WO2024232682A1 - Mir-6880-5p as target for diagnosis and treatment of prostate cancer - Google Patents

Abstract

The present invention relates to a use of miR-6880-5p for the treatment of prostate cancer. The present invention increases cytotoxicity by regulating the expression level of miR-6880-5p in prostate cancer, inhibits the occurrence, proliferation, and metastasis of prostate cancer, and exhibits a high cell growth inhibitory effect even in prostate cancer cells having resistance to hormone therapy, thereby being expected to be used for the treatment of prostate cancer.

Description

MIR-6880-5P as a diagnostic and therapeutic target for prostate cancer

The present invention relates to miR-6880-5p as a diagnostic and therapeutic target for prostate cancer, and specifically provides the use of miR-6880-5p in exosomes derived from cancer cells for prostate cancer diagnosis and the use of miR-6880-5p for prostate cancer treatment.

Prostate cancer (PCa) is the second most common malignant tumor among male cancers worldwide. According to the 2019 National Cancer Registry Statistics, the cancer incidence rate increased by 3.6% compared to the previous year, and prostate cancer was reported to be the 6th most common cancer (16,803 cases) when divided by cancer type. The incidence rates of stomach cancer, colon cancer, and liver cancer, which are more common than prostate cancer, have been decreasing for the past 10 years, but prostate cancer has been reported to have been continuously increasing since 1999.

Prostate cancer has various intratumor heterogeneities. Tumor heterogeneity is observed differently depending on the location of the information on the genotype and phenotype of cells within the tumor, and depending on the degree, it can affect drug response, treatment resistance, etc. Therefore, it is very important to understand the formation and progression pattern of the tumor through the degree of tumor heterogeneity in selecting a treatment method for the patient. Currently, prostate cancer is diagnosed and predicted through PSA (Prostate Specific Antigen) measurement, ultrasound examination, CT examination, MRI examination, and pathological tissue examination using tissue samples.

PSA is an enzyme secreted from prostate cells that is secreted into semen and a small amount of the enzyme is secreted into the blood. Therefore, the test is performed through the blood PSA level, and most normal people show a level of less than 4ng/ml. If this level is abnormally elevated, it is considered that there are diseases such as benign prostatic hyperplasia, prostatitis, and prostate cancer. However, since the accuracy of prostate cancer diagnosis in the PSA gray zone is about 30%, overdiagnosis and overtreatment may occur. Therefore, a tissue examination is required for an accurate diagnosis, and an appropriate treatment method is selected by considering the clinical stage, malignancy of cancer cells, and other diseases through a pathological tissue examination. However, a tissue examination is not only difficult to perform depending on the location, size, and condition of the tumor, but also invasive to the human body, so it is risky for patients and doctors, so a technology that can diagnose more simply and accurately is needed.

The present invention provides a method for diagnosing and predicting the prognosis of prostate cancer containing miR-6880-5p in exosomes. As a diagnostic method using blood, it reduces the risk of tissue biopsy and overcomes the low accuracy rate in the PSA gray zone of the conventional technology to diagnose prostate cancer more accurately and quickly.

In addition, it is an object of the present invention to provide a biomarker capable of predicting the prognosis of a therapeutic response to endocrine therapy.

In addition, the present invention aims to provide a kit for predicting prostate cancer metastasis or prognosis comprising the composition.

In addition, the present invention was completed by confirming the reduced expression pattern of miR-6880-5p in prostate cancer and confirming that the proliferation, migration, and invasion ability of cancer can be reduced by positively regulating the expression of miR-6880-5p.

Accordingly, the present invention provides miR-6880-5p as a target for preventing and treating prostate cancer and inhibiting its metastasis, and provides a pharmaceutical composition for preventing or treating prostate cancer comprising the miRNA itself or a vector expressing the same as an active ingredient, and a pharmaceutical composition for preventing prostate cancer metastasis.

However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

The present invention provides miR-6880-5p in exosomes separated from plasma as a biomarker for diagnosing prostate cancer and predicting the prognosis for treatment response.

In addition, the present invention provides a biomarker composition for diagnosing prognosis and predicting treatment response of prostate cancer, comprising miR-6880-5p in exosomes separated from plasma as an active ingredient.

