WO2023013970A1 - Composition containing mirna derived from extracellular vesicles of t cells as active ingredient and use thereof - Google Patents

Abstract

The present invention relates to: a composition containing, as an active ingredient, miRNA derived from extracellular vesicles of T cells; and a use thereof. It was confirmed that extracellular vesicles expressing IL-2 on the surface are produced in T cells transfected with a vector containing IL-2, a linker, and a transmembrane protein, miR-101-3p, miR-181a-3p, miR-223-3p, miR-619-5p, miR-1246, miR-3182, miR-4787-5p, and miR-5787 among the miRNAs contained in the extracellular vesicles expressing IL-2 on the surface inhibit the expression of PD-L1 and Rab 27a genes in cancer cells, thus exhibiting anti-cancer activity, and miR-17-5p, miR-29a-3p, miR-92a-1-5p, miR-125a-5p, miR-181a-3p and miR-223-3p exhibit an immunity-enhancing effect of increasing the expression levels of Ki-67, IFN-γ and Granzyme B genes in T cells. Therefore, a composition containing the miRNAs is provided as an anticancer composition and a composition for enhancing immunity.

Description

Composition containing miRNA derived from extracellular endoplasmic reticulum of T cells as an active ingredient and uses thereof

The present invention relates to a composition comprising miRNA derived from extracellular endoplasmic reticulum of T cells as an active ingredient and a use thereof.

Cancer is a product of uncontrolled and disorderly cell proliferation caused by an excess of abnormal cells, and can be said to be a disease caused by genetic mutations from a molecular biological point of view. There are dozens of types of cancer that have been identified so far, and they are mainly classified according to the location of the onset tissue. Cancer is divided into benign tumors and malignant tumors. Benign tumors grow relatively slowly and do not metastasize from the original tumor site to other tissues, whereas malignant tumors leave the primary site and invade other tissues rapidly. It has growing properties and is life threatening. Most cancers are asymptomatic in their early stages, and even if symptoms are mild, most people tend to overlook them, which causes an increase in cancer mortality.

Surgery, chemotherapy, radiation therapy, etc. are used for the treatment of cancer, but despite many studies, it is reported that more than 50% of all cancer patients die without being cured. The reason for this is that even if surgical resection is performed, the cancer recurs due to the inability to remove finely metastasized cancer cells, or the death of cancer cells to anticancer drugs is not induced, or the tumor seems to shrink due to a response at the beginning, but during treatment or after treatment is finished. This is because cancer cells that have developed resistance to anticancer drugs rapidly increase. Today, about 60 different types of anticancer drugs are being used, and research on the development of new anticancer drugs is being actively conducted as a lot of knowledge about the occurrence of cancer and the characteristics of cancer cells is known. However, most anticancer drugs cause serious side effects such as nausea and vomiting, hair loss, discoloration of skin and nails, and side effects of the nervous system. There is a downside to In addition, radiation therapy induces the death of cancer cells by irradiating high-energy radiation to cancer tissue, but has a disadvantage of causing side effects by damaging normal tissues around the cancer tissue.

On the other hand, small extracellular vesicles (sEVs) are membrane-structured small vesicles secreted from most cells. The diameter of sEVs is approximately 30-10 nm, and sEVs contain various types of proteins, genetic materials (DNA, RNA, miRNA), lipids, etc. derived from the cell. sEVs are not directly released from the plasma membrane, but originate from specific intracellular compartments called multivesicular bodies (MVBs) and are released and secreted outside the cell. That is, when the fusion of the polycystic body and the plasma membrane occurs, the vesicles are released into the extracellular environment, which is called sEV. Although it has not been precisely identified by what mechanism sEVs are produced, it is known that they are separated and released from multiple cell types in both normal and diseased conditions.

