to the stabilization of NRF2 in a tumor and to provide a means for utilizing them for cancer diagnosis and treatment. .
- the present inventors screened 470 types of microRNAs in the miRNA library using the ARE reporter system.
- the ARE activity value was calculated by measuring the luciferase activity in HeLa cells transfected with 470 types of miRNAs, and the 8 types of miRNAs in which the ARE activity value was greatly reduced compared to the control miRNA ( hsa-miR-507, hsa-miR-634, hsa-miR-450a, hsa-miR-129-5p, hsa-miR-639, hsa-miR-337, hsa-miR-153, and hsa-miR- 556) and 8 types of miRNAs (hsa-miR-26a, hsa-miR-17-3p, hsa-miR-190, hsa-miR-567, hsa-miR-125b, hs
- microRNAs it is possible to detect the activation of NRF2 in the living body, in particular, tumor cells, whereby the malignancy of the tumor or the prognosis of the cancer patient, It was found that can be distinguished. Furthermore, it discovered that the nucleic acid containing the microRNA sequence concerning the reduction
- the present invention provides the following inventions.
- the present invention primarily relates to hsa-miR-507, hsa-miR-634, hsa-miR-450a, hsa-miR-129-5p, hsa-miR-639, hsa-miR-337, hsa in human specimens.
- microRNAs that are the subject of the low value measurement method of the present invention, in particular, hsa-miR-507, hsa-miR-634, hsa-miR-450a, and hsa-miR-129-5p
- the four microRNAs are characterized by low quantitative values in malignant tumors.
- the present invention secondly relates to hsa-miR-26a, hsa-miR-17-3p, hsa-miR-190, hsa-miR-567, hsa-miR-125b, hsa-miR-125a, hsa in human specimens.
- Both the low value measuring method and the high value measuring method of the present invention further detect 1 to 3 types selected from the group consisting of NRF2 gene mutation, KEAP1 gene mutation, and p62 protein accumulation in tumors. It is possible to improve discrimination accuracy by adding the above element as an index.
- the total number of individual indicators indicating that the malignancy of the tumor is increased, NRF2 activation, or the prognosis of the cancer patient is deteriorated is used as a score indicator. It is preferable to differentiate between malignant grade, NRF2 activation, or patient prognosis.
- the tumor (cancer) to be differentiated such as the malignancy of cancer by the low value measurement method and the high value measurement method of the present invention is a tumor (cancer) in which the activation of NRF2 is a problem in malignancy and the like. It is not particularly limited, and can be applied to all malignant tumors such as esophageal cancer, lung cancer, breast cancer, oral cancer, stomach cancer, colon cancer, liver cancer, uterine cancer, osteosarcoma, skin cancer, etc. .
- the present invention relates to hsa-miR-507, hsa-miR-634, hsa-miR-450a, hsa-miR-129-5p, hsa-miR-639, hsa-miR-337, hsa-miR-153.
- a cancer therapeutic agent hereinafter also referred to as the cancer therapeutic agent of the present invention
- a cancer therapeutic agent of the present invention comprising a nucleic acid comprising one or more selected from the group consisting of hsa-miR-556. It is.
- microRNAs that are the subject of the cancer therapeutic agent of the present invention, in particular, hsa-miR-507, hsa-miR-634, hsa-miR-450a, and hsa-miR-129-5p
- the four types of microRNAs are excellent in general anticancer effects.
- cancer therapeutic agent of the present invention is suitable for use in combination with means for imparting stress to cancer cells.
- the present invention provides a pharmaceutical composition for treating cancer (hereinafter also referred to as the pharmaceutical composition of the present invention), which comprises the cancer therapeutic agent of the present invention. .
- the tumor (cancer) to which the cancer therapeutic agent and the pharmaceutical composition of the present invention are administered is particularly limited as long as the activation of NRF2 is a problem in malignancy or the like (cancer). Instead, it can be applied to all malignant tumors such as esophageal cancer, lung cancer, breast cancer, oral cancer, stomach cancer, colon cancer, liver cancer, uterine cancer, osteosarcoma, skin cancer and the like. In particular, it is preferable that the cancer can be administered locally.
- Table 1-1 shows eight types of miRNAs (hereinafter also referred to as low-value miRNAs) to be measured in the low value measurement method of the present invention. SEQ ID NOs: 1 to 8 are listed in order from the top of Table 1-1. Allocated.
- Table 1-2 shows 8 types of miRNAs (hereinafter also referred to as high level miRNAs) to be measured in the high value measurement method of the present invention, and SEQ ID NOs: 9 to 16 are assigned in order from the top of Table 1-2. .
- a measurement method capable of differentiating NRF2 activation and differentiating tumor malignancy and cancer patient prognosis by measuring specific miRNA. Furthermore, a cancer therapeutic agent exhibiting an anti-tumor effect that inhibits activation and stabilization of NRF2 in a tumor using a nucleic acid containing a specific miRNA, and a pharmaceutical composition for cancer treatment are provided. .
- the cancer therapeutic agent and pharmaceutical composition of the present invention are associated with various cancer treatments and tests, for example, anticancer drug treatment, radiation therapy, surgical operation, and “stress on cancer cells” given by biopsy. It is effective to administer.
- FIG. 6 shows the results of Western blot analysis for NRF2 expression in cells transfected with miRNA.
- FIG. 4 shows the expression analysis of four miRNAs and the effect of transfection of hsa-miR-507 on cell survival under cell stress caused by cisplatin treatment in A549 cells.
- 1 is a drawing showing the tumor suppressive effect of hsa-miR-507 in vivo.
- 1 is a drawing showing the tumor suppressive effect of hsa-miR-634 in vitro in esophageal cancer cell line KYSE170. It is a drawing showing the tumor suppressive effect of hsa-miR-634 in vivo in esophageal cancer cell line KYSE170.
- the low value measuring method of the present invention is “quantifying“ low value miRNA ”in a human specimen, and using the decrease in the quantitative value as an index, A method for measuring microRNA characterized by distinguishing NRF2 activation or prognosis of cancer patients.
- the low-value miRNA to be measured includes hsa-miR-507, hsa-miR-634, hsa-miR-450a, hsa-miR-129-5p, hsa-miR-639, hsa- one or more selected from the group consisting of miR-337, hsa-miR-153, and hsa-miR-556, in particular hsa-miR-507, hsa-miR-634, One type or two or more types selected from the group consisting of hsa-miR-450a and hsa-miR-129-5p are characterized by a low quantitative value in malignant tumors. It is preferred to select a species of microRNA as the measurement target.
- Human specimens are literally human (human) specimens, and specimen donors are often cancer patients, but it is not clear whether they have cancer or not. can do.
- the type of specimen is not particularly limited as long as it is a specimen capable of reflecting a change in low-value miRNA in tumor cells. It is a tumor specimen that is directly a tumor itself, and specifically includes tumor specimens, biopsy specimens, etc. removed by surgery or endoscopy. As long as that is the case, any human tissue can be used as the sample tissue.
- blood samples such as serum, plasma and whole blood, urine samples, lung lavage fluid samples, sputum samples, lymph fluid samples, spinal fluid samples and the like can be used as necessary.
- the low-value miRNA quantification means is not particularly limited, and can be used including existing means and means developed in the future.
- a typical example is an RNA quantification method based on a gene amplification method such as RT-PCR. From the viewpoint of mass processing and rapidity, the real-time RT-PCR method using an automated detection device can be exemplified as one of the more preferable methods. In addition, the Northern blot method etc. are mentioned.
- the specific threshold can be set individually and specifically, it is preferable to set a reduction of at least 30% as a threshold, and a reduction of 50% or more is set as a threshold as disclosed in this embodiment described later. Is more preferable.
- a decrease in individual quantitative values can also be used as an index.
- the more miRNAs that are “decreased quantitative values” the stronger the malignancy of the tumor. NRF2 is activated and the patient's prognosis is differentiated.
- the above-mentioned four types “hsa-miR-507, hsa-miR-634, hsa-miR-450a, and hsa-miR-129-5p” It is more preferable to narrow down to “one or two or more selected from microRNAs” and perform the low value measurement method of the present invention. The results of verifying the malignancy of tumors using these four types of microRNAs were disclosed in the examples described later.
- NRF2 gene mutation 1 to 3 species selected from the group consisting of NRF2 gene mutation, KEAP1 gene mutation, and p62 protein accumulation in tumors are further detected, and these gene mutations are detected.
- Tumor malignancy particularly activation of NRF2 in the tumor, and can be included as an indicator of patient prognosis.
- the NRF2 gene mutation and / or the KEAP1 gene mutation is any of a missense mutation including a point mutation, a nonsense mutation, and a frameshift mutation. , Shows an example of a missense mutation.
- the method for detecting the gene mutation is not particularly limited.
- PCR-dependent methods such as pyrosequencing method, MALDI-TOF MS method, RFLP method, SSCP method, SSOP method, RNase protection method, RDA method, RAPD method, AFLP method, etc.
- Typing method PCR-independent typing method such as TaqMan PCR method and Invader method can be used.
- a “low score group” is set, and two or more are set as a “high score group”.
- Score can be used as an index such as the grade of malignancy of the tumor, and is preferable.
- how to set the threshold value of the score differs depending on the type of target cancer, the content of the selected index, and the like, and is not limited to the content of the examples described later.
- the essential significance of the low value measurement method of the present invention is closely related to the activation of NRF2 that leads to an increase in the malignancy of cancer, in particular, the “decrease” in the quantitative value of miRNA serving as a differentiation index, This has been found to enable accurate prognosis of cancer prognosis, and there is no major significance in formal elements such as threshold setting.
- the low value measurement method of the present invention makes it possible to perform therapeutic measures such as administering the cancer therapeutic agent of the present invention described later to a patient whose cancer has been found to be at high risk.
- the method for measuring high value of the present invention is as follows: “Quantifying“ high-value miRNA ”in a human specimen and using the increase in the quantitative value as an index, the malignancy of the tumor, the activity of NRF2” Or a method for measuring microRNA, characterized in that the prognosis of a cancer patient is differentiated.
- high-level miRNAs to be measured include hsa-miR-26a, hsa-miR-17-3p, hsa-miR-190, hsa-miR-567, hsa-miR-125b, hsa-miR.
- the quantitative value of each high value miRNA is relatively higher than the internal standard control in the threshold value, it is determined that “the quantitative value of the individual miRNA has increased”.
- a specific threshold can be set individually and specifically, it is preferable to set an increase of at least 30% as a threshold, and an increase of 50% or more is set as a threshold as disclosed in this embodiment described later. Is more preferable.
