KR102320430B1 - Use of miR-135b, miR-135a and their target gene AMOTL2 for medulloblastoma - Google Patents

Abstract

The present invention provides a pharmaceutical for the prevention or treatment of medulloblastoma comprising miR-135b and miR-135a for predicting the possibility of metastasis or recurrence of medulloblastoma, the miRNA target gene AMOTL2 for predicting the prognosis, and an inhibitor of the miRNA as active ingredients When using the biomarker for predicting metastasis or recurrence of medulloblastoma and the biomarker for predicting the prognosis according to the present invention as a pharmaceutical composition, etc., the expression level of miR-135b or miR-135a in treating an incurable medulloblastoma patient By measuring , the possibility of tumor metastasis and recurrence can be predicted, and the prognosis of a patient can be predicted by measuring the expression level of AMOTL2, a target gene of the miRNA. Furthermore, by artificially suppressing the expression of the miRNA to induce an increase in the expression of AMOTL2, it is expected to be able to effectively prevent and treat medulloblastoma.

Description

{Use of miR-135b, miR-135a and their target gene AMOTL2 for medulloblastoma}

The present invention relates to a pharmaceutical composition for preventing or treating medulloblastoma, comprising the miRNA for predicting the possibility of metastasis or recurrence of medulloblastoma, the use of the miRNA target gene AMOTL2 for predicting the prognosis, and an inhibitor of the miRNA as an active ingredient, etc. will be.

Brain tumors contain a small subpopulation of cells with stem cell characteristics, which play an important role in tumor development, progression, metastasis and recurrence. In addition, these subpopulation cells have the ability to form spheroids and can be said to be an enrichment of cancer stem cells (CSCs), also called tumor initiating cells (TIC). They are known to be capable of differentiation into self-renewal and heterogeneous cancer cell lineages. Studies on such cells have been reported in medulloblastoma, the most common malignant pediatric brain tumor.

With advances in surgery and adjuvant therapies, the cure rate for medulloblastoma is increasing. In particular, the recent 5-year survival rate of medulloblastoma patients has reached 75%. However, the remaining 25% who do not respond to conventional therapy eventually worsen again due to recurrence and metastasis. In addition, the survivors are not free from the serious side effects of chemotherapy and the sequelae of long-term radiotherapy. Therefore, a better understanding of the molecular and cell biology of medulloblastoma is needed to identify the causes of treatment resistance and develop safe and effective treatment methods.

The clinical complexity of many brain tumors is related to the central nervous system (CNS)-specific microenvironment and intracellular/intercellular interactions. This contributes to making brain tumors a medical challenge.

Extracellular vesicles (EVs) are one of the forms of cell interaction and can provide an environment for the development, metastasis and recurrence of cancer. Tumor-secreting EVs containing exosomes play a pivotal role in communication between tumor cells and stromal cells in local and remote microenvironments. EVs contain selected microRNAs (miRNAs) that can modulate biological functions such as cell proliferation, differentiation, apoptosis, metabolism and drug resistance. Through previous studies, it was found that cancer stem cell-derived extracellular vesicles act as mediators that transmit genetic information and create a microenvironment favorable for cancer development, and that they contain tumor cell-specific miRNAs.

Although molecular genetic studies of medulloblastoma are currently extensive and continue to make progress, the study of the extracellular endoplasmic reticulum of medulloblastoma remains an area of research to be further explored. The major EVs-miRNAs and their biological roles in brain tumor spheroid-forming cells (BTSCs) of medulloblastoma have not yet been elucidated.

J. Wu, Z. Qu, Z. W. Fei, J. H. Wu, C.P. Jiang, Role of stem cell-derived exosomes in cancer, Oncol Lett 13(5) (2017) 2855-2866.

The present invention has been devised to solve the above problems, and an object of the present invention is to predict the possibility of metastasis or recurrence of medulloblastoma, including any one or more selected from the group consisting of miR-135b and miR-135a as an active ingredient To provide a biomarker composition.

Another object of the present invention is to provide a biomarker composition for predicting the prognosis of a medulloblastoma patient, comprising the AMOTL2 gene as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating medulloblastoma, comprising an inhibitor for at least one selected from the group consisting of miR-135b and miR-135a as an active ingredient.

However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description. will be.

In order to achieve the object of the present invention as described above, the present invention provides a biomarker composition for predicting metastasis or recurrence of medulloblastoma, comprising as an active ingredient any one or more selected from the group consisting of miR-135b and miR-135a. to provide.

In addition, the present invention provides a composition for predicting the possibility of metastasis or recurrence of medulloblastoma, comprising an agent for measuring the expression level of any one or more selected from the group consisting of miR-135b and miR-135a.

Furthermore, the present invention provides a kit for predicting the possibility of metastasis or recurrence of medulloblastoma, comprising an agent for measuring the expression level of any one or more selected from the group consisting of miR-135b and miR-135a.

In one embodiment of the present invention, the miR-135b or miR-135a is specifically in brain tumor spheroid-forming cells of medulloblastoma or extracellular vesicles derived therefrom. The expression may be increased, but is not limited thereto.

In another embodiment of the present invention, the miR-135b or miR-135a may inhibit the expression of angiomotin-like 2 (AMOTL2) gene, but is not limited thereto.

In another embodiment of the present invention, the agent may include one or more selected from the group consisting of antisense oligonucleotides, primer sets, probes, and antibodies that specifically bind to miR-135b or miR-135a. However, the present invention is not limited thereto.

In another embodiment of the present invention, the kit may be an RT-PCR kit or a DNA chip, but is not limited thereto.

In addition, the present invention provides a biomarker composition for predicting the prognosis of a medulloblastoma patient, comprising the AMOTL2 gene as an active ingredient.

Furthermore, the present invention provides a composition for predicting the prognosis of a medulloblastoma patient, comprising an agent for measuring the expression level of the AMOTL2 gene.

In addition, the present invention provides a kit for predicting the prognosis of a medulloblastoma patient, comprising an agent for measuring the expression level of the AMOTL2 gene.

In one embodiment of the present invention, the medulloblastoma may be a group 3 medulloblastoma or a group 4 medulloblastoma, but is not limited thereto.

In another embodiment of the present invention, the AMOTL2 gene may be specifically reduced in expression in medulloblastoma brain tumor spheroid-forming cells, but is not limited thereto.

In another embodiment of the present invention, the expression of the AMOTL2 gene may be suppressed by any one or more selected from the group consisting of miR-135b and miR-135a, but is not limited thereto.

In another embodiment of the present invention, the agent may include one or more selected from the group consisting of antisense oligonucleotides, primer sets, probes, and antibodies that specifically bind to the AMOTL2 gene, but is limited thereto it is not

In addition, the present invention provides a pharmaceutical composition for preventing or treating medulloblastoma, comprising an inhibitor for at least one selected from the group consisting of miR-135b and miR-135a as an active ingredient.

Furthermore, the present invention provides a method for preventing or treating medulloblastoma, comprising administering to a subject an inhibitor for at least one selected from the group consisting of miR-135b and miR-135a.

In addition, the present invention provides the use of an inhibitor for any one or more selected from the group consisting of miR-135b and miR-135a for preventing or treating medulloblastoma.

In one embodiment of the present invention, the miR-135b or miR-135a may be derived from brain tumor spheroid-forming cells of medulloblastoma or extracellular vesicles derived therefrom, but is not limited thereto.

In another embodiment of the present invention, the composition may inhibit the formation of tumor spheres, but is not limited thereto.

In another embodiment of the present invention, the composition may reduce the viability or regeneration of medulloblastoma or inhibit recurrence, but is not limited thereto.

In another embodiment of the present invention, the composition may increase the expression of the AMOTL2 gene, but is not limited thereto.

In another embodiment of the present invention, the inhibitor is an antisense oligonucleotide complementary to miR-135b or miR-135a, double-stranded siRNA (small interfering RNA), shRNA (short hairpin RNA) and a ribozyme consisting of It may be one or more selected from the group, but is not limited thereto.

In another embodiment of the present invention, the antisense oligonucleotide is ribonucleotide, deoxyribonucleotide, 2'-O-modified oligonucleotide, phosphorothioate-backbone deoxyribonucleotide, PNA (peptide nucleic acid) or LNA (locked nucleic acid) acid), but is not limited thereto.

By using the biomarker for predicting the metastasis or recurrence of medulloblastoma and the biomarker for predicting the prognosis according to the present invention, tumor metastasis by measuring the expression level of miR-135b or miR-135a in treating an incurable medulloblastoma patient And the possibility of recurrence can be predicted, and the prognosis of the patient can be predicted by measuring the expression level of AMOTL2, which is the target gene of the miRNA. Furthermore, by artificially suppressing the expression of the miRNA to induce an increase in the expression of AMOTL2, it is expected to be able to effectively prevent and treat medulloblastoma.

