KR20230039624A - Composition for diagnising cancer, kit for diagnosing, pharmaceutical composition for preventing or treating and method for measuring of cancer using the same - Google Patents

Abstract

The present invention relates to a composition for diagnosing cancer, a kit for diagnosing cancer including the same, a pharmaceutical composition for preventing or treating cancer, and a method for determining whether or not cancer has occurred. By identifying the mechanisms of proliferation, migration and invasion of cells based on the association of miR-146a and NRP2 in circulating tumor cells or mimetic floating cells thereof, the composition for diagnosing cancer according to the present invention can inhibit cancer through the miRNA-146a. Therefore, the composition can be usefully used in fields such as cancer diagnosis and treatment, and specifically, the composition can be usefully applied to the cancer diagnosis kit, the pharmaceutical composition for preventing or treating cancer, and the method for determining whether or not cancer has occurred.

Description

A composition for diagnosing cancer, a kit for diagnosing cancer containing the same, a pharmaceutical composition for preventing or treating cancer, and a method for measuring the occurrence of cancer USING THE SAME}

The present invention relates to a composition for diagnosing cancer, a kit for diagnosing cancer including the same, a pharmaceutical composition for preventing or treating cancer, and a method for determining whether cancer occurs.

Recently, research is being conducted to discover therapeutic targets through research on the function of genes related to cancer generation and treatment worldwide and to use them for diagnosis and development of therapeutic agents. Along with the activation of genome research, research on human gene DNA chip or proteome analysis has been actively carried out, and as genes related to cancer have been discovered in large numbers, lists of many genes and related databases have been established, but most of them have been specifically identified in cells for these genes. Biological functions and relevance to cancer have not yet been studied or are uncertain, so there are considerable difficulties in discovering genes that can effectively treat cancer as well as actual cancer relevance or diagnosis and utilization as target genes. Therefore, it is necessary to discover new genes in addition to the cancer-related genes that have been identified so far.

On the other hand, cancer metastasis is a major cause of cancer-related death, and the propagation of tumor cells through the vascular system, which is a major mechanism of cancer metastasis, is an important intermediate step in converting a typical local disease to a systemic disease. Approximately 3 to 4 × 10 ⁹ tumor cells are shed from 1 gram (109 cells) of cancer patient's tumor tissue every day and enter blood vessels. Most of these cells are mostly eliminated in the hostile environment in the blood, but a very small number of about 1 per 10 ⁶ to 10 ⁷ normal peripheral mononuclear blood cells (PBMC) survives, and circulating tumor cells (circulating tumor cells) , CTCs). Therefore, early detection and analysis of circulating tumor cells in the blood of cancer patients is an important strategy for both monitoring and preventing metastasis.

Methods such as flow cytometry, magnetic cell separation, and di-electrophoresis have been proposed as methods for detecting circulating tumor cells in blood. However, despite years of development, these methods are still only useful in research, and clinical applications are limited due to lack of efficiency and reproducibility of results.

Patent Literature 1 proposes a technique for providing disease information about a tumor using the aforementioned circulating tumor cells.

More specifically, in the case of the technique proposed in Patent Document 1, receiving genetic data of "cancer" tissue from a first terminal of a cancer patient; Receiving, from a second terminal of the institution, the gene data of the "circulating tumor cells" of the "cancer" patient; generating tumor heterogeneity data by comparing the "cancer" tissue genetic data and the "circulating tumor cell" genetic data; and transmitting the tumor heterogeneity data to the first terminal or the second terminal; a technique for a method for providing tumor heterogeneity data based on circulating tumor cells is proposed.

However, the technology for Patent Document 1 proposed above requires analysis of genes of cancer tissues from biological samples of cancer patients and comparative analysis of tumor heterogeneity, which takes a long time for diagnosis and confirmation of cancer occurrence. However, since it does not contain clear biomarkers related to cancer metastasis, there is a problem in that it is a diagnosis of heterogeneity of a tumor rather than a diagnosis of the tumor itself, or a level of providing data therefor.

