JP6073775B2 - MicroRNA having glioma formation inhibitory action - Google Patents

Description

  The present invention relates to a therapeutic agent for brain tumor using microRNA. The present invention also relates to microRNA for identifying a therapeutic agent for brain tumor and use thereof.

  Glioma (glioma) is a kind of malignant tumor and is a common primary brain tumor in Japanese. Among them, the treatment of malignant glioma is based on surgical resection, but radiotherapy and chemotherapy are used in combination depending on the degree of malignancy. Specifically, malignant gliomas are classified into four grades (grades I-VI) according to WHO criteria, for example, the least aggressive WHO grade I, such as ciliary astrocytoma that occurs in the cerebellum of children. Surgery only treatment is selected. In addition, WHO grade II or higher glioma often recurs only by excision by surgery, and therefore, chemotherapy or immunotherapy with radiation or an anticancer agent is used in combination. In particular, WHO grade VI glioma is called glioblastoma and is one of the most malignant tumors among brain tumors. Glioblastoma is a disease that is difficult to treat, and because surgery alone recurs within months, postoperative radiation therapy and chemotherapy are essential.

  By the way, if it is cancer or tumor of tissues other than the brain, it is possible to remove and cure the cancer or tumor by treatment such as surgical therapy, radiation therapy, and chemotherapy. However, because glioma is a brain tumor, applying either of these therapies will cause significant damage to normal cells and the function of the brain itself if it tries to completely remove the wet tumor. For this reason, the average survival time due to surgery alone is short, and even if radiation therapy or chemotherapy is used as an adjunct therapy for the residual tumor after surgery, the effect is often transient. Therefore, since glioma is a cancer that is extremely difficult to treat, there is a need for a therapeutic agent that has little adverse effect on brain function and suppresses glioma growth.

  The present invention has been made in view of such circumstances, and an object of the present invention is to provide a therapeutic agent that has little adverse effect on brain function in brain tumor patients and suppresses the growth of brain tumors.

  Another object of the present invention is to provide a microRNA used for the therapeutic agent.

  The present inventors have obtained the knowledge that human and mouse GIC (glioblastoma-initiating cells) and microRNAs (miRNA, miR) whose expression is suppressed in human glioblastoma tissue exist. The possibility that RNA acts as a tumor suppressor gene was obtained. In addition, the present inventors have reported that overexpression of this microRNA not only suppresses GIC growth and motility, but also prevents their tumor formation, suggesting that this microRNA suppresses glioma growth. We focused on the possibility of becoming a therapeutic drug.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors indirectly inhibited tumor formation by suppressing the expression of the target protein (PLAT: plasminogen activator, tissue). It was found that it can be suppressed.

  Specifically, according to the first main aspect of the present invention, there is provided a therapeutic agent for brain tumor having miR340 or a precursor thereof, or a vector expressing them.

  According to such a configuration, it is possible to suppress the growth of the brain tumor by simply administering the drug without adversely affecting the brain function of the patient.

  Moreover, according to one Embodiment of this invention, said miR340 has a sequence shown by sequence ID number 1.

  According to another embodiment of the present invention, the miR340 has a sequence represented by SEQ ID NO: 2.

  Furthermore, in one embodiment of the present invention, the brain tumor is glioma.

  According to another embodiment of the present invention, the miR340 treats the brain tumor by reducing the expression level of PLAT.

  According to a second main aspect of the present invention, there is provided a pharmaceutical composition for treating a brain tumor, comprising the above therapeutic agent and a pharmaceutically acceptable carrier.

  In addition, according to a third main aspect of the present invention, there is provided use of miR340 or a precursor thereof, or a vector expressing them for the manufacture of a medicament for treating a brain tumor in a mammal.

  Furthermore, according to a fourth main aspect of the present invention, there is provided a pharmaceutical composition for reducing PLAT expression level in a mammal, comprising miR340 or a precursor thereof, or a vector expressing them. Certain compositions are provided.