As one embodiment of the present invention, the exosome may be a cancer-derived exosome, and the cancer-derived exosome may be isolated using a CD63 specific antibody.

In addition, the present invention provides a composition for diagnosing prostate cancer and predicting the prognosis for treatment response, comprising as an active ingredient an agent capable of measuring the expression level of miR-6880-5p in exosomes separated from plasma.

As one embodiment of the present invention, the agent capable of measuring the expression level of the miR-6880-5p may be an oligonucleotide comprising a nucleotide sequence complementary to part or all of the miR-6880-5p, and a non-limiting example thereof may be a primer or probe specific for the miR-6880-5p.

In addition, the present invention provides a method for diagnosing prostate cancer and a method for providing information for diagnosing prostate cancer, including the steps of isolating exosomes from a liquid biopsy isolated from a subject requiring diagnosis of prostate cancer; measuring the expression level of miR-6880-5p in the exosomes; and determining that the subject has prostate cancer if the expression level of miR-6880-5p is lower than that of a normal subject.

In addition, the present invention provides a method for determining the suitability of a treatment method in a prostate cancer patient by performing the following steps before and after the cancer treatment step: a step of isolating exosomes from a liquid biopsy isolated from the patient; a step of measuring the expression level of miR-6880-5p in the exosomes; and a step of determining that the treatment prognosis is likely to be good if the expression level of miR-6880-5p after the treatment is significantly higher than the expression level of miR-6880-5p before the treatment, and a step of determining that the treatment prognosis is likely to be poor if the expression level of miR-6880-5p is significantly lower than the expression level of miR-6880-5p before the treatment.

In addition, the present invention provides a pharmaceutical composition for preventing or treating prostate cancer, comprising miR-6880-5p or a vector expressing the same as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing prostate cancer metastasis, comprising miR-6880-5p or a vector expressing the same as an active ingredient.

The present invention provides miR-6880-5p as a biomarker capable of diagnosing prostate cancer more simply and quickly by a non-invasive method, and provides the miRNA as a target for the prevention and treatment of prostate cancer and inhibition of its metastasis, so that by increasing the level of miR-6880-5p in cancer cells, the survival, proliferation, migration, and invasion abilities of prostate cancer can be reduced, and further, it exhibits an anticancer effect even in malignant prostate cancer that exhibits resistance to hormone therapy, and therefore, it is expected to be utilized in various ways for the treatment of prostate cancer.

Figure 1 shows the results of analyzing the characteristics of cancer cell-derived exosomes isolated from the blood of prostate cancer patients.

Figure 2 shows differentially expressed genes in exosomes derived from the blood of prostate cancer patients and normal people.

Figure 3 is a graph comparing the expression levels of miR-6880-5p in exosomes derived from the blood of prostate cancer patients and normal people.

Figure 4 is a graph showing the change in the expression level of miR-6880-5p before and after endocrine therapy in the PR group with a good treatment response and the PD group with a poor treatment response.

Figure 5 compares the expression levels of miR-6880-5p in various types of prostate cancer cells.

Figure 6a confirms that proliferation is reduced when miR-6880-5p is transfected in PC3 cells.

Figure 6b confirms that proliferation is reduced when miR-6880-5p is transfected in DU145 cells.

Figure 6c shows the results of a wound healing assay confirming the migration ability of PC3 cells following miR-6880-5p transfection.

Figure 6d shows the results of a wound healing assay confirming the migration ability of DU145 cells following miR-6880-5p transfection.

Figure 6e shows the results of a cell invasion analysis confirming the invasiveness of PC3 cells following miR-6880-5p transfection.

Figure 6f shows the results of a cell invasion analysis confirming the invasiveness of DU145 cells following miR-6880-5p transfection.

Figure 7a shows the results of differential expression gene analysis between the untreated control group and DU145 cells transfected with miR-6880-5p mimic.

Figure 7b shows the results of differential expression gene analysis between DU145 cells transfected with negative control or miR-6880-5p mimic.

Figure 7c is a mosaic map showing the expression patterns of genes related to angiogenesis and cell migration by expression of miR-6880-5p.

Figures 7d, 7f, and 7g show signaling pathways downregulated by expression of miR-6880-5p.