MicroRNA (miRNA), one of the substances derived from extracellular endoplasmic reticulum, is a non-coding RNA with 22 base sequences or less, which degrades target mRNA or inhibits the transcription of target mRNA. to regulate gene expression. Its gene expression regulation is related to cell differentiation and cell proliferation. In particular, during embryogenesis, miRNAs are expressed at elusive times, which play an important role in each stage of embryonic development. However, although the important function of regulating miRNA differentiation continues to be discovered, the precise mechanism regulating miRNA expression remains unknown. Recently, the possibility of epigenetic expression of miRNAs with anticancer functions in human cancer cells has been suggested, and various evidences for the epigenetic regulation of miRNA clusters have been reported. In detail, the anticancer miRNA encoding various DNA regions is abnormally hypermethylated in human breast cancer cell lines, so the gene is not activated, and DNMT (DNA methylatransferase) 5-Asa in AGS gastric cancer cell lines. Studies have shown that when treated with -dC (5-Asadeoxycytidine), DNA is demethylated and the expression of a specific miRNA cluster is restored.

An object of the present invention is to provide an anticancer composition comprising miRNAs exhibiting anticancer activity among miRNAs derived from extracellular endoplasmic reticulum of T cells as active ingredients.

In addition, another object of the present invention is to provide a composition for enhancing immunity comprising, as an active ingredient, miRNAs that enhance the proliferation and activity of immune cells among miRNAs derived from the extracellular endoplasmic reticulum of T cells.

The present invention is any one selected from the group consisting of miR-101-3p, miR-181a-3p, miR-223-3p, miR-619-5p, miR-1246, miR-3182, miR-4787-5p and miR-5787. Provided is an anti-cancer composition comprising at least one miRNA as an active ingredient.

In addition, the present invention comprises the steps of transfecting T cells with a vector containing IL-2, a linker and a transmembrane protein; isolating extracellular vesicles expressing IL-2 on the surface from the transfected T cells; Collecting miRNA from the extracellular vesicles; and selecting a miRNA that inhibits the expression of PD-L1 in cancer cells from among the collected miRNAs.

In addition, the present invention provides any one or more selected from the group consisting of miR-17-5p, miR-29a-3p, miR-92a-1-5p, miR-125a-5p, miR-181a-3p and miR-223-3p. Provided is a composition for enhancing immunity comprising miRNA as an active ingredient.

In addition, the present invention comprises the steps of transfecting T cells with a vector containing IL-2, a linker and a transmembrane protein; isolating extracellular vesicles expressing IL-2 on the surface from the transfected T cells; Collecting miRNA from the extracellular vesicles; and selecting miRNAs that promote the expression of Ki-67, IFN-γ and Granzyme B genes in T cells from among the collected miRNAs.

According to the present invention, T cells transfected with a vector containing IL-2, a linker, and a transmembrane protein produce extracellular vesicles expressing IL-2 on their surface, and extracellular vesicles expressing IL-2 on their surface. Among the miRNAs contained in miR-101-3p, miR-181a-3p, miR-223-3p, miR-619-5p, miR-1246, miR-3182, miR-4787-5p and miR-5787 are cancer cells. It suppresses the expression of PD-L1 and Rab 27a genes to show anticancer activity, miR-17-5p, miR-29a-3p, miR-92a-1-5p, miR-125a-5p, miR-181a-3p and miR -223-3p showed an immune enhancing effect by increasing the expression levels of Ki-67, IFN-γ and Granzyme B genes in T cells, so that the composition containing the miRNAs could be used as an anti-cancer composition and an immune enhancing composition. can be provided.

Figure 1 shows the results of RNA-Seq data analysis of the activity of miRNAs isolated from the extracellular vesicles of T cells expressing IL-2 on the surface.

2 shows the results of classifying miRNAs that regulate PD-L1 expression in cancer cells among miRNAs isolated from the extracellular vesicles of T cells expressing IL-2 on their surface.

3 is a result of classifying miRNAs that increase immune cell activity among miRNAs isolated from extracellular endoplasmic reticulum of T cells expressing IL-2 on the surface.

4 is a result of evaluating the expression changes of PD-L1 and Rab 27a genes by transfecting cancer cells with miRNA that regulates PD-L1 expression in cancer cells.