Therefore, there is an urgent need for a technology for more clearly diagnosing cancer related to circulating tumor cells at the molecular genetic level.

Patent Document 1: Korean Patent Publication No. 10-2020-0136093

In order to solve the above-mentioned problems of the prior art, the present inventors identified miRNAs that change the molecular biological characteristics of circulating tumor cell-like floating cells, and through them, specific genes and proteins capable of controlling the proliferation, migration, and invasion of the cells. By confirming the association with cancer diagnosis, in particular, the mechanism related to the diagnosis of metastasis cancer was identified.

Accordingly, an object of the present invention is to provide a composition for diagnosing cancer.

Another object of the present invention is to provide a kit for diagnosing cancer comprising the composition for diagnosing cancer.

In addition, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising the composition for diagnosing cancer.

In addition, an object of the present invention is to provide a method for determining whether or not cancer has occurred, including the composition for diagnosing cancer.

As one embodiment for achieving the above object,

The present invention provides a method for determining the occurrence of cancer, comprising measuring the level of NRP2 protein in a biological sample.

In addition, in the present invention, the biological sample is a method for determining whether cancer occurs, characterized in that the biological sample is a circulating-tumor cell (CTC) or a circulating-tumor cell-mimicking suspension cell thereof. provides

In addition, the present invention provides a method for determining whether cancer occurs, further comprising the step of measuring the mRNA level of the NRP2 before expression of the NRP2 protein.

In addition, the present invention provides a method for determining whether cancer occurs, further comprising the step of injecting the NRP2 protein expression inhibitor into the biological sample.

In addition, the present invention provides a method for determining whether cancer occurs, characterized in that the NRP2 protein expression inhibitor is miR-146a.

The cancer diagnosis composition according to the present invention identifies the mechanism of proliferation, migration, and invasion of miR-146a and NRP2 based on the association between circulating tumor cells or their mimic floating cells, and through the miRNA-146a Can inhibit cancer development. Therefore, it can be usefully used in fields such as cancer diagnosis and treatment, and specifically, it can be usefully applied to a cancer diagnosis kit, a pharmaceutical composition for preventing or treating cancer, and a method for determining whether or not cancer has occurred.

The accompanying drawings are intended to explain the contents of the present invention in more detail to those skilled in the art, but the technical spirit of the present invention is not limited thereto.
1 and 2 are diagrams showing some of the results of Experimental Example 1 according to the present invention.
3 is a diagram showing the results of Experimental Example 2 according to the present invention.
4 is a diagram showing the results of Experimental Example 3 according to the present invention.
5 is a diagram showing the results of Experimental Example 4 according to the present invention.
6 is a diagram showing the results of Experimental Example 5 according to the present invention.
7 is a diagram showing the results of Experimental Example 6 according to the present invention.

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

The present invention provides a composition for diagnosing cancer. More specifically, the present invention provides a composition for diagnosing cancer containing the miR-146a represented by SEQ ID NO: 1 in a cell, and in this case, the miR-146a is related to a protein that can act as an expression promoter or expression inhibitor. The above "agent for measuring protein level" may be included.

The term "miR-146a" in the present specification may be used as a term referring to "microRNA-146a".

In addition, the term "agent for measuring protein level" in the present specification refers to an agent used in a method for measuring the level of a target protein contained in a sample, preferably Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay , Complement Fixation Assay, FACS, and protein chip assay.

The term "antibody" as used herein refers to a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. Such an antibody is obtained by cloning each gene into an expression vector according to a conventional method, A protein encoded by the marker gene is obtained, and can be produced from the obtained protein by a conventional method. The form of the antibody is not particularly limited, and as long as it has polyclonal antibody, monoclonal antibody, or antigen-binding properties, a part thereof is also included in the antibody of the present invention, and all immunoglobulin antibodies may be included, as well as humanized antibodies, etc. may include special antibodies of In addition, the antibody includes functional fragments of the antibody molecule as well as complete forms having two full-length light chains and two full-length heavy chains.