  According to a fifth main aspect of the present invention, there is provided a method for identifying a therapeutic agent for a brain tumor, comprising the step of bringing a test agent into contact with the brain tumor cell, and measuring the expression level of miR340 in the brain tumor cell. And an increase in the expression level of miR340 in the brain tumor cells as compared to control cells indicates that the test agent is a therapeutic agent for brain tumors.

  According to an embodiment of the present invention, the method further comprises a step of measuring the expression level of PLAT in the brain tumor cell, wherein the expression level of PLAT in the brain tumor cell is compared with the control cell. A decrease indicates that the test agent is a therapeutic agent for brain tumors.

  It should be noted that the features of the present invention other than those described above and the remarkable actions and effects will become clear to those skilled in the art with reference to the following embodiments and drawings of the present invention.

FIG. 1 is a graph showing that miR-340 expression is reduced in mouse and human GIC in one embodiment of the invention. FIG. 2 is a photograph and graph showing that miR-340 overexpression inhibits cell proliferation, invasion and migration in one embodiment of the invention. FIG. 3 is a photograph and graph showing that miR-340 overexpression inhibits tumor formation in vivo in one embodiment of the invention. FIG. 4 is a photograph and graph showing that PLAT is a target of miR-340 in one embodiment of the present invention.

Hereinafter, an embodiment and an example according to the present invention will be described with reference to the drawings.
In one embodiment according to the present invention, miR340 or a precursor thereof, or a vector expressing them is used as a brain tumor therapeutic agent.

  Here, in this embodiment, “miR340” or “miR-340” refers to one type of microRNA (miRNA, miR). miRNA is a protein non-translated RNA (small non-coding RNA) of about 20 to about 25 bases, and it is suggested that there are about 1000 or more types in humans.

  miRNA attracts attention as a molecule that suppresses the expression of various genes in vivo. A region of each miRNA gene exists on the genome and is transcribed by RNA polymerase II to form a miRNA initial transcript of about several hundred bases. The miRNA early transcript is processed by two RNase III enzymes called Drosha in the nucleus and Dicer in the cytoplasm to form mature miRNA. It is known that a mature miRNA interacts with mRNAs of a plurality of target genes having complementary sequences and cooperates with a regulatory protein complex (RNA-induced silencing complex: RISC) to suppress gene expression.

  In the therapeutic agent according to the present invention, “miR340” can use miRNAs of SEQ ID NOs: 1 and 2 as biomarkers for detecting brain tumor diseases, particularly glioma. In gene expression regulation by miRNA, 8 nucleotides on the 5 ′ side of miRNA constituting RISC are called seed sequence, and this sequence recognizes and binds to the sequence of 3′-UTR region of target mRNA. Suppresses translation of Accordingly, in the present invention, the full-length sequence of miR340, SEQ ID NO: 1, and SEQ ID NO: 2, which is 8 nucleotides on the 5 'side of SEQ ID NO: 1, can be used in the same manner.

  In the therapeutic agent according to the present invention, miR340 may be a mature form or a precursor (pre-miRNA).

  In the present invention, PLAT is plasminogen activator, tissue, which promotes degradation of extracellular matrix (fibrin etc.), cell motility and cell proliferation through activation of functional factors including plasmin, Works in migration and cancer metastasis.

  The molecular mechanism in the present invention is as follows. That is, miR340 in tumor cells is expected to be associated with tumorigenesis and some of its functions are performed via PLAT. Specifically, miR340 regulates PLAT expression, which is associated with tumorigenesis. It is considered that PLAT expression decreases due to increased miR340 expression, and tumor formation is suppressed.

  The pharmaceutical composition of the present invention comprises a therapeutic agent mixed with a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention may also contain at least one therapeutic agent encapsulated by liposomes or a pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carrier include water, buffered water, physiological saline, 0.4% by weight physiological saline, 0.3% by weight glycine, hyaluronic acid and the like.