5 is a result of evaluating the expression level of PD-L1 protein by transfecting cancer cells with miRNA that regulates PD-L1 expression in cancer cells.

6 is a result of evaluating expression levels of Ki-67, IFN-γ, and Granzyme B genes by transfecting T cells with miRNAs that increase immune cell activity.

The terms used in this specification have been selected from general terms that are currently widely used as much as possible while considering the functions in the present invention, but these may vary depending on the intention of a person skilled in the art, precedent, or the emergence of new technologies. In addition, in a specific case, there is also a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, not simply the name of the term.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in this application, it should not be interpreted in an ideal or excessively formal meaning. don't

Numerical ranges are inclusive of the values defined therein. Every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written. Every numerical limitation given throughout this specification will include every better numerical range within the broader numerical range, as if the narrower numerical limitations were expressly written.

Hereinafter, the present invention will be described in more detail.

The present invention is any one selected from the group consisting of miR-101-3p, miR-181a-3p, miR-223-3p, miR-619-5p, miR-1246, miR-3182, miR-4787-5p and miR-5787. Provided is an anti-cancer composition comprising at least one miRNA as an active ingredient.

The miR-101-3p consists of the nucleotide sequence represented by SEQ ID NO: 7, the miR-181a-3p consists of the nucleotide sequence represented by SEQ ID NO: 8, and the miR-223-3p consists of the nucleotide sequence represented by SEQ ID NO: 9 The miR-619-5p consists of the nucleotide sequence represented by SEQ ID NO: 10, the miR-1246 consists of the nucleotide sequence represented by SEQ ID NO: 11, and the miR-3182 consists of the nucleotide sequence represented by SEQ ID NO: 10 12, the miR-4787-5p may consist of the nucleotide sequence represented by SEQ ID NO: 13, and the miR-5787 may consist of the nucleotide sequence represented by SEQ ID NO: 14.

The miRNA has the effect of suppressing the expression of PD-L1 and Rab 27a genes in cancer cells, and thus can exhibit anticancer activity.

The miRNA is derived from the extracellular endoplasmic reticulum of T cells, and the extracellular endoplasmic reticulum can express IL-2 on its surface.

The anticancer composition is melanoma, colon cancer, lung cancer, skin cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, perianal cancer, breast cancer, fallopian tube carcinoma, endometrium Carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hawkins' disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma , Bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary gland adenoma to prevent or treat any one or more cancer diseases selected from the group consisting of may be for

In addition, the present invention comprises the steps of transfecting T cells with a vector containing IL-2, a linker and a transmembrane protein; isolating extracellular vesicles expressing IL-2 on the surface from the transfected T cells; Collecting miRNA from the extracellular vesicles; and selecting a miRNA that inhibits the expression of PD-L1 in cancer cells from among the collected miRNAs.

The extracellular vesicles are small extracellular vesicles (sEVs) having a diameter of 30 to 100 nm, and may include exosomes, microvesicles, and the like. As the linker, a linker sequence commonly used in the art may be used, and specifically, a linker having an amino acid sequence represented by SEQ ID NO: 27 (GSTSGSGKPGSGEGSTKG) may be used. The transmembrane protein is epidermal growth factor receptor, insulin receptor, platelet-derived growth factor (PDGF) receptor, vascular endothelial growth factor receptor, fibroblast growth factor receptor, cholecystokinin (CCK) receptor, neurotrophic factor Any one selected from the group consisting of (Neurotrophic factor; NGF) receptor, Hepatocyte growth factor (HGF) receptor, Ephrin (Eph) receptor, angiopoietin receptor, and RTK (Related to receptor tyrosine kinase) receptor It may be the transmembrane domain of one or more receptors.

In addition, the present invention provides any one or more selected from the group consisting of miR-17-5p, miR-29a-3p, miR-92a-1-5p, miR-125a-5p, miR-181a-3p and miR-223-3p. Provided is a composition for enhancing immunity comprising miRNA as an active ingredient.