Figures 7e, 7h, and 7i show signaling pathways upregulated by expression of miR-6880-5p.

Figure 8 shows the results of an MTT assay confirming a decrease in cell viability following miR-6880-5p transfection.

The present inventors have conducted a preliminary study on a method for diagnosing prostate cancer by a noninvasive method, identified low levels of miR-6880-5p in exosomes isolated from the blood of prostate cancer patients, and provided miR-6880-5p as a biomarker for diagnosing prostate cancer.

Meanwhile, the inventors of the present invention confirmed the change in the expression level of miR-6880-5p before and after endocrine therapy in groups with a good and poor prognosis for endocrine therapy. As a result, in the group with a good prognosis, a low level of miR-6880-5p was found to increase after treatment, and in the group with a poor prognosis, a relatively high level of miR-6880-5p was found and its expression level decreased after treatment.

Accordingly, the present inventors provide miR-6880-5p as a biomarker for predicting treatment response in prostate cancer patients.

The present invention is characterized by using microRNA biomarkers as noninvasive tools for diagnosing prostate cancer and predicting therapeutic response to endocrine therapy in prostate cancer patients.

More specifically, the present invention provides a technology for determining that a patient in a sample has prostate cancer by measuring the expression level of miRNA-6880-5p present in the sample and determining that the patient in the sample has prostate cancer when miRNA-6880-5p is relatively underexpressed.

In the present invention, the transcript base sequence of miRNA-6880-5p is 5'-UGGUGGAGGAAGAGGGCAGCUC-3' (SEQ ID NO: 1) (Accession Number: MIMAT0027660).

In the present invention, the case where miRNA-6880-5p is relatively underexpressed is based on the expression level of miRNA-6880-5p in exosomes isolated from the plasma of a normal person. In order to determine that the patient in question has prostate cancer, the expression level should be about 0.8 times or less than the expression level of miRNA-6880-5p in a normal person, and preferably, an underexpression of about 0.6 times or less should be confirmed.

Various agents capable of detecting microRNA can be used to measure the expression level of miRNA-6880-5p present in a sample.

In the present invention, “diagnosis” includes determining the susceptibility of an individual to a specific disease or condition, determining whether an individual currently has a specific disease or condition, determining the prognosis of an individual with a specific disease or condition, or therametrics (e.g., monitoring the condition of an individual to provide information about the efficacy of a treatment).

In addition, the prostate cancer diagnostic kit of the present invention may include an agent capable of measuring the expression level of miRNA-6880-5p from a biological sample, wherein the agent means a molecule that can be used for detecting the biomarker of the present invention by confirming the expression level of miRNA-6880-5p.

In the present invention, an antisense oligonucleotide, primer, probe, and/or antibody that specifically binds to miRNA-6880-5p can be used as a miRNA-6880-5p expression level measuring agent, and the expression level of miRNA-6880-5p can be measured by a known method used to measure the level of gene expression, for example, performing PCR using a primer, or performing a hybridization reaction using a probe. In addition, in addition to the measuring agent, the kit may include various tools and reagents known in the art that facilitate detection, for example, a suitable carrier, a labeling substance capable of generating a detectable signal, a stabilizer, etc.

The biological sample used to measure the expression level of miRNA-6880-5p in the present invention may be a non-invasive sample, and the non-invasive sample may be a liquid biopsy, i.e., blood, serum, plasma, urine, or saliva, preferably blood, serum, or plasma, and more preferably plasma, but is not limited thereto.

In the present invention, miRNA-6880-5p is a concept that includes functional equivalents of the oligonucleotides constituting it, for example, variants and mimics that can perform functionally the same function as the microRNA-6880-5p nucleic acid molecule, although some of the base sequences of the miRNA-6880-5p oligonucleotide have been modified by deletion, substitution, or insertion.

In the present invention, the “primer” refers to a short nucleic acid sequence having a short free hydroxyl group, which can form base pairs with a complementary template and serves as a starting point for copying the template strand. The primer of the present invention can be chemically synthesized using a method known in the art, such as, for example, a phosphoramidite solid support method.

In the present invention, the “probe” refers to a nucleic acid fragment such as RNA or DNA consisting of several to several hundred bases that can specifically bind to miRNA and is labeled so that the presence or absence of a specific miRNA can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc., and can be labeled with biotin, FITC, rhodamine, DIG, etc., or labeled with a radioisotope, etc.