1A to 1F are diagrams analyzing the characteristics of bulk tumor cells (BTCs) or brain tumor spheroid forming cells (BTSCs) and extracellular vesicles (BTCs-EVs or BTSCs-EVs) released therefrom: ( FIG. 1A ) Representative microscopic images showing the phenotypes of adherent BTCs and spheroid-forming BTSCs (scale bar, 100 μm); (FIG. 1B) A diagram showing the expression of stem cell markers nestin (nestin; green) and musashi (red) (scale bar, 100 μm); (FIGS. 1c and 1f) Western blot results comparing the expression of flotillin-1 and calnexin; (FIG. 1d) Transmission electron microscopy (TEM) image of EVs shown as isolated vesicles with a characteristic circular exosome structure; (FIG. 1e) Nanoparticle tracking analysis (NTA) results showing a size distribution ranging from 10 to 200 nm in diameter.
2A to 2D are diagrams analyzing the expression of miRNA in BTCs and BTSCs of medulloblastoma patients: ( FIGS. 2A and 2B ) Heat map of miRNA expression analysis of BTSCs compared to BTCs and BTSCs- compared to BTCs-EVs miRNA expression analysis heat map of EVs (red or green indicates relatively high or low expression, respectively, significant difference at P <0.05); (Fig. 2c) Venn diagram showing the number of differentially expressed miRNAs in BTSCs (blue) and BTSCs-EVs (pink); (Fig. 2d) RT-qPCR analysis of the expression levels of miR-135b and miR135a in BTSCs compared to BTCs (***P<0.001).
3A to 3D are diagrams analyzing the functions of miR-135b and miR-135a in BTSCs: ( FIG. 3A ) Transfection with anti-miR-135b, anti-miR-135a or anti-miR-NC (negative control) RT-qPCR analysis of miR-135b or miRNA-135a levels in infected BTSCs; (FIG. 3B) a graph comparing cell viability according to the inhibition of miR-135b or miR-135a; ( FIG. 3C ) a graph showing the ability to form tumorspheres according to the inhibition of miR-135b or miR-135a; ( FIG. 3D ) Images showing the expression of nestin (Nestin; green fluorescence) and Musashi (MSI1; red fluorescence) according to the inhibition of miR-135b or miR-135a by immunofluorescence.
4a and 4b are diagrams showing the results of the integrated analysis and verification of mRNA-targeted miRNAs in BTSCs: ( FIG. 4a ) a heat map analyzing differential gene expression between BTCs and BTSCs; (FIG. 4B) RT-qPCR analysis of the expression of the predicted target genes AMOTL2, STAT6 and GPX8 of miR-135b or miR-135a (*** P<0.001).
5A to 5E are diagrams showing the correlation with target genes AMOTL2, STAT3 and GPX8 by miR-135b and/or miR-135a and signaling pathways of these genes: ( FIGS. 5A and 5B ) miR-135b and A diagram showing the correlation coefficient between miR-135a and its target gene AMOTL2 for each subgroup of medulloblastoma (miR-135b, R=-0.92006, P<0.0001; miR-135a R=-0.82885, P<0.0001); (FIG. 5c) a graph analyzing the expression of AMOTL2 according to the inhibition of miR-135b or miR-135a; (FIGS. 5D and 5E) Visualization of AMOTL2 expression patterns of hippo signaling and tight junction pathways in BTSCs.
6A and 6B are roads showing the expression level of AMOTL2 and the survival probability of pediatric medulloblastoma in two independent cohorts using Kaplan-Meier survival analysis through log-rank test; Figure 6a is a result of analysis of 30 Korean patients (SNUH group), Figure 6b is a target MAGIC (medulloblastoma advanced genomics international consortium) group.

Extracellular vesicles (EVs) secreted from tumors, including exosomes, are considered to be important factors regulating intercellular interactions with respect to tumor development, progression, metastasis and recurrence. Although studies on EVs have been actively conducted, the biological understanding of EVs-miRNAs derived from brain tumor spheroid-forming cells (BTSCs) of medulloblastoma is generally ambiguous. We have explored specific cellular miRNAs and EVs-miRNAs in medulloblastoma BTSCs through comparison with bulk tumor cells (BTCs), and completed the present invention by revealing their potential biological functions.

BTCs and BTSCs were isolated from medulloblastoma tissue (N = 10) and cultured under different conditions. Differentially expressed miRNAs were analyzed by analyzing specific miRNAs derived from cells and EVs in BTSCs compared to those in BTCs via NanoString ^{® .} miRNA analysis results were verified by rt-PCR (real-time polymerase chain reaction). In order to inhibit the up-regulated miRNA, a specific inhibitor for each was transfected with BTSCs. Analysis of characteristics and functions was evaluated by cell survival, limiting dilution, immunofluorescence and western blot analysis. The target mRNAs of miRNAs, their correlations and predicted signaling pathways were determined by bioinformatics-based integrated analysis (see Experimental Examples).

In one embodiment of the present invention, compared to BTCs, 24 miRNAs that were simultaneously increased in both cells and EVs derived from BTSCs were identified. As a result, miR-135b and miR135a were most significantly increased in BTSCs (see Example 2).

In another embodiment of the present invention, it was confirmed that cell viability, self-renewal and expression of stem cell markers were significantly reduced after inhibition of miR-135b or miR-135a (see Example 3).

In another embodiment of the present invention, through integrated analysis of mRNA and miRNAs data, AMOTL2 (angiomotin-like 2) targeted by both miR-135b and miR-135a and STAT6 and GPX8 targeted only by miR-135a was found to be significantly reduced (see Example 4).

In another example of the present invention, it was confirmed that low expression of AMOTL2 in group 3 and group 4 pediatric medulloblastoma patients was significantly associated with poor overall survival (see Example 5).

Summarizing the above results, miR-135b or miR-135a can inhibit the stem cell ability of BTSCs through the regulation of AMOTL2. This suggests that it plays an important role in proliferation.

Accordingly, the present invention can provide a biomarker composition for predicting the possibility of metastasis or recurrence of medulloblastoma, comprising at least one selected from the group consisting of miR-135b and miR-135a as an active ingredient.

As used herein, the term “biomarker” refers to a molecule quantitatively or qualitatively related to the presence of a biological phenomenon, and the biomarker of the present invention is a criterion for predicting or diagnosing the metastatic potential or recurrence potential of medulloblastoma. It refers to a gene, more specifically miRNA. Medulloblastoma brain tumor spheroid-forming cells (BTSCs) play a key role in tumor self-renewal and proliferation. is a cell that plays a pivotal role in metastasis as it can differentiate into other types of cells because it has stem cell ability, i.e., pluripotency. The present inventors found that the expression of miR-135b and miR-135a specifically increased only in the BTSCs. Therefore, the higher the expression level of the miRNA, the higher the ratio of BTSCs, and the higher the ratio of BTSCs, the more the tumor's self-renewal ability, proliferative power, and metastatic ability increase overall, so the possibility of tumor metastasis or recurrence increases. will become

“miR-135b” according to the present invention refers to mature has-miR-135b-5p, and may include the nucleotide sequence shown in SEQ ID NO: 1 or may consist of the nucleotide sequence shown in SEQ ID NO: 1.

“miR-135a” according to the present invention refers to mature has-miR-135a-5p, and may include the nucleotide sequence shown in SEQ ID NO: 2 or may consist of the nucleotide sequence shown in SEQ ID NO: 2.

As used herein, the term “metastasis” refers to the movement of cancer cells from the original tumor site through blood vessels and lymphatic vessels to form cancer in other tissues. Metastasis is also a term used for secondary cancers that grow at distant sites.

As used herein, the term “relapse” refers to the reappearance of cancer after a period of remission. This may be due to incomplete removal of cells from the initial cancer, locally (same site as the initial cancer), locally (near the initial cancer, possibly in lymph nodes or tissues), and/or distant as a result of metastasis can occur

As another aspect of the present invention, the present invention provides a composition for predicting the possibility of metastasis or recurrence of medulloblastoma, comprising an agent for measuring the expression level of any one or more selected from the group consisting of miR-135b and miR-135a.

As another aspect of the present invention, the present invention provides a kit for predicting the possibility of metastasis or recurrence of medulloblastoma, comprising an agent for measuring the expression level of any one or more selected from the group consisting of miR-135b and miR-135a.

In the present invention, the miR-135b or miR-135a is specifically increased in expression in brain tumor spheroid-forming cells of medulloblastoma or extracellular vesicles derived therefrom. may be, but is not limited thereto.

As used herein, the term “extracellular vesicles and EVs” refers to vesicles with a size of about 10 nm to 10 μm consisting of a lipid bilayer or fluid, macro-molecules, solutes, and metabolites from cells contained by micelles. refers to EVs referred to herein are cell-derived EVs. The term “EVs” also includes lipid vesicles engineered to contain bioactive molecules found in cell-derived EVs, such as neuronal EVs. These terms include both exosomes and etosomes. Exosomes are released onto exocytosis of the multivesicular body (MVB). Etosomes are vesicles that assemble at the plasma membrane and are released from it.

In the present invention, the miR-135b or miR-135a may inhibit the expression of their target gene AMOTL2 (angiomotin-like 2).

As used herein, the term “AMOTL2” refers to an “Angiomotin-like 2” gene, from which four isoform proteins are known to be expressed. The AMOTL2 gene includes, for example, a nucleotide sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, or a base represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 It may consist of a sequence, and has a sequence homology of 80% or more, more preferably 90% or more, even more preferably 95% or more to the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 It may consist of a sequence. For example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology It includes a nucleotide sequence having The "% of sequence homology" for a base sequence is determined by comparing two optimally aligned sequences with a comparison region, and a portion of the sequence in the comparison region is a reference sequence (including additions or deletions) to the optimal alignment of the two sequences. additions or deletions (ie, gaps) compared to not).

In the present invention, the agent may include one or more selected from the group consisting of antisense oligonucleotides, primer sets, probes and antibodies that specifically bind to miR-135b or miR-135a, but is limited thereto it is not

In the present invention, the kit may be an RT-PCR kit or a DNA chip, but is not limited thereto.

As another aspect of the present invention, the present invention provides a method for predicting metastasis or recurrence of medulloblastoma or an information providing method for predicting the same, comprising the steps of:

(a) measuring the expression level of any one or more selected from the group consisting of miR-135b and miR-135a in a sample from a medulloblastoma patient; and

(b) determining that the higher the expression level of any one or more selected from the group consisting of miR-135b and miR-135a, the higher the possibility of metastasis or recurrence.

As another aspect of the present invention, the present invention provides a biomarker composition for predicting the prognosis of a medulloblastoma patient, comprising the AMOTL2 gene as an active ingredient.

As another aspect of the present invention, the present invention provides a composition for predicting the prognosis of a medulloblastoma patient, comprising an agent for measuring the expression level of the AMOTL2 gene.

As another aspect of the present invention, the present invention provides a kit for predicting the prognosis of a medulloblastoma patient, comprising an agent for measuring the expression level of the AMOTL2 gene.

In the present invention, the medulloblastoma may be a group 3 medulloblastoma or a group 4 medulloblastoma, but is not limited thereto.

In the present invention, the AMOTL2 gene may be specifically reduced in expression in medulloblastoma brain tumor spheroid-forming cells, but is not limited thereto.

In the present invention, the expression of the AMOTL2 gene may be inhibited by any one or more selected from the group consisting of miR-135b and miR-135a, but is not limited thereto.

In the present invention, the agent may include one or more selected from the group consisting of antisense oligonucleotides, primer sets, probes, and antibodies that specifically bind to the AMOTL2 gene, but is not limited thereto.

As used herein, the term “primer” refers to an oligonucleotide synthesized for use in diagnosis, DNA sequencing, etc. as a short gene sequence serving as a starting point of DNA synthesis. The primers can be synthesized and used with a length of typically 15 to 30 base pairs, but may vary depending on the purpose of use, and may be modified by methylation, capping, etc. by a known method. Primers are capable of initiating DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures.

As used herein, the term “probe” refers to a nucleic acid capable of specifically binding to RNA having a length of several bases to several hundreds of bases produced through an enzymatic chemical separation, purification or synthesis process. The presence or absence of RNA can be checked by labeling radioactive isotopes or enzymes, and can be designed and modified by a known method.