The miR-17-5p consists of the nucleotide sequence represented by SEQ ID NO: 21, the miR-29a-3p consists of the nucleotide sequence represented by SEQ ID NO: 22, and the miR-92a-1-5p consists of the nucleotide sequence represented by SEQ ID NO: 23 The miR-125a-5p consists of the nucleotide sequence represented by SEQ ID NO: 24, the miR-181a-3p consists of the nucleotide sequence represented by SEQ ID NO: 25, and the miR-181a-3p consists of the nucleotide sequence represented by SEQ ID NO: 25. 223-3p may consist of the nucleotide sequence represented by SEQ ID NO: 26.

The miRNA can increase the expression levels of Ki-67, IFN-γ and Granzyme B genes in T cells.

The miRNA is derived from the extracellular endoplasmic reticulum of T cells, and the extracellular endoplasmic reticulum can express IL-2 on its surface.

In addition, the present invention comprises the steps of transfecting T cells with a vector containing IL-2, a linker and a transmembrane protein; isolating extracellular vesicles expressing IL-2 on the surface from the transfected T cells; Collecting miRNA from the extracellular vesicles; and selecting miRNAs that promote the expression of Ki-67, IFN-γ and Granzyme B genes in T cells from among the collected miRNAs.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Example 1. Cell culture

HEK-293FT cells (human embryonic kidney) were cultured in DMEM medium (Hyclone) supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin, and Jurkat cells (human T lymphocytes) were cultured in 10% fetal bovine serum, 1% It was cultured in RPMI 1640 medium (Hyclone) supplemented with penicillin and streptomycin.

Example 2. lentivirus manufacturing

Construct a lentiviral vector containing the transmembrane domain of antibody-linker-PDGF receptor, construct and clone it into a lentiviral vector containing the transmembrane domain of IL-2-linker (GSTSGSGKPGSGEGSTKG, SEQ ID NO: 27)-PDGF receptor did For transfection, HEK-293FT cells were seeded in a 6-well plate at a density of approximately 1 × 10 ⁶ cells/well and cultured in Lipofectamine 2000 Reagent (ref. 11668-027) under Opti-MEM (ref. 31985-070; Gibco) medium. ; Thermo fisher scientific), pCMVD (# PS100001; Origene) and pVSVg (# 1733; Addgene), which are lentiviral vectors expressing the above IL-2, mixed at a ratio of 1: 1: 1 and treated with 37 Incubated overnight at °C. The next day, the supernatant was removed and replaced with a fresh medium supplemented with 10% fetal calf serum. After 48 hours, the supernatant containing the virus was collected, and cell debris was removed using centrifugation and surfactant-free cellulose acetate (SFCA) membrane filtration device (0.22 μm; Corning). Lentivirus titers were determined using the Lenti-X p24 Rapid Titer Kit (# 632200; Takara). Lentivirus was aliquoted and frozen at -80°C.

Example 3. the lentivirus used Jurkat transduction into cells

Lentiviruses expressing 10 μg/mL polybrene and 500 μg/mL IL-2 were added to 0.3 mL RPMI medium and treated with Jurkat cells (1×10 ⁶ cells/ml). Spinoculation was performed by centrifuging the lentivirus and cell mixture at 30° C. at 1,200×g for 90 minutes. Cells were then incubated overnight at 37°C with lentivirus. Excess virus was removed and fresh medium supplemented with 10% fetal bovine serum was added the next day.

Example 4. small extracellular endoplasmic reticulum (small extracellular vesicles; sEVs ) refine

Control Jukat cells or IL-2-expressing Jukat cells were seeded at a density of about 2 × 10 ⁶ cells/well, and cultured for 24 hours in RPMI medium without addition of fetal calf serum. Subsequently, in order to isolate small extracellular vesicles (sEV) or sEV ^{IL -2} (IL-2 surface-expressed extracellular vesicles), each supernatant obtained from each cell was centrifuged at 300xg, 2500xg, and 10,000xg consecutively. . The supernatant was then filtered through a 0.2 μm syringe filter and centrifuged at 120,000×g. The sEV pellet was resuspended in PBS and centrifuged again at 120,000xg. The purified sEV pellet was resuspended in PBS or 1X cell lysis buffer for the next experiment.