  The pharmaceutical composition of the present invention may comprise conventional pharmaceutical excipients and / or additives. Examples of the excipient include a stabilizer, an antioxidant, a molar osmotic pressure adjusting agent, a buffering agent, and a pH adjusting agent. Examples of additives include physiologically biocompatible buffers (for example, tromethamine hydrochloride), chelating agents (for example, DTPA and DTPA-bisamide), calcium chelate complexes (for example, calcium DTPA, CaNaDTPA-bisamide) The additive etc. can be mentioned.

  In the present invention, the sample for measuring the expression of miRNA is not particularly limited as long as it is a sample collected from a brain tumor patient and may contain brain tumor cells. For example, blood samples (peripheral blood, plasma, serum, etc.), cerebrospinal fluid, urine, lymph, saliva, and other body fluids can be used. Such a sample can also use an extracted component derived from a pathological tissue collected by biopsy. The sample collection method can be appropriately selected according to the type of sample and the like, and RNA can be prepared from the sample by a generally known method.

  Moreover, the expression level of miRNA can be measured according to known gene expression level measurement methods such as the miRNA microarray method, the Northern blot method, and the RT-PCR method using a probe or primer that specifically hybridizes with miRNA.

  Such probes and primers are known probes so as to specifically hybridize with a continuous base sequence of at least 15 base lengths to all base lengths, preferably 18 base lengths to all base lengths in the base sequence of miRNA. Alternatively, it is designed as a polynucleotide having the above-mentioned base lengths according to the primer design method.

  The probe need not be completely complementary as long as it specifically hybridizes with each miRNA. Such a polynucleotide preferably has a nucleotide sequence of 70% or more, preferably 80% or more compared to a polynucleotide consisting of at least 15 or more consecutive nucleotide sequences in the nucleotide sequence of each miRNA or a complementary polynucleotide thereof. The polynucleotide is preferably 90% or more, more preferably 95% or more, particularly preferably 98% or more.

  The probe or primer may be labeled with a commonly used radioactive substance, fluorescent substance, chemiluminescent substance, or enzyme so that miRNA can be easily detected.

  In the present invention, it is possible to identify a therapeutic agent for a brain tumor using the expression level of miRNA measured as described above. In this case, a test agent that is a therapeutic drug candidate is brought into contact with a brain tumor cell, and then the expression level of miR340 is measured. If the expression level of miR340 is increased from the normal state, the test agent is used as a brain tumor. Indicates that it is likely to be used as a therapeutic agent.

  Similarly, the PLAT expression level of brain tumor cells is measured. If the PLAT expression level is lower than the normal state, it indicates that the test agent is likely to be used as a therapeutic agent for brain tumors.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
(Experimental methods and materials)
Hereinafter, experimental methods and materials used in the present invention will be described. In the present embodiment, the following experimental method is used, but the same result can be obtained by using other experimental methods.

1. Cell Culture Seven human glioma samples (glioblastoma line E1-5, anaplastic oligodendroma (AO) line, and diffuse astrocytoma (DA) line) were used. Tumor samples were separated using the Papain dissociation system and the dissociated cells were human basic fibroblast growth factor (bFGF; 10 nM), human epidermal growth factor (EGF; 10 nM), heparin (5 μM), N2 supplement. (Wako) Serum-free Dulbecco's Modified Eagle's Medium (DMEM) / Ham'sF-12 containing 10 μg / ml insulin (Wako), GlutaMAX ™ supplement, 100 units / ml penicillin G, and 100 μg / ml streptomycin. Wako) was cultured as a suspension culture cell mass. GIC was cultured with poly-D-lysine (PDL, 15 μg / ml, Sigma) and fibronectin (1 μg / ml) and seeded on 8-well chamber slides (Nunc) for immunostaining. Human glioma cell lines (U251 and U87) and mouse GIC NSCL61 were cultured according to the method described in Hide and Kondo, Cancer Res, 2009. Human neural stem cells (derived from H9 human embryonic stem cells) were cultured. In all experiments, the cells were maintained at 37 ° C. and humidity 5% CO ₂ /95% air.