- an increase in individual quantitative values can also be used as an index.
- the greater the number of miRNAs that are “increased quantitative values” the stronger the malignancy of the tumor, and particularly the NRF2 in the tumor. Is activated and the patient's prognosis is differentiated.
- An increase in one or more quantitative values selected from the group of microRNAs consisting of -miR-29 is used as an indicator of increased tumor malignancy, NRF2 activation, or worsening patient prognosis It is possible.
- 1 to 3 species selected from the group consisting of NRF2 gene mutation, KEAP1 gene mutation, and p62 protein accumulation in tumors are detected, and these gene mutations are detected.
- the malignancy of the tumor, in particular, the activation of NRF2 in the tumor and being able to join as a prognostic indicator of the patient is the same as the above-mentioned “low value measuring method of the present invention”.
- the high value measurement method of the present invention it is detected whether or not it means an increase in the malignancy of a tumor at each level of an element that is an index, and the number of elements that mean the increase (
- the score can be used as an index such as the grade of malignancy of the tumor, and is preferable.
- how to set the threshold value of the score differs depending on the type of target cancer, the content of the selected index, and the like.
- the essential significance of the high value measurement method of the present invention is that the “increase” in the quantitative value of miRNA, which is an indicator for differentiation, is closely related to the activation of NRF2 that leads to an increase in the malignancy of cancer.
- the high value measurement method of the present invention makes it possible to perform therapeutic measures such as administering an antagonistic cancer therapeutic agent to a patient whose cancer has been found to be at high risk.
- the antagonistic cancer therapeutic agent is, for example, a cancer comprising, as an essential component, a synthetic oligo that can antagonize miRNA that has been observed to have increased expression by the high-value measurement method of the present invention and inhibit its function. It is a therapeutic agent.
- the cancer therapeutic agent of the present invention includes “SEQ ID NO: 1 which is the base sequence of hsa-miR-507, SEQ ID NO: which is the base sequence of hsa-miR-634” 2, the base sequence of hsa-miR-450a, SEQ ID NO: 3, the base sequence of hsa-miR-129-5p, SEQ ID NO: 4, the base sequence of hsa-miR-639, the base sequence 5, hsa-miR- 1 selected from the group consisting of SEQ ID NO: 6, the base sequence of 337, SEQ ID NO: 7, the base sequence of hsa-miR-153, and SEQ ID NO: 8, the base sequence of hsa-miR-556. It is a “cancer therapeutic agent comprising a nucleic acid comprising two or more species”.
- each of the microRNAs having the nucleotide sequences of SEQ ID NOS: 1 to 8 has enhanced or stabilized NRF2 activity, and its expression is suppressed in cancer cells having high therapeutic resistance and high malignancy.
- the therapeutic agent for cancer according to the present invention administers a nucleic acid that acts as a microRNA whose expression is suppressed to a cancer having such therapeutic resistance and high malignancy, so that the NRF2 It is a cancer therapeutic agent intended to inactivate cancer, weaken the vitality of cancer, and treat cancer.
- NRF2 activity in cancer cells is high from the beginning, but in many cases, it is activated by applying stress to cancer cells.
- This stress factor includes treatment for cancer. Specific examples include anticancer drug treatment, radiation therapy, and surgical operation. These actions, on the other hand, are actions that can cure cancer, but conversely, they are actions that take the lives of cancer cells. It is believed that NRF2 activity in cancer cells is enhanced by stress due to such actions. Therefore, using the cancer therapeutic agent of the present invention in combination with the application of stress to cancer such as the anticancer drug treatment mentioned here is one of the preferred modes of use of the cancer therapeutic agent.
- the stimulation with respect to the cancer cell by a biopsy also gives the physical stress with respect to a cancer cell similarly to a surgical operation, it can become a combination object with the cancer therapeutic agent of this invention.
- “combining” includes temporal concepts of “before”, “simultaneous”, and “after”. That is, it is also possible to administer the cancer therapeutic agent of the present invention “before” the administration of other anticancer agents, or stress act such as surgical operation or biopsy. Further, at the same time, for example, it can be taken simultaneously with other anticancer agents, or as a mixture (pharmaceutical composition) with other anticancer components. And it is also possible to administer the cancer therapeutic agent of the present invention “after” the stress imparting action such as administration of other anticancer agents, surgical operation or biopsy.
- nucleic acid containing the above-mentioned eight kinds of miRNA base sequences constituting the essential component of the cancer therapeutic agent of the present invention is as long as it contains the base sequences and can function as miRNA in cancer cells. It is not limited. “MiRNA itself” present in the living body as a single-stranded RNA may be used, but in this case, from the viewpoint of stability in the living body, a double-stranded nucleic acid is preferable.
- a double-stranded nucleic acid is a “double-stranded RNA” composed of a pair of miRNA sequences and complementary RNA, also “complex double-stranded RNA and DNA”, and “RNA And “complex double strand of PNA” and “complex double strand of RNA and LNA”.
- PNA peptide nucleic acid
- PNA is a nucleic acid analog having a skeleton in which the deoxyribose-phosphate skeleton, which is the skeleton structure of the nucleic acid skeleton, is replaced with (2-aminoethyl) glycine.
- LNA locked nucleic acid
- BNA bridged nucleic acid
- RNA synthesis methods include, for example, the phosphoramidite method and its improved method (M. Kataoka, Y. Hayakawa, J. Org. Chem., 64, 6087 (1999)), the H-phosphonate method and its improved method (T Wada, F. Handa, Y. Sato, S. Kawahara, M. Sekine, Nucleic® Acids® Symp. Ser., 37, 19 (1997)), enzyme synthesis method (in vitro transcription method) and the like.
- the nucleic acid containing the base sequence of the specific miRNA thus obtained can be used as an essential component of the cancer therapeutic agent of the present invention.
- the cancer therapeutic agent of the present invention is administered to the human body, it is in principle administered as a pharmaceutical composition of the present invention, which will be described later.
- the dose when the cancer therapeutic agent of the present invention is administered to the human body Is about 0.01 ⁇ g to 10 mg per adult per day. This administration can be performed once to 2 to 5 times a day and every several days.
- cancers to which the cancer therapeutic agent of the present invention can be applied are specific miRNAs (hsa-miR-507, hsa-miR-634, hsa-miR-450a, hsa-miR) rather than their types.
- hsa-miR-639, hsa-miR-337, hsa-miR-153, and hsa-miR-556, more typically hsa-miR-507, hsa-miR-634 , Hsa-miR-450a, and hsa-miR-129-5p) are suppressed, and NRF2 activity in the cancer cells is enhanced or stabilized. is there. That is, administration of the cancer therapeutic agent of the present invention to a cancer patient identified as having suppressed the expression of the specific microRNA and suppressing NRF2 by the above-described low value measurement method of the present invention. Is actively considered.
- the nucleic acid containing the base sequence of miRNA which is an essential component of the cancer therapeutic agent of the present invention, is a microRNA of an off-the-shelf combination (typically a nucleic acid containing the above four types of miRNA base sequences).
- the cancer therapeutic agent of the present invention comprising a nucleic acid containing a miRNA base sequence for which expression suppression is specified as an essential component.
- the specific miRNA which is an essential component of the cancer therapeutic agent of the present invention can be used alone or in combination of two or more.
- the cancer therapeutic agent of the present invention can be applied, the above esophageal cancer, lung cancer, breast cancer, oral cancer, stomach cancer, colon cancer, liver cancer, uterine cancer, skin cancer, or Including, but not limited to, osteosarcoma.
- Agents ibritumomab tiuxetan, imatinib, everolimus, erlotinib, gefitinib, gemtuzumab ozogamicin, sunitinib, cetuximab, sorafenib, dasatinib, tamivarotene, trastuzumab, tretizumab, panitumimab, panitumimab
- Molecular targeting drugs Enocitabine, capecitabine, carmofur, cladribine, gemcitabine, cytarabine, cytarabine ocphosphate, tegafur, tegafur uracil
- Antimetabolites such as tegafur, gimeracil, oratesil potassium, doxyfluridine, nelarabine, hydroxycarbamide, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine, methotrex
- oxaliplatin cal Platinum preparations such as platin, cisplatin (CDDP), nedaplatin; anastrozole, exemestane, estramustine, ethinylestradiol, chlormadinone, tamoxifen, dexamethasone, toremifene, bicalutamide, flutamide, prednisolone, phosesthol, mitotane, methyl testosterone, med Hormone preparations such as roxiprogesterone, mepithiostane, leuprorelin, letrozole; biological response modifiers such as interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , interleukin, ubenimex, dry BCG, lentinan, etc.
- CDDP cisplatin
- nedaplatin anastrozole, exemestane, estramustine, ethinylestradiol, chlormadinone, tam
- NRF2 activation of NRF2
- the radiation therapy in addition to the conventional radiation therapy, particle beam therapy and the like are also targeted. And as mentioned above, surgical operation and biopsy are also targeted.
- the cancer therapeutic agent of the present invention can also be applied when aiming for “coexistence with cancer”. That is, it is considered that the cancer therapeutic agent of the present invention is suitable for preventing malignant transformation of cancer by suppressing the activation of NRF2 in cancer and aiming for full life.
- the pharmaceutical composition of the present invention is a “pharmaceutical composition for cancer treatment characterized by containing the above-mentioned cancer therapeutic agent”.
- both of the cancer therapeutic agents of the present invention are administered to the human body as an active ingredient of a “pharmaceutical composition”.
- a pharmaceutical composition In the case of direct administration of the cancer therapeutic agent, since an injection is mixed at the time of use, it is also included in the pharmaceutical composition.
- the pharmaceutical composition of the present invention is prepared in the form of a pharmaceutical composition by blending an appropriate pharmaceutical preparation carrier with the cancer therapeutic agent of the present invention.
- an appropriate pharmaceutical preparation carrier such as a filler, a bulking agent, a binder, a moistening agent, a disintegrant, and a surfactant are used. can do.
- the form of the composition is not particularly limited as long as it can effectively contain the cancer therapeutic agent of the present invention, and may be a solid or ointment such as a tablet, powder, granule, or pill. Usually, it is preferably in the form of an injection such as a solution, suspension or emulsion.
- the cancer therapeutic agent of the present invention can be made into a dry product that can be made liquid at the time of use by adding an appropriate carrier.
- a drug delivery system such as nanoparticles composed of cyclodextrin-containing polymers, polymer micelles, stable nucleic acid lipid particles (SNALP), multifunctional envelope nanostructures (MEND) is utilized. The effect of the cancer therapeutic agent of the present invention can be further improved.