In the present invention, the kit may be a kit including essential elements necessary for performing RT-PCR, but is not limited thereto. As an example, in the case of an RT-PCR kit, in addition to each primer pair specific for a marker gene, the kit may include a test tube or other appropriate container, a reaction buffer, deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase, DNase, RNase inhibitor, DEPC-water, sterile water, and the like.

In addition, the present invention can provide a method for diagnosing prostate cancer and a method for providing information for diagnosing prostate cancer, including the steps of isolating exosomes from a liquid biopsy isolated from a subject requiring diagnosis of prostate cancer; measuring the expression level of miR-6880-5p in the exosomes; and determining that the subject has prostate cancer if the expression level of miR-6880-5p is lower than that of a normal subject.

The measurement of the above miRNA expression level can be performed by, but is not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, quantitative RT-PCR, RNase protection assay (RPA), Northern blotting, or DNA chip method.

In the present invention, prevention means all acts of delaying the occurrence of prostate cancer by administering the composition according to the present invention, treatment means suppressing the survival and proliferation of prostate cancer by administering the pharmaceutical composition according to the present invention, and “metastasis” in the present invention means that prostate cancer moves away from the site of occurrence, and prevention or suppression of metastasis means all acts of reducing the movement and invasiveness of prostate cancer cells to reduce the departure of cancer cells from the site of occurrence.

In addition, the subject to be administered the pharmaceutical composition of the present invention is not limited to a mammal, but may preferably be a human, and more specifically, may be a prostate cancer patient.

The pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions.

In the present invention, the term "carrier", also called a vehicle, means a compound that facilitates the addition of a protein or peptide into a cell or tissue. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic substances into the cells or tissues of a living organism.

In the present invention, a "diluent" is defined as a compound that is diluted in water, which not only stabilizes the biologically active form of the target protein or peptide, but also dissolves the protein or peptide. A salt dissolved in a buffer solution is used as a diluent in the art. A commonly used buffer solution is phosphate buffered saline, because it mimics the salt state of human solutions. Since the buffer salt can control the pH of the solution at low concentrations, it is rare for a buffer diluent to modify the biological activity of the compound. The compounds containing azelaic acid used herein can be administered to a human patient as such, or as a pharmaceutical composition mixed with other ingredients or with suitable carriers or excipients, as in combination therapy.

In addition, the pharmaceutical composition for preventing or treating non-alcoholic steatohepatitis according to the present invention can be formulated and used in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. and sterile injection solutions according to conventional methods, and carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing the compound with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenterally, and in the case of parenteral administration, can be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc.

The appropriate dosage of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity.

The pharmaceutical composition of the present invention can be manufactured in the form of a unit dose or can be manufactured by placing it in a large-capacity container by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and by being formulated. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granules, tablets or capsules, and may additionally contain a dispersant or stabilizer.

The present invention can be modified in various ways and has various embodiments, and thus specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention are included. In describing the present invention, if it is determined that a specific description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

[Example]

Example 1. Plasma separation

Blood from normal subjects and prostate cancer patients was centrifuged at 2500 rpm for 7 minutes to separate plasma. The separated plasma was dispensed into cryovial tubes (550 ㎕ each) and stored at -80℃ until use in the experiment.

Example 2. Exosome isolation

The separated plasma samples were preprocessed in three steps.

1) After centrifugation at 1,200 g, 20 minutes, and 4℃, the supernatant was collected.

2) The supernatant was centrifuged once more at 10,000 g, 30 minutes, and 4°C, and then the supernatant was collected.

3) Finally, the separated supernatant was filtered using a 0.2 ㎛ syringe to remove large molecules.

The preprocessed samples were reacted with immuno-magnetic beads conjugated with CD63 antibody, an exosome surface marker, using a roller for 2 hours, and exosomes were separated using elution buffer. The separated exosomes were stored at -80℃ until used in the experiment.

Example 3. Exosome identification

The size and concentration of the isolated exosomes were analyzed using NanoSight LM10. After diluting 1:10 in PBS, the exosomes were injected into the sample measurement chamber using a 1 ㎖ syringe while minimizing bubble formation. Data analysis was performed using NTA Version 2.3 Build 0034 software.

