Pharmaceutical composition for enhancing radiation sensitivity comprising miR-184 as an active ingredient

Abstract

The present invention relates to a pharmaceutical composition for enhancing radiation sensitivity comprising miR-184 as an active ingredient. By confirming that the anticancer effect is enhanced through the inhibition of SLC25A22 expression, increase in reactive oxygen species, inhibition of glutathione synthesis, inhibition of proline synthesis, and inhibition of collagen synthesis when miR-184 is used in combination with radiation anticancer therapy for glioblastoma, the composition can be usefully utilized as a composition for enhancing radiation sensitivity; a composition for adjuvant radiation anticancer therapy; and a composition for preventing, treating, or improving cancer diseases.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition for enhancing radiation sensitivity comprising miR-184 as an active ingredient. Background Technology

[0002] Glioblastoma is a malignant brain tumor classified as stage 4. Although it is a type of cancer highly dependent on radiation therapy due to the limitations of surgical removal, it is classified as a malignant brain tumor with a 5-year survival rate of only about 8%. This is attributed to the acquisition of radiation resistance by cancer cells, which reduces the efficiency of radiation therapy. Accordingly, there is a need for research on anticancer sensitizers to enhance the efficiency of radiation therapy.

[0003] Meanwhile, previous studies have shown that glioblastoma acquires radiation resistance through various metabolic reprogramming mechanisms during radiation therapy. Through this metabolic reprogramming, the tumor obtains energy or generates antioxidants to evade toxicity from cytotoxic substances produced by radiation therapy. Among various antioxidants, glutathione (GSH) is known to be synthesized using glutamate (Glu) as a material, and it is known that the distribution of this antioxidant is increased in radiation-resistant glioblastoma. Furthermore, cytoplasmic glutamate is known to be used for the synthesis of non-essential amino acids (NEAAs) in addition to glutathione synthesis, and NEAA metabolism is also known to play a multifaceted role in radiation resistance. However, there are no cases of drugs applied in anticancer therapy that reduce generated glutathione or target NEAA metabolism, and there is a need for the development of new anticancer radiosensitizers by fundamentally inhibiting the aforementioned mechanisms. Prior art literature

[0004] 1. Republic of Korea Published Patent KR 10-2018-0092378 (Published Aug. 20, 2018) The problem to be solved

[0005] The object of the present invention is to provide a pharmaceutical composition for enhancing radiation sensitivity comprising miR-184 as an active ingredient.

[0006] Another objective of the present invention is to provide a pharmaceutical composition for adjuvant radiation anticancer therapy comprising miR-184 as an active ingredient.

[0007] Another objective of the present invention is to provide a method for preventing or treating cancer, comprising the step of treating an individual other than a human with miR-184 and radiation in combination.

[0008] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer diseases comprising miR-184 as an active ingredient.

[0009] Another objective of the present invention is to provide a health functional food composition for the prevention or improvement of cancer diseases comprising miR-184 as an active ingredient. means of solving the problem

[0010] To achieve the above objective, the present invention provides a pharmaceutical composition for enhancing radiation sensitivity comprising miR-184 as an active ingredient.

[0011] In addition, the present invention provides a pharmaceutical composition for adjuvant radiation anticancer therapy comprising miR-184 as an active ingredient.

[0012] In addition, the present invention provides a method for preventing or treating cancer, comprising the step of treating an individual other than a human with miR-184 and radiation in combination.

[0013] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising miR-184 as an active ingredient.

[0014] In addition, the present invention provides a health functional food composition for preventing or improving cancer, comprising miR-184 as an active ingredient. Effects of the invention

[0015] According to the present invention, when miR-184 is used in combination with radiation anticancer therapy for glioblastoma, it is confirmed that the anticancer effect is increased through the inhibition of SLC25A22 expression, increase in reactive oxygen species, inhibition of glutathione synthesis, inhibition of proline synthesis, and inhibition of collagen synthesis, thereby making it useful as a composition for enhancing radiation sensitivity; a composition for adjuvant radiation anticancer therapy; and a composition for preventing, treating, or improving cancer diseases. Brief explanation of the drawing