2. Microarray Hybridization and Data Processing Total RNA was extracted from cell lines and glioma tissues using TRIzol® Plus RNA Purification System (Invitrogen). MicroRNA microarrays are manufactured by Agilent Technologies, labeled with 100 ng of total RNA using microRNA Microarray Kit protocol, hybridized, and Human miRNA Microarray Release 16.0, or Mouse miRNA Micro0 Micro16 Micro16 MicroRNA Micro. Hybridization signals were detected with a DNA microarray scanner (Agilent Technologies), and the scanned images were analyzed using an Agilent feature extraction software. Total RNA was amplified for gene expression analysis and labeled with Cyanine 3 (Cy3) using an Agilent Low Input Quick Amp Labeling Kit-color (Agilent Technologies). Labeled cRNA was fragmented and hybridized to Agilent Human GE 8x60K Microarray. After washing, the microarray was scanned using an Agilent DNA microarray scanner. The intensity of each scanned feature was quantified using an Agilent feature extraction software that performs a background removal method. Normalization was performed using Agilent GeneSpring GX version 11.0.2. After normalization, hierarchical samples were clustered with expressed genes (DEGs) using the Euclidean distance and the mean binding method (Agilent GeneSpring GX). GenMAPP 2.1 (http://www.genmapp.org/) and ConPath® navigator were used for pathway analysis.

3. Real-time PCR
Total RNA was extracted from cell lines and glioma tissues using Isogen (Nippon gene, Japan) and reverse transcribed using MMLV RT (Invitrogen). The expression of miR-340 was analyzed using TaqMan small RNA assays (Applied Biosystems) from the MiniOpticon Real Time PCR System (BIO-RAD). PLAT expression was analyzed using the MiniOpticon Real Time PCR System (BIO-RAD) SYBR Green (Roche). The following oligonucleotide DNA primers were synthesized:

5 'primer for plat: 5'-CCATTTTGGAAGTTTTCAGG-3'
3 ′ primer: 5′-GCTTTTATTTTGTGGTCCT-3 ′

4). Transfection miR-340 precursor or control miR was overexpressed in glioma cells using miRNA lenticular particles (pEZX-MR03, Genecopoia). Glioma cells were cultured with recombinant virus for 12 hours at 37 ° C. and cultured for 3 days in the presence of puromycin (2 μg / ml, Sigma). Infection efficiency was confirmed by GFP expression. The expression of miR-340 was confirmed by RT-PCR after selection. In some experiments, glioma cells were transfected with the miR-340 expression vector (pBApoCMV-Neo-miR-340, Takara) using Nucleofector device (Lonza) and in the presence of neomycin (300 μg / ml, Sigma). Cultured for 10 days. After selection, cells were immunolabeled with goat anti-rabbit IgG-Alexa488 (1: 500; molecular probe), then immunolabeled with rabbit anti-PLAT antibody (1:50, Sigma), and counterstained with DAPI (1 μg / ml) did.

5. Cell proliferation Glioma cells (1 × 10 ⁴ cells / well) were transfected with control or miR-340 expression vectors and cultured for 72 hours. Cells were collected on days 1, 2 and 3 and counted with a hemocytometer. BrdU staining was performed according to Kondo and Raff, EMBO J, 2000.

6). Cell Invasion and Migration Analysis Cell invasion and migration were assessed using BioCoat Matrigel Invasion Chamber (Becton-Dickinson) and BioCoat Tumor Invasion System (Becton-Dickinson), respectively. Transfected glioma cells (5-10 × 10 ⁴ ) were resuspended in DMEM / Ham's F-12 and 1 mg / ml bovine serum albumin (BSA; Sigma) and placed in the upper chamber of the well. 750 μl of 10% FCS medium was placed in the lower chamber. After 12 hours of culture, cells on the upper membrane surface were mechanically removed. Wet or migrated cells under the membrane were fixed, stained with 0.1% crystal violet, and counted microscopically in 5 or more randomly selected areas.