As used herein, the term “antibody” refers to a specific immunoglobulin directed against an antigenic site. The antibody is an antibody that specifically binds to each of miR-135b or miR-135a, which are biomarkers for predicting metastasis or recurrence of medulloblastoma, or an antibody that specifically binds to the AMOTL2 gene, which is a biomarker for predicting the prognosis of medulloblastoma patients. means The antibody can be prepared according to a conventional method in the art. The form of the antibody includes a polyclonal antibody or a monoclonal antibody, and all immunoglobulin antibodies are included. The antibody does not have the structure of a complete antibody with two full-length light chains and two full-length heavy chains, as well as a complete form with two light and two heavy chains, but is directed against an antigenic site. It also includes a functional fragment of an antibody molecule having a specific antigen-binding site (binding domain) and retaining an antigen-binding function.

Since the nucleotide sequence of the miR-135b, miR-135a or AMOTL2 gene is known, a person skilled in the art can design a primer or probe that specifically binds to the sequence of these genes based on the sequence according to a conventional method in the art. can

The kit of the present invention may additionally include other components in addition to the above components. For example, when the kit of the present invention is applied to a PCR amplification process, the kit of the present invention is optionally a reagent necessary for PCR amplification, such as a buffer, a DNA polymerase [eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or thermostable DNA polymerase obtained from Pyrococcus furiosus (Pfu)], DNA polymerase cofactors and dNTPs. The kit of the present invention may be manufactured in a number of separate packaging or compartments containing the reagent components described above. According to a preferred embodiment of the present invention, the kit of the present invention may be a microarray. According to a preferred embodiment of the present invention, the kit of the present invention is a gene amplification kit. When the kit of the present invention is a microarray, a probe is immobilized on the solid surface of the microarray. When the kit of the present invention is a gene amplification kit, primers are included.

As another aspect of the present invention, the present invention provides a method for predicting the prognosis of a medulloblastoma patient, comprising the steps of:

(a) measuring the expression level of the AMOTL2 gene or protein in a sample from a medulloblastoma patient; and

(b) determining that the higher the measured expression level of AMOTL2, the higher the remaining life expectancy of the medulloblastoma patient.

As another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating medulloblastoma, comprising an inhibitor for at least one selected from the group consisting of miR-135b and miR-135a as an active ingredient.

As used herein, the term “prevention” refers to any action that blocks medulloblastoma symptoms or suppresses or delays medulloblastoma symptoms by administering the composition according to the present invention.

As used herein, the term “treatment” refers to any action in which medulloblastoma symptoms are improved or beneficially changed by administration of the composition according to the present invention.

On the other hand, the pharmaceutical composition according to the present invention may further include an appropriate carrier, excipient and/or diluent commonly used to prepare a pharmaceutical composition in addition to the active ingredient. In addition, according to a conventional method, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions.

Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose , microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, and mineral oil. When formulating the composition, it can be prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant usually used.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , sensitivity to drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs, and other factors well known in the medical field.

In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be determined by a person skilled in the art. Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient in the body, inactivation rate and excretion rate, disease type, and drugs used in combination.

The pharmaceutical composition of the present invention may be administered to an individual by various routes. For example, it may be administered by oral administration, intranasal administration, transbronchial administration, arterial injection, intravenous injection, subcutaneous injection, intramuscular injection or intraperitoneal injection, and may be administered by direct injection into tumor tissue. The daily dosage may be administered once or divided into several times a day.

In the present invention, the miR-135b or miR-135a may be derived from brain tumor spheroid-forming cells of medulloblastoma or extracellular vesicles derived therefrom, but is not limited thereto.

In the present invention, the composition may inhibit the formation of tumor spheres, but is not limited thereto.

As used herein, the term “tumorsphere” refers to a cell aggregate formed by some cells having characteristics similar to stem cells among cells constituting cancer or tumor tissue.

In the present invention, the composition may reduce the viability or regeneration of medulloblastoma or inhibit recurrence, but is not limited thereto.

In the present invention, the composition may increase the expression of the AMOTL2 gene, but is not limited thereto.

In the present invention, the inhibitor is an antisense oligonucleotide complementary to miR-135b or miR-135a, double-stranded siRNA (small interfering RNA), shRNA (short hairpin RNA) and ribozyme 1 selected from the group consisting of It may be more than a species, but is not limited thereto.

As used herein, the term “antisense oligonucleotide” refers to a nucleic acid-based molecule capable of forming a duplex with a miRNA by having a sequence complementary to a seed sequence of a target miRNA, particularly miRNA. encompasses Accordingly, the term “antisense oligonucleotide” herein may be described as a “complementary nucleic acid-based inhibitor”.

As used herein, the term “complementary” when referring to an antisense oligonucleotide means that the antisense oligonucleotide is sufficiently complementary to selectively hybridize to the miRNA target under predetermined hybridization or annealing conditions, preferably physiological conditions. It means that it has a meaning encompassing both substantially complementary and perfectly complementary, and preferably means completely complementary.

Antisense oligonucleotides in the present invention include various molecules. The antisense oligonucleotide is a DNA or RNA molecule, more preferably an RNA molecule. Optionally, the antisense oligonucleotide used in the present invention is ribonucleotide (RNA), deoxyribonucleotide (DNA), 2'-O-modified oligonucleotide, phosphorothioate-backbone deoxyribonucleotide, PNA (peptide nucleic acid) or LNA (locked nucleic acid). The 2'-O-modified oligonucleotide may be, for example, a 2'-O-alkyl oligonucleotide, a 2'-O-C1-3 alkyl oligonucleotide, or a 2'-O-C1-3 methyl oligonucleotide.

As used herein, the terms “siRNA, shRNA and miRNA” refer to nucleic acid molecules that bind to mRNA transcribed from a target gene mainly to mediate RNA interference or gene silencing, thereby inhibiting the translation of the mRNA. do. Since the miRNA, siRNA and shRNA can inhibit the expression of a target gene at a translational level, they can be used in an efficient gene knockdown method or gene therapy method.

As used herein, the term “ribozyme” may inhibit protein expression of a target gene by recognizing a specific nucleotide sequence in a target RNA molecule and cleaving it site-specifically.

As used herein, the term "PNA" refers to a nucleic acid mimic, e.g., a DNA mimic, wherein the deoxyribose phosphate backbone is substituted with a pseudopeptide backbone and only the original four nucleobases are retained. . The neutral backbone of PNA is known to provide specific hybridization to DNA and RNA under conditions of low ionic strength, inducing transcriptional or translational repression, or inhibiting replication, resulting in antisense or antisense to sequence-specific regulation of gene expression. It can be used as an antigen preparation.

In another aspect of the present invention, the present invention provides a method for screening a therapeutic agent for medulloblastoma, comprising the steps of:

(a) processing the test substance in the sample;

(b) measuring the expression level of miR-135b, miR-135a or AMOTL2 gene in the sample; and

(c) when the expression level of miR-135b or miR-135a decreases or when the expression level of AMOTL2 gene increases, determining that the test material has an effect on treating medulloblastoma.

The terms or words used in the present specification and claims should not be construed as being limited to their ordinary or dictionary meanings, and the inventor may properly define the concept of the term in order to best describe his invention. It should be interpreted as meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Experimental materials and methods

1. Patient and Sample

Fresh tumor tissue (N=10) was obtained from a medulloblastoma patient who underwent surgery at Seoul National University Children's Hospital. Tumor tissue samples were used with written consent from appropriate patients and/or their parents. For primary cell culture, the tumor tissue was excised and processed within 6 hours. Snap-frozen tissues were used for subgroup analysis. Among the 10 patient samples, patient numbers 1-9 were used for all analyses, and number 10 was used only for functional analysis after verifying the expression levels of miR-135b, miR-135a and AMOTL2 (angiomotin-like 2) according to the analysis results. was used

2. Cell Culture

Fresh tumor tissue (N=10) was obtained and dissociated into single cells using enzymes. Single cells obtained from one patient were separated into bulk tumor cells (BTCs) and brain tumor spheroid-forming cells (BTSCs) according to a known method and cultured individually. BTCs were cultured in DMEM (Dulbecco's modified eagle's medium; Invitrogen, USA) supplemented with 10% Exo-FBS™ (exosome-depleted fetal bovine serum; System Biosciences, USA) and penicillin-streptomycin (Life Technologies, USA). did. BTSCs contain 2 mM L-glutamine, N2 supplement (Invitrogen), B27 supplement (Invitrogen), 20 ng/ml human recombinant epidermal growth factor (EGF; R&D system, USA) and basic fibroblast growth factor (basic fibroblast growth factor). Growth factor, bFGF; R&D system) containing a neurobasal medium (neurobasal medium; Invitrogen) was cultured. Subculture of each cell was performed by treating BTCs with TrypLE™ Express (Invitrogen) and BTSCs with Accutase™ (Invitrogen). All cells were only used for the initial passage (<4) experiments and were maintained at 37° C. with _{5% CO 2 in a humidified atmosphere.} In additional functional studies, only BTSCs corresponding to group 4 were used.

3. Immunofluorescence

Cells were plated on 8-well Lab-Tek chamber slides (Nunc) and using primary antibodies: Nestin (1:200; Chemicon, USA) and Musashi (1:100; Neuromics, USA) Immunofluorescence studies were performed. Goat anti-mouse IgG conjugated with secondary antibody Alexa Fluor 488 and goat anti-rabbit IgG conjugated with Alexa Fluor 594 (1:200; Invitrogen), followed by DAPI (4′,6-diamidino-2 -phenylindole) containing antifading solution (Vector Laboratories, USA) was used for counterstaining. Fluorescence images were obtained by confocal microscopy (Leica, Germany).

4. Isolation of Extracellular Vesicles (EVs) Containing Exosomes

EVs containing exosomes were isolated from conditioned media using the miRCURY exosome kit (Exiqon, Denmark) according to the manufacturer's instructions. To remove cells and cell fragments, samples were centrifuged at 3,200 g for 5 minutes. Precipitation buffer was added to the supernatant and cooled at -20 °C for 12 hours to precipitate EVs. The EVs pellet collected by centrifugation at 3,200 g for 30 minutes was dissolved in 20 μL of phosphate-buffered saline (PBS).