The "functional fragment of an antibody molecule" refers to a fragment having at least an antigen-binding function, and may be, for example, Fab, F(ab'), F(ab') 2 and Fv.

In addition, the term “diagnosis” in this specification means confirming the presence or characteristics of a pathological state. In the present invention, the "diagnosis" can be interpreted as observing and confirming whether or not a cancer disease has occurred.

The miR-146a may be represented by the sequence of SEQ ID NO: 1, and may be a sequence having 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more homology with SEQ ID NO: 1.

In the present invention, the miR-146a, that is, the miRNA nucleic acid molecule may have a length of 18 to 100 nt (Nucleotide), preferably 19 to 25 nt in length, more preferably 21, 22 or 23 nt in length.

In addition, miR-146a used in the present invention is a functional equivalent of the nucleic acid molecule constituting it, for example, a part of the nucleotide sequence of the miR-146a nucleic acid molecule is subject to deletion, substitution, or insertion. However, it is a concept that includes variants that can functionally have the same action as the miR-146a nucleic acid molecule. In the present invention, the mutants are also referred to as Mimics.

In addition, miR-146a of the present invention may exist in a single-stranded or double-stranded form. While mature miRNA molecules are predominantly single-stranded, precursor miRNA molecules are predominantly at least partially self-complementary (eg stem- and loop-structures) capable of forming double-stranded regions. In addition, the nucleic acid molecule of the present invention may be configured in the form of RNA, DNA, peptide nucleic acids (PNA) or locked nucleic acid (LNA).

The nucleic acid molecules of the present invention can be isolated or prepared using standard molecular biology techniques, such as chemical synthesis methods or recombinant methods, or commercially available ones can be used. In addition, the composition of the present invention may include not only the miRNA nucleic acid molecule itself, but also other substances capable of increasing the expression rate of the miRNA of the present invention in cells, such as compounds, natural products, novel proteins, and the like.

Meanwhile, the miR-146a nucleic acid molecule in the composition of the present invention may be included in a vector for intracellular expression.

The miR-146a nucleic acid molecule of the present invention can be introduced into cells using various transformation techniques, such as a complex of DNA and DEAE-dextran, a complex of DNA and nucleoprotein, and a complex of DNA and lipid. To this end, the miR-146a nucleic acid molecule can be introduced into cells. The -146a nucleic acid molecule may be in a form incorporated in a delivery vehicle that allows for efficient introduction into cells. The delivery vehicle is preferably a vector, and both viral vectors and non-viral vectors can be used. As the viral vector, for example, lentivirus, retrovirus, adenovirus, herpes virus, and Avipox virus vectors can be used.

In addition, the vector containing the miR-146a nucleic acid molecule preferably further includes a selectable marker. The term "selection marker" in the present specification may be used to facilitate the selection of transformed cells into which miR-146a nucleic acid molecules are introduced. Selectable markers that can be used in the vector of the present invention are not particularly limited as long as they are genes that can easily detect or measure whether or not the vector has been introduced, but are typically drug resistance, nutrient requirements, resistance to cytotoxic agents, or Markers conferring selectable phenotypes such as expression of surface proteins, e.g. GFP (Green Fluorescent Protein), Puromycin, Neomycin (Neo), Hygromycin (Hyg), Histi Histidinol dehydrogenasegene (hisD) and guanine phosphoribosyltransferase (Gpt), and the like, preferably use GFP (green fluorescent protein) and puromycin markers.

Meanwhile, the miR-146a nucleic acid molecule in the composition of the present invention may be introduced into cells.

The term "introduction" in the present specification may mean introduction of foreign DNA into cells by transfection or transduction. Transfection can be carried out using sugars such as calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofectamine and protoplast fusion. This can be done by several methods known in the art. In addition, transduction may mean the transfer of a gene into a cell using a virus or viral vector particles by means of infection.

In addition, the present invention provides a composition for diagnosing cancer, characterized in that the cells are circulating-tumor cells (CTC) or circulating-tumor cell-mimicking suspension cells thereof.