Through this experiment, the size, concentration, and shape of the separated exosomes were confirmed to confirm whether they were exosomes (Figure 1).

Example 4. Selection of miRNAs in exosomes for prostate cancer diagnosis

-80℃ stored plasma was thawed and exosomes were isolated using CD63 antibody-conjugated immunomagnetic beads. miRNA from exosomes was isolated using Invitrogen (Cat No. 4478545) according to the manufacturer's instructions. Total RNA extracted from exosomes was labeled with fluorescent dye using FlsachTag Biotin RNA Labeling Kit to synthesize cDNA, and double-stranded DNA was converted to single-strand for hybridization. Then, it was hybridized to GeneChip miRNA 4.0 microarray. All arrays were measured by Affymetrix Gene Chip Scanner 3000, and data were analyzed by Transcriptome Analysis ConsoleTM (TAC) software. Differentially expressed genes were identified in the normalized miRNA data set using the R programming language (Fig. 2).

Through this experiment, we were able to identify miRNAs in plasma-derived exosomes that are differentially expressed between normal individuals and prostate cancer. In this result, PR is an abbreviation for Partial Response and refers to patients who respond well to treatment. PD is an abbreviation for Progressive disease and refers to a case in which one or more new lesions develop despite treatment. However, in this result, the samples of prostate cancer patients used the blood of patients before endocrine therapy. The experimental results showed that miR-6880-5p showed a significantly low expression level in the PD group and a relatively high expression level in the normal and PR groups. Therefore, it can be seen that the expression of miR-6880-5p in exosomes can distinguish prostate cancer patients from normal individuals and is related to the response to endocrine therapy in prostate cancer patients (Fig. 4).

Example 5. Confirmation of miRNA expression level in exosomes: qRT-PCR (Quantitative real-time polymerase chain reaction)

Total RNA in exosomes was isolated, and cDNA was synthesized using the TaqMan MicroRNA Reverse Transcription Kit and a primer with the sequence of has-miR-6880-5p (assay ID 467246_mat; 5'-UGGUGGAGGAAGAGGGCAGCUC-3'). 7 ㎕ of the reverse transcription kit mixture (100 mM dNTPs, MultiScribe Reverse Transcriptase, 10X Reverse Transcription Buffer, RNase inhibitor, Nuclease-free water), 3 ㎕ of 5X RT primer, and 5 ㎕ of RNA were added per sample (Table 1). Then, PCR was performed under the conditions of 16℃ for 30 min, 42℃ for 30 min, 85℃ for 5 min, and 4℃ ∞. The kit and primers for cDNA synthesis were purchased from Applied Biosystems, and PCR was performed according to the manufacturer's instructions. 1.33 ㎕ of synthesized cDNA, TaqMan master mix (2x), has-miR-6880-5p probe (TaqMan assay (20x)), and Ultra pure water were adjusted to a total of 20 ㎕ and dispensed into a 96-well standard (0.2 ㎖) plate (Table 2). qRT-PCR was performed for 1 cycle of 50℃ for 2 minutes, 1 cycle of 95℃ for 10 minutes, and 40 cycles of 95℃ for 15 seconds and 60℃ for 60 seconds to measure the expression of miR-6880-5p.

Through this experiment, the expression level of miR-68805p in blood-derived exosomes of prostate cancer patients was confirmed compared to that of normal people. By confirming that the expression of miR-6880-5p in prostate cancer patients was decreased compared to normal people, it was found that the expression of miR-6880-5p in exosomes is related to prostate cancer (Fig. 3). In addition, the expression of miR-6880-5p before and after endocrine therapy in the PR and PD groups of prostate cancer was confirmed. In the PR group, the expression of miR-6880-5p increased after treatment (FU, follow-up) compared to before treatment (BL, baseline), and in the PD group, the expression of miR-6880-5p decreased after treatment compared to before treatment (Fig. 4).