- the obtained pharmaceutical composition is administered in an appropriate administration route according to its form, for example, an injectable pharmaceutical composition is administered in a solid form by intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal administration, etc.
- the pharmaceutical composition is administered orally or enterally.
- the amount of the therapeutic agent for cancer of the present invention in the pharmaceutical composition is appropriately selected according to the administration method, administration mode, purpose of use, patient symptom, etc. of the composition, but is usually constant according to the present invention.
- the therapeutic agent is prepared in the form of a composition containing about 0.1 to 95% by mass, and the above-mentioned dose (about 0.01 ⁇ g to 10 mg per adult per day, once to 2 to 5 times a day) It is also possible to carry out administration at a time, every few days).
- HeLa, Lk-2, A549 and JHH-5 cells were purchased from American Culture Collection (USA). The cells are obtained by adding 10% fetal bovine serum, penicillin, and Dulbecco's modified Eagle medium (for HeLa, Lk-2 cells), RPMI 1640 medium (for A549 cells), or William E medium (for JHH-5 cells), and Streptomycin was added and cultured at 5% CO 2 ⁇ 37 ° C.
- ESCC primary esophageal squamous cell carcinoma
- control non-cancerous esophageal mucosa were treated between April 2005 and June 2007 at Tokyo Medical and Dental University Hospital.
- the samples were obtained from patients who underwent rapid freezing in liquid nitrogen and stored at ⁇ 80 ° C. until DNA and RNA were extracted. The letter of consent is always officially obtained.
- Patient sample collection and analysis was approved by the Tokyo Medical and Dental University Trial Review Board (Authorization # 2010-5-2). Tumor samples fixed in formalin and embedded in paraffin were used for immunohistochemical analysis. Relevant clinical and survival data were obtained from all patients. None of the patients had received chemotherapy and radiation therapy prior to surgery.
- Antibodies and reagents Anti-NRF2 rabbit polyclonal antibody (Santa Cruz Biotechnology) for Western blotting, anti-NRF2 rabbit polyclonal antibody (Santa Cruz Biotechnology) for immunohistochemical analysis, anti-KEAP1 rabbit polyclonal antibody (Proteintech) Anti-ME-1 mouse monoclonal antibody (Santa Cruz Biotechnology), anti-XIAP rabbit antibody (Cell signaling), anti-APIP rabbit antibody (Abcam), anti-OPA1 rabbit antibody (Abcam), anti-TFAM rabbit antibody (Sigma) ) And anti- ⁇ -actin mouse monoclonal antibody (Sigma). Hydrogen peroxide (Wako Pure Chemical Industries) or cisplatin (Wako Pure Chemical Industries) was used for the treatment of the cultured cells.
- Cell survival assay Cell survival was assessed by a crystal violet (CV) staining assay. Cells were washed with PBS and fixed in 10% formaldehyde PBS containing 0.2% CV for 3 minutes. Excess CV solution was removed and after complete air drying, the stained cells were lysed by shaking the plate for 1 hour while in contact with 2% SDS solution. Optical density (OD) absorbance was measured at 560 nm using a microplate reader (ARVOmx; Perkin Elmer), and the absorbance percentage was calculated for each well. The OD absorbance value of the cells in the control well was arbitrarily set to 100% to determine the percentage of viable cells.
- CV crystal violet
- the luciferase reporter plasmid is a pGL3 vector containing a human NQO-1 promoter region “5′-CTCAGCCTTCCAAATCGCAGTCACAGTGACTCAGCAGAATC-3 ′” (SEQ ID NO: 19) containing an antioxidant response element (ARE).
- ARE antioxidant response element
- a double-stranded DNA having a sticky end complementary to the Mlu1 / Xho1 site was prepared using Mlu1 and Xho1. This was inserted into the Mlu1 / Xho1 site to produce the desired recombinant pGL3 (reporter plasmid).
- the reporter plasmid was transiently transfected into HeLa cells (1 ⁇ 10 7 cells / dish) on a 10 centimeter dish. Twenty-four hours after transfection, the cells are seeded in 96-well plates (1 ⁇ 10 4 cells / well) and are derived from Pre-miRmiRNA precursor library-HumanV3 (Ambion) or control miRNA the next day. 20 nmol / L of each of 470 double-stranded RNAs were transfected. Two days later, firefly luciferase activity was measured with a microplate reader (ARVOmx: Perkin Elmer) using a Bright-Glo luciferase assay system (Promega). At the same time, viable cells were detected by CV staining assay. The ARE activity was calculated from the luciferase activity for each viable cell.
- microRNAs and small interference RNAs (siRNAs) 20 nmol / L miRNAs or siRNAs were individually transfected into cells using Lipofectamin RNAiMAX (Invitrogen) according to the operating manual. Double-stranded RNA mimicking human mature miRNA (hsa-miR-507 (PM10509), hsa-miR-129-5p (PM10195), hsa-miR-450a (PM11192), and hsa-miR-634 (PM11538) ), And control miRNA (negative control # 1) were obtained from Ambion.
- SiRNA against NRF2 (siGENOME SMARTpool; M-003755-02-0005) and siRNA against KEAP1 (siGENOME SMARTpool; M-012453-00-0005) were obtained from Thermo Scientific Dalmacon.
- the luciferase reporter plasmid is a 3 ′ untranslated region (UTR: SEQ ID NO: NRF2, ME1, or NQO1 downstream of the luciferase gene of pmirGlo Dual-Luciferase miRNA Target Expression Vector (Promega). 49-51). All site-specific mutations were performed using GeneTailor site-directed mutagenesis system (Invitrogen) or KOD mutagenesis kit (TOYOBO). The luciferase reporter plasmid and the pTK plasmid as an internal standard control were both transfected into HeLA cells and transfected the next day with double-stranded RNA mimicking human mature miRNA or control miRNA.
- Quantitative RT-PCR Total RNA was isolated using TRIzol® reagent (Invitrogen) by standard methods. Single-stranded cDNA prepared from the total RNA was amplified with primers specific to each gene. Quantitative real-time RT-PCR (qRT-PCR) was performed according to the operation manual using KAPA SYBR System (Kapa Biosystems) and ABI PRISM 7500 sequence detection system (Applied Biosystems). Gene expression values are given by the ratio between the genes of interest and the internal standard control (GAPDH) or U6 (difference in Ct values) and are subsequently normalized together with the values in control cells ( Relative expression level). See Tables 3 and 4 for information on primers and TaqMan probes used.
- GPDH internal standard control
- U6 difference in Ct values
- SEQ ID NOS: 22 to 32 are assigned in order from the top as the base sequence number in the left column indicating the forward primer, and the base sequence ID numbers in the right column indicating the reverse primer are listed in order from the top. 33-43 were allocated.
- SEQ ID NOS: 44 to 48 were assigned in order from the top as the SEQ ID NOs of the TaqMan probe base sequences.
- Real-time reverse transcription PCR (RT-PCR) for miRNA is ABI Prizm 7500 Fast Real-time PCR System (Applied Biosystems), Taqman Universal PCR Master Mix (Applied Biosystems), Taqman Reverse Transcription kit (Applied Biosystems) ) And Taqman® MicroRNA® Assays (Applied Biosystems), and according to the operation manual.
- the expression level of the miRNA gene is based on the amount of target message associated with the amount of RNU6B transcript as a normalization control of the initial amount of total RNA.
- IPA ingenuity pathway analysis
- mice In vivo tumor growth assay and administration of miRNA Seven-week-old female BALB / c nude mice were purchased from Oriental Yeast Co., Ltd. and kept sterile. 200 ⁇ l of PBS containing 1 ⁇ 10 7 cells was injected subcutaneously into the flank of the mice. A mixture of 1 nmol of double stranded RNA (Ambion) and 200 ⁇ l of AteroGene (KOKEN) was administered into the space between the tumor and the skin. Mice were given intraperitoneal administration of 5 mg / kg body weight of cisplatin. 35 days after cell administration, mice were euthanized and tumors were removed. The experimental procedures performed on all mice were approved by the Animal Care and Use Committee of Tokyo Medical and Dental University.
- FIG. 1 is a drawing showing the results of verification of an ARE reporter system using siRNA.
- HeLa cells were co-transfected with an ARE luciferase reporter plasmid and an internal standard control vector, and after 24 hours, control siRNA, NRF2-siRNA (a), or KEAP1-siRNA ( Transfection was performed in b). 48 hours after transfection with siRNA, SDS-PAGE of the cell lysate was performed, and an immune reaction with each antibody was performed (upper panel). On the other hand, firefly or Renilla luciferase activity was measured, and the ARE activity relative to the ARE activity in Mock was shown on the vertical axis (lower panel). Bars are standard deviation (SD).
- SD standard deviation
- FIG. 2-1 is a drawing showing the results of screening for miRNA that negatively regulates the transcriptional activity of NRF2.
- FIG. 2-1 (a) shows a miRNA library screening method using the ARE reporter system. To screen for 470 double stranded RNAs, each miRNA was serially transfected into HeLa cells with a reporter plasmid and transduced as luciferase activity per viable cell as measured by crystal violet (CV) staining assay. The ARE activity in the infected cells was calculated.
- CV crystal violet
- each miRNA derived from the ARE luciferase reporter plasmid and library was sequentially transfected into HeLa cells, and 48 hours later, luciferase activity or viable cell counts were individually determined using the Bright-GIo luciferase assay system or CV staining assay. Was measured. The ARE activity of the cells transfected with each miRNA was calculated as the luciferase activity for each viable cell.
- FIG. 2-1 (b) shows a summary of the results of ARE activity in cells transfected with miRNA.
- the ARE activity for cells transfected with control miRNA is shown on the vertical axis.
- Eight kinds of miRNAs having a relative value of 0.5 or less were identified as candidates by this screening system.
- 8 types of miRNAs having a relative value of 1.5 or more were also identified as candidates by this screening system.
- FIG. 2-1 (c) shows the results of verification by luciferase assay of 4 types of candidate miRNAs.
- HeLa cells were transfected with ARE luciferase reporter plasmid and an internal standard control vector, and 24 hours later, hsa-miR-507, -634, -450a, -129-5p, or control miRNA were transfected. Firefly luciferase activity or Renilla luciferase activity was measured 48 hours after transfection of miRNA.
- the ARE activity for cells transfected with control miRNA is shown on the vertical axis. Bars are standard deviation (SD).
- FIG. 2-1 (d) shows the results of NQO1 mRNA expression analysis in cells transfected with miRNA.
- HeLa cells are transfected with hsa-miR-507, -634, -450a, -129-5p, or control miRNA.