7). Intracranial cell transplantation into the brain of NOD / SCID mice Control cells and miR-340 expressing cells were suspended in ⁵ μl (1 × 10 ⁵ ) medium and anesthetized with 10% pentobarbital. Injection into the brain of SCID mice. The stereotaxic coordinates of the injection site are 2 mm forward from the lambda, 2 mm outside the sagittal suture, and 5 mm deep.

8). Brain fixation and histopathology Dissected mouse brains were fixed in 4% paraformaldehyde overnight at 4 ° C. After fixation, the brain was frozen with 12-18% sucrose / PBS, embedded in Tissue-Tek OCT compound (Miles, Elkhart, IN), or embedded in paraffin. Coronal sections (thickness 6-10 μm) were prepared from cerebral cortex and stained with hematoxylin-eosin (H & E) following standard techniques.

9. Vector structure Full-length mouse plats were prepared by RT-PCR and KOD-Plus-Ver. Amplification from human glioma cDNA using 2 polymerase (TOYOBO, Japan), pCDNA3-2xFLAG-c vector (pCDNA3-hPLAT-2xFLAG-c) and p3xFLAG-CMV10 vector (p3xFLAG-CMV10- hPLAT). The following oligonucleotide DNA was synthesized for full length human plat:
5 ′ primer: 5′-TGAATTCGCCACCCATGACAGAATACAAGCTTGTGTG-3 ′
3 ′ primer: 5′-ACTCGAGTCAGGACAGCACACATTTGCAG-3 ′
In order to knock down human plats, their hairpin sequences were inserted into a psiRNA-h7SKhygro G1 expression vector (InvivoGen) to create psiRNA-h7SKhygro-platsh. The siRNA target sequence is 5′-GAATTCGATGATGACTACT-3 ′. To construct the firefly luciferase-human PLAT 3′UTR expression vector, human plat 3 ′ genomic DNA was amplified using KOD and DNA polymerase and cloned into the pT7Blue-2 vector (Novagen). Plat3′UTR genomic DNA and firefly luciferase cDNA were inserted into the pcDNA3.1-hyg vector to obtain pcDNA3.1-hyg-Luc-PLAT 3′UTR. The following oligonucleotide DNA was synthesized for the full-length human plat 3′UTR:
5 ′ primer: 5′-CTCTAGACCAGGAACACCCCACTCCTC-3 ′
3 'primer: 5'-ACTCGAGCAGAAGTCAATTAAGTCCAAAC-3'
The nucleotide sequences of the cloned cDNA and genomic DNA were confirmed using BigDye Terminator Kit version 3.1 (Applied Biosystems) and ABI sequencer model 3130xl (Applied Biosystems).

10. Luciferase assay E3 was transfected into pEF-Rluc, pcDNA3.1-hyg-Luc-PLAT3′UTR, and any control vector, or pBApoCMV-Neo-miR-340, and their luciferase activity was measured.

11. Statistical analysis The distribution of each continuous variable was summarized by its mean, standard deviation and range. The distribution of each categorical variable was summarized by its frequency and percentage. Continuous variables were compared between treatment groups by the Mann-Whitney test. The Mann-Whitney test is performed when comparing two paired groups. The Kaplan-Meier curve was used to estimate the unadjusted time for the event variable. The log rank test was used to compare each time and event variable between groups. P values less than 0.05 (two-sided) were considered statistically significant. All statistical analyzes were performed using Statmate. Error bars indicate SD.

(Experimental result)
The experimental results will be described below with reference to the drawings.