Component Volume (1 reaction) 100mM dNTPs (with dTTP) 0.15 μL MutiScribe ^™ Reverse Transcriptase, 50 U/μL 1.00 μL 10 1.50 μL RNase Inhibitor, 20 U/μL 0.19 μL Nuclease-free Water 4.16 μL Total RT Reaction Mix volume 7.00 μL

Component Volume per reaction 384-well or 96-well fast [0.1 mL] plate 96-well standard [0.2mL] plate TaqMan ^TM Small RNA Assay [20X] 0.50 μL 1.00 μL PCR Master Mix 5.00 μL 10.00 μL Nuclease-free water 3.84 μL 7.67 μL Total PCR Reaction Mix volume 9.34 μL 18.67 μL

Example 6. Cell culture

In this study, the normal prostate epithelial cell line RWPE-1, the hormone-responsive prostate cancer cell line LNCaP, and the castration-resistant prostate cancer cell lines DU145 and PC3 were used. RWPE-1 was cultured in Keratinocyte Serum-Free Medium containing 0.05 mg/mL BPD, 0.005 μg/mL Epidermal Growth Factor, and 1% Penicillin-Streptomycin, and LNCaP, DU145, and PC3 cell lines were cultured in RPMA-1640 medium containing 10% FBS and 1% Penicillin-Streptomycin. All cells were cultured in an incubator at 5% CO2 and 37℃.

Example 7. Confirmation of miRNA expression level in prostate cancer cells

To confirm the expression of miR-6880-5p by prostate cancer type, miRNA was isolated from normal prostate epithelial cells and prostate cancer cells using QIAzol reagent, miRNeasy Mini kit (Qiagen, Cat No. 217004) according to the manufacturer's instructions. Then, cDNA was synthesized using the same method as the method for confirming the expression of miRNA obtained from exosomes, and the expression of miR-6880-5p in prostate cancer cells was confirmed through qRT-PCR.

As a result, it was found that the expression of miR-6880-5p was low in the order of RWPE-1, LNCaP, DU145, and PC3 (Fig. 5). The results above show that the expression level of miR-6880-5p can be used to predict the prognosis of each type of prostate cancer.

Example 8. Confirmation of cell proliferation, migration, and invasion ability according to miR-6880-5p regulation in prostate cancer cells

To determine whether the level of miR-6880-5p expression affects the characteristics of prostate cancer cells, miR-6880-5p mimic and inhibitor were transfected into prostate cancer cell lines, DU145 and PC3 cells, using Lipofectamin RNAimax (Cat No. 13778075). Specifically, transfection of miR-6880-5p mimic and inhibitor was performed using Cationinc lipid-based reagent of Lipofectamin RNAimax (Cat No. 13778075) according to the manufacturer's guidelines. The miRNA to be transfected and Lipofectamin RNAimax were diluted using Opti-MEM Medium. miRNA mimic was diluted to 1, 5, or 10 nM, and miRNA inhibitor was diluted to 50 nM. NC was performed in the same way. Next, the diluted miRNA and NC were mixed with Lipofectamin RNAimax in a 1:1 ratio to form miRNA-lipid complexes. The formed complexes were injected into cells that were 60-80% full in a 6-well plate and cultured in a 37°C incubator for 24 hours.

Product information of miR-6880-5p inhibitor/mimic is as shown in Table 3 below.

miRBase ID Assay ID Product Type Catalog No. hsa-miR-6880-5p AM27166 Ambion®　Anti-miR™　miRNA Inhibitor AM17000 PM27166 Ambion®　Pre-miR™　miRNA Precursor AM17100

- Cell viability assay

The transfected cells were seeded into 96-well plates. After culturing in an incubator for 24, 48, and 72 hours, they were stained with 3-(4,5 dimethylthiazol-2-yl)-2,5-disphenyltetrazolium bromide. Then, the cell viability was measured at a wavelength of 595 nm using a microplate reader.

- Cell wound healing assay

The transfected cells were seeded in a 24-well plate and stabilized in an incubator for 24 hours. Then, the cell layer was scratched with a sterile 200 ㎕ plastic pipette tip to create a wound, and images were taken using an optical microscope immediately (0 h). After an additional 24 hours of culture, images were taken using an optical microscope and changes in cell migration were confirmed compared to each negative control group.