- 48 hours after transfection mRNA level of NQO1 gene was measured by qRT-PCR.
- GAPDH gene expression was used as an internal standard control.
- the expression level for cells transfected with control miRNA is shown on the vertical axis. Bars are standard deviation (SD).
- FIG. 2-2 is a drawing showing the results of expression analysis of the NRF2 target gene.
- Transfect HeLa cells using siRNA (control, NRF2, or KEAP1) (a), or miRNA (control miRNA, hsa-miR-507, -634, -450a, or -129-5p) (b) went. Forty-eight hours after the transfection, mRNA expression levels of the four NRF2 target genes NQO1, GPX2, and TXNRD1 were measured by qRT-PCR. GAPDH expression was used as an internal standard control. The expression level relative to the expression level in cells transfected with control siRNA or control miRNA is shown on the vertical axis. Bars are standard deviation (SD). The results of Western blotting and NQO1 expression shown in (b) are also shown in FIGS. 3a and 2-1 (d), respectively.
- SD standard deviation
- FIG. 2-1 (c), FIG. 2-1 (d) d, and FIG. 2-2 the top four miRNAs (hsa-miR-507, -634, -450a, and -129-5p). These transfections were confirmed to actually reduce ARE activity and to down-regulate the transcriptional activity of known NRF2 target genes “NQO1, HO1, GPX2, and TXNRD1”. It was.
- Example 2 Identification of NRF2 as a functional target of candidate miRNAs of four candidate miRNAs (hsa-miR-507, hsa-miR-634, hsa-miR-450a, and hsa-miR-129-5p)
- IPA Ingenuity Systems Pathway Analysis, Redwood City, CA
- TargetScan program http://www.targetscan.org
- FIG. 3 (b) shows the design of the reporter plasmid and the homologous sequence of the seed sequences of four candidate miRNAs within the 3′UTR sequence of NRF2.
- the seed sequences of hsa-miR-507 (2 sites), hsa-miR-634 (1 site), and hsa-miR-129-5p (2 sites) were mapped in the 3′UTR of NRF2. Since the seed sequence of hsa-miR-450a is highly homologous to hsa-miR-507 (6 of 7 seed sequences), the seed sequence of hsa-miR-450a is shown as the same site as hsa-miR-507 Has been. Region 1 (R1), region 2 (R2) or mutation R2 was used in the luciferase assay. Black crosses indicate mutation sites inserted in each seed sequence.
- FIG. 3 (a) shows the result of Western blot analysis of NRF2 expression in cells transfected with miRNA.
- HeLa cells have been transfected with hsa-miR-507, -634, -450a, -129-5p, or control miRNA. Forty-eight hours after transfection, Western blotting was performed using cell lysates, and immunoreactivity was performed with the indicated antibodies.
- FIG. 3 (a) The negative regulation of NRF2 by miRNA overexpression shown in FIG. 3 (a) is due to the NRF2 stabilized cancer cell line, LK2 with NRF2 mutation (NSCLC), A549 with KEAP1 mutation (NSCLC), and p62 protein. It is shown in FIG. 4 that it was also observed in JHH-5 (HCC) with aggregation.
- FIG. 4 shows the results of Western blot analysis for NRF2 expression in cells transfected with miRNA, hsa-miR-507, -634, -450a, or -129-5p, or control miRNA in LK2 cells.
- FIG. 3 (c) shows the results of a luciferase assay using a reporter plasmid having each region shown in FIG. 3 (b).
- HeLa cells were transfected with a reporter plasmid and an internal standard control vector, and 24 hours later, hsa-miR-507, -634, -450a, -129-5p, or control miRNA was transfected. 48 hours after miRNA transfection, firefly or Renilla luciferase activity was measured. The luciferase activity corresponding to cells transfected with control miRNA is indicated on the vertical axis. The horizontal line at the bottom of the graph shows the mutant plasmids that are relevant in cells transfected with miRNA. Bars are standard deviation (SD).
- the luciferase activity for the R2 vector is significantly reduced compared to the vacancy in the cells introduced with each miRNA and the luciferase activity for the RA vector, and suddenly in the R2 seed sequence. It has been shown to be completely repaired with the vector carrying the mutation.
- FIG. 3 (d) shows the result of examination on the effect of miRNA transfection using cell survival under cell stress caused by cisplatin (CDDP) (left) or oxidative stress (hydrogen peroxide) (right) as an index.
- CDDP cisplatin
- oxidative stress hydrogen peroxide
- FIG. 3 (d) shows the result of examination on the effect of miRNA transfection using cell survival under cell stress caused by cisplatin (CDDP) (left) or oxidative stress (hydrogen peroxide) (right) as an index.
- CDDP cisplatin
- oxidative stress hydrogen peroxide
- FIG. 3 (d) shows that transfection of each miRNA not only showed expression of the NRF2 protein, but also significantly increased sensitivity to treatment with cisplatin or exposure to hydrogen peroxide.
- Example 2 results indicate that all four candidate miRNAs bind directly to the 3′-UTR, making NRF2 a functional target, and survival of cancer cells involving NRF2 despite increased cellular stress. It is suggested to suppress.
- FIG. 5 is a drawing in which the clinical significance in the primary tumor sample derived from esophageal cancer (esophageal squamous cell carcinoma: ESCC) and the relationship between the negative regulation of miRNA are examined.
- FIG. 5 (b) is a Kapian-Meier curve showing the survival rate of all 30 patients with ESCC in light of the abnormal score.
- FIG. 5 (c) is a representative of immunostaining of NRF2 protein in primary ESCC of high score group (cases 1, 4, 7) (upper panel) and low score group (cases 21, 25, 26) (lower panel). Shows an image.
- the black bar is 100 ⁇ m on the scale bar.
- FIG. 5-2 is a drawing showing the results of mutation analysis of NRF2 and KEAP1 genes in primary tumor samples derived from esophageal cancer (esophageal squamous cell carcinoma: ESCC).
- ESCC esophageal squamous cell carcinoma
- mutations detected in five specimens in which amino acid substitution mutations were observed in NRF2 or KEAP1 were shown by sequence chromatography.
- Three samples shown in the upper row a are examples in which a mutation in the NRF2 gene was observed, and two samples shown in the lower row b were examples in which a mutation in the KEAP1 gene was observed.
- the up arrow indicates the wild-type sequence in the corresponding normal tissue, and the down arrow indicates the mutation and amino acid sequence changes in the primary tumor tissue.
- D77G shown at the bottom indicates that “a mutation in which the 77th D (aspartic acid) in the wild-type amino acid sequence encoded by the NRF2 gene is replaced with G (glycine)”.
- G glycine
- R arginine
- Q glutamine
- L leucine
- M methionine
- the miRNA level decreased by 50% or more compared with the corresponding non-tumor tissue in the primary tumor tissue in 9 cases (30.0%) in hsa-miR-507, in hsa-miR-634.
- 9 cases (30.0%)
- 2 cases 60.0%)
- 6 cases 60.0%) for hsa-miR-129-5p.
- FIG. 5-1 (a) mutation analysis revealed NRF2 missense mutations in 3 cases (10%) and KEAP1 missense mutations in 2 cases (6.7%) (FIGS. 5-1 (a) and 5-2).
- an “abnormal score” is defined as an index indicating the decrease in the four miR levels and the corresponding number of missense mutations in the two genes as a score (number). Based on the negative regulation of NRF2 or KEAP1, and / or each miRNA, 30 cases were assigned to two groups. That is, 14 primary ESCC cases with “abnormal score 2-4” were assigned as “high score group”, and 16 primary ESCC cases with “abnormal score 0 or 1” were assigned as “low score group” (FIG. 5-1 (a)). ).
- the 30 samples were subjected to gene amplification by PCR using each extracted DNA as a template and the primers listed in Table 3, and the PCR products were sequenced by the Sanger method.
- a sequence chromatography chart showing the results is shown in FIG.
- a function-gaining gene mutation (D77G; 1 case, D29G; 2 cases) with a known amino acid substitution was detected in a total of 3 cases concerning the NRF2 gene.
- KEAP1 gene gene mutation with amino acid substitution was observed in a total of 2 cases, one of which is a known loss-of-function gene mutation (R320Q), and in case 4 with an abnormal score of “3”, a novel gene A mutation (L276M) was detected.
- Example 4 Tumor suppressive effect in vivo by administration of hsa-miR-507 Since one miRNA can target multiple genes that contribute to one cancerous pathway, It is thought to be effective against tumors involving the cancerous pathway. Then, using hs-miR-507 among the four miRNAs, whether or not it can functionally target a gene whose transcription is regulated by NRF2 is examined using an expression array and pathway analysis, In these analyses, 8 types of transcription target genes of NRF2 that were suppressed by transfection of hsa-miR-507 were found. Among them, ME1 was confirmed to be a direct target of hsa-miR-507 through binding of NRF2 to 3′-UTR (FIG. 6). FIG.
- FIG. 6 is a drawing showing the results of identification of a transcription target gene of NRF2 as a target of hsa-miR-507.
- FIG. 6 (a) shows the result of verification of the transcription target gene of NRF2 negatively regulated by hsa-miR-507.
- 181 indicates that it was predicted as a target of hsa-miR-507 by the TargetScan program.
- FIG. 6 (a) shows the result of verification of the transcription target gene of NRF2 negatively regulated by hsa-miR-507.
- 181 indicates that it was predicted as a target of hsa-miR-507 by the TargetScan program.
- FIG. 6 (b) shows that, using IPA, eight transcription targets of NRF2 (ME1, PSMB6, SLC7A11, MGLL, DNAJB5, LGALS8, B4GALNT1, EIF4G2) among 181 genes are expressed by hsa-miR-507. It shows that it was predicted to be targeted.
- FIG. 6 (c) shows the mapping of the design for the seed sequence and reporter plasmid for hsa-miR-507 in ME1 3′-UTR. The results of examining whether hsa-miR-507 binds directly to ME1 3′-UTR by performing luciferase analysis are shown. The seed sequence for hsa-miR-507 is mapped in the ME1 3′UTR.
- FIG. 6 (d) shows the results of luciferase analysis using a reporter plasmid. The results of co-transfection of HeLa cells with a reporter plasmid and an internal standard control vector and transfection with hsa-miR-507 or control miRNA after 24 hours are shown. 48 hours after transfection with miRNA, the firefly or Renilla luciferase activity was measured.
- FIG. 6 (e) shows the results of Western blot analysis for ME1 expression in cells transfected with miRNA. The results of transfection of HeLa cells with hsa-miR-507 or control miRNA are shown.