5. Nanoparticle tracking analysis (NTA)

An NTA equipped with a NanoSight NS3000 system (NanoSight Technology Malvern, UK) was used to analyze the size, concentration and distribution of EVs. Samples were diluted to match 20-100 objects per frame and gently injected into the nanosite sample chamber. To ensure accurate and consistent detection of small particles, the detection threshold was kept at 7 as previously reported. Data were analyzed using NTA analysis software (ver. 2.3).

6. Transmission electron microscopy (TEM)

For morphological investigations, TEM (HT7700; Hitachi Ltd., Japan) was performed by Seoul National University Imaging Core Facility. Briefly, EVs were mixed with 4% paraformaldehyde and embedded in a formvar/carbon-coated grid at room temperature (RT) for 20 min. The embedded EVs were washed with PBS, fixed in 1% glutaraldehyde for 5 minutes, and then stained with saturated aqueous uranyl oxalate (uranyl oxalate). Then, the sample was embedded in 0.4% uranyl acetate and 1.8% methylcellulose on ice for 10 minutes. Samples were dried at room temperature prior to visualization by TEM (JEOL, USA). EVs were quantified by micro BCA protein assay (Thermo Fisher Scientific, USA).

7. Western Blotting

Cellular proteins and EVs were extracted using RIPA buffer (GenDEPOT, USA) containing a protease inhibitor cocktail. Protein concentration was quantified by BCA Protein Assay Kit (Pierce, Thermo Fisher Scientific). Equal amounts of protein were separated using 4-12% gradient SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and nitrocellulose membranes were separated by iBlot 2 blotting system (Life Technologies) according to the manufacturer's instructions. was transferred to After blocking the membrane with 5% skim milk, primary antibodies: flotilin-1 (1:500; Abcam, USA), calnexin (1:500; Cell signaling Technology, USA) and β-actin ( 1:10,000; Sigma-Aldrich, USA). Then, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody, and the immunoreactive protein was visualized using Novex™ ECL Chemiluminescent Substrate Reagent Kit (Invitrogen) enriched for immune response.

8. RNA Isolation

Total RNA including miRNA was isolated using the miRNasy kit (Qiagen, Germany) according to the manufacturer's instructions. The quality of purified RNA was confirmed by Nanodrop 200 and Agilent 2100 Bioanalyzer (Agilent Technologies, USA).

9. NanoString nCounter Analysis

miRNA profiling assays were assessed by a NanoString nCounter (NanoString Technologies, Inc., USA) using an 800 human v3 miRNA expression assay according to the manufacturer's protocol. In brief, the NanoString nCounter platform includes the process of mixing total RNA and capture and reporter probe pairs tailored for each miRNA, hybridization, washing excess probes, and binding of probes to the surface. It includes the process of fixing the miRNA and scanning the color-coded bar tags in the reporter probe. 80-100 ng of total RNA per sample from snap-frozen tissue was used as input material with a total volume of 3 μL per sample. All hybridization reactions were incubated at 65° C. for 18 hours, and a maximum-density scan (555 field of view) was selected.

To identify molecular subgroups for medulloblastoma, nCounter Elements TagSets were applied as previously described. Class prediction analysis was performed using the algorithm provided by Dr. M. Taylor (Toronto, Canada).

10. mRNA Profiling via RNA Sequencing Analysis

Total RNA extracted from BTSCs and BTCs was used together with the TruSeq Stranded mRNA LT Sample Prep Kit to prepare paired-end libraries. Specifically, purified RNAs were randomly fragmented, and the fragmented RNAs were converted into cDNA by reverse transcription. Both ends of the cDNA fragment were ligated with adapters. After they were amplified by PCR in an amount suitable for sequencing, cDNA fragments with an insertion size of 200-400 bp were selected and sequenced in a paired-end manner in a NovaSeq 6000 system.

After trimming the adapter and low quality sequences from the raw data, the trimmed RNAseq data were mapped to the human genome (hg19) using Bowtie2 and HISAT2 software. Using StringTie software, transcript assembly was performed and Fragment Per Kilobase of transcript per Million mapped reads (FPKM) values were calculated.

11. Analysis of differentially expressed mRNA and miRNA

Differentially expressed genes were identified in normalized mRNA and miRNA data sets using the limma package written in the R programming language (version 3.5.1). mRNAs and miRNAs with a log2 fold change of ≥2 or ≤-2 and a false discovery rate (FDR) <0.05 were considered differentially expressed.

12. Bioinformatics analysis for the prediction of miRNA target genes

Twelve representative miRNA target databases, PhenomiR, DIANA-microT, ElMMo, MicroCosm, miRBase, miRanda, miRDB, PicTar, PITA, TargetScan, miRecords, and miRTarBase were used to select candidate target genes for differentially expressed miRNAs. To avoid bias or insufficiency when analyzing target genes, we utilized the comprehensive resources of the 12 miRNA target databases, rather than relying on one or two separate databases. In selecting candidate target genes, we maintained the following criteria: target genes predicted simultaneously by at least three of the aforementioned representative miRNA target databases or confirmed by study validation in at least one previous investigation. target gene.

13. Integrated analysis of miRNA and mRNA

In the RNAseq data set for the sample, the target gene was finally selected by comparing the candidate miRNA target gene selected above with the mRNA (log2 fold change ≥2 or ≤-2, p value <0.05 and FDR <0.05). To ensure confidence in the target gene, priority (for validation tests) to target genes that exhibit a statistically significant negative correlation (Pearson correlation coefficient <-0.7) in expression between miRNA and mRNA in the sample was given. Gene ontology (GO) and pathway analysis were performed on target genes using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.

14. RT-qPCR (Reverse transcription and quantitative real-time polymerase chain reaction)

Total RNA was extracted and cDNA was generated using an RT kit (Qiagen) according to the manufacturer's instructions. RT-qPCR using the ABI real-time PCR detection system and the SYBR Green master mix kit with specific primers was performed to measure the expression of selected genes. All reagents and primers were supplied from Qiagen. RUN6B was used to normalize miRNA expression in cells. Since there are no known control or housekeeping miRNAs for EVs, we used spiked-in Caenorhabditis elegans miRNAs directly into QIAzol prior to RNA extraction as a control for normalization. strategy was adopted. The relative expression of each gene was calculated using a comparative threshold cycle method according to a known method.

15. miRNA inhibitors in vitro transfection

For transfection of miRNA inhibitors, BTSCs at 50% confluency were prepared, followed by 20 nM of anti-miR-135b (Qiagen; catalog number, MIN0000758; catalog number, 219300) and anti-miR- 135a (Qiagen; catalog number, MIN0000428; product number, 219300) was transfected into BTSCs to inhibit each miRNA. A miScript inhibitor negative control (anti-miR-NC) was used as a control for miRNA inhibition experiments under the same concentration and conditions. Transient transfection was performed using RNAimax reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. 4 hours after transfection, cells were replaced with normal growth medium and incubated for 48 hours. Transformation efficiency was assessed by RT-qPCR as described above, and all functional studies were performed at 48 hours.

16. Cell viability assay

Cell viability assays after transfection of miRNA inhibitors were evaluated using a colorimetric cell counting kit-8 (CCK; Dojindo Molecular Technologies, USA). Quantification of viable cells by reading the ultraviolet (UV) absorption spectrum at 450 nm was performed on a Versamax microplate according to the manufacturer's instructions. All experiments were repeated three times. Cell viability was calculated as the ratio of absorbance of control cells to miRNA inhibitor-treated cells.

17. Limiting dilution assay

In order to evaluate the self-renewal ability, limiting dilution analysis was performed according to a known method. After transfection with miRNA inhibitors, cells (1×10 ³ ) were cultured on a 48-well ultra-law cluster plater (Costar). For limiting dilution analysis, tumorspheres were dissociated into single cells and transferred to 96-well plates with various seeding densities (5-300 cells/well in 100 μL). Data were analyzed using Extreme Limiting Dilution Analysis (ELDA) software web interface (http://bioinf.wehi.edu.au/software/elda/).

18. Survival analysis according to AMOTL2 gene expression in two juvenile medulloblastoma cohorts

To investigate the association of AMOTL2 expression levels with overall survival outcomes, two independent pediatric medulloblastoma cohorts were obtained from 30 Korean patients and 530 Medulloblastoma Advanced Genomic International Consortium (MAGIC) patients (<18 years of age). A log-rank test was performed. In the log-rank test, patients were classified into high and low expression groups at one of three cutoff points, 0.25, 0.5, or 0.75 quantiles for AMOTL2 expression across samples in each cohort. The survival distributions were then compared between the two groups, and the cutoff point with the largest p-value was chosen for each final log-rank test. All analyzes were repeated in each subgroup of medulloblastoma. Subgrouping of 30 Korean medulloblastoma populations was performed using unsupervised hierarchical clustering and partitioning around medoids (PAM) based on the expression profiles of 22 marker genes from CodeSet.

19. Statistical Analysis

All statistical analyzes were performed using SPSS 19.0 software (SPSS, USA) and GraphPad Prism 5.0 (GraphPad Software, Inc., USA). Data are presented as mean±SD. To check the differences between two or more groups, a one-way analysis of variance followed by a Tukey post-hoc test was used, and the t-test was It was used to identify differences between groups. All experiments were independently repeated at least three times. A P-value of <0.05 was considered statistically significant.

Example 1: Characterization of primary cultured cells and extracellular vesicles from medulloblastoma patients

Prior to all experiments, we performed characterization of isolated cells and cell-derived extracellular vesicles (EVs) containing exosomes. The morphology of BTCs and BTSCs cultured under different culture conditions was adhered to adherent cells in the former and to floating spheroid-forming cells in the latter, respectively, at passage number 5 and lower (Fig. 1a). Suspended BTSCs are believed to be rich in cancer stem/progenitor cells. Expression of stem cell markers such as nestin and musashi was confirmed in BTCs and BTSCs. In particular, as shown in FIG. 1b , nestin was expressed only in a very small number of BTCs cells, and the expression of Musashi was not found. In contrast, it was confirmed that BTSCs were very strongly expressed in both nestin and musashi. In addition, as shown in FIG. 1c , the results of immunoblotting showed positive calnexin expression and negative floatillin-1 in both BTCs and BTSCs.

The characteristics of EVs derived from BTCs and BTSCs were confirmed. TEM showed circular double-lipid membrane vesicles (Fig. 1d). NTA showed a particle size distribution in the range of 10-200 nm in diameter, and most of the particles were detected in the size range corresponding to the exosome dimension (85.8 ± 6.8 nm) (Fig. 1e). Immunoblotting revealed vesicular enrichment of flotilin-1 and the absence of calnexin and β-actin ( FIG. 1f ).