Example 5. IL-2 surface expression extracellular Small RNA sequencing of the endoplasmic reticulum

5-1. RNA isolation

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA quality was assessed by an Agilent 2100 bioanalyzer using an RNA 6000 Pico Chip (Agilent Technologies, Amstelveen, The Netherlands) and RNA quantification was performed using a NanoDrop 2000 Spectrophotometer system (Thermo Fisher Scientific, Waltham, MA, USA) .

5-2. Library preparation and sequencing

For control and test RNA, library construction was performed using the NEB Next Multiplex Small RNA Library Prep kit (New England BioLabs, Inc., USA) according to the manufacturer's instructions. For library construction, adapters were ligated with 1 μg of total RNA from each sample, and then cDNA was synthesized using reverse transcriptase with adapter-specific primers. PCR was performed for library amplification and library cleanup using the QIAquick PCR Purification Kit (Quaigen, Inc, German) and AMPure XP beads (Beckmancoulter, Inc., USA). The yield and size distribution of small RNA libraries were evaluated with an Agilent 2100 Bioanalyzer instrument for high-sensitivity DNA analysis (Agilent Technologies, Inc., USA). High-throughput sequences were generated by the NextSeq 500 system using single-end 75 sequencing (Illumina, San Diego, CA., USA).

5-3. data analysis

Sequence reads were mapped with the bowtie2 software tool to obtain Bam files (alignment files). Mature miRNA sequences were used as references for mapping. The number of reads mapped to mature miRNA sequences were extracted from alignment files using bedtools (v2.25.0) and Bioconductor (R development Core Team, 2011) using the R (version 3.2.2) statistical programming language. Read counts were used to determine the expression levels of miRNAs. Quantile normalization method was used for comparison between samples.

Through RNA-seq data analysis of differentially expressed genes (FIG. 1), miRNAs regulating PD-L1 expression in cancer cells (FIG. 2) and miRNAs increasing immune cell activity (FIG. 3) were identified.

Example 6. human origin melanoma cells microRNA mimic oligo transfection

In a 6 well plate (REF; 3513, Corning), 1 × 10 ⁶ cells/ml of human-derived melanoma cells (SK-MEL-28) in MEM medium supplemented with 10% fetal bovine serum were dispensed in 2 ml. The next day ^, 100 nM hsa-miRNA mimic oligos (hsa-miR-10a ^- 5p, hsa-miR-92a-1-5p, hsa-miR-101-3p, hsa-miR- 146a-5p, hsa-miR-150-5p, hsa-miR-181a-3p, hsa-miR-200c-3p, hsa-miR-hsa-miR-223-3p, hsa-miR-619-5p, hsa- miR-1246p, hsa-miR-1285-5p, hsa-miR-1908-5p, hsa-miR-1972, hsa-miR-3138, hsa-miR-3182, hsa-miR-3195, hsa-miR-3654, hsa-miR-4259, hsa-miR-4448, hsa-miR-4488, hsa-miR-4497, hsa-miR-4530, hsa-miR-4634, hsa-miR-4787-5p, hsa-miR-5787, hsa-miR-6510-5p, hsa-miR-6734-3p and hsa-miR-7851-3p) were respectively transfected. RNA was isolated after 48 hours.

Example 7. hsa - miRNAs mimic oligos After transfection human origin PD-L1 in melanoma cells and RAB 27a gene expression analysis

7-1. real-time polymerase chain reaction

After transfecting human-derived melanoma cells with hsa-miRNA mimic oligos in Example 6, mRNA (Cat; R2062, ZYMO RESEARCH) was extracted from the cells, and then nanodrop (DS-11 Series Spectrophotometer; DeNovix) was used. to measure the concentration of mRNA. After synthesizing cDNA (PrimeScriptTM 1st strand cDNA Synthesis Kit, # 6110A; TaKaRa) with 100ng of mRNA, gene expression was detected using TB GreenTM Premix Ex TaqTM (Tli RNaseH Plus) kit (# RR420A; TaKaRa). Primers used in this experiment are shown in Table 1 below.