- FIG. 7 is a drawing showing the expression analysis of four miRNAs and the effect of transfection of hsa-miR-507 on cell survival under cell stress caused by cisplatin (CDDP) treatment in A549 cells.
- FIG. 7 (a) is an expression analysis of four miRNAs, hsa-miR-507, -634, -450a, and -129-5p, by qRT-PCR.
- the expression level of RNU6B was used as an internal standard control.
- the vertical axis shows the expression level in A549 cells relative to the expression level in normal lung tissue. Bars are standard deviation (SD).
- FIG. 7 (b) shows cell proliferation analysis in A549 cells transfected with hsa-miR-507.
- A549 cells were transfected with hsa-miR-507 or control miRNA and the number of living cells was shown. The results measured by CV staining analysis on the day are shown. The relative cell growth rate is shown on the vertical axis. Bars are standard deviation (SD). Significant differences were analyzed by Student's t-test.
- FIG. 7 (c) shows the effect of transfection of hsa-miR-507 on cell survival under cell stress caused by cisplatin (CDDP) in A549 cells. Specifically, transfection of the cells with hsa-miR-507 or control miRNA, and treatment with PBS or cisplatin (2 ⁇ M) for 48 hours the next day, for the transfected cells with control miRNA or miRNA, Cell viability relative to cell viability in untreated cells was assessed by CV staining analysis. Bars are standard deviation (SD). Significant differences were analyzed by two-way ANOVA (two factor analysis of variance).
- FIG. 8 is a drawing showing the tumor suppressive effect of hsa-miR-507 in vivo.
- FIG. 8 (a) is an experimental schedule of combined administration of hsa-miR-507 and cisplatin (CDDP). Tumors were formed by subcutaneous injection of A549 cells into nude mice. Control miRNA or hsa-miR-507 was administered a total of four times around tumors formed subcutaneously from A549 cells (7, 14, 21, and 28 days after injection of A549 cells). In addition, mice received intraperitoneal administration of PBS or cisplatin three times the next day (8, 15, 22 days after A549 cell injection). Mice were euthanized 35 days after injection of A549 cells and tumors were removed.
- CDDP cisplatin
- FIG. 8 (b) shows a typical image (left) of a mouse in which a tumor was formed and a tumor (right) removed 35 days after injection of A549 cells.
- the two white letters “miR-507” are both “hsa-miR-507”.
- FIG. 8 (c) shows the weight of the extracted tumor. Mice were euthanized 35 days after A549 cell injection and the weight of each excised tumor was weighed. The graph shows “mean ⁇ SD value” in mice using 4 PBS and 3 mice using cisplatin.
- FIG. 8 (d) shows the expression analysis of hsa-miR-507 in the excised tumor.
- the expression level of hsa-miR-507 mRNA was measured by qRT-PCR.
- RNU6B expression was used as an internal standard control.
- Expression levels corresponding to mouse tumors to which control miRNA was administered among mice in which PBS was used are shown on the vertical axis. Bars indicate standard deviation (SD).
- FIG. 8 (e) is a typical image of NRF2 and ME1 immunostaining in the excised tumor. The scale bar indicates 100 ⁇ m.
- the present invention provides a cancer therapeutic agent comprising a combination of a cancer therapeutic agent comprising a nucleic acid comprising SEQ ID NO: 1 which is the base sequence of hsa-miR-507 and a platinum preparation such as cisplatin.
- a cancer therapeutic agent comprising a nucleic acid comprising SEQ ID NO: 1 which is the base sequence of hsa-miR-507 and a platinum preparation such as cisplatin.
- the mode of combination of a cancer therapeutic agent comprising a nucleic acid comprising SEQ ID NO: 1 which is the base sequence of hsa-miR-507 and a platinum preparation such as cisplatin is not particularly limited. Both agents may be contained in the same composition, or both agents may be administered separately.
- the administration mode of the cancer therapeutic agent comprising the nucleic acid comprising SEQ ID NO: 1 which is the base sequence of hsa-miR-507 is as described above.
- the administration mode of cisplatin follows a known method. Specifically, it is usually 70-80 mg / m 2 (body surface area) per adult per day, about once a day, with a dosing period of 2-5 months, and a dosing frequency (approximately 1 to 4 weeks). Usually administered by infusion.
- FIG. 9 is a drawing showing the results of in vitro studies on the apoptosis-inducing effect of hsa-miR-634 on tumor cells.
- double-stranded RNA imitating hsa-miR-634 or control miRNA was introduced into cervical cancer cell line HeLa cells by lipofection, and then 3 Cultured for days.
- FIG. 9 (A) is a microscopic image 2 days after introduction. When Hesa cells were transfected with hsa-miR-634 and incubated for 48 hours at 5% CO 2 and 37 ° C. Morphological changes are represented by micrographs.
- FIG. 9 (B) shows the results of pursuing the effect of inhibiting cell proliferation over time, and the tendency of cell proliferation after 1 day, 2 days and 3 days while performing the incubation of FIG. 9 (A), It is the drawing examined using the fluorescence intensity by CV staining as an index. The results are shown in a cell group using only Mock, a cell group transfected with control miR, and a cell group transfected with miR-634.
- FIG. 10 Tumor cell death induction effect in vitro by administration of hsa-miR-634 and cisplatin (2) (FIG. 10) Double-stranded RNA imitating hsa-miR-634 or control miRNA was introduced into esophageal cancer cell line KYSE170 by the lipofection method.
- the upper part of FIG. 10 shows the in vitro experimental schedule of the combined administration of hsa-miR-634 and cisplatin (CDDP).
- the amount of double-stranded RNA imitating hsa-miR-634 or control miRNA was 0.2 nM, respectively, and a group added with 2.5 ⁇ M cisplatin and 5.0 ⁇ M The added group was incubated and verified. Incubation time is 72 hours.
- the effect of inducing cell death was evaluated by counting the number of cells dead by trypan blue staining, and the frequency (%) of the number of cells dead relative to the total number of cells.
- the lower graph of FIG. 10 shows the results.
- the vertical axis shows the frequency.
- cell death was induced at a high frequency in cells coadministered with hsa-miR-634 and cisplatin. Thereby, the synergistic anticancer effect by combined use of the therapeutic agent of this invention and cisplatin was clarified.
- FIG. 11 Tumor cell death induction effect in vivo by administration of hsa-miR-634 and cisplatin.
- FIG. 11 shows the in vivo experimental schedule of the combined administration of double-stranded RNA imitating hsa-miR-634 or control miRNA and cisplatin (CDDP). The incubation period for tumor formation is 7 days.
- control miRNA or hsa-miR-634 was administered by a total of 5 injections around tumors formed subcutaneously in nude mice by KYSE170 cells.
- intraperitoneal administration of PBS or cisplatin in mice was performed twice at the first and fourth time of miRNA administration and simultaneously with miRNA administration.
- Mice were euthanized 21 days after injection of KYSE170 cells, and tumors were removed.
- the lower graph of FIG. 11 shows the weight (vertical axis) of the extracted tumor.
- the lower graph shows the “mean ⁇ SD value” of tumor weights in mice using 7 PBSs and mice using 8 cisplatins. Significant differences between the two groups were revealed by analysis with t-test.
- FIG. 12 shows the introduction of hsa-miR-634 into U2OS cells, an osteosarcoma cell line. It shows the expression suppression effect of the target gene.
- the luciferase assay whose results are shown in the graphs on the left side of the respective drawings in FIGS. That is, by inserting the 3 ′ untranslated region (UTR) of each target gene downstream of the luciferase gene of pmirGlo Dual-Luciferase miRNA Target Expression Vector (Promega), a luciferase reporter plasmid used for each study was prepared.
- Each target gene is (a) XIAP (X-linked inhibitor of apoptosis protein: Fig. 12-1), (b) APIP (APAF1 interacting protein: Fig. 12-2), (c) OPA1 (optic atrophy 1: Fig. 12) 12-3) and (d) TFAM (transcription factor A, mitochondrial: FIG. 12-4).
- the UTR seed sequences of the genes encoding these four proteins are shown in the sequence listing at SEQ ID NOs: 52 (XIAP), 53 (APIP), 54 (OPA1), and 55 (TFAM). All site-specific mutations were performed using KOD mutagenesis kit (TOYOBO).
- “mt” in each graph represents each mutation insertion plasmid (mutant: mt).
- the luciferase reporter plasmid or the mutant insertion plasmid (mt) and the pTK plasmid as an internal standard control were both transfected into U2OS cells and the next day double-stranded RNA mimicking hsa-miR-634, control miRNA was transfected. Two days later, firefly luciferase activity and Renilla luciferase activity were measured using Dual-Luciferase Reporter Assay System (Promega), and relative luciferase activity was read with the corresponding internal standard control Renilla luciferase activity. Was calculated by standardizing.
- hsa-miR-634 has a function of directly binding to the seed sequence of the 3′UTR region of each of the above target genes in tumor cells and suppressing the expression of these genes. It became clear.
- the enhancement of the expression of these target genes increases the malignancy of cancer cells, and the expression state of these genes can be added as an indicator of the measurement method of the present invention to further increase the measurement reliability. It became clear that it was possible.
- the membrane was washed and exposed for 2 hours with HRP-conjugated anti-mouse or anti-rabbit IgG antibody (both diluted 1/2000). The bound antibody was visualized according to the operating manual of HRP staining solution or ECL Western blotting kit (Cell Signaling Technology).
- NRF2 is of course not only XIAP, APIP , OPA1, or TFAM gene expression is particularly useful for cancer cells that are enhanced or stabilized.
- the present invention provides a cancer therapeutic agent comprising a combination of a cancer therapeutic agent comprising a nucleic acid comprising SEQ ID NO: 2 which is the base sequence of hsa-miR-634 and a platinum preparation such as cisplatin.
- a cancer therapeutic agent comprising a nucleic acid comprising SEQ ID NO: 2 which is the base sequence of hsa-miR-634 and a platinum preparation such as cisplatin.
- the mode of combination of a cancer therapeutic agent comprising a nucleic acid comprising SEQ ID NO: 2 which is the base sequence of hsa-miR-634 and a platinum preparation such as cisplatin is not particularly limited. Both agents may be contained in the same composition, or both agents may be administered separately.
- the administration mode of the cancer therapeutic agent comprising the nucleic acid comprising SEQ ID NO: 2 which is the base sequence of hsa-miR-634 is as described above.
- the administration mode of cisplatin follows a known method.
- the administration mode is as described above.