Example 2: Comparative analysis of cells and EVs-miRNA between BTCs and BTSCs

First, we performed gene profiling of 22 subgroup-specific signature genes to identify subgroups of medulloblastoma. Class prediction analysis identified WNT, SHH, group 3 and group 4 medulloblastoma. Next, miRNA profiling of cells and EVs in individuals with BTSCs compared to BTCs was performed using the NanoString nCounter miRNA array.

As a result, as shown in FIGS. 2a and 2b, 86 miRNAs were statistically significantly expressed in BTSCs compared to BTCs through differentially expressed miRNA (hereinafter referred to as 'DEmiR') analysis. gave. In the case of EVs, 48 miRNAs were differentially expressed in BTSCs-derived EVs (BTSCs-EVs) compared to BTCs-derived EVs (BTCs-EVs). In both BTSCs-EVs and BTSCs, it was confirmed that 25 miRNAs were significantly altered, with the interesting observation that 24 miRNAs were upregulated (see Venn diagram in Fig. 2c). Of these, miR-1246 was upregulated in BTSCs but downregulated in BTSCs-EVs. For further functional analysis, we selected miR-135b and miR-135a because they showed the highest statistical significance and fold change in both BTSCs and BTSCs-EVs. Differential expression of the two miRNAs was confirmed by RT-qPCR in BTCs and BTSCs. That is, it was confirmed that both miR-135b and miR-135a were significantly upregulated in BTSCs compared to BTCs, as shown in FIG. 2d .

Example 3: Functional regulation of BTSCs by miR-135b or miRNA-135a inhibition

Next, it was investigated whether the secreted miR-135b and miR-135a had a function at the microenvironment level by affecting the stemness of BTSCs. To determine the effect of miR-135b and miR-135a on the function, mechanism and signaling pathway of BTSCs, we performed inhibition experiments on group 4 BTSCs. After anti-miR (miRNA inhibitor) treatment, miR-135b and miR-135a expression levels were significantly decreased compared to anti-miR-NC (control), respectively ( FIG. 3A ).

In addition, as shown in FIG. 3b , it was confirmed that cell viability was significantly reduced at 48 hours after miR-135b or miR-135a inhibition in all BTSCs through cell viability analysis.

Furthermore, after anti-miRNA treatment, the ability to form spheres was reduced (FIG. 3C) and the expression of stem cell-related markers was decreased (FIG. 3D).

Taken together, these results suggest that inhibition of miR-135b or miR-135a can inhibit the self-renewal ability of BTSCs and the expression of stem cell markers.

Example 4: Integrated analysis of miRNA and mRNA expression through miRNA target prediction

The present inventors analyzed the predicted target mRNA of up-regulated miRNA as down-regulated mRNA in a microarray, and on the contrary, the predicted target mRNA of down-regulated miRNA with up-regulated mRNA was analyzed. Based on this method, a total of 325 significant miRNA-mRNA pairs were predicted, consisting of 109 differentially expressed miRNAs and 1,640 mRNAs. Of the 750 significantly reduced mRNAs, 199 genes were predicted targets of 23 up-regulated miRNAs (Fig. 4a).

Target mRNAs of miR-135b and/or miR-135a were selected, identified and analyzed (N=10). As a result, as shown in Figure 4b, miR-135b targets only one gene, AMOLT2, and miR-135a targets AMOTL2, STAT6 (signal transducer and activator of transcription 6) and GPX8 (glutathione peroxidase 8). found that it does. In particular, AMOTL2 was targeted by both miR-135b and miR-135a, and the expression level of AMOTL2 was found in miR-135b (R=-0.92006; P<0.0001) and miR-135a (R=-0.82885; P<0.0001). showed a negative correlation with the expression level of ( FIGS. 5A and 5B ). In addition, STAT6 (R=-0.85386; P<0.0001) and GPX8 (R=-0.81841; P<0.0001) were associated with miR-135a ( FIGS. 5A and 5B , N=10). However, there was no significant difference according to the subgroups of medulloblastoma, and a similar pattern of association was observed in all groups. These results suggest that high expression levels of miR-135b and miR-135a can target AMOTL2 and downregulate its expression.

To further demonstrate whether AMOTL2 is regulated by miR-135b or miR-135a, the expression level of AMOTL2 after inhibition of miR-135b and miR-135a was investigated by RT-PCR. As a result, as shown in FIG. 5c, it was confirmed that the expression of AMOTL2 was 15.7-fold and 5.3-fold higher by inhibition of miR-135b and miR-135a, respectively, compared to the control group. These results support the fact that miR-135a and miR-135a on BTSCs function, at least in part, by upregulating the expression of AMOTL2.

To explore the biological pathway associated with AMOTL2, the target of miR-135b, we used KEGG pathway analysis. As a result, as shown in FIGS. 5d and 5e, Hippo and tight junction pathways related to AMOTL2 were found. Based on the RNA-seq results, up-regulated genes (red) and down-regulated genes (green) were identified.

Example 5: Relationship between overall survival outcome and AMOTL2 expression in medulloblastoma patients

The association between the expression level of AMOTL2 and clinical outcome in two independent populations (SNUH and MAGIC) was evaluated. Survival analysis showed that high expression of AMOTL2 was significantly associated with good prognosis in group 3 and group 4 pediatric medulloblastoma patients, which was consistently observed in these two independent populations (see FIGS. 6A and 6B ).