As used herein, the term "circulating-tumor cell (CTC)" refers to a tumor cell separated from a primary tumor and flowing into the bloodstream, which spreads from the original tumor to another location and leads cancer metastasis. It may mean including. In addition, as used herein, the term "circulating-tumor cell-mimicking suspension cell" refers to a medium other than adherent cells having characteristics similar to those of the aforementioned circulating tumor cells. It may mean to include all cells that live while floating in

The composition for diagnosis of cancer according to the present invention was completed by identifying mechanisms of proliferation, migration, and invasion of circulating tumor cells or their mimetic floating cells based on their association.

In addition, the cancers that can be diagnosed by the cancer diagnosis composition according to the present invention are not particularly limited, and examples include brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, and lung cancer. , bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, perianal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, Esophageal cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureteral cancer, renal cell carcinoma, kidney It may be any one or more cancers selected from the group consisting of pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and cancers caused by metastases thereof.

In addition, the cancer that can be diagnosed by the composition for diagnosing cancer according to the present invention may be a cancer induced by overexpression of NRP2 represented by SEQ ID NO: 2.

As used herein, the term "NRP2 (Nuropilin2)" is expressed by various cell types, and may mean an NRP2 protein or NRP2 protein receptor that may be encoded or encoded by the NRP2 gene.

The miR-146a included in the composition for diagnosing cancer according to the present invention targets NRP2 and interferes with the p38 signaling process. More specifically, the miR-146a can inhibit the expression of NRP2 by binding to the 3' UTR region of the NRP2. The details of this mechanism will be described in detail in experimental examples to be described later.

The composition for diagnosing cancer according to the present invention may be a composition for diagnosing cancer caused by metastasis.

Cancers caused by the metastases, for example, as described above, include brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, uterus Cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, proximal anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphatic cancer, bladder cancer, gallbladder cancer, endocrine cancer, Thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureteric cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma , spinal cord tumor, brainstem glioma, and pituitary adenoma, but may be one selected from the group consisting of, but is not limited thereto.

In addition, if necessary, an expression inhibitor capable of suppressing the expression of the miR-146a-expressed protein or NRP2 protein at the mRNA level before the expression of the miR-146a-expressed protein or the NRP2 protein. can include

The term "capable of suppressing at the mRNA level" in the present specification may mean suppressing the level of mRNA transcribed from the target gene in order to determine the expression of the target gene included in the sample, and the agent used for this Zero is preferably RT-PCR, competitive RT-PCR (Competitive RT-PCR), real-time RT-PCR (Real-time RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blotting ), a probe or primer capable of specifically binding to a target gene used in methods such as DNA chip analysis, but is not particularly limited thereto.

The term "primer" in the present specification has the same meaning as a primer in a conventional sense, and means, for example, a nucleic acid sequence having a short free 3' hydroxyl group, and base pairing with a complementary template A short nucleic acid sequence that can form a base pair and serves as a starting point for copying the template strand. These primers can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature.

The term "probe" in the present specification may have the same meaning as a probe in a conventional sense, and for example, RNA or DNA corresponding to a few bases to several hundred bases that can specifically bind to a gene or mRNA It can mean a nucleic acid fragment such as, etc., and can be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, etc., and can be more easily It can be labeled for detection.

In addition, the present invention provides a kit for diagnosing cancer comprising the composition for diagnosing cancer.

In addition, the present invention provides a kit for diagnosing cancer, characterized in that the kit is an RT-PCR kit, a DNA chip kit, or a protein chip kit.

The kit of the present invention measures the protein level of a protein for which miR-146a can act as an expression promoter or expression inhibitor from a sample of a patient suspected of having cancer or a metastatic cancer disease from the cancer, to determine whether or not cancer disease has occurred. It can be used to diagnose cancer diseases by identifying, but is not particularly limited, suitable for analysis methods as well as primers, probes or antibodies for measuring the level of a protein that can act as an expression promoter or expression inhibitor of the miR-146a. One or more other component compositions, solutions or devices may also be included.