- the administration mode of cisplatin follows a known method. Specifically, it is usually 70-80 mg / m 2 (body surface area) per adult per day, about once a day, with a dosing period of 2-5 months, and a dosing frequency (approximately 1 to 4 weeks). Usually administered by infusion.
- NRF2 is structurally stable in many types of human cancers by virtue of a survival mechanism that includes gain-of-function mutations in NRF2, loss-of-function mutations in KEAP1, and functional inactivation of KEAP1 by p62 protein accumulation. It has become.
- the inventors have shown that certain miRNAs are found to have abnormally low level adjustments in cancer cases. Importantly, mutations in NRF2 or KEAP1 and the abnormally low level of one or more miRNAs that coincide with the accumulation of p62 protein are closely linked to the stability of NRF2 in tumors and poor prognosis in cancer patients. It was related.
- the inventors have examined the expression status of specific miRNAs that directly target NRF2, and the low level regulation of these miRNAs helps to increase basal NRF2 activity levels and in tumors Together with or separately from NRF2 or KEAP1 gene abnormalities and p62 protein accumulation, it was revealed that NRF2 protein is synergistically stabilized.
- the present inventors have provided the measurement method of the present invention that can distinguish the malignancy of cancer and the prognosis of cancer patients.
- NRF2 Accumulation or stabilization of NRF2, KEAP1, or p62 protein, and further enhancement or stabilization of XIAP, APIP, OPA1, or TFAM gene is an index for screening tumors stabilized by NRF2. At the same time, inhibiting tumor NRF2 activity itself is a very reasonable approach to the treatment of tumors in which NRF2 is stabilized.
- Non-patent Document 25 Previous studies have also shown that one miRNA can inhibit the expression of multiple targets by directly binding to the 3'-UTR of each gene. This suggests that one miRNA simultaneously targets several genes involved in one information transmission system. In fact, miR-16 has been reported to negatively regulate cell cycle by directly targeting CDK1 and CDK2 (Non-patent Document 26).
- miR-34a is known to act as a negative regulator of tumor growth and metastasis by directly targeting the complex genes CCND1, CDK4, CDK6, and MYC (Non-patent Document 27).
- the present inventors have found that the administration of hsa-miR-507 has the effect of actually inhibiting the growth of tumors formed from A549 cells in nude mice by targeting NRF2 and ME1, which are transcriptional targets of NRF2. It was shown that. In vivo analysis by the present inventors revealed that transfection of hsa-miR-507 leads to increased sensitivity of cell growth inhibition of platinum preparations such as cisplatin in A549 cells.
- NRF2 In addition to acquiring resistance to cellular stress during chemotherapy, NRF2 also boosts tumor cell growth through metabolic regulation.
- hsa-miR-507 inhibits tumor growth both in vitro and in vivo. This growth inhibition by hsa-miR-507 may be due to changes in internal metabolism through suppression of NRF2 and its target gene.
- the present inventors further clarified the tumor suppressive effect of hsa-miR-634 in vitro and in vivo. This tumor suppressive effect was realized by leading tumor cells to apoptosis. Furthermore, it has been found that the use of hsa-miR-634 in combination with a platinum preparation such as cisplatin significantly enhances it. This fact also strongly supports the effectiveness of the cancer therapeutic agent of the present invention.
- the present invention provides a new means of molecular diagnosis based on miRNA and treatment of tumors stabilized by NRF2 or the like.
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Abstract
Description
(1)本発明の低値測定方法
上記の通り本発明の低値測定方法は、「ヒト検体における『低値miRNA』を定量して、当該定量値の低下を指標として、腫瘍の悪性度、NRF2の活性化、又は、がん患者の予後、を鑑別することを特徴とするマイクロRNAの測定方法」である。
上記の通り本発明の高値測定方法は、「ヒト検体における『高値miRNA』を定量して、当該定量値の上昇を指標として、腫瘍の悪性度、NRF2の活性化、又は、がん患者の予後、を鑑別することを特徴とするマイクロRNAの測定方法」である。
(1)本発明のがん治療剤
上述のように本発明のがん治療剤は、「hsa-miR-507の塩基配列である配列番号1、hsa-miR-634の塩基配列である配列番号2、hsa-miR-450aの塩基配列である配列番号3、hsa-miR-129-5pの塩基配列である配列番号4、hsa-miR-639の塩基配列である塩基配列5、hsa-miR-337の塩基配列である配列番号6、hsa-miR-153の塩基配列である配列番号7、及び、hsa-miR-556の塩基配列である配列番号8、からなる群の塩基配列から選ばれる1種又は2種以上を含む核酸からなるがん治療剤」である。
上述のように本発明の医薬組成物は「上記のがん治療剤を含有することを特徴とする、がん治療のための医薬組成物」である。
1.材料と方法
実施例の結果の開示に先立ち、それに用いられた材料と試験方法について説明する。
HeLa,Lk-2,A549及びJHH-5細胞は、アメリカンカルチャーコレクション(米国)から購入した。当該細胞は、ダルベッコ改変イーグル培地(HeLa,Lk-2細胞用)、RPMI1640培地(A549細胞用)、又は、ウイリアムE培地(JHH-5細胞用)に、10%ウシ胎児血清、ペニシリン、及び、ストレプトマイシンを添加して、5%CO2・37℃にて培養した。
ウェスタンブロッティング用の抗NRF2ウサギポリクローナル抗体(サンタクルズバイオテクノロジー社)、免疫組織化学解析用の抗NRF2ウサギポリクローナル抗体(サンタクルズバイオテクノロジー社)、抗KEAP1ウサギポリクローナル抗体(プロテインテック社)、抗ME-1マウスモノクローナル抗体(サンタクルズバイオテクノロジー社)、抗XIAPウサギ抗体(Cell signaling社)、抗APIPウサギ抗体(Abcam社)、抗OPA1ウサギ抗体(Abcam社)、抗TFAMウサギ抗体(シグマ社)、及び、抗β-アクチンマウスモノクローナル抗体(シグマ社)、を用いた。培養細胞の処理のために過酸化水素(和光純薬社)又はシスプラチン(和光純薬社)を用いた。
細胞の生存は、クリスタルバイオレット(CV)染色アッセイにより評価した。細胞はPBSで洗浄され、0.2%CV含有10%ホルムアルデヒドPBSにおいて3分間固定した。過剰なCV溶液は除去され、完全に空気乾燥した後、染色細胞は2%SDS溶液に接触させつつ、プレートを1時間振とうすることにより溶解した。光学密度(OD)吸光度は、マイクロプレートリーダー(ARVOmx;ペルキンエルマー社)を用いて560nmにて計測し、吸光度百分率はウエル毎に算出した。コントロールウエルにおける細胞のOD吸光度値は、生存細胞の百分率を決定するために、任意に100%にセットした。
ルシラーゼレポータープラスミドは、抗酸化応答エレメント(ARE)を含んだヒトNQO-1プロモーター領域「5’-CTCAGCCTTCCAAATCGCAGTCACAGTGACTCAGCAGAATC-3’」(配列番号19)を、pGL3ベクター(プロメガ社)のMlu1/Xho1サイトに挿入することを目的として、下記表2の互いに相補的な合成オリゴの組(上段は配列番号20、下段は配列番号21)をアニールして二本鎖DNAとし、Mlu1とXho1を用いて当該Mlu1/Xho1サイトに相補的な粘着末端を有する2本鎖DNAを調製した。これを当該Mlu1/Xho1サイトに挿入することにより、所望の組換えpGL3(レポータープラスミド)を作出した。