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> SEOUL NATIONAL UNIVERSITY HOSPITAL <120> Use of miR-135b, miR-135a and their target gene AMOTL2 for medulloblastoma <130> MP19-280 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> RNA <213> Homo sapiens <400> 1 uauggcuuuu cauuccuaug uga 23 <210> 2 <211> 23 <212> RNA <213> Homo sapiens <400> 2 uauggcuuuu uauuccuaug uga 23 <210> 3 <211> 4991 <212> DNA <213> Homo sapiens <400> 3 ctcactgtgt gtgactctgg gcacgctgct taagctctct gagccctggt gtcctcattt 60 acaacacagg gatgataaca gaactccgtt ggagggtgct tggcagggct gaatcggatg 120 actgggcgaa aagccagcgg tggcacacct tgcactctgc gtaaaggggc gcctattatc 180 accttgggga agaactggac agagcgcctg gcggcgggtg attcagttgg gtgctctggg 240 gccaggtgcc accggccatt gtccaggcag ctgtgtgcaa gccaaagaag catgaggaca 300 ctggaagact cctcggggac agtcctgcac cgcctcatcc aggagcagct gcgctacggc 360 aacctgactg agacgcgcac gctgctagcc atccagcagc aggccctgag gggtggggct 420 ggaactgggg gtacagggag cccccaggcc tccctggaga tcctggcccc agaggacagt 480 caggtgctgc agcaggccac caggcaggag ccccagggcc aggagcacca gggcggtgag 540 aaccacctgg cagagaacac cctctaccgg ctatgcccac agcccagcaa gggagaggag 600 ctgcccacct atgaggaggc caaagcccac tcgcagtact atgcggccca gcaggcaggg 660 acccggccac atgcggggga ccgagatccc cgtggggccc cgggaggcag tcggaggcag 720 gacgaggccc tgcgggagct gaggcatggg cacgtgcgct cgttgagtga acggctcctt 780 cagttgtccc tggagaggaa cggcgcccgg gcccccagcc acatgagctc ctcccacagc 840 ttcccacagc tggcccgcaa ccagcagggc cccccactga ggggcccccc tgctgagggc 900 ccagagtccc gaggaccccc acctcagtac cctcatgttg tactagctca tgagaccacc 960 actgctgtca ctgacccacg gtaccgtgcc cgcggcagcc cgcacttcca gcatgctgaa 1020 gtcaggatcc tgcaggccca ggtgcctcct gtgttcctcc aacagcagca gcagtaccag 1080 tacctgcagc aatctcagga gcacccccct cccccacatc cagctgctct cggccatggc 1140 cccctgagct ccctcagtcc acctgctgtg gaggggccag tgagtgccca ggcctcctca 1200 gccacctcgg gcagtgccca cctggcccag atggaggccg tgctgaggga gaatgccagg 1260 ctgcagagag acaatgagcg gctgcagagg gagctggaga gctctgcgga gaaggctggc 1320 cgcattgaga agctggaaag cgaaatccag cggctctctg aggcccatga 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tgcagtgatc 2220 aaggtccttc agcagcgctc caggagagac cctggcaagg ccatccaggg ctccctgcgg 2280 cctgccaagt cggtgccatc tgttttcgcg gctgcggcag caggaaccca gggctggcaa 2340 gggctctctt ctagtgagcg acaaacagca gacgcccctg ctcggctgac tacagacaga 2400 gcacccacag aggagccagt ggtcacagct ccccctgctg cccatgccaa acacgggagc 2460 agagatggga gcacccagac tgagggcccc ccagacagca cctccacctg cctgccaccg 2520 gagcctgaca gccttctggg gtgcagcagt agccagagag cagcctctct ggactctgta 2580 gctacaccca gagtccagga cttgtcagac atggtggaga tactgatctg aaggaggtgg 2640 tgcttcagga ctctgagcca ttctctcccc tcctctgccc tgtgccactc tcagccattt 2700 cagcagcccc gtcaaccgct gctccgtccc tttccccagc cagacactca ttcccattga 2760 ccatctggtc ccaggagctc aggaggagga ccccagggga gaggagagct gtgagagcac 2820 cggcaccccc agaagactct gcttcttagc ccacattcct ccgggcctta tggagaatga 2880 ggattcagcc ttgacttctt gcccaaggcc tgctactggg gtagcaactg acagctcaga 2940 aaggagctga gctccctctg ccctgccagt tgtcagtcag gcagggaggg agtggctgtg 3000 ttggtttggg gaactaattt ccacggacgg ctgcccgtgg acaccaggtg gactggttca 3060 ctaatcaagt cagccatatt gttctctggc taagtttggt tccagccaac gtcatctgct 3120 cttcagttcc tcactgcctt cttgggatac taagacttga attttttggg gactattaag 3180 ggtgttagtc ttggagaaga cacagcctca ccttctcact tgctgtgggt gaggggccat 3240 ttaagtggac tgggagacag tgcgcagttt gtatattaatt ccctttcttg tggaacagaa 3300 gactgaggcc tgcaggttcc gatgtgtctc catgggctgt gctcccctct tcctactgtc 3360 agtttctgaa acttctgact ggcctcccag ttatgcctcc tcctcaagtt cctggcccgt 3420 ggatgttaaa gctgctcgat tcccaggatc tcggctgcct tttcctctat cttgagccct 3480 ataaatgccc acgggacccc caccaccagc ctcttgaagt ggctccacag ctcctgtccc 3540 tggaacatcc tgtcagtttg gtcataaacc ctgagccaga tgaaatgagc caccgtgaac 3600 agacatctgc catgccccca ggtgggcttc ggtggcccta cccggtacca gttctttctg 3660 agaaactgga gatgtcttgt tagcataagt gtcttcattc ccacctggag ggtttgggag 3720 aggagcaaag cagttgaaaa ctagttaatg agctacaaga gtcaaatagt cctctgaatg 3780 gagcccccat cacaaaacag tgcccaggag gctggctcct caagctaccc atgcccagcg 3840 ccctaaagca gcaccagatg ctttggaatt ggggtgaaac acccacatgg cagcctgcta 3900 gcagcagtga ctttgacttc tggtcttaaa gagtccctca cttcagcccc aggagctatt 3960 ggtgggtttt agcagttttg tctttaccgt ttttagttct ccttgattct ttgttttctt 4020 cctttatcgt ttttaggttt ggtatgtgtt gttttatttc catggttcct caagtttcct 4080 ttttaaacat ttgcatttgc tggacaattg caattttttt taaaaaattc ccctacccct 4140 gtttaaagct gaaaaataca tttggttcat gtgcattgtt tacaaagcaa aaagaaaaaa 4200 gaggaaaaaa aggcaaaaaa tattgtgaaa gaaaaaaaac aacttaatat attttggatt 4260 aatatttggt atttctttta aagtattttt tgtgctgtga acattttctg ccaaagacca 4320 tgatgtgtgt ctgtatgttt aagttatcgt aaatatttaa aatgtaaaca tggctgtttt 4380 gttatgccac cctgtaccag gattgctgcc gcattccact gggtataaca gtattttaat 4440 taaaaaaaaa taattaaaag tgatgtcttt gcacaggtga gactttgcac tgagggaagg 4500 gaaagggtga ggattttggg gggccaccca agtcatagtc caatctcttc aaggacccaa 4560 gatagaattt aggaattttg tgaagctgca ttaaggatag agttggaatg aggatgtggc 4620 tggcattttt cccctttcat agataacatt tcttgaaggc tgggagggga aggagggcct 4680 ggcagatggc agcctcccga ggtagggctg tgttctaatc agctttgttc ggccacacgc 4740 tgtggttatc cacagtggga gtgggtgtcc aaagatggct ttatccttcc cccactcctt 4800 ttcccactcc cacctagcac ataaagtttt gtccgaaacc atccagaagg ccaggcacca 4860 tgcagaggac attcaagaat gtgcatgttc ttagccaccg cctccctcct ttgctgtgac 4920 atcaccactc tttcctgatg ctcaggaaag catattgctc caattcaaaa ttaaagaaag 4980 ccattctcag t 4991 <210> 4 <211> 4868 <212> DNA <213> Homo sapiens <400> 4 agagcgtcgg cgccacggcc gagaacacat cttcgccgcc gagctgagct gggccgagcc 60 gggaggttgtg gtgtctgact gcgctgggca ccctcgggcc gcagcggtgc tctggggcca 120 ggtgccaccg gccattgtcc aggcagctgt gtgcaagcca aagaagcatg aggacactgg 180 aagactcctc ggggacagtc ctgcaccgcc tcatccagga gcagctgcgc tacggcaacc 240 tgactgagac gcgcacgctg ctagccatcc agcagcaggc cctgaggggt ggggctggaa 300 ctgggggtac agggagcccc caggcctccc tggagatcct ggccccagag gacagtcagg 360 tgctgcagca ggccaccagg caggagcccc agggccagga gcaccagggc ggtgagaacc 420 acctggcaga gaacaccctc taccggctat gcccacagcc cagcaaggga gaggagctgc 480 ccacctatga ggaggccaaa gcccactcgc agtactatgc ggcccagcag gcagggaccc 540 ggccacatgc gggggaccga gatccccgtg gggccccggg aggcagtcgg aggcaggacg 600 aggccctgcg ggagctgagg catgggcacg tgcgctcgtt gagtgaacgg ctccttcagt 660 tgtccctgga gaggaacggc gcccgggccc ccagccacat gagctcctcc cacagcttcc 720 cacagctggc ccgcaaccag cagggccccc cactgagggg cccccctgct gagggcccag 780 agtcccgagg acccccacct cagtaccctc atgttgtact agctcatgag accaccactg 840 ctgtcactga cccacggtac cgtgcccgcg gcagcccgca cttccagcat gctgaagtca 900 ggatcctgca ggcccaggtg cctcctgtgt tcctccaaca gcagcagcag taccagtacc 960 tgcagcaatc tcaggagcac ccccctcccc cacatccagc tgctctcggc catggccccc 1020 tgagctccct cagtccacct gctgtggagg ggccagtgag tgcccaggcc tcctcagcca 1080 cctcgggcag tgcccacctg gcccagatgg aggccgtgct gagggagaat gccaggctgc 1140 agagagacaa tgagcggctg cagagggagc tggagagctc tgcggagaag gctggccgca 1200 ttgagaagct ggaaagcgaa atccagcggc tctctgaggc ccatgagagc ctgaccagag 1260 cctcctccaa gcgtgaggcc ctggagaaga ccatgcggaa caagatggac agtgaaatga 1320 ggaggctgca agacttcaac cgggatctta gagagagatt ggaatctgca aatcgccgcc 1380 tggcaagcaa gacacaggag gcccaggccg gcagtcagga catggtggcc aagctgcttg 1440 ctcagagcta cgaacagcag caggagcaag agaagctgga gcgagagatg gcactgctgc 1500 gcggcgccat cgaggaccag cggcggcgtg ccgagctgct ggagcaggct ctgggcaatg 1560 cgcagggccg ggcagctcga gccgaagagg agctgcgcaa gaagcaggcc tatgtggaga 1620 aagtggagcg gctgcagcag gcgctcgggc agctgcaggc agcctgtgag aagcgggagc 1680 agctggagct gcgtctgcgg actcgcctgg agcaggaact caaggccctg cgtgcacagc 1740 agagacaggc aggtgcccca ggtggtagca gtggcagtgg tgggtctcca gagctcagcg 1800 ccctgcgact gtcagaacaa ctgcgagaga aggaggagca gatcctggcg ctggaggccg 1860 acatgaccaa gtgggagcag aagtatttgg aggaacgtgc catgaggcag tttgccatgg 1920 atgcggctgc cacggctgct gctcagcgtg acaccactct catccgacat tccccccagc 1980 cctcacccag cagcagcttc aatgagggtc tgctcactgg tggccacagg catcaggaga 2040 tggaaagcag gttaaaggtg ctccatgccc agatcctgga gaaggatgca gtgatcaagg 2100 tccttcagca gcgctccagg agagaccctg gcaaggccat ccagggctcc ctgcggcctg 2160 ccaagtcggt gccatctgtt ttcgcggctg cggcagcagg aacccagggc tggcaagggc 2220 tctcttctag tgagcgacaa acagcagacg cccctgctcg gctgactaca gcagacagag 2280 cacccacaga ggagccagtg gtcacagctc cccctgctgc ccatgccaaa cacgggagca 2340 gagatgggag cacccagact gagggccccc cagacagcac ctccacctgc ctgccaccgg 2400 agcctgacag ccttctgggg tgcagcagta gccagagagc agcctctctg gactctgtag 2460 ctacatccag agtccaggac ttgtcagaca tggtggagat actgatctga aggaggtggt 2520 gcttcaggac tctgagccat tctctcccct cctctgccct gtgccactct cagccatttc 2580 agcagccccg tcaaccgctg ctccgtccct ttccccagcc agacactcat tcccattgac 2640 catctggtcc caggagctca ggaggaggac cccaggggag aggagagctg tgagagcacc 2700 ggcaccccca gaagactctg cttcttagcc cacatcctc cgggccttat ggagaatgag 2760 gattcagcct tgacttcttg cccaaggcct gctactgggg tagcaactga cagctcagaa 2820 aggagctgag ctccctctgc cctgccagtt gtcagtcagg cagggaggga gtggctgtgt 2880 tggtttgggg aactaatttc cacggacggc tgcccgtgga caccaggtgg actggttcac 2940 taatcaagtc agccatattg ttctctggct aagtttggtt ccagccaacg tcatctgctc 3000 ttcagttcct cactgccttc ttgggatact aagacttgaa ttttttgggg actattaagg 3060 gtgttagtct tggagaagac acagcctcac cttctcactt gctgtgggtg aggggccatt 3120 taagtggact gggagacagt gcgcagtttg tatataattc cctttcttgt ggaacagaag 3180 actgaggcct gcaggttccg atgtgtctcc atgggctgtg ctcccctctt cctactgtca 3240 gtttctgaaa cttctgactg gcctcccagt tatgcctcct cctcaagttc ctggcccgtg 3300 gatgttaaag ctgctcgatt cccaggatct cggctgcctt ttcctctatc ttgagcccta 3360 taaatgccca cgggaccccc accaccagcc tcttgaagtg gctccacagc tcctgtccct 3420 ggaacatcct gtcagtttgg tcataaaccc tgagccagat gaaatgagcc accgtgaaca 3480 gacatctgcc atgcccccag gtgggcttcg gtggccctac ccggtaccag ttctttctga 3540 gaaactggag atgtcttgtt agcataagtg tcttcattcc cacctggagg gtttgggaga 3600 ggagcaaagc agttgaaaac tagttaatga gctacaagag tcaaatagtc ctctgaatgg 3660 agcccccatc acaaaacagt gcccaggagg ctggctcctc aagctaccca tgcccagcgc 3720 cctaaagcag caccagatgc tttggaattg gggtgaaaca cccacatggc agcctgctag 3780 cagcagtgac tttgacttct ggtcttaaag agtccctcac ttcagcccca ggagctattg 3840 gtgggtttta gcagttttgt ctttaccgtt tttagttctc cttgattctt tgttttcttc 3900 ctttatcgtt tttaggtttg gtatgtgttg ttttatttcc atggttcctc aagtttcctt 3960 tttaaacatt tgcatttgct ggacaattgc aatttttttt aaaaaattcc cctacccctg 4020 tttaaagctg aaaaatacat ttggttcatg tgcattgttt acaaagcaaa aagaaaaaag 4080 aggaaaaaaa ggcaaaaaat attgtgaaag aaaaaaaaca acttaatata ttttggatta 4140 atatttggta tttcttttaa agtatttttt gtgctgtgaa cattttctgc caaagaccat 4200 gatgtgtgtc tgtatgttta agttatcgta aatatttaaa atgtaaacat ggctgttttg 4260 ttatgccacc ctgtaccagg attgctgccg cattccactg ggtataacag tattttaatt 4320 aaaaaaaaat aattaaaagt gatgtctttg cacaggtgag actttgcact gagggaaggg 4380 aaagggtgag gattttgggg ggccacccaa gtcatagtcc aatctcttca aggacccaag 4440 atagaattta ggaattttgt gaagctgcat taaggataga gttggaatga ggatgtggct 4500 ggcatttttc ccctttcata gataacattt cttgaaggct gggaggggaa ggagggcctg 4560 gcagatggca gcctcccgag gtagggctgt gttctaatca gctttgttcg gccacacgct 4620 gtggttatcc acagtgggag tgggtgtcca aagatggctt tatccttccc ccactccttt 4680 tcccactccc acctagcaca taaagttttg tccgaaacca tccagaaggc caggcaccat 4740 gcagaggaca ttcaagaatg tgcatgttct tagccaccgc ctccctcctt tgctgtgaca 4800 tcaccactct ttcctgatgc tcaggaaagc atattgctcc aattcaaaat taaagaaagc 4860 cattctca 4868 <210> 5 <211> 4859 <212> DNA <213> Homo sapiens <400> 5 agagcgtcgg cgccacggcc gagaacacat cttcgccgcc gagctgagct gggccgagcc 60 gggaggttgtg gtgtctgact gcgctgggca ccctcgggcc gcagcggtgc tctggggcca 