[0016] Figure 1 shows the results of analyzing genes among the amino acid transmembrane transporter and mitochondrial glutamate transporter that show specific changes in expression in radiation-resistant GBM cells compared to glioblastoma (GBM) cells. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 2 shows the results of analyzing SLC25A22 expression in GBM cells and patient-derived GBM cells (BCL20-HP02) after irradiation. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 3 shows the results of analyzing the effect on cell viability after irradiation or regulation of SLC25A22 expression in GBM cells. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 4 shows the results of analyzing glutamate (Glutamte; hereinafter referred to as Glu) concentrations in the mitochondria and cytoplasm of GBM cells after regulating SLC25A22 expression in GBM cells. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 5 shows the inside of the mitochondria of GBM cells after regulating SLC25A22 expression in GBM cells. 13 This is the result of confirming the concentration of Glu through the analysis of the distribution pattern of Glu labeled with C. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 6 shows the results of analyzing the involvement of Glu in the energy metabolism of mitochondria in GBM cells after regulating SLC25A22 expression in GBM cells. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 7 shows the results of analyzing glutathione (hereinafter referred to as GSH) concentrations after irradiating GBM cells. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 8 shows the results of analyzing GSH synthase concentrations after irradiation of GBM cells. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 9 shows the results of analyzing gamma-glutamylcysteine ​​(γ-glutamylcysteine) and GSH concentrations in GBM cells after irradiation, regulation of SLC25A22 expression, or Glu treatment. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 10 shows the results of analyzing reactive oxygen species (hereinafter referred to as ROS) in GBM cells after irradiation, regulation of SLC25A22 expression, or treatment with Glu. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 11 shows the results of analyzing the cell viability and apoptosis rates of GBM cells after irradiation, regulation of SLC25A22 expression, or Glu treatment. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 12 shows the results of analyzing amino acids whose expression specifically changes in GBM cells; and the results of analyzing proline synthase concentrations after irradiating GBM cells. Ala; Alanine, Asn; Asparagine, Asp; Aspartic acid, Ser; Serine and Pro; Proline; ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 13 shows the results of analyzing proline synthesis in GBM cells after irradiation, regulation of SLC25A22 expression, or Glu treatment. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 14 shows the results of analyzing collagen synthesis in GBM cells after irradiation, regulation of SLC25A22 expression, or Glu treatment. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 15 shows the results of analyzing malignancy through the patterns of GBM cell invasion and migration after irradiation, regulation of SLC25A22 expression, or Glu treatment on GBM cells. Figure 16 shows the results of analyzing SLC25A22 promoter activity after irradiation of GBM cells. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 17 shows the results of analyzing the expression of three miRNAs (miR-1-3p, miR-184, and miR-206) selected to specifically target SLC25A22 and irradiated into GBM cells. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 18 shows the results of analyzing SLC25A22 expression in GBM cells after treatment with each of the three types of miRNAs (miR-1-3p, miR-184, and miR-206). ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 19 shows the results of analyzing SLC25A22 expression in GBM cells after miR-184 treatment. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 20 shows the results of analyzing SLC25A22 expression in GBM cells after irradiation or miR-184 treatment. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 21 shows the results of analyzing the concentration of Glu in the mitochondria and cytoplasm of GBM cells after irradiation or miR-184 treatment of GBM cells. Figure 22 shows the results of analyzing GSH concentrations in GBM cells after irradiation or miR-184 treatment. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 23 shows the results of ROS analysis after irradiation or miR-184 treatment of GBM cells. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 24 shows the results of analyzing apoptosis in GBM cells after irradiation or miR-184 treatment. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 25 shows the results of analyzing proline and collagen synthesis in GBM cells after irradiation or miR-184 treatment. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 26 shows the results of analyzing cell malignancy after irradiation or miR-184 treatment of GBM cells. Figure 27 shows the results of analyzing malignancy through the patterns of cell invasion and migration after irradiation or miR-184 treatment of GBM cells. Figure 28 shows the results of analyzing whether miR-184 was successfully delivered to the tumor via Cy5 fluorescence after intranasal administration of miR-184 to a GBM xenograft mouse model. Figure 29 shows the results of analyzing GBM tumor size after irradiation, modulation of SLC25A22 expression, and intranasal administration of miR-184 in a GBM xenograft mouse model. ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. Figure 30 shows the results of analyzing mouse survival rates after radiation, regulation of SLC25A22 expression, and intranasal administration of miR-184 in a GBM xenograft mouse model. Specific details for implementing the invention

[0017] The present invention will be described in more detail below.

[0019] The present invention provides a pharmaceutical composition for enhancing radiation sensitivity comprising miR-184 as an active ingredient.

[0020] The above miR-184 may have a nucleotide sequence represented by SEQ ID NO. 1.

[0021] [Sequence No. 1]

[0022] 5'-UGGACGGAGAACUGAUAAGGGU-3'

[0023] The above miR-184 can inhibit glioblastoma activity or expression, and the glioblastoma may be a general glioblastoma or a radiation-resistant glioblastoma.

[0024] In addition, the above miR-184 can inhibit SLC25A22 expression.