20nmol/LのmiRNAs又はsiRNAsが個別にLipofectamin RNAiMAX(インビロトロゲン社)を用いて細胞に操作用マニュアルに従ってトランスフェクトされた。ヒト成熟miRNAに模した二本鎖RNA(hsa-miR-507(PM10509)、hsa-miR-129-5p(PM10195)、hsa-miR-450a(PM11192)、及び、hsa-miR-634(PM11538))、並びに、コントロールmiRNA(ネガティブコントロール#1)は、アンビオン社から入手した。NRF2に対するsiRNA(siGENOME SMARTpool;M-003755-02-0005)とKEAP1に対するsiRNA(siGENOME SMARTpool;M-012453-00-0005)は、サーモサイエンティフィックダルマコン社から入手した。
ルシフェラーゼリポータープラスミドは、pmirGlo Dual-Luciferase miRNA Target Expression Vector(プロメガ社)のルシフェラーゼ遺伝子の下流に、NRF2、ME1、又は、NQO1の3’非翻訳領域(UTR:配列番号49~51)を挿入することで作出した。全ての部位特異性変異は、GeneTailor site-directed mutagenesis system(インビトロゲン社)、又は、KOD mutagenesis kit(TOYOBO社)を用いて行った。ルシフェラーゼレポータープラスミドと、内部標準コントロールとしてのpTKプラスミドには、共にHeLA細胞にトランスフェクトされ、次の日にヒト成熟miRNAに模した二本鎖RNA又はコントロールmiRNAをトランスフェクトした。2日後、ホタルルシフェラーゼ活性とウミシイタケルシフェラーゼ活性を、Dual-Luciferase Reporter Assay System(プロメガ社)を用いて計測し、相対的ルシフェラーゼ活性は、対応する内部標準コントロールのウミシイタケルシフェラーゼで読み取りながらホタルルシフェラーゼ活性を標準化することにより算出された。
全細胞の溶解物はSDS-PAGEに付されて、蛋白はPVDFメンブレン(GEヘルスケア社)に転写された。0.05%のTween20と5%のスキムミルクを含有するTBSで1時間ブロッキングを行った後、当該膜を抗体と共に一晩反応した。初期の抗体の希釈は、抗NRF2ウサギ抗体(1/1000)、抗KEAP1ウサギ抗体(1/1000)、抗ME1マウス抗体(1/1000)、及び、抗βアクチンマウス抗体(1/5000)であった。当該膜は洗浄され、HRP結合抗マウス又は抗ウサギIgG抗体(共に1/2000)で2時間曝露した。結合した抗体は、HRP染色溶液又はECLウェスタンブロッティングキット(セルシグナリングテクノロジー社)の操作用マニュアルに従って、視覚化された。
全RNAは、TRIzol(登録商標)試薬(インビトロゲン社)を標準的方法にて用いて分離した。当該全RNAから調製された一本鎖cDNAは、各々の遺伝子に特異的なプライマーで増幅された。定量リアルタイムRT-PCR(qRT-PCR)は、KAPA SYBR System(カパバイオシステム社)とABI PRISM 7500配列検出システム(アプライドバイオシステム社)を用いて、操作用マニュアルに従い行った。遺伝子発現値は、遺伝子産物(the genes of interest)と内部標準コントロール(GAPDH)若しくはU6の間の比率(Ct値の差違)により与えられ、そして引き続いてコントロール細胞における値と一緒に標準化される(相対的発現レベル)。用いられたプライマーとTaqManプローブの情報は表3と表4を参照のこと。表3において、フォワードプライマーを示す左側の欄の塩基配列の配列番号として、上から順番に配列番号22~32を割り振り、リバースプライマーを示す右側の欄の塩基配列の配列番号として、上から順番に33~43を割り振った。また、表4においてTaqManプローブの塩基配列の配列番号として、上から順に配列番号44~48を割り振った。
腫瘍検体は、10%のホルムアルデヒド含有PBSによって固定化し、パラフィン包埋を行い、4μm厚の切片へと薄片化し、アビジン-ビオチン-ペルオキシダーゼ法でNRF2又はME1の免疫組織化学的染色を行った。パラフィンで包埋された腫瘍検体からの切片はキシレンで脱パラフィンを行い、エタノール中で再水和を行った。10mMのクエン酸緩衝液(pH6.0)中での煮沸による抗原の回復の後、当該切片は、内因性のペルオキシダーゼを不活性化するために0.3%過酸化水素含有メタノールで処理した。そして当該切片は、抗NRF2抗体(1/1000希釈)又は抗ME1抗体(1/500希釈)と一緒に4℃で一晩インキュベートした。結合した抗体は、色原体としてのジアミノベンディジン(VECTASTAIN-EluteABC Kit,ベクターラボラトリー社)を用いて可視化し、当該切片は素早くヘマトキシリンにて対比染色した。
NRF2のエクソン2又はKEAP1の全てのコーディング領域を含む遺伝子領域は、KOD-plus(TOYOBO社)を用いたPCRによって増幅した。PCR産物は、ExoSAP-IT(GEヘルスケア社)を用いて精製され、配列解析を行った。プライマーの情報は、上記表3を参照のこと。
遺伝子発現のアレイ解析のために、Agilent4×44K遺伝子発現アレイ(アジレントテクノロジーズ社)を、操作用マニュアルに従い用いた。各々の遺伝子アレイ実験は、二重に行われ、データはGeneSpring(アジレントテクノロジー社)で解析した。
7週齢の雌のBALB/cヌードマウスをオリエンタル酵母工業株式会社から購入し、無菌状態を保った。1×107cellsを含むPBS200μlをマウスの横腹に皮下注射した。1nmolの2本鎖RNA(アンビオン社)と200μlのAteroGene(KOKEN社)の混合物を、腫瘍と皮膚の間の空隙に投与した。マウスに、5mg/kg体重のシスプラチンの腹腔内投与を施した。細胞投与35日間後、マウスを安楽死させ腫瘍を摘出した。全てのマウスに対して行った実験の手順は、東京医科歯科大学の動物保護と利用委員会の承認を受けた。
サブグループ間の差違は、Student's t-testによって検討した。対応する患者に関係する臨床病理学的な変化は、Χ2テスト、又は、フィッシャーの抽出テストによって解析された。生存の解析を行うために、Kaplan-Meierカーブが一変量の予測に基礎付けられた群のために構築され、群間の差違はlog-rank testによって検討した。インビボの実験の結果とA549細胞における細胞生存試験は、変化に対するtwo-way ANOVA(二要因の分散分析)を用いて統計学的有意性を解析した。計算されたP値が0.05未満(<0.05)の場合に統計学的な有意性有りと判断した。
[実施例1] NRF2の転写活性をネガティブ制御しているmiRNAのスクリーニング
NRF2の転写活性をネガティブ制御しているmiRNAを確認するために、我々はルシフェラーゼレポーターシステムを用いてmiRNAライブラリーをスクリーニングした。当該システムは、antioxidative responsible element(ARE)を動かすことでルシフェラーゼの発現の計測が可能である。このレポーターシステムを用いて、当該ARE活性が、HeLa細胞におけるNRF2蛋白レベルに依存して変化することを確認した(図1)。図1は、siRNAを用いたAREレポーターシステムの検証の結果を示す図面である。具体的には、AREルシフェラーゼレポータープラスミド及び内部標準コントロールベクターを用いて、HeLa細胞に共トランフェクションを行い、24時間後、コントロールsiRNA、又は、NRF2-siRNA(a)、若しくは、KEAP1-siRNA(b)でトランフェクションを行った。siRNAでのトランフェクションの48時間後、当該細胞溶解物のSDS-PAGEを行い、各々の抗体での免疫反応を行った(上部パネル)。一方で、ホタル又はウミシイタケルシフェラーゼ活性を測定し、Mock中のARE活性に対するARE活性を縦軸に示した(下部パネル)。バーは、標準偏差(SD)である。
4つの候補miRNA(hsa-miR-507、hsa-miR-634、hsa-miR-450a、及び、hsa-miR-129-5p)のための機能的標的を同定するために、まず、NRF2経路に関連していることが知られている遺伝子が、IPA(Ingenuity Systems Pathway Analysis, Redwood City, CA)(http://www.ingenity.com)を用いて、TargetScanプログラム(http://www.targetscan.org)により予測された標的の中に存在するかを調査した。コンピュータ内での分析により、意外にも、4つの候補miRNA全てが、NRF2それ自体を機能的に標的にしていることを見出した。なぜならば、hsa-miR-450aではない3つのmiRNAのためのシード配列は、NRF2の3’-UTRの中にマップされたからである(hsa-miR-507において2箇所、hsa-miR-634において1箇所、hsa-miR-129-5pにおいて2箇所)(図3(b))。図3(b)は、NRF2の3’UTR配列内における、4種の候補miRNAのシード配列(seed sequence)の相同配列とレポータープラスミドのデザインを示している。hsa-miR-507(2カ所)、hsa-miR-634(1カ所)、及び、hsa-miR-129-5p(2カ所)のシード配列が、NRF2の3’UTRにおいてマップされた。hsa-miR-450aのシード配列はhsa-miR-507と高度に相同している(7シード配列のうち6)から、hsa-miR-450a のシード配列はhsa-miR-507と同じサイトとして示されている。領域1(R1)、領域2(R2)又は変異R2はルシフェラーゼアッセイにおいて用いられた。黒い×印は、各々のシード配列に挿入された変異サイトを示している。
原発性の食道扁平上皮癌患者からの30検体においてqRT-PCR解析による各々のmiRNAのレベルを検討した。図5は、食道がん(食道扁平上皮癌:ESCC)由来の原発腫瘍検体における臨床的な意義とmiRNAの負の調節の関わりに関して検討した図面である。
一つのmiRNAは、一つのがん性経路に貢献する複数の遺伝子をターゲットにすることができるから、当該miRNAはこのがん性経路を伴う腫瘍に対して効果的であると考えられる。そして、当該4つのmiRNAのうちhsa-miR-507について、それがNRF2によって転写制御されている遺伝子を機能的にターゲットとすることができるかどうかを、発現アレイと経路解析を用いて検討し、これらの解析においてhsa-miR-507のトランスフェクションによって抑制されている、8種類のNRF2の転写標的遺伝子を見出した。そしてそれらの中でME1は、NRF2の3’-UTRへの結合を通じhsa-miR-507の直接的な標的であることを確認した(図6)。図6は、hsa-miR-507の標的としてのNRF2の転写標的遺伝子の同定の結果を示す図面である。図6(a)は、hsa-miR-507により負に制御されたNRF2の転写標的遺伝子の検証の結果を示している。hsa-miR-507又はNRF2-siRNAのトランスフェクションにより発現低下した遺伝子のうち、181は、TargetScanプログラムによりhsa-miR-507の標的として予測されたことを示している。図6(b)は、IPAを用いて、181の遺伝子の中で、NRF2の8つの転写標的(ME1,PSMB6,SLC7A11,MGLL,DNAJB5,LGALS8,B4GALNT1,EIF4G2)が、hsa-miR-507の標的とされることが予測されたことを示している。図6(c)は、ME1 3’-UTR中のhsa-miR-507のためのシード配列及びレポータープラスミドのための設計のマッピングを示している。ルシフェラーゼ分析を行うことによって、hsa-miR-507が、ME1 3’-UTRに直接的に結合するかを調べた結果を示している。hsa-miR-507のためのシード配列は、ME1 3’UTR中にマッピングされている。hsa-miR-507(野生型;WT)のためのシード配列又はその突然変異体のゲノム領域を有するレポータープラスミドをルシフェラーゼ分析において用いた。黒色の十字形は変異部位を示している。図6(d)は、レポータープラスミドを用いたルシフェラーゼ分析の結果を示している。レポータープラスミド及び内部標準コントロールベクターでHeLa細胞の共トランスフェクションを行い、24時間後、hsa-miR-507又はコントロールmiRNAでのトランスフェクションを行った結果を示している。miRNAでのトランスフェクションの48時間後、ホタル又はウミシイタケのルシフェラーゼ活性を測定した。コントロールmiRNAでのトランスフェクト細胞のルシフェラーゼ活性に対するルシフェラーゼ活性を縦軸に示す。空ベクターのルシフェラーゼ活性よりWT領域を持つベクターのルシフェラーゼ活性が弱かったが、シード配列中に変異を挿入することにより完全に修復された。バーは標準偏差(SD)である。図6(e)は、miRNAでのトランスフェクト細胞中のME1発現に関するウェスタンブロット分析の結果を示している。hsa-miR-507又はコントロールmiRNAでHeLa細胞のトランスフェクションを行った結果を示している。トランスフェクションの48時間後、細胞溶解物のSDS-PAGEを行い、指示された抗体での免疫反応を行い、hsa-miR-507のトランスフェクションが、実際に、ME1タンパク質に関する発現レベルの低下を引き起こすことを確認した。