120 ggtgccaccg gccattgtcc aggcagctgt gtgcaagcca aagaagcatg aggacactgg 180 aagactcctc ggggacagtc ctgcaccgcc tcatccagga gcagctgcgc tacggcaacc 240 tgactgagac gcgcacgctg ctagccatcc agcagcaggc cctgaggggt ggggctggaa 300 ctgggggtac agggagcccc caggcctccc tggagatcct ggccccagag gacagtcagg 360 tgctgcagca ggccaccagg caggagcccc agggccagga gcaccagggc ggtgagaacc 420 acctggcaga gaacaccctc taccggctat gcccacagcc cagcaaggga gaggagctgc 480 ccacctatga ggaggccaaa gcccactcgc agtactatgc ggcccagcag gcagggaccc 540 ggccacatgc gggggaccga gatccccgtg gggccccggg aggcagtcgg aggcaggacg 600 aggccctgcg ggagctgagg catgggcacg tgcgctcgtt gagtgaacgg ctccttcagt 660 tgtccctgga gaggaacggc gcccgggccc ccagccacat gagctcctcc cacagcttcc 720 cacagctggc ccgcaaccag cagggccccc cactgagggg cccccctgct gagggcccag 780 agtcccgagg acccccacct cagtaccctc atgttgtact agctcatgag accaccactg 840 ctgtcactga cccacggtac cgtgcccgcg gcagcccgca cttccagcat gctgaagtca 900 ggatcctgca ggcccaggtg cctcctgtgt tcctccaaca gcagcagcag taccagtacc 960 tgcagcaatc tcaggagcac ccccctcccc cacatccagc tgctctcggc catggccccc 1020 tgagctccct cagtccacct gctgtggagg ggccagtgag tgcccaggcc tcctcagcca 1080 cctcgggcag tgcccacctg gcccagatgg aggccgtgct gagggagaat gccaggctgc 1140 agagagacaa tgagcggctg cagagggagc tggagagctc tgcggagaag gctggccgca 1200 ttgagaagct ggaaagcgaa atccagcggc tctctgaggc ccatgagagc ctgaccagag 1260 cctcctccaa gcgtgaggcc ctggagaaga ccatgcggaa caagatggac agtgaaatga 1320 ggaggctgca agacttcaac cgggatctta gagagagatt ggaatctgca aatcgccgcc 1380 tggcaagcaa gacacaggag gcccaggccg gcagtcagga catggtggcc aagctgcttg 1440 ctcagagcta cgaacagcag caggagcaag agaagctgga gcgagagatg gcactgctgc 1500 gcggcgccat cgaggaccag cggcggcgtg ccgagctgct ggagcaggct ctgggcaatg 1560 cgcagggccg ggcagctcga gccgaagagg agctgcgcaa gaagcaggcc tatgtggaga 1620 aagtggagcg gctgcagcag gcgctcgggc agctgcaggc agcctgtgag aagcgggagc 1680 agctggagct gcgtctgcgg actcgcctgg agcaggaact caaggccctg cgtgcacagc 1740 aggcaggtgc cccaggtggt agcagtggca gtggtgggtc tccagagctc agcgccctgc 1800 gactgtcaga acaactgcga gagaaggagg agcagatcct ggcgctggag gccgacatga 1860 ccaagtggga gcagaagtat ttggaggaac gtgccatgag gcagtttgcc atggatgcgg 1920 ctgccacggc tgctgctcag cgtgacacca ctctcatccg acattccccc cagccctcac 1980 ccagcagcag cttcaatgag ggtctgctca ctggtggcca caggcatcag gagatggaaa 2040 gcaggttaaa ggtgctccat gcccagatcc tggagaagga tgcagtgatc aaggtccttc 2100 agcagcgctc caggagagac cctggcaagg ccatccaggg ctccctgcgg cctgccaagt 2160 cggtgccatc tgttttcgcg gctgcggcag caggaaccca gggctggcaa gggctctctt 2220 ctagtgagcg acaaacagca gacgcccctg ctcggctgac tacagacaga gcacccacag 2280 aggagccagt ggtcacagct ccccctgctg cccatgccaa acacgggagc agagatggga 2340 gcacccagac tgagggcccc ccagacagca cctccacctg cctgccaccg gagcctgaca 2400 gccttctggg gtgcagcagt agccagagag cagcctctct ggactctgta gctacatcca 2460 gagtccagga cttgtcagac atggtggaga tactgatctg aaggaggtgg tgcttcagga 2520 ctctgagcca ttctctcccc tcctctgccc tgtgccactc tcagccattt cagcagcccc 2580 gtcaaccgct gctccgtccc tttccccagc cagacactca ttcccattga ccatctggtc 2640 ccaggagctc aggaggagga ccccagggga gaggagagct gtgagagcac cggcaccccc 2700 agaagactct gcttcttagc ccacattcct ccgggcctta tggagaatga ggattcagcc 2760 ttgacttctt gcccaaggcc tgctactggg gtagcaactg acagctcaga aaggagctga 2820 gctccctctg ccctgccagt tgtcagtcag gcagggaggg agtggctgtg ttggtttggg 2880 gaactaattt ccacggacgg ctgcccgtgg acaccaggtg gactggttca ctaatcaagt 2940 cagccatatt gttctctggc taagtttggt tccagccaac gtcatctgct cttcagttcc 3000 tcactgcctt cttgggatac taagacttga attttttggg gactattaag ggtgttagtc 3060 ttggagaaga cacagcctca ccttctcact tgctgtgggt gaggggccat ttaagtggac 3120 tgggagacag tgcgcagttt gtatataatt ccctttcttg tggaacagaa gactgaggcc 3180 tgcaggttcc gatgtgtctc catgggctgt gctcccctct tcctactgtc agtttctgaa 3240 acttctgact ggcctcccag ttatgcctcc tcctcaagtt cctggcccgt ggatgttaaa 3300 gctgctcgat tcccaggatc tcggctgcct tttcctctat cttgagccct ataaatgccc 3360 acgggacccc caccaccagc ctcttgaagt ggctccacag ctcctgtccc tggaacatcc 3420 tgtcagtttg gtcataaacc ctgagccaga tgaaatgagc caccgtgaac agacatctgc 3480 catgccccca ggtgggcttc ggtggcccta cccggtacca gttctttctg agaaactgga 3540 gatgtcttgt tagcataagt gtcttcattc ccacctggag ggtttgggag aggagcaaag 3600 cagttgaaaa ctagttaatg agctacaaga gtcaaatagt cctctgaatg gagcccccat 3660 cacaaaacag tgcccaggag gctggctcct caagctaccc atgcccagcg ccctaaagca 3720 gcaccagatg ctttggaatt ggggtgaaac acccacatgg cagcctgcta gcagcagtga 3780 ctttgacttc tggtcttaaa gagtccctca cttcagcccc aggagctatt ggtgggtttt 3840 agcagttttg tctttaccgt ttttagttct ccttgattct ttgttttctt cctttatcgt 3900 ttttaggttt ggtatgtgtt gttttatttc catggttcct caagtttcct ttttaaacat 3960 ttgcatttgc tggacaattg caattttttt taaaaaattc ccctacccct gtttaaagct 4020 gaaaaataca tttggttcat gtgcattgtt tacaaagcaa aaagaaaaaa gaggaaaaaa 4080 aggcaaaaaa tattgtgaaa gaaaaaaaac aacttaatat attttggatt aatatttggt 4140 atttctttta aagtattttt tgtgctgtga acattttctg ccaaagacca tgatgtgtgt 4200 ctgtatgttt aagttatcgt aaatatttaa aatgtaaaca tggctgtttt gttatgccac 4260 cctgtaccag gattgctgcc gcattccact gggtataaca gtattttaat taaaaaaaaa 4320 taattaaaag tgatgtcttt gcacaggtga gactttgcac tgagggaagg gaaagggtga 4380 ggattttggg gggccaccca agtcatagtc caatctcttc aaggacccaa gatagaattt 4440 aggaattttg tgaagctgca ttaaggatag agttggaatg aggatgtggc tggcattttt 4500 cccctttcat agataacatt tcttgaaggc tgggagggga aggagggcct ggcagatggc 4560 agcctcccga ggtagggctg tgttctaatc agctttgttc ggccacacgc tgtggttatc 4620 cacagtggga gtgggtgtcc aaagatggct ttatccttcc cccactcctt ttcccactcc 4680 cacctagcac ataaagtttt gtccgaaacc atccagaagg ccaggcacca tgcagaggac 4740 attcaagaat gtgcatgttc ttagccaccg cctccctcct ttgctgtgac atcaccactc 4800 tttcctgatg ctcaggaaag catattgctc caattcaaaa ttaaagaaag ccattctca 4859 <210> 6 <211> 4852 <212> DNA <213> Homo sapiens <400> 6 acacacacag ccggagtccc ctcccgcagc aaccccagga cacgcgtggt acagcccttg 60 ctgtcgtccc gtcgatgggt ttagggatga agggtgctct ggggccaggt gccaccggcc 120 attgtccagg cagctgtgtg caagccaaag aagcatgagg acactggaag actcctcggg 180 gacagtcctg caccgcctca tccaggagca gctgcgctac ggcaacctga ctgagacgcg 240 cacgctgcta gccatccagc agcaggccct gaggggtggg gctggaactg ggggtacagg 300 gagcccccag gcctccctgg agatcctggc cccagaggac agtcaggtgc tgcagcaggc 360 caccaggcag gagccccagg gccaggagca ccagggcggt gagaaccacc tggcagagaa 420 caccctctac cggctatgcc cacagcccag caagggagag gagctgccca cctatgagga 480 ggccaaagcc cactcgcagt actatgcggc ccagcaggca gggacccggc cacatgcggg 540 ggaccgagat ccccgtgggg ccccgggagg cagtcggagg caggacgagg ccctgcggga 600 gctgaggcat gggcacgtgc gctcgttgag tgaacggctc cttcagttgt ccctggagag 660 gaacggcgcc cgggccccca gccacatgag ctcctcccac agcttcccac agctggcccg 720 caaccagcag ggccccccac tgaggggccc ccctgctgag ggcccagagt cccgaggacc 780 cccacctcag taccctcatg ttgtactagc tcatgagacc accactgctg tcactgaccc 840 acggtaccgt gcccgcggca gcccgcactt ccagcatgct gaagtcagga tcctgcaggc 900 ccaggtgcct cctgtgttcc tccaacagca gcagcagtac cagtacctgc agcaatctca 960 ggagcacccc cctccccccac atccagctgc tctcggccat ggccccctga gctccctcag 1020 tccacctgct gtggaggggc cagtgagtgc ccaggcctcc tcagccacct cgggcagtgc 1080 ccacctggcc cagatggagg ccgtgctgag ggagaatgcc aggctgcaga gagacaatga 1140 gcggctgcag agggagctgg agagctctgc ggagaaggct ggccgcattg agaagctgga 1200 aagcgaaatc cagcggctct ctgaggccca tgagagcctg accagagcct cctccaagcg 1260 tgaggccctg gagaagacca tgcggaacaa gatggacagt gaaatgagga ggctgcaaga 1320 cttcaaccgg gatcttagag agagattgga atctgcaaat cgccgcctgg caagcaagac 1380 acaggaggcc caggccggca gtcaggacat ggtggccaag ctgcttgctc agagctacga 1440 acagcagcag gagcaagaga agctggagcg agagatggca ctgctgcgcg gcgccatcga 1500 ggaccagcgg cggcgtgccg agctgctgga gcaggctctg ggcaatgcgc agggccgggc 1560 agctcgagcc gaagaggagc tgcgcaagaa gcaggcctat gtggagaaag tggagcggct 1620 gcagcaggcg ctcgggcagc tgcaggcagc ctgtgagaag cgggagcagc tggagctgcg 1680 tctgcggact cgcctggagc aggaactcaa ggccctgcgt gcacagcaga gacaggcagg 1740 tgccccaggt ggtagcagtg gcagtggtgg gtctccagag ctcagcgccc tgcgactgtc 1800 agaacaactg cgagagaagg aggagcagat cctggcgctg gaggccgaca tgaccaagtg 1860 ggagcagaag tatttggagg aacgtgccat gaggcagttt gccatggatg cggctgccac 1920 ggctgctgct cagcgtgaca ccactctcat ccgacattcc ccccagccct cacccagcag 1980 cagcttcaat gagggtctgc tcactggtgg ccacaggcat caggagatgg aaagcaggtt 2040 aaaggtgctc catgcccaga tcctggagaa ggatgcagtg atcaaggtcc ttcagcagcg 2100 ctccaggaga gaccctggca aggccatcca gggctccctg cggcctgcca agtcggtgcc 2160 atctgttttc gcggctgcgg cagcaggaac ccagggctgg caagggctct cttctagtga 2220 gcgacaaaca gcagacgccc ctgctcggct gactacagac agagcaccca cagaggagcc 2280 agtggtcaca gctccccctg ctgcccatgc caaacacggg agcagagatg ggagcaccca 2340 gactgagggc cccccagaca gcacctccac ctgcctgcca ccggagcctg acagccttct 2400 ggggtgcagc agtagccaga gagcagcctc tctggactct gtagctacat ccagagtcca 2460 ggacttgtca gacatggtgg agatactgat ctgaaggagg tggtgcttca ggactctgag 2520 ccattctctc ccctcctctg ccctgtgcca ctctcagcca tttcagcagc cccgtcaacc 2580 gctgctccgt ccctttcccc agccagacac tcattcccat tgaccatctg gtcccaggag 2640 ctcaggagga ggaccccagg ggagaggaga gctgtgagag caccggcacc cccagaagac 2700 tctgcttctt agcccacatt cctccgggcc ttatggagaa tgaggattca gccttgactt 2760 cttgcccaag gcctgctact ggggtagcaa ctgacagctc agaaaggagc tgagctccct 2820 ctgccctgcc agttgtcagt caggcaggga gggagtggct gtgttggttt ggggaactaa 2880 tttccacgga cggctgcccg tggacaccag gtggactggt tcactaatca agtcagccat 2940 attgttctct ggctaagttt ggttccagcc aacgtcatct gctcttcagt tcctcactgc 3000 cttcttggga tactaagact tgaatttttt ggggactatt aagggtgtta gtcttggaga 3060 agacacagcc tcaccttctc acttgctgtg ggtgaggggc catttaagtg gactgggaga 3120 cagtgcgcag tttgtatata attccctttc ttgtggaaca gaagactgag gcctgcaggt 3180 tccgatgtgt ctccatgggc tgtgctcccc tcttcctact gtcagtttct gaaacttctg 3240 actggcctcc cagttatgcc tcctcctcaa gttcctggcc cgtggatgtt aaagctgctc 3300 gattcccagg atctcggctg ccttttcctc tatcttgagc cctataaatg cccacgggac 3360 ccccaccacc agcctcttga agtggctcca cagctcctgt ccctggaaca tcctgtcagt 3420 ttggtcataa accctgagcc agatgaaatg agccaccgtg aacagacatc tgccatgccc 3480 ccaggtgggc ttcggtggcc ctacccggta ccagttcttt ctgagaaact ggagatgtct 3540 tgttagcata agtgtcttca ttcccacctg gagggtttgg gagaggagca aagcagttga 3600 aaactagtta atgagctaca agagtcaaat agtcctctga atggagcccc catcacaaaa 3660 cagtgcccag gaggctggct cctcaagcta cccatgccca gcgccctaaa gcagcaccag 3720 atgctttgga attggggtga aacacccaca tggcagcctg ctagcagcag tgactttgac 3780 ttctggtctt aaagagtccc tcacttcagc cccaggagct attggtgggt tttagcagtt 3840 ttgtctttac cgtttttagt tctccttgat tctttgtttt cttcctttat cgtttttagg 3900 tttggtatgt gttgttttat ttccatggtt cctcaagttt cctttttaaa catttgcatt 3960 tgctggacaa ttgcaatttt ttttaaaaaa ttcccctacc cctgtttaaa gctgaaaaat 4020 acatttggtt catgtgcatt gtttacaaag caaaaagaaa aaagaggaaa aaaaggcaaa 4080 aaatattgtg aaagaaaaaa aacaacttaa tatattttgg attaatattt ggtatttctt 4140 ttaaagtatt ttttgtgctg tgaacatttt ctgccaaaga ccatgatgtg tgtctgtatg 4200 tttaagttat cgtaaatatt taaaatgtaa acatggctgt tttgttatgc caccctgtac 4260 caggattgct gccgcattcc actgggtata acagtatttt aattaaaaaa aaataattaa 4320 aagtgatgtc tttgcacagg tgagactttg cactgaggga agggaaaggg tgaggatttt 4380 ggggggccac ccaagtcata gtccaatctc ttcaaggacc caagatagaa tttaggaatt 4440 ttgtgaagct gcattaagga tagagttgga atgaggatgt ggctggcatt tttccccttt 4500 catagataac atttcttgaa ggctgggagg ggaaggaggg cctggcagat ggcagcctcc 4560 cgaggtaggg ctgtgttcta atcagctttg ttcggccaca cgctgtggtt atccacagtg 4620 ggagtgggtg tccaaagatg gctttatcct tcccccactc cttttcccac tccccacctag 4680 cacataaagt tttgtccgaa accatccaga aggccaggca ccatgcagag gacattcaag 4740 aatgtgcatg ttcttagcca ccgcctccct cctttgctgt gacatcacca ctctttcctg 4800 atgctcagga aagcatattg ctccaattca aaattaaaga aagccattct ca 4852