[0025] In addition, the miR-184 may exhibit one or more activities selected from the group consisting of inhibition of glutathione synthesis, promotion of reactive oxygen species, inhibition of proline synthesis, and inhibition of collagen synthesis in glioblastoma, but is not limited thereto.

[0026] The pharmaceutical composition of the present invention may be manufactured in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier according to a method that can be easily carried out by a person skilled in the art to which the invention belongs.

[0027] The above-mentioned pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0028] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0029] The above pharmaceutical composition may be formulated in the form of one or more external skin preparations selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, and cataplasms, but is not limited thereto.

[0030] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starch, sugars, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinylpyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbate, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically affinities for the target. Examples of diluents include, but are not limited to, saline solution, aqueous buffer solution, solvent, and / or dispersion media.

[0031] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. In the case of oral administration, it may be formulated into tablets, troches, lozenges, water-soluble suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc. In the case of parenteral administration, it may be formulated into injectable solutions, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for topical application, oils, creams, etc.

[0032] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition, weight, age, gender, health status, dietary constitutional specificity, properties of the formulation, degree of disease, time of administration of the composition, method of administration, duration or interval of administration, excretion rate, and form of the drug, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day.

[0033] The above pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutical effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, method of administration, time of administration, route of administration, etc., and a person skilled in the art can easily determine and prescribe a dosage effective for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0035] In addition, the present invention provides a pharmaceutical composition for adjuvant radiation anticancer therapy comprising miR-184 as an active ingredient.

[0037] In addition, the present invention provides a method for preventing or treating cancer, comprising the step of treating an individual other than a human with miR-184 and radiation in combination.

[0038] The above-mentioned cancer may be glioblastoma.

[0040] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising miR-184 as an active ingredient.

[0041] The above-mentioned cancer may be glioblastoma.

[0043] In addition, the present invention provides a health functional food composition for preventing or improving cancer, comprising miR-184 as an active ingredient.

[0044] The present invention can be generally used as a commonly used food.

[0045] The food composition of the present invention may be used as a health functional food. The term “health functional food” refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body in accordance with the Health Functional Foods Act, and the term “functional properties” refers to consuming the food for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0046] The above-mentioned health functional food composition may include ordinary food additives, and unless otherwise specified, suitability as a “food additive” shall be determined in accordance with the specifications and standards for the relevant item, based on the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety.

[0047] Examples of items listed in the above “Food Additives Codex” include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline noodle additives, preservative preparations, and tar dye preparations.

[0048] The food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. For example, among health functional foods in capsule form, hard capsules can be manufactured by mixing and filling a conventional hard capsule with the composition according to the present invention and additives such as excipients, and soft capsules can be manufactured by mixing the composition according to the present invention with additives such as excipients and filling it into a capsule base such as gelatin. The soft capsules may contain plasticizers such as glycerin or sorbitol, coloring agents, preservatives, etc., as needed.

[0049] The definitions of terms regarding the above excipients, binders, disintegrants, lubricants, synergists, flavoring agents, etc., are those described in literature known in the art and include those with identical or similar functions. There are no special restrictions on the types of food mentioned above, and they include all health functional foods in the conventional sense.

[0050] In the present invention, the term “prevention” refers to any act of suppressing or delaying cancer disease through the administration of a composition according to the present invention.

[0051] In the present invention, the term “treatment” refers to any act of improving or beneficially altering the symptoms of cancer through the administration of a composition according to the present invention.

[0052] In this invention, the term “improvement” refers to any act of administering or ingesting the composition of this invention to an individual to improve a poor condition of cancer.

[0054] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0056] [ Experimental Example 1] Experiment Preparation

[0057] 1-1. Cells

[0058] U87MG cells, which are GBM (glioblastoma) cells, were obtained from Severance Hospital, Yonsei University. Radiation-resistant U87MG cells (hereinafter referred to as U87MG-RR cells) were produced in this laboratory using U87MG cells for repetitive in vivo It was produced using selection techniques. Specifically, in vivo The selection method involved implanting U87MG cells subcutaneously into 6-week-old male Balb / c nude mice purchased from Yeongnam Bio Co., Ltd., and obtaining only the cells that survived after irradiation. in vitro They were cultured on a substrate. Subsequently, the surviving cells were transplanted back subcutaneously into Balb / c nude mice, and only the cells that survived after irradiation were obtained. in vitro The cells were cultured in a cell. Subsequently, the surviving cells were transplanted into the brains of mice, and cells were obtained after irradiation to create a cell line. BCL20-HP02 (patient-derived GBM cells) are patient-derived cancer cells grown in a sphere shape from a 38-year-old male patient's GBM cancer provided by Inje University Haeundae Paik Hospital.

[0060] 1-2. miR

[0061] miR-1-3p, miR-184, and miR-206 were purchased from AcceGene Biotech and used.