(1)hsa-miR-634の投与によるインビトロでの腫瘍の細胞死誘導効果(1)(図9)
図9は、hsa-miR-634の腫瘍細胞に対するアポトーシス誘導効果についてインビトロで検討した結果を示す図面である。hsa-miR-634のインビトロでの腫瘍抑制効果を調べるために、hsa-miR-634を模した二本鎖RNA若しくはコントロールmiRNAをリポフェクション法により子宮頸癌細胞株HeLa細胞に導入し、その後、3日間培養した。導入1、2、3日後、それぞれ1%クリスタルバイオレットを含むPBS溶液で1分間染色し、水洗浄後、2%SDSを含むPBSバッファーにより1時間反応させた。その後、マルチプレートリーダーにより、染色強度を測定することにより、生存細胞の定量を行った。その結果、hsa-miR-634を発現させた細胞では、顕著な細胞増殖抑制および細胞死の誘導が確認された。ここで図9(A)は導入2日後の顕微鏡像であり、HeLa細胞に対して、hsa-miR-634をトランスフェクトして、これを5%CO2・37℃で48時間インキュベートした際の形態変化を顕微鏡写真にて表している。上段がコントロールmiRをトランスフェクトした細胞であり、下段がhsa-miR-634をトランスフェクトした細胞である。右側の写真は左側の写真の拡大図である。図9(B)は経時的な細胞増殖の抑制効果を追った結果を示しており、上記図9(A)のインキュベートを行いつつ、1日後、2日後及び3日後における細胞増殖の傾向を、CV染色による蛍光強度を指標に検討した図面である。Mockのみ用いた細胞群、コントロールmiRをトランスフェクトした細胞群、及び、miR-634をトランスフェクトした細胞群における結果を示している。
hsa-miR-634を模した二本鎖RNA若しくはコントロールmiRNAをリポフェクション法により食道癌細胞株KYSE170に導入した。図10の上段には、このhsa-miR-634とシスプラチン(CDDP)の組み合わせ投与のin vitro実験スケジュールを示した。このスケジュールに記載されているように、hsa-miR-634を模した二本鎖RNA若しくはコントロールmiRNAの添加量は、それぞれ0.2nMであり、シスプラチンを2.5μM添加した群と、5.0μM添加した群についてインキュベートを行い、検証した。インキュベート時間は72時間である。細胞死の誘導効果は、トリパンブルー染色により、細胞死した細胞をカウントし、全細胞数に対する細胞死した細胞数の頻度(%)で評価した。図10の下段のグラフはその結果を示している。縦軸が前記頻度を示している。特に、hsa-miR-634とシスプラチンを同時投与された細胞では、高頻度に細胞死が誘導されていることを表している。これにより、本発明の治療剤とシスプラチンとの併用による相乗的な抗がん効果が明らかになった。
インビボでのhsa-miR-634とシスプラチンの併用効果の検討は、ヌードマウスにKYSE170細胞を皮下注射することによって腫瘍を形成させることにより行った。図11の上段には、hsa-miR-634を模した二本鎖RNA若しくはコントロールmiRNAと、シスプラチン(CDDP)の組み合わせ投与のin vivo実験スケジュールを示した。腫瘍を形成させるためのインキュベート期間は7日間である。インキュベート7日目に、コントロールmiRNA又はhsa-miR-634を、KYSE170細胞によりヌードマウスの皮下に形成された腫瘍の周囲に計5回注射により投与した。加えてマウスにおける、PBS又はシスプラチンの腹腔内投与は、miRNA投与の1回目と4回目に、miRNA投与と同時に計2回行った。KYSE170細胞の注射から21日後にマウスを安楽死させ、腫瘍を摘出した。図11の下段のグラフは、摘出された腫瘍の重量(縦軸)を示している。当該下段のグラフにおいては7個体のPBSが用いられたマウスと、8個体のシスプラチンが用いられたマウスにおける、腫瘍の重量の「平均±SD値」を示している。両群間における顕著な差違がt-testによる解析により明らかになった。すなわち、p=0.0182がシスプラチン処理群でコントロールmiRNA群とhsa-miR-634投与間で認められ、p=0.00813がhsa-miR-634投与群でPBSとシスプラチン間で認められた。
図12(図12-1~4)は、骨肉腫細胞株であるU2OS細胞へのhsa-miR-634の導入による標的遺伝子の発現抑制効果を示している。図12-1~4の各図面の左側のグラフに結果を示したルシフェラーゼアッセイは、前述の「伝統的なルシフェラーゼアッセイ」の手法に従って行った。すなわち、各標的遺伝子の3’非翻訳領域(UTR)をpmirGlo Dual-Luciferase miRNA Target Expression Vector(プロメガ社)のルシフェラーゼ遺伝子の下流に挿入することにより、各々の検討に用いるルシフェラーゼリポータープラスミドを調製した。各々の標的遺伝子は、(a)XIAP (X-linked inhibitor of apoptosis protein:図12-1)、 (b)APIP (APAF1 interacting protein:図12-2)、 (c)OPA1(optic atrophy 1:図12-3)、及び、(d)TFAM(transcription factor A, mitochondrial:図12-4)、である。これらの4種の蛋白をコードする遺伝子のUTRのシード配列を、配列番号52(XIAP)、53(APIP)、54(OPA1)、及び、55(TFAM)にて、配列表において示した。また全ての部位特異性変異は、KOD mutagenesis kit(TOYOBO社)を用いて行った。また、各グラフの「mt」は、各々の変異挿入プラスミド(mutant:mt)を示している。
Claims (20)
- ヒト検体における、hsa-miR-507、hsa-miR-634、hsa-miR-450a、hsa-miR-129-5p、hsa-miR-639、hsa-miR-337、hsa-miR-153、及び、hsa-miR-556、からなる群のマイクロRNAから選ばれる1種又は2種以上を定量して、当該定量値の低下を指標として、腫瘍の悪性度、NRF2の活性化、又は、がん患者の予後、を鑑別することを特徴とする、マイクロRNAの測定方法。
- ヒト検体における、hsa-miR-507、hsa-miR-634、hsa-miR-450a、及び、hsa-miR-129-5p、からなる群のマイクロRNAから選ばれる1種又は2種以上を定量して、当該定量値の低下を指標として、腫瘍の悪性度、NRF2の活性化、又は、がん患者の予後、を鑑別することを特徴とする、マイクロRNAの測定方法。
- ヒト検体における、hsa-miR-26a、hsa-miR-17-3p、hsa-miR-190、hsa-miR-567、hsa-miR-125b、hsa-miR-125a、hsa-miR-432*、及び、hsa-miR-29、からなる群のマイクロRNAから選ばれる1種又は2種以上を定量して、当該定量値の上昇を指標として、腫瘍の悪性度、NRF2の活性化、又は、患者の予後、を鑑別することを特徴とする、マイクロRNAの測定方法。
- さらに腫瘍におけるNRF2遺伝子の変異、KEAP1遺伝子の変異、及び、p62蛋白の蓄積、からなる群から選ばれる1~3種を検出して、これらの遺伝子変異を指標として加入することを特徴とする、請求項1~3のいずれかに記載のマイクロRNAの測定方法。
- さらに腫瘍におけるXIAP、APIP、OPA1、及び、TFAMからなる群から選ばれる1~4種の遺伝子の発現の亢進又は安定化を検出して、これらを指標として加入することを特徴とする、請求項1~4のいずれかに記載のマイクロRNAの測定方法。
- 腫瘍の悪性度の増大、NRF2の活性化、又は、がん患者の予後の悪化、に向かうことを示す個別指標の総数をスコア指標として、腫瘍の悪性度、NRF2の活性化、又は、患者の予後、を鑑別することを特徴とする、請求項1~5のいずれかに記載のマイクロRNAの測定方法。
- ヒト検体は腫瘍検体又は血液検体であることを特徴とする、請求項1~6のいずれかに記載のマイクロRNAの測定方法。
- 食道がん、肺がん、乳がん、口腔がん、胃がん、大腸がん、肝臓がん、子宮がん、若しくは、皮膚がんにおける、腫瘍の悪性度、NRF2の活性化、又は、患者の予後、を鑑別することを特徴とする、請求項1~7のいずれかに記載のマイクロRNAの測定方法。
- hsa-miR-507の塩基配列である配列番号1、hsa-miR-634の塩基配列である配列番号2、hsa-miR-450aの塩基配列である配列番号3、hsa-miR-129-5pの塩基配列である配列番号4、hsa-miR-639の塩基配列である塩基配列5、hsa-miR-337の塩基配列である配列番号6、hsa-miR-153の塩基配列である配列番号7、及び、hsa-miR-556の塩基配列である配列番号8、からなる群の塩基配列から選ばれる1種又は2種以上を含む核酸からなるがん治療剤。
- hsa-miR-507の塩基配列である配列番号1、hsa-miR-634の塩基配列である配列番号2、hsa-miR-450aの塩基配列である配列番号3、hsa-miR-129-5pの塩基配列である配列番号4、からなる群の塩基配列から選ばれる1種又は2種以上を含む核酸からなるがん治療剤。
- 核酸は二本鎖RNAであることを特徴とする、請求項9又は10に記載のがん治療剤。
- がん細胞に対するストレスの付与手段と組み合わせて用いることを特徴とする、請求項9~11のいずれかに記載のがん治療剤。
- がん細胞に対するストレスの付与手段は、抗癌剤、放射線療法、外科的手術、又は、生検、であることを特徴とする、請求項9~12のいずれかに記載のがん治療剤。
- がん細胞は、NRF2が亢進又は安定化したがん細胞である、請求項9~13のいずれかに記載のがん治療剤。
- がん細胞は、NRF2に加えて、XIAP、APIP、OPA1、及び、TFAMからなる群から選ばれる1~4種が亢進又は安定化したがん細胞である、請求項14に記載のがん治療剤。
- がん治療剤の本質成分は、hsa-miR-634の塩基配列である配列番号2を含む核酸からなる、請求項15に記載のがん治療剤。
- (1)プラチナ製剤、及び、(2)hsa-miR-507の塩基配列である配列番号1を含む核酸又はhsa-miR-634の塩基配列である配列番号2を含む核酸、を組み合わせてなる、がん治療剤。
- プラチナ製剤はシスプラチンである、請求項17に記載のがん治療剤。
- がん細胞は、食道がん、肺がん、乳がん、口腔がん、胃がん、大腸がん、肝臓がん、子宮がん、骨肉腫、又は、皮膚がんのがん細胞であることを特徴とする、請求項9~18のいずれかに記載のがん治療剤。
- 請求項9~19のいずれかに記載のがん治療剤を含有することを特徴とする、がん治療のための医薬組成物。
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US20190367915A1 (en) | 2019-12-05 |
US9994843B2 (en) | 2018-06-12 |
EP2963125A4 (en) | 2016-11-02 |
DK2963125T3 (da) | 2020-10-12 |
JPWO2014126233A1 (ja) | 2017-02-02 |
EP2963125A1 (en) | 2016-01-06 |
JP6587142B2 (ja) | 2019-10-09 |
EP2963125B1 (en) | 2020-09-30 |
ES2828510T3 (es) | 2021-05-26 |
US20160053257A1 (en) | 2016-02-25 |
JP2018143248A (ja) | 2018-09-20 |
JP6745071B2 (ja) | 2020-08-26 |
US10876115B2 (en) | 2020-12-29 |
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