- Cell invasion assay (Trans-well assay)

To analyze the invasion ability of cells, a 24-well trans well chamber (Costar) was used. 200 μL of serum-free medium and transfected cells were dispensed into the upper chamber (8.0 μm pore size), and 750 μL of complete medium was dispensed into the lower chamber. After culturing in an incubator for 48 h, the upper and lower chambers were washed with PBS, and the cells on the lower membrane surface of the upper chamber were fixed with 4% paraformaldehyde and methanol and stained with 0.1% crystal violet. Then, the cells on the upper membrane surface were removed with a cotton swab. Invasion images were captured using an optical microscope, and the number of invaded cells in four random areas was measured.

Both PC3 and DU145 cells mimicking miR-6880-5p showed significantly inhibited cell proliferation, migration, and invasion compared to the negative control, whereas cells in which miR-6880-5p was suppressed showed significantly increased proliferation compared to the negative control. These results confirmed that the expression of miR-6880-5p directly regulates the proliferation (Fig. 6a and Fig. 6b), migration (Fig. 6c and Fig. 6d), and invasion (Fig. 6e and Fig. 6f) abilities of cancer cells.

Example 9. Analysis of gene functions regulated by miR-6880-5p expression

Total RNA was isolated from DU145 cells transfected with miR-6880-5p mimic (6880_mi), negative control (NC), and untreated (Con) DU145 cells using Trizol reagent according to the manufacturer's instructions. Total RNA concentration was measured using Nanodrop (Thermo Scientific0). RNA quality was assessed using an Agilent 2100 bioanalyzer with an RNA 6000 nano chip (Agilent Technologies). The library for RNA sequencing analysis was prepared using the QunatSeq Library Prep kit (Lexogen), and high-throughput sequencing was performed using NextSeq 500 (Illumina). The entire transcriptome was used to identify differentially expressed genes (DEGs) that were more than twice as high in the cell group transfected with miR-6880-5p mimic. DEGs were analyzed using ExDEA software (Ebiogen), and gene classification was analyzed based on DAVID, Kyoto Encyclopedia of Gens and Genomes (KEGG) pathway, and STRING. Heatmaps were analyzed using MultiExperiment Viewer software (MeV, V4.9.0).

All significantly differentially expressed genes (DEGs) were classified into 14 GO categories by ExDEA software. Genes involved in angiogenesis and cell migration by miR-6880-5p expression were down-regulated at the highest rate compared to the control and negative controls (Fig. 7a to Fig. 7c). In addition, the signaling pathways of DEGs according to the overexpression of miR-6880-5p were analyzed using KEGG pathway. Compared to the control and negative controls, the genes down-regulated by the overexpression of miR-6880-5p were related to eight major signaling pathways (Ras signaling pathway, MAPK signaling pathway, cancer pathway, cell senescence, ErbB signaling pathway, EGFR tyrosine kinase inhibitor resistance, and AMPK signaling pathway) (Fig. 7d, 7f, and 7g). Angiogenesis is known to promote cancer metastasis by regulating cell invasion, proliferation, and migration (Melegh Z et al., 2019, Int J Mol. 31;20(11):2676). In addition, Ras and MAPK have been reported to play important roles in the development of metastatic prostate cancer, and ErbB promotes tumor growth by reactivating androgen receptor expression in patients with castration-resistant prostate cancer (Kojima M et al., 2012, Cancer Res. 72: 1878-89; Gao S et al., 2016, Clin Cancer. 22: 3672-82). And the genes upregulated by overexpression of miR-6880-5p were related to nine major signaling pathways (cell cycle, autophagy, FoxO signaling pathway, cancer pathway, Hippo signaling pathway, ferroptosis, mitophagy, HIF-1 signaling pathway, and cellular senescence) (Figures 7e, 7h, and 7i). FoxO signaling pathway, ferroptosis, and mitophagy are closely related to apoptosis and antitumor immunity (Reference [Ashrafizadeh M et al., 2022, J Exp Clin Cancer Res. 41: 105]; Reference [Wang Y et al., 2022, Prostate Cancer and Prostate Diseases. doi:10.1038/s41391-022-00583-w]). In particular, FoxO is known as a tumor suppressor gene associated with cell differentiation, apoptosis, cell cycle arrest, DNA damage and repair (Shan Z et al., 2019, J Cell Mol Med. 23: 3130-9).