gene base sequence sequence number PD-L1 F GGTCATCCCAGAACTACCTC One R AGTGCTACACCAAGGCATAA 2 Rab27a F AAAAGGAGAAAGGGGCATGT 3 R TAATGGGGATGGTGAGAAGC 4 Gapdh F GAATTTGGCTACAGCAACAG 5 R TGAGGGTCTCTCTCTTCCTC 6

Real-time polymerase chain reaction was performed using the Step One Plus Real-Time PCR System (Applied Biosystems). The relative mRNA level of the sample was calculated by Ct (comparative threshold cycle) analysis after normalization for the amount of Gapdh in the same sample, and was expressed as a 2 ^-ΔΔCt value modified from the initial Ct value.

As a result, as shown in Figure 4, among the above 28 hsa-miRNA mimic oligos, 8 miRNAs (hsa-miR-101-3p , hsa-miR-181a-3p, hsa-miR-223-3p, hsa-miR-619-5p, hsa-miR-1246, hsa-miR-3182, hsa-miR-4787-5p and hsa-miR-5787 ) was selected. These results indicate that the anticancer effect can be enhanced by suppressing PD-L1 and Rab 27a gene expression in human-derived melanoma cells by overexpressing 8 miRNAs.

Example 8. western blot selected using hsa - miRNAs mimic oligos 8 types of transfection

48 hours after transfection with the selected 8 types of hsa-miRNA mimic oligos, cell lysate was obtained for changes in PD-L1 protein expression, and primary antibodies (ab10931; abcam/PD-L1, #4970, Cell signaling/β- actin) and incubated with HRP-conjugated secondary antibody. Images were visualized using enhanced chemiluminescence (ECL) detection reagent (# 34095; Thermo Scientific, # RPN2209; GE Healthcare) and quantified using ECL hyperfilm (AGFA, Morstel).

As a result, as shown in FIG. 5 , it was confirmed that PD-L1 expression in human melanoma cells transfected with hsa-miRNA mimic oligos was significantly reduced compared to the control group. In particular, it was confirmed that miR-101-3p and miR-181a-3p are key miRNAs that significantly suppress PD-L1 expression in human-derived melanoma cells. The genetic sequence of key miRNAs that inhibit melanoma PD-L1 expression selected in this experiment is as follows.

(1) hsa-miR-101-3p: UACAGUACUGUGAUAACUGAA (SEQ ID NO: 7)

(2) hsa-miR-181a-3p: ACCAUCGACCGUUGAUUGUACC (SEQ ID NO: 8)

(3) hsa-miR-223-3p: UGUCAGUUUGUCAAAUACCCCA (SEQ ID NO: 9)

(4) hsa-miR-619-5p: GCUGGGAUUACAGGCAUGAGCC (SEQ ID NO: 10)

(5) hsa-miR-1246: AAUGGAUUUUUGGAGCAGG (SEQ ID NO: 11)

(6) hsa-miR-3182: GCUUCUGUAGUGUAGUC (SEQ ID NO: 12)

(7) hsa-miR-4787-5p: GCGGGGGUGGCGGCGGCAUCCC (SEQ ID NO: 13)

(8) hsa-miR-5787: GGGCUGGGGCGCGGGGAGGU (SEQ ID NO: 14)