[0063] [ Experimental Example 2] Analysis of Cancer Cell Death Effect

[0064] To confirm the apoptotic effect of miR-184 on GBM cells, 10,000 cells were seeded per well in a 96-well plate, and after 24 hours, radiation was administered alone or in combination with miR-184 mimics. After 24 hours, cell viability was measured using the CellTiter-Glo® Luminescent Viability Assay kit (#G7570, Promega).

[0066] [ Experimental Example 3] Glu analyze

[0067] Intracellular and extracellular Glu amounts were measured using the Glutamate Assay Kit. 2×10 6 GBM cells were harvested, and cell lysates were obtained according to the protein extraction protocol. 50 μL of cell lysate was transferred to a 96-well plate, and 100 μL of reaction mix, consisting of Glutamate Assay Buffer, Glutamate Developer, and Glutamate Enzyme Mix, was added to each well. Subsequently, the plates were incubated at room temperature for 1 hour under light blocking conditions. The amount of Glu was determined by measuring absorbance at 450 nm using a GloMax® Discover Microplate Reader.

[0069] [ Experimental Example 4] Mitochondria isolation

[0070] 5×10 6Mitochondria were isolated using GBM cells. The cells were centrifuged at 900g for 3 minutes, the supernatant was discarded, and the cells were mixed with PBS (phosphate-buffered saline) containing a protease inhibitor. Then, the cells were centrifuged at 600g for 5 minutes, and the obtained supernatant was centrifuged at 7,000g for 10 minutes to obtain the supernatant. The obtained supernatant was discarded, and the cells were mixed with PBS and centrifuged at 7,000g for 10 minutes. Afterward, the supernatant was discarded, and the obtained mitochondrial suspension was centrifuged at 10,000g for 10 minutes to obtain purified mitochondria.

[0072] [ Experimental Example 5] Glu Carbon labeling and transport analysis

[0073] To measure the movement of Glu through mitochondria, isolated mitochondria were resuspended in 50 ml of PBS. The mitochondria were transferred to a 96-well plate coated with poly-L-lysine (Sigma), and the plate was attached to a swinging bucket microplate adapter and centrifuged at 2,000 g for 20 minutes. Afterward, the supernatant was removed, and warmed PBS was added back to each well. Radiolabeled glutamine (Perkin Elmer) (3 mCi / mL) was added to each well for Glu migration analysis, and the entire process was performed at 37°C. The migrated amino acids were extracted using lysis buffer (0.2% SDS), mixed with Microscint PS (Perkin Elmer), and measured using scintillation spectrophotometry with a MicroBeta2® Microplate Counter (Perkin Elmer). Mitochondrial protein concentration for quantification was measured using a BCA assay kit (Intron).

[0075] [ Experimental Example 6] GSH analyze

[0076] The amount of GSH was measured using the GSH / GSSG Ratio Detection Assay Kit. 1×10 7 Experiments were conducted using GBM cells. After appropriate treatment, cells were acquired, mixed with 100 μL of lysis buffer, and centrifuged at 4°C for 15 minutes to obtain the supernatant containing GSH. 50 μL of the supernatant was dispensed into a 96-well plate, and 50 μL of either GSH assay mixture (GAM) or Total GSH assay mixture (TGAM) was added to each sample well. The plates were incubated at room temperature for 1 hour after blocking light. The amount of GSH was measured by fluorescence measurement at 490 / 520 nm Ex / Em (excitation / emission) using a GloMax® Discover Microplate Reader.

[0078] [ Experimental Example 7] ROS analyze

[0079] The amount of ROS was measured using the DCFDA (dichlorodihydrofluorescein diacetate) Cellular ROS assay kit. GBM cells were placed in a 96-well plate at a ratio of 1 × 10⁶ 4 Samples were dispensed individually and treated appropriately. Subsequently, diluted DCFDA solution and Hoechst 33342 were added, and after blocking light, the samples were incubated at 37°C for 1 hour. The amount of ROS was measured by fluorescence measurement at 490 / 520 nm Ex / Em using a GloMax® Discover Microplate Reader.

[0081] [ Experimental Example 8] Matrigel My 3D culture

[0082] 50 μL of Matrigel was dispensed into μ-Slide 8-well high plates (ibiTreat) and incubated at 37°C for 1 hour. Subsequently, 25,000 GBM cells / mL were placed in growth medium containing 4% Matrigel, and 200 μL was injected into each well. The growth medium was replaced every 3 days.