Therefore, we found that genes down-regulated by miR-6880-5p overexpression were closely related to tumor promotion, whereas up-regulated genes were related to regulating cell proliferation and apoptosis. These results indicate that miR-6880-5p can function as a tumor suppressor miRNA.

Example 10. Confirmation of antitumor effect through regulation of miR-6880-5p expression

The apoptotic effect in prostate cancer was confirmed by regulating the expression of miR-6880-5p. The experiment was performed in the time sequence shown in Table 4 below.

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Seeding
(2.5*10 ⁵ cells/well) Transfection (24h) Re-seeding
(2.5*10 ⁵ cells/well) Enz 10uM, 20uM treat (48h) MTT treat (0.5mg/ml, 1h

Enzalutamide, a drug used to treat prostate cancer, was used as a positive control to confirm the therapeutic effect of miR-6880-5p mimic.

As a result of performing MTT assay, it was confirmed that the survival rate of prostate cancer cells transformed with miR-6880-5p was significantly reduced (Fig. 8). In the case of miR-6880-5p mimic, the survival rate of cancer cells was significantly reduced compared to the case of enzalutamide treatment alone, and it was confirmed that the antitumor effect of miR-6880-5p mimic was independent of enzalutamide. Meanwhile, the antitumor effect of miR-6880-5p mimic was shown to be more excellent in the highly malignant PC3 cell line.

Although the embodiments have been described with limited drawings as described above, those skilled in the art can apply various technical modifications and variations based on the above. For example, even if the described techniques are performed in a different order than the described method, and/or the components of the described system, structure, device, circuit, etc. are combined or combined in a different form than the described method, or are replaced or substituted by other components or equivalents, appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are also included in the scope of the claims described below.

Claims (13)

A pharmaceutical composition for preventing or treating prostate cancer, comprising miR-6880-5p or a vector expressing the same as an active ingredient. In the first paragraph, A pharmaceutical composition wherein the above miR-6880-5p is an oligonucleotide consisting of the base sequence of sequence number 1. In the first paragraph, A pharmaceutical composition, wherein the vector expressing the above miR-6880-5p comprises a base sequence of sequence number 1 or a base sequence complementary thereto. In the first paragraph, A pharmaceutical composition, wherein the above prostate cancer is a prostate cancer resistant to hormone therapy. A pharmaceutical composition for preventing prostate cancer metastasis, comprising miR-6880-5p or a vector expressing the same as an active ingredient. A biomarker composition for diagnosing prostate cancer comprising miR-6880-5p in cancer cell-derived exosomes as an active ingredient. In Article 6, The above cancer cell-derived exosomes were isolated from plasma. A biomarker composition wherein the above cancer cell-derived exosomes express CD63. A composition for diagnosing prostate cancer, comprising as an active ingredient an agent capable of measuring the expression level of miR-6880-5p in cancer cell-derived exosomes. In Article 8, A composition for diagnosing prostate cancer, wherein the agent capable of measuring the expression level of the above miR-6880-5p is an oligonucleotide containing a nucleotide sequence complementary to part or all of the above miR-6880-5p. (1) A step of isolating cancer cell-derived exosomes from a liquid biopsy isolated from an individual requiring diagnosis of prostate cancer; (2) a step of measuring the expression level of miR-6880-5p in the exosome; and (3) A method for diagnosing prostate cancer and a method for providing information for diagnosing prostate cancer, comprising: a step of determining that the patient has prostate cancer if the expression level of the miR-6880-5p is lower than that of a normal person. In Article 10, An information providing method, wherein the step (1) above is performed using an immuno-magnetic bead combined with a CD63 antibody. In Article 10, A method of providing information, wherein the liquid biopsy is blood, plasma, or serum. How to determine the appropriateness of treatment in patients with prostate cancer by performing the following steps before and after cancer treatment: (a) isolating cancer cell-derived exosomes from a liquid biopsy isolated from the patient; (b) a step of measuring the expression level of miR-6880-5p in the exosome; and (c) a step of determining that the treatment prognosis is good if the expression level of the miR-6880-5p after treatment is significantly higher than that before treatment, and determining that the treatment prognosis is poor if the expression level is significantly lower than that before treatment.

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