Example 9. In isolated primary CD8+ T cells microRNA mimic oligo transfection

1.5 ml of RPMI 1640 medium supplemented with 10% fetal calf serum was dispensed into a 12 well plate (REF; 3513, Corning). Thereafter, the isolated primary CD8+ T cells were mixed with a solution of 0.5mL Supplement 1 (Cat; V4XP-3024, Lonza) and 2.25mL P3 Primary Cell Nucleofector Solution (Cat; V4XP-3024, Lonza), and 1~2 × 10 in 100μl. After making it at the rate of ⁶ cells, it was divided by dispensing 100 μl into each new e-tube. 10 μM hsa-miRNA mimic oligos (hsa-miR-17-5p, hsa-miR-29a-3p, hsa-miR-92a-1-5p, hsa-miR-125a-5p, hsa-miR-125a-5p, hsa-miR-125a-5p, hsa-miR-29a-3p, miR-181a-3p and hsa-miR-223-3p) were dispensed at 1 μl each. Then, 100 μl of each hsa-microRNA mimic oligo was added to a 100 μl Nucleocuvette Vessel (Cat; PCK-2005, Lonza), and 4D Nucleofector Core unit (Serial no: 510B0263, Lonza) and 4D Nucleofector X unit (Serial no: 510 X 0441 , Lonza) for transfection. After the procedure, add 500 μl of RPMI 1640 medium supplemented with 10% fetal bovine serum to the corresponding Nucleocuvette, suck it up with single use pipettes, and place 12 well plate ( REF; 3513, Corning) and cultured for 24 hours in a 5% CO ₂ incubator at 37°C.

Example 10. hsa - miRNAs mimic oligos Evaluation of cytotoxic ability of primary CD8+ T cells after transfection

10-1. real-time polymerase chain reaction

Six types of hsa-miRNA mimic oligos (hsa-miR-17-5p, hsa-miR-29a-3p, hsa-miR-92a-1-5p, hsa-miR-125a-5p, hsa-miR-181a-3p and hsa-miR-223-3p) into primary CD8+ T cells, and then mRNA (Cat; R2062, ZYMO RESEARCH) was extracted from the cells and mRNA was measured using nanodrop (DS-11 Series Spectrophotometer; DeNovix). The concentration of was measured. After synthesizing cDNA (PrimeScriptTM 1st strand cDNA Synthesis Kit, # 6110A; TaKaRa) with 100ng of mRNA, gene expression was detected using TB GreenTM Premix Ex TaqTM (Tli RNaseH Plus) kit (# RR420A; TaKaRa). Primers used in this experiment are shown in Table 2 below.

gene base sequence sequence number Ki-67 F AATCATCAAGGAACGGCCCC 15 R TGCCTGATCTGCGTCTTTGA 16 IFN-γ F CTGTTACTGCCAGGACCCAT 17 R TCTGTCACTCTCCTCTTTCAA 18 Granzyme B F AGTCTCTGAAGAGGTGCGGT 19 R TTATGGAGCTTCCCCAACAGTG 20 Gapdh F GAATTTGGCTACAGCAACAG 5 R TGAGGGTCTCTCTCTTCCTC 6

Real-time polymerase chain reaction was performed using the Step One Plus Real-Time PCR System (Applied Biosystems). The relative mRNA level of the sample was calculated by Ct (comparative threshold cycle) analysis after normalization for the amount of Gapdh in the same sample, and was expressed as a 2 ^-ΔΔCt value modified from the initial Ct value.

As a result, as shown in FIG. 6, it was confirmed that the mRNA expression levels of Ki-67, a marker for T cell proliferation, IFN-γ, and Granzyme B, an activation marker, were increased under the influence of the six types of hsa-miRNA mimic oligos. In particular, proliferation and activation genes of primary CD8+ T cells were significantly increased by miR-181a-3p. This means that the ability of primary CD8+ T cells to attack cancer cells is promoted by the above 6 types of miRNAs, which can enhance anticancer effects. The genetic sequences of key miRNAs involved in proliferation and activity of primary CD8+ T cells selected in this experiment are as follows.