[0084] [ Experimental Example 9] SLC25A22 Promoter activity analysis

[0085] The SLC25A22 promoter (from -1265 to +136 bp of the gene's transcription start site) or a genomic region containing SLC25A22 was cloned into the pGL3-NFAT luciferase vector cleaved by MluI and HindIII. Luciferase activity was measured using Promega's Luciferase Assay System. GBM cells were placed in 3.0 × 10⁶ 60 mm culture dishes. 5 The cells were aliquoted, treated with radiation or miR-184 mimics, and then transfected with the cloned vector. After 48 hours, the medium was removed and washed with PBS, then 400 μl of lysis buffer was added and the cells were centrifuged. The supernatant was mixed with Luciferase Assay Reagent, placed in a 96-well plate, and luminescence was measured using a GloMax Discover Microplate Reader.

[0087] [ Experimental Example 10] miRNA Screening

[0088] We screened miRNAs with potential to target SLC25A22 using three miRNA databases (miRDB, TargetScan, and miRWalk). Through the screening, we selected three miRNA candidates with high scores, including miR-184.

[0090] [ Experimental Example 11] Lucifer Race Activation analysis

[0091] The 3'UTR of the SLC25A22 gene containing the miR-184 binding site was amplified and cloned, then inserted into the psiCHECK-2 vector (Promega). Mutations in the miR-184 binding site were generated using the QuickChange II XL Site-Directed Mutagenesis Kit (Agilent). Wild-type SLC25A22 3'UTR or mutant SLC25A22 3'UTR were co-transfected into cells along with miR-184 mimics, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) after 48 hours.

[0093] [ Experimental Example 12] Orthotopic Xenotransplantation ( xenograft ) Mouse model creation

[0094] Extracellular ( in vivo For the experiment, 6-week-old male Balb / c nude mice purchased from Yeongnam Bio Co., Ltd. were used. Sixteen mice were used per experimental group. All mice were housed in cages providing a constant temperature (23 ± 1℃) and a 12-hour day / night cycle, and all experiments were conducted in compliance with animal ethics (Busan National University Animal Experiment Approval No.: PNU-2022-0060). 1.5×10 5Acquire 4.5×10⁶ / μL of U87MG-RR or U87MG-RR shSLC25A22 cells 5 U87MG-RR or U87MG-RR shSLC25A22 cells were injected into the mouse subventricular zone over a period of 10 minutes.

[0096] [ Experimental Example 13] Radiation irradiation

[0097] in vivo After confirming that the tumor had grown properly through bioluminescence imaging, radiation therapy was performed. Using the TrueBeam STx (Varian Medical Systems, USA) located at Inje University Haeundae Paik Hospital, 2 Gy of radiation was administered daily for 5 days at a dose rate of 600 MU / min to the mouse brains.

[0099] [ Experimental Example 14] Intranasal administration

[0100] An aqueous solution of AgomiR was prepared by dissolving it in 250 μl of RNase-free water. Droplets of 4 μl of the solution were prepared and injected alternately into both nostrils of mice. After 4 minutes had passed since the injection into one side, the solution was injected into the opposite side, and the process was repeated a total of 6 times. To evaluate the delivery efficiency of AgomiR into cancer tissue, U87MG-RR-GFP cells were xenografted in the same manner, and brain tissue was obtained after 24 hours following the injection of Cy5-labeled AgomiR in the same way. The obtained mouse brain tissue was prepared into paraffin blocks through fixation, dehydration, and paraffinization processes, and fluorescence was observed after slicing into 4 μm thick slices.

[0102] [ Experimental Example 15] Extracellular Imaging

[0103] Luciferin solution (3 mg / mL in PBS, dose of 15 mg / kg) was administered intraperitoneally to an orthotopic xenograft mouse model, and 10 minutes later, anesthesia was performed via inhalation anesthesia using isoflurane. After anesthesia, using the VISQUE In vivo Smart LF device in vivo Bioluminescence imaging was performed.

[0105] [ Examples 1] GBM Radiation irradiation in cells and SLC25A22 Analysis of impact ( in vitro )

[0106] 1-1. SLC25A22 Expression analysis

[0107] As a result of analyzing genes whose expression specifically changed among the intracellular amino acid transmembrane transporter and mitochondrial glutamate transporter in radiation-resistant GBM cells, SLC25A22 expression was specifically increased in U87MG-RR cells compared to U87MG cells as shown in Figure 1, and SLC25A22 expression increased when other types of GBM cells (A172, BCL20-HPL02 and GSC11) were irradiated as shown in Figure 2.

[0109] 1-2. Cell Viability Analysis

[0110] After inhibiting SLC25A22 expression in GBM cells by treating them with SLC25A22 siRNA and measuring cell viability, as shown in Figure 3, cell viability decreased when SLC25A22 expression was inhibited in GBM (U87MG, U87MG-RR, and BCL20-HPL02) cells.