The term "sample" as used herein refers to a direct object to measure the expression level of a protein that is isolated from a patient with cancer and miR-146a can act as an expression promoter or expression inhibitor, and is, for example, a tissue sample of a cancer patient. It may be, but is not limited thereto.

The kit of the present invention may be a kit for protein chip analysis for measuring a protein in which miR-146a can act as an expression promoter or expression inhibitor. The kit is not particularly limited thereto, but is described for immunological detection of antibodies. , a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, and the like.

The substrate is not particularly limited thereto, but a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, and a slide glass made of glass may be used, and the color-developing enzyme is not particularly limited thereto. However, peroxidase and alkaline phosphatase may be used, and the fluorescent material may be, but is not particularly limited to, FITC, RITC, etc., and the color-developing substrate solution is not particularly limited thereto, but ABTS (2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) or OPD (o-phenylenediamine), TMB (tetramethyl benzidine).

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising miR-146a as an active ingredient.

Even in the case of the above cancers, as described above, brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, and ovarian cancer , colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, proximal anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphatic cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer , adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureteric cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, It may be any one or more selected from the group consisting of brainstem glioma, pituitary adenoma, and cancer due to metastasis thereof, but is not particularly limited thereto.

The pharmaceutical composition for preventing or treating cancer of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, and colloidal. Silicon Dioxide, Calcium Hydrogen Phosphate, Lactose, Mannitol, Taffy, Gum Arabic, Pregelatinized Starch, Corn Starch, Powdered Cellulose, Hydroxypropyl Cellulose, Opadry, Sodium Starch Glycolate, Carnauba Lead, Synthetic Aluminum Silicate, Stearic Acid, Magnesium stearate, aluminum stearate, calcium stearate, white sugar, dextrose, sorbitol, talc, and the like can be used. Pharmaceutically acceptable additives that can be used in the present invention are preferably included in an amount of 0.1 part by weight to 90 parts by weight based on the composition, but are not limited thereto.

That is, the pharmaceutical composition for preventing or treating cancer of the present invention may be administered in various oral and parenteral formulations during actual clinical administration. When formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, It may be prepared using diluents or excipients such as surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient such as starch, calcium carbonate, water, etc. It may be prepared by mixing sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used. Liquid formulations for oral use include suspensions, solutions for oral use, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. . Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The pharmaceutical composition for preventing or treating cancer of the present invention may be administered orally or parenterally, depending on the desired method, and in the case of parenteral administration, external skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection Injection or intrathoracic injection is preferred. The dosage varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.

The dosage of the pharmaceutical composition for preventing or treating cancer of the present invention varies in its range depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of the disease, and daily administration The amount is 0.0001 mg/kg to 100 mg/kg, preferably 0.001 mg/kg to 10 mg/kg, based on the amount of the soybean extract or fraction thereof, and may be administered once to six times a day.

The pharmaceutical composition for preventing or treating cancer of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

The present invention also provides a method for determining the occurrence of cancer, comprising measuring the level of NRP2 protein in a biological sample.

The method for determining the occurrence of cancer according to the present invention relates to a method for determining the occurrence of cancer using the composition for diagnosing cancer according to the present invention described above, and will be omitted if it is the same as the above description.

The biological sample may be a circulating-tumor cell (CTC) or a circulating-tumor cell-mimicking suspension cell thereof.

In addition, the present invention provides a method for determining whether cancer occurs, further comprising the step of measuring the mRNA level of the NRP2 before expression of the NRP2 protein.

In addition, the present invention provides a method for determining whether cancer occurs, further comprising the step of injecting the NRP2 protein expression inhibitor into the biological sample.