  FIG. 1 is a graph showing that miR-340 expression is decreased in mouse and human GIC. (A) shows the results of hierarchical cluster analysis of miRNA expressed in mouse (left) and human (right) GIC, control cells, AO, DA, and human glioma cell lines (U87 and U251). (B) represents changes in miR-340 expression of human GIC (E1-4, E6), AO, DA, human glioma cell line, and NSCL61 when compared to normal NSCs. (C) is the relative ratio of miR-340 expression between GBM tissue and normal brain tissue. Mean ± SEM from 3 or more experiments. D. It is. In all graphs, * P <0.05.

FIG. 2 is a graph showing that miR-340 overexpression inhibits cell proliferation, invasion and migration. (A) Fluorescence images of control (top) and miR-340 overexpressing hGIC (bottom) (scale is 200 μm). (B) is a graph showing the ratio of BrdU positive cells to miR-340 overexpressing cells in the control and the mean ± S.D. Of 5 experiments. D. As shown.
(C) is a graph showing the relative infiltration ratio between miR-340 overexpressing cells and control cells, and the mean ± S.D. Of 9 experiments. D. As shown. (D) is a graph showing the relative cell migration ratio between miR-340 overexpressing cells and control cells, and the mean ± S.D. Of 9 experiments. D. As shown. In both graphs, ** P <0.01 and *** P <0001.

  FIG. 3 is a graph showing that miR-340 overexpression inhibits tumor formation in vivo. (A) H & E staining of whole brain slice (left) and injection area (right) cut 5 weeks after injection of hGIC (E3 control) and miR-340 overexpressing cells (E3 miR-340). It is. The scale is 1 mm for the left photo and 100 μm for the right photo. (B) and (c) are photographs showing wet regions (b) and mitotic cells (arrow c) in brain slices stained with H & E injected with hGIC. The scale (b) is 50 μm and (c) is 20 μm. (D) is a survival curve of mice injected with hGIC (black dotted line) and miR-340 expressing cells (red solid line).

  FIG. 4 is a graph showing that PLAT is a target of miR-340. (A) is a photograph immunolabeled for miR-340 overexpressing GIC and their parental cells PLAT (green). All nuclei were counterstained with DAPI (blue). The scale is 25 μm. (B) is a graph showing the relative luciferase activity between miR-340 overexpressing hGIC and control cells. Mean of 4 experiments ± S. D. Indicates. (C) is a graph showing PLAT expression in the control brain and GBM. Mean of three or more experiments ± S. D. Indicates. (D) is a graph showing cell growth of control and miR-340 overexpressing cells. Mean of 4 experiments ± S. D. As shown. (E) is a graph showing the ratio of BrdU positive cells in the control and PLAT shRNA-expressing GIC. D. As shown. (F) and (g) are graphs showing the ratio of relative wet (f) and migration (g) between PLAT shRNA-expressing GIC and control cells, the mean ± S.D. Of 5 experiments. D. As shown. (H) is a scheme showing the relationship between miR-340 function and PLAT in glioma development. In either case, * P <0.05 and ** P <0.01.

  In addition, it cannot be overemphasized that this invention can be deform | transformed variously, It is not limited to one Embodiment mentioned above, A various deformation | transformation is possible in the range which does not change the summary of invention.

Claims (5)

  A pharmaceutical composition for reducing the expression level of PLAT in a mammal, comprising miR340 or a precursor thereof, or a vector that expresses them.   The composition according to claim 1, wherein the miR340 has a sequence represented by SEQ ID NO: 1.   The composition according to claim 1, wherein the miR340 has a sequence represented by SEQ ID NO: 2.   Use of miR340 or a precursor thereof, or a vector that expresses them for the manufacture of a medicament for reducing the expression level of PLAT in a mammal. A method for identifying a therapeutic agent for glioma or glioblastoma comprising the steps of:
Contacting the test agent with glioma or glioblastoma cells;
And a step of measuring the expression level of PLAT of the glioma or glioblastoma cells, compared to control cells, decreased expression of PLAT of the glioma or glioblastoma cells is, the test agent A method which indicates that it is a therapeutic agent for glioma or glioblastoma .

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