[0112] 1-3. Glu analyze

[0113] As a result of measuring Glu concentration in GBM cells, as shown in Figure 4, Glu was present in greater quantities in the cytoplasm than in the mitochondria in U87MG-RR cells compared to U87MG cells, and when SLC25A22 expression was inhibited in radiation-resistant GBM cells, the Glu concentration in the mitochondria increased and the Glu concentration in the cytoplasm decreased. From the above results, it was confirmed that SLC25A22 transports Glu from the mitochondria to the cytoplasm in radiation-resistant GBM cells.

[0114] In addition, mitochondria from GBM cells were isolated, carbon-labeled Glu was applied to the isolated mitochondria, and Glu migration was analyzed after 12 hours. As shown in Figure 5, it was confirmed that the concentration of Glu in the mitochondria was lower in U87MG-RR cells compared to U87MG cells. In addition, when the expression or function of SLC25A22 was regulated through siRNA treatment and SLC25A22 functional mutations (SLC25A22 P206A and SLC25A22 G236W), the mitochondrial Glu concentration increased (SLC25A22 G236W is in which glycine at position 236 of SLC25A22 is replaced with tryptophan, and SLC25A22 P206A is in which proline at position 236 of SLC25A22 is replaced with alanine).

[0115] In addition, as a result of analyzing the degree of involvement of Glu in energy metabolism within mitochondria in U87MG-RR cells, as shown in Figure 6, it was confirmed that the cells synthesize and use only a certain amount of Glu as an intermediate product of energy metabolism regardless of a decrease in Glu concentration within mitochondria.

[0117] 1-4. GSH analyze

[0118] As a result of measuring GSH concentration in GBM cells, as shown in Figure 7, the basal-GSH concentration was higher in U87MG-RR cells compared to U87MG cells, and the GSH concentration increased when GBM cells were irradiated.

[0119] In addition, when the concentration of GSH synthetase (GLCL; Glutamate-Cysteine ​​Ligase Catalytic Subunit, GCLM; Glutamate-Cysteine ​​Ligase Modifier Subunit, and GS; Glutamine synthetase) was measured in GBM cells, it was confirmed that the GSH synthesis pathway was increased according to the expression level of GSH synthetase in U87MG-RR cells compared to U87MG cells, as shown in Figure 8, and the concentration of GSH synthetase increased when GBM cells were irradiated.

[0120] In addition, gamma-glutamylcysteine ​​and GSH concentrations were measured in GBM cells. Buthionine sulfoximine (BSO), known as a GSH production inhibitor, was used as a positive control. As a result, as shown in Figure 9, gamma-glutamylcysteine ​​and GSH concentrations were higher in U87MG-RR cells compared to U87MG cells, and gamma-glutamylcysteine ​​and GSH concentrations decreased when SLC25A22 was inhibited in U87MG-RR and BCL20-HPL02 cells. Furthermore, gamma-glutamylcysteine ​​and GSH concentrations increased when SLC25A22 was overexpressed or Glu was supplemented in U87MG cells with low SLC25A22 expression and U87MG-RR cells with inhibited SLC25A22 (U87MG-RR shSLC25A22).

[0122] 1-5. ROS measurement

[0123] ROS in U87MG-RR and BCL20-HP02 cells was analyzed. BSO (Buthionine sulfoximine), known as a GSH production inhibitor, was used as a positive control. As a result, as shown in Figure 10, the ROS that increases upon irradiation in the cells increased further when SLC25A22 expression was inhibited. In addition, ROS decreased when SLC25A22 was overexpressed or Glu was supplemented in U87MG cells with low SLC25A22 expression and U87MG-RR cells with inhibited SLC25A22 (U87MG-RR shSLC25A22).

[0125] 1-6. Cell viability and cells mortality rate measurement

[0126] Cell viability and apoptosis rates were measured in U87MG-RR and BCL20-HP02 cells upon irradiation or inhibition of SLC25A22 expression. BSO (Buthionine sulfoximine), known as a GSH production inhibitor, was used as a positive control. As a result, as shown in Figure 11, cell viability decreased and apoptosis rates increased in the cells upon irradiation or inhibition of SLC25A22. Additionally, cell viability increased and apoptosis rates decreased in U87MG cells with low SLC25A22 expression and U87MG-RR cells with inhibited SLC25A22 (U87MG-RR shSLC25A22) upon SLC25A22 overexpression or Glu supplementation.