In addition, the present invention provides a method for determining whether cancer occurs, characterized in that the NRP2 protein expression inhibitor is miR-146a.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

[Experiment method]

1. Cell culture

MDA-MB-468 breast cancer cells were cultured in an incubator at 37° C., 5% CO ₂ , and a medium in which 10% fetal bovine serum (FBS) was added to RPMI-1640 was used. Circulating tumor cell-like MDA-MB-468 floating cells were treated with 0.1 μg/ml nocodazole for 16 hours to induce cell division simultaneously. Thereafter, the cells were collected, washed with PBS, and maintained in a non-adhesive dish (Coring). More than 160 cells were used for the passage of MDA-MB-468 floating cells used in the experiment, and HeLa cells were cultured in DMEM supplemented with 10% FBS (fetal bovine serum), 1% penicillin-streptomycin, and mycozap. .

2. Small-RNA, mRNA sequencing analysis

Small RNA sequencing was performed using Theragen Etex on total RNA extracted from MDA-MB-468 adherent and floating cells. Data were analyzed by the Theragen Etex bioinformatics team. Analysis of miRNA-mRNA interactions showing expression differences was performed. The mRNA sequencing was reanalyzed to confirm the difference in expression patterns between adherent and floating cells using log2 difference.

3. Transfection

In MDA-MB-468, siRNA and miRNA mimics (analogues) were harvested 48 hours after transfection using lipofectamine RNAiMAX. HeLa was transfected with plasmid and miRNA mimic using Lipofectamine 2000. SEMA3C (R&D systems) was used at 50 ng/ml after 24 hours of starvation.

4. Gene expression analysis

Total RNA was extracted using the miRNeasy RNA isolation kit and then reverse transcribed using the TaqMan miRNA Reverse Transcription kit. miRNA real-time polymerase chain reaction was performed using TaqMan Universal Master Mix II, no UNG product, and miR-146a was detected with a Taqman probe. Data were normalized using small ribonucleic acid 48 as a control.

The extracted mRNA was reverse transcribed with the qPCRBIO cDNA Synthesis Kit product. Real-time polymerase chain reaction was performed using qPCRBIO SyGreen Mix Hi-ROX product. Ribosome 18S was used as a control.

5. Western blot

MDA-MB-468 was dissolved in RIPA buffer containing protease and phosphatase inhibitors. Thereafter, proteins were extracted by centrifugation at 13000 RPM and 4° C. for 15 minutes. For protein quantification, Pierce BCA Protein assay kit was used. Equal amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis and transferred to a polyvinyl-difluoride (PVDF) membrane. After treatment with 3% bovine serum albumin (BSA) in 0.2% PBST for 1 hour, the primary antibody was shaken and stored until the next day. Primary antibodies used are NRP2 (1:800, R&D systems), p38 (1:1000, Cell signaling), phosphor-p38 (Thr180/Tyr18) (1:800, Cell signaling), GAPDH (1:10000, Cell signaling) ) was The amount of protein was detected using an HRP-conjugated secondary antibody (1:4000, Cell Signaling), (1:4000, Abcam) and a chemiluminescence detection method.

6. Cell invasion assay

Matrigel (Coring) was diluted in serum free media and coated on the upper frame of a 24-well plate (8.0 μm pore size; SPL). Transfected cells were spread on the upper frame with medium without serum and medium with serum on the lower frame. After culturing in an incubator at 37° C. and 5% CO ₂ for 48 to 72 hours, the medium of the upper frame was removed and washed with PBS. Cells were fixed with 4% formaldehyde and permeabilized with 100% methanol. After staining the cells with 0.5% crystal violet diluted in 20% methanol, the number of cells infiltrating the coated membrane was measured using ImageJ. In the cell invasion assay to see the effect of SEMA3C, SEMA3C was treated 24 hours before removing the medium.

7. Cell migration assay

MDA-MB-468 adherent cells were spread in each frame using SPLScar block (SPL) in a 12-well plate. The next day after removing the block, it was cultured in a medium containing 10% fetal bovine serum. The distance traveled by the cells was taken with a microscope equipped with a camera, and images were taken at regular intervals. In this case, the distance traveled was calculated using ImageJ.