Claims (23)

delete delete delete delete delete delete delete delete AMOTL2 gene as an active ingredient,
The medulloblastoma is a biomarker composition for predicting the prognosis of a medulloblastoma patient, characterized in that it is a group 3 medulloblastoma or a group 4 medulloblastoma.
delete 10. The method of claim 9,
The AMOTL2 gene is characterized in that the expression is specifically reduced in brain tumor spheroid-forming cells of medulloblastoma, a biomarker composition for predicting the prognosis of medulloblastoma patients.
10. The method of claim 9,
The AMOTL2 gene is a biomarker composition for predicting the prognosis of medulloblastoma patients, characterized in that the expression is inhibited by any one or more selected from the group consisting of miR-135b and miR-135a.
A composition for predicting the prognosis of a medulloblastoma patient, comprising an agent for measuring the expression level of AMOTL2 gene.
14. The method of claim 13,
The composition for predicting the prognosis of a medulloblastoma patient, characterized in that it comprises at least one selected from the group consisting of antisense oligonucleotides, primer sets, probes and antibodies that specifically bind to the AMOTL2 gene.
A kit for predicting the prognosis of a medulloblastoma patient, comprising an agent for measuring the expression level of the AMOTL2 gene.
16. The method of claim 15,
The agent is a kit for predicting the prognosis of a medulloblastoma patient, characterized in that it comprises at least one selected from the group consisting of antisense oligonucleotides, primer sets, probes, and antibodies that specifically bind to the AMOTL2 gene.
delete delete delete delete delete delete delete

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