[0128] 1-7. Proline Analysis

[0129] As a result of analyzing whether there were amino acids whose expression specifically changed in GBM cells, as shown in Figure 12, proline (Pro) expression was specifically increased in U87MG-RR cells, and the concentration of proline synthase (P5CS; Pyrroline-5-carboxylate synthase and PYCRL; Pyrroline-5-carboxylate reductase) was higher in U87MG-RR cells compared to U87MG cells, and the concentration of proline synthase increased when GBM cells were irradiated.

[0130] In addition, when proline concentrations were measured upon irradiation or inhibition of SLC25A22 expression in GBM cells, as shown in Figure 13, proline synthesis increased upon irradiation of U87MG-RR and BCL20-HP02 cells, and decreased upon inhibition of SLC25A22 expression (siSLC25A22 and siPYCRL, siPYCRL is siRNA that inhibits the pyrroline-5-carboxylate reductase family of enzymes). Furthermore, proline synthesis increased upon overexpression of SLC25A22 or supplementation of Glu in U87MG cells with low SLC25A22 expression and U87MG-RR cells with inhibited SLC25A22 (U87MG-RR shSLC25A22).

[0132] 1-8. Collagen Analysis

[0133] Collagen concentrations were measured upon irradiation of GBM cells or inhibition of SLC25A22 expression. CHP (collagen hybridizing peptide) was used as a positive control. As a result, as shown in Figure 14, irradiation of U87MG-RR and BCL20-HP02 cells increased collagen mRNA levels, leading to increased collagen protein synthesis. In inhibition of SLC25A22 expression, it was confirmed that while collagen mRNA levels remained unchanged, the amount of collagen protein decreased. Furthermore, when SLC25A22 overexpression or Glu supplementation was applied to U87MG cells with low SLC25A22 expression and U87MG-RR cells with inhibited SLC25A22 (U87MG-RR shSLC25A22), collagen mRNA levels remained unchanged, but the amount of collagen protein increased.

[0135] 1-9. GBM Analysis of cell malignancy

[0136] The malignancy of GBM cells was analyzed upon irradiation of GBM cells or inhibition of SLC25A22 expression. CHP (collagen hybridizing peptide) was used as a positive control. As a result, as shown in Figure 15, when U87MG-RR and BCL20-HP02 cells were irradiated, the expression of the epithelial marker (E-cadherin) of GBM cells decreased, and the expression of mesenchymal markers (Twist, N-cadherin, and Vimentin) increased, leading to increased malignancy; conversely, when SLC25A22 expression was inhibited, the expression pattern of the markers showed an opposite trend, resulting in decreased malignancy. In addition, when SLC25A22 overexpression or Glu supplementation was applied to U87MG cells with low SLC25A22 expression and U87MG-RR cells with inhibited SLC25A22 (U87MG-RR shSLC25A22), the expression of epithelial markers (E-cadherin) decreased, and the expression of mesenchymal markers (Twist, N-cadherin, and Vimentin) increased, leading to increased malignancy of GBM cells.

[0138] 1-10. SLC25A22 Promoter activity analysis

[0139] When U87MG-RR cells were irradiated, SLC25A22 promoter activity was analyzed, and as shown in Figure 16, no significant change in SLC25A22 promoter activity was observed. From the above results, it was confirmed that the increase in SLC25A22 expression caused by irradiation was not due to promoter activity.

[0141] [ Examples 2] GBM In cells miR Analysis of the impact of -184 in vitro )

[0142] 2-1. SLC25A22 specific Target miR Selection

[0143] To select miRs capable of specifically targeting SLC25A22, three miR candidates (miR-1-3p, miR-184, and miR-206) were selected based on three miR databases (TargetScan, miRDB, and miRWalk). After irradiating U87MG-RR cells, the expression of the miRs was analyzed, and as shown in Figure 17, it was confirmed that miR-184 expression decreased.

[0145] 2-2. SLC25A22 Expression analysis

[0146] After treating the above GBM cells with each of the three types of miRs (miR-1-3p, miR-184, and miR-206) selected in Example 2-1, SLC25A22 expression was analyzed. As shown in Fig. 18, miR-1-3p promoted SLC25A22 expression in U87MG-RR cells, while miR-184 and miR-206 significantly inhibited SLC25A22 expression. Additionally, as shown in Fig. 19, miR-184 inhibited the activity of the SLC25A22 promoter in U87MG-RR and BCL20-HP02 cells. Furthermore, as shown in Fig. 20, SLC25A22 expression, which had increased after irradiation in U87MG-RR and BCL20-HP02 cells, decreased upon treatment with miR-184.

[0148] 2-3. Glu Concentration measurement

[0149] After irradiating GBM cells or treating them with miR-184, the concentrations of Glu in the mitochondria and cytoplasm of GBM cells were analyzed. As shown in Figure 21, the concentration of Glu in the cytoplasm of U87MG-RR cells, which had increased after irradiation, decreased upon treatment with miR-184.