8. Cell proliferation assay

The transfected MDA-MB-468 cells were plated in a 6-well plate. After culturing in an incubator at 37°C, 5% CO ₂ for 2 to 3 days, PBS washing, treatment with 0.25% trypsin EDTA (Wellgene) at 37°C for 2 minutes, neutralization, and the number of cells using trypan blue was counted every set day. measured. To see the effect of SEMA3C, the cells were spread in serum-free medium and the number of cells was measured in the same way after SEMA3C treatment for 24 hours. In this case, 10% fetal bovine serum was used as a positive control factor.

In the WST-1 experiment, 1 × 10 ⁴ cells per well of transfected MDA-MB-468 adherent cells were spread in a 96-well plate. After culturing in a 37°C, 5% CO ₂ incubator for 2 to 3 days, 10 μl of EZ-Cytox (Daeil Lab), a WST-1 reagent, was treated at 37°C for 30 minutes. After that, the absorbance at a wavelength of 450 nm was measured every designated day.

9. Luciferase reporter assay

The Human NRP2 3'UTR (3075bp) part containing the seed sequence predicted to bind to miR-146a with the 3'UTR luciferase reporter assay was cloned into the NotI and XhoI sites of the psiCHECK-2 vector. The seed sequence AGTTCTC of NRP2 was mutated to AACTCTA (Mutant #1) and AATCACC (Mutant #2) using the QuikChange II Site-Directed Mutagenesis Kit (Agilent). HeLa was transfected with a luciferase reporter vector and miR-146a mimic, negative control miRNA using lipofectamine 2000 (Invitrogen). After 48 hours, cells were lysed and luciferase activity was measured using a Dual-Luciferase Reporter Assay kit (Promega).

10. In silico analysis

The overall survival and distant metastasis-free survival of breast cancer patients according to the expression of NRP2 in the Kaplan-Meier plotter dataset were analyzed.

11. Statistical analysis

All experiments were performed at least three times, and analysis was performed using GraphPad Prism 5.0 software. Unpaired two-tailed Student's t-test was used, and a value of p<0.05 was judged to be significant.

[Experimental Example]

[Experimental Example 1] mRNA and small RNA sequencing

In order to find miRNAs that change the molecular biological characteristics of circulating tumor cell-like floating cells, small RNA sequencing was performed on MDA-MB-468 adherent cells and floating cells, and shown in FIGS. 1 and 2 .

It will be described with reference to FIGS. 1 and 2 below.

As a result, 46 increased and 43 decreased miRNAs were found in floating cells (Fig. 1A), and among these results, 5 increased and 6 decreased miRNAs showed significant changes in expression level as shown in Table 1 below. .

miRNAs Log2 Fold Change p-value Upregulated miRNAs - miR-187 3.90 1.92E-41 miR-224 1.27 9.69E-06 miR-488 5.38 1.91E-03 miR-582 2.63 1.01E-03 miR-1298 4.53 4.43E-02 Downregulated miRNAs - miR-135a -2.21 1.46E-05 miR-146a -5.00 3.33E-16 miR-149a -1.60 2.06E-12 miR-181d -1.35 2.58E-06 miR-486 -1.49 8.06E-04 miR-1271 -2.48 2.57E-04

In particular, miR-146a showed the greatest decrease (Fig. 1B). Next, RNA sequencing was analyzed to find miRNA targets, and 209 increased and 392 decreased genes appeared in floating cells (Fig. 1C), and GO (gene ontology) analysis using the increased 209 genes was performed. Through this, it was confirmed that the semaphorin-plexin signaling process was significantly enhanced in circulating tumor cell-like floating cells (Fig. 1D). In addition, 24 increased genes involved in the semaphorin-plexin signaling process were identified (Fig. 1E). In addition, as shown in Table 2 below, expression was increased in floating cells through integrated analysis of mRNA and miRNA sequencing, and as a result of TargetScan, six genes predicted to have miR-146a attachment sites in the 3'UTR were identified as miR. It was confirmed that it showed a great correlation with -146a (Fig. 1F).