(1) hsa-miR-17-5p: CAAAGUGCUUACAGUGCAGGUAG (SEQ ID NO: 21)

(2) hsa-miR-29a-3p: UAGCACCAUCUGAAAUCGGUUA (SEQ ID NO: 22)

(3) hsa-miR-92a-1-5p: AGGUUGGGAUCGGUUGCAAUGCU (SEQ ID NO: 23)

(4) hsa-miR-125a-5p: UCCCUGAGACCCUUUAACCUGUGA (SEQ ID NO: 24)

(5) hsa-miR-181a-3p: ACCAUCGACCGUUGAUUGUACC (SEQ ID NO: 25)

(6) hsa-miR-223-3p: UGUCAGUUUGUCAAAUACCCCA (SEQ ID NO: 26)

As above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (13)

Any one or more miRNAs selected from the group consisting of miR-101-3p, miR-181a-3p, miR-223-3p, miR-619-5p, miR-1246, miR-3182, miR-4787-5p and miR-5787 An anti-cancer composition comprising as an active ingredient. According to claim 1, wherein the miR-101-3p consists of the nucleotide sequence represented by SEQ ID NO: 7, the miR-181a-3p consists of the nucleotide sequence represented by SEQ ID NO: 8, and the miR-223-3p consists of the nucleotide sequence represented by SEQ ID NO: 9, the miR-619-5p consists of the nucleotide sequence represented by SEQ ID NO: 10, the miR-1246 consists of the nucleotide sequence represented by SEQ ID NO: 11, and the miR-3182 consists of the nucleotide sequence represented by SEQ ID NO: 12, the miR-4787-5p consists of the nucleotide sequence represented by SEQ ID NO: 13, and the miR-5787 consists of the nucleotide sequence represented by SEQ ID NO: 14 Anticancer composition, characterized in that. The anticancer composition according to claim 1, wherein the miRNA has an effect of suppressing the expression of PD-L1 and Rab 27a genes in cancer cells. The anticancer composition according to claim 1, wherein the miRNA is derived from the extracellular endoplasmic reticulum of T cells. The anticancer composition according to claim 4, wherein the extracellular cell body expresses IL-2 on its surface. The method of claim 1, wherein the anticancer composition is melanoma, colon cancer, lung cancer, skin cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, proximal anal cancer, breast cancer , fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hawkins' disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or any one or more cancers selected from the group consisting of acute leukemia, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. An anticancer composition characterized in that for preventing or treating a disease. Transfecting T cells with a vector comprising IL-2, a linker and a transmembrane protein; isolating extracellular vesicles expressing IL-2 on the surface from the transfected T cells; Collecting miRNA from the extracellular vesicles; and A method for preparing an anti-cancer composition comprising the step of selecting a miRNA that inhibits the expression of PD-L1 in cancer cells from among the collected miRNAs. Any one or more miRNAs selected from the group consisting of miR-17-5p, miR-29a-3p, miR-92a-1-5p, miR-125a-5p, miR-181a-3p and miR-223-3p as an active ingredient A composition for enhancing immunity comprising: According to claim 8, wherein the miR-17-5p consists of the nucleotide sequence represented by SEQ ID NO: 21, the miR-29a-3p consists of the nucleotide sequence represented by SEQ ID NO: 22, and the miR-92a-1 -5p consists of the nucleotide sequence represented by SEQ ID NO: 23, the miR-125a-5p consists of the nucleotide sequence represented by SEQ ID NO: 24, and the miR-181a-3p consists of the nucleotide sequence represented by SEQ ID NO: 25 And the miR-223-3p is a composition for enhancing immunity, characterized in that consisting of the nucleotide sequence represented by SEQ ID NO: 26. The composition for enhancing immunity according to claim 8, wherein the miRNA increases expression levels of Ki-67, IFN-γ and Granzyme B genes in T cells. The composition for enhancing immunity according to claim 8, wherein the miRNA is derived from the extracellular endoplasmic reticulum of T cells. The composition for enhancing immunity according to claim 11, wherein the extracellular cell body expresses IL-2 on its surface. Transfecting T cells with a vector comprising IL-2, a linker and a transmembrane protein; isolating extracellular vesicles expressing IL-2 on the surface from the transfected T cells; Collecting miRNA from the extracellular vesicles; and A method for preparing a composition for enhancing immunity comprising selecting miRNAs that enhance the expression of Ki-67, IFN-γ and Granzyme B genes in T cells from among the collected miRNAs.

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