[0151] 2-4. GSH Concentration measurement

[0152] As a result of analyzing GSH concentrations after irradiating GBM cells or treating them with miR-184, as shown in Figure 22, GSH concentrations were significantly reduced when U87MG-RR and BCL20-HP02 cells were treated with radiation and miR-184 in combination.

[0154] 2-5. ROS analyze

[0155] As a result of analyzing ROS after irradiating GBM cells or treating them with miR-184, as shown in Figure 23, ROS increased when U87MG-RR and BCL20-HP02 cells were irradiated, and ROS increased more significantly when radiation and miR-184 were combined.

[0157] 2-6. Cell Death Analysis

[0158] Analysis of apoptosis after irradiating or treating GBM cells with miR-184 was performed. As shown in Figure 24, apoptosis increased when U87MG-RR and BCL20-HP02 cells were irradiated, and apoptosis increased more significantly when radiation and miR-184 were combined.

[0160] 2-7. Analysis of Proline and Collagen

[0161] As a result of analyzing proline and collagen synthesis after irradiating or treating GBM cells with miR-184, as shown in Figure 25, when U87MG-RR and BCL20-HP02 cells were irradiated, proline and collagen synthesis increased, whereas when treated with miR-184, proline and collagen synthesis, which had increased due to irradiation, decreased.

[0163] 2-8. GBM Analysis of cell malignancy

[0164] As a result of analyzing cell malignancy after irradiating GBM cells or treating them with miR-184, as shown in Figure 26, when U87MG-RR and BCL20-HP02 cells were irradiated, the expression of epithelial markers (E-cadherin) in GBM cells decreased and the expression of mesenchymal markers (Twist, N-cadherin, and Vimentin) increased, indicating increased malignancy. When treated with miR-184, the expression pattern of the markers showed an opposite trend, confirming that it reduced malignancy.

[0166] 2-9. GBM Cell migration and infiltration analysis

[0167] Analysis of cell invasion and migration after irradiation or miR-184 treatment of GBM cells showed that, as shown in Figure 27, cell migration and migration increased when U87MG-RR cells were irradiated, and cell migration and migration significantly decreased when radiation and miR-184 were combined.

[0169] [ Examples 3] GBM In xenograft mouse models miR Analysis of the impact of -184 in vivo )

[0170] 3-1. miR -184 Transfer Analysis

[0171] To confirm whether miR-184 was successfully delivered to the GBM (brain tumor) after intranasal administration of miR-184 to a GBM xenograft mouse model, Cy5-labeled agomiR-184 was administered intranasally and fluorescence was checked. As shown in Figure 28, Cy5 fluorescence was detected in the tumors of the miR-184 treatment group. From the above results, it was confirmed that miR-184 was successfully delivered to the tumor.

[0173] 3-2. GBM Size analysis

[0174] After radiation and intranasal administration of miR-184 to a GBM xenograft mouse model, the size of the GBM tumor was analyzed. As shown in Figure 29, radiation alone did not have a significant effect on the change in GBM size, but radiation combined with miR-184 treatment; or radiation treatment with SLC25A22 inhibition, the size of the GBM was significantly reduced.

[0176] 3-3. GBM Mouse model survival rate analysis

[0177] After irradiating and administering miR-184 intranasally to a GBM xenograft mouse model, mouse survival rates were analyzed. As shown in Figure 30, survival rates were significantly increased when radiation and miR-184 were combined, or when radiation was combined with SLC25A22 inhibition.

[0179] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A composition for enhancing radiation sensitivity to glioblastoma comprising miR-184 as an active ingredient. Claim 2 A composition according to claim 1, characterized in that the miR-184 has a nucleotide sequence represented by SEQ ID NO.

1. Claim 3 A composition according to claim 1, wherein the miR-184 inhibits glioblastoma activity or expression. Claim 4 A composition characterized in that, in paragraph 3, the glioblastoma is a general glioblastoma or a radiation-resistant glioblastoma. Claim 5 A composition according to claim 1, wherein the miR-184 inhibits SLC25A22 expression. Claim 6 A composition according to claim 1, wherein the miR-184 exhibits one or more activities selected from the group consisting of inhibition of glutathione synthesis, promotion of reactive oxygen species, inhibition of proline synthesis, and inhibition of collagen synthesis in glioblastoma. Claim 7 A pharmaceutical composition for adjuvant radiation anticancer therapy comprising miR-184 as an active ingredient, wherein the pharmaceutical composition is characterized by being used to treat glioblastoma. Claim 8 A method for the prevention or treatment of glioblastoma comprising the step of treating an individual other than a human with miR-184 and radiation in combination. Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete

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