Symbol Gene Name Log2 Fold Change STC1 Stanniocalcin 1 1.20 SBSPON Somatomedin B and thrombospondin type 1 domain containing 2.20 RIMS2 Regulating synaptic membrane exocytosis 2 1.54 RCSD1 RCSD domain containing 1 1.98 NRP2 Neuropilin 2 3.02 MAPT Microtubule associated protein tau 3.30

As can be seen in Table 2, among these genes, NRP2 and STC1 were involved in the semaphorin-plexin signaling process, and NRP2 showed a large increase in expression.

In addition, an inverse correlation between miR-146a and NRP2 was observed in floating cells, that is, the expression pattern of miR-146a decreased and NRP2 increased in floating cells (Fig. 1G).

From the results of Experimental Example 1, potential targets related to molecular biological traits of circulating tumor cell-like flow cells were identified through RNA and miRNA sequencing.

[Experimental Example 2] Confirmation of the inverse correlational expression pattern of NRP2 and miR-146a

From Experimental Example 1, it was confirmed that MDA-MB-468 floating cells showed increased expression of NRP2 and decreased miR-146a compared to adherent cells.

In order to more specifically confirm the decorrelational expression pattern of NRP2 and miR-146a, real-time quantitative PCR and Western blotting were performed in MDA-MB-468 adherent and floating cells.

The results for this are shown in Figure 3.

As can be seen in Figure 3, NRP2 was increased in both mRNA and protein levels in floating cells (Figures 2A and 2B), whereas the expression level of miR-146a was rapidly decreased in floating cells (Figure 2C).

From the results of Experimental Example 2, it was confirmed that NRP2 and miR-146a showed a strong inverse correlation in adherent cells and floating cells.

[Experimental Example 3] Confirmation of direct correlation between NRP2 and miR-146a

To examine whether miR-146a directly regulates NRP2 expression, we first analyzed whether there is an attachment site for miR-146a in the 3'UTR of NRP2 mRNA.

The results for this are shown in Figure 4.

As can be seen in Figure 4, it was confirmed that there are two sites where the seed sequence of miR-146a attaches to the 3'UTR of NRP2, indicating that miR-146a plays a role as an important regulator of NRP2. were present (Figure 3A). Next, when the wild type NRP2 3'UTR vector and the miR-146a analogue were transfected together, the degree of luciferase was significantly reduced, but the mutant type in which both sequences predicted to be attached to miR-146a were changed was transfected It was confirmed that the level of luciferase did not change in one sample (Fig. 3B). In addition, overexpression of miR-146a in floating cells decreased both mRNA and protein expression of NRP2 (Fig. 3C and 3D). From these results, it was confirmed that miR-146a directly regulates NRP2 in floating cells.

In addition, it was confirmed that p-p38 (Phosphorylated p38), a phosphorylated form of p38 MAPK known to be associated with breast cancer metastasis, was increased in floating cells (Fig. 3E). In addition, the level of p-p38 was decreased in floating cells after overexpression of miR-146a (Fig. 3F), and the activation of p-p38 was increased after treatment with SEMA3C, one of the ligands that activates the downstream signaling of NRP2 (Fig. 3G ), it was confirmed that p-p38 induced by SEMA3C was reduced by miR-146a (Fig. 3H). From these results, it was confirmed that miR-146a plays an important role in regulating the p38 signaling process by targeting NRP2.

From the results of Experimental Example 3, it was confirmed that MiR-146a directly targets NRP2 and regulates SEMA3C-induced p38 phosphorylation.

[Experimental Example 4] Confirmation of the role of NRP2 in circulating tumor cells

To investigate the specific role of NRP2, NRP2 was knocked down and tested.

The results for this are shown in FIG. 5 .

Referring to Figure 5, both mRNA and protein levels of NRP2 were significantly decreased after siRNA transfection of NRP2 in MDA-MB-468 adherent and floating cells (Figures 4A and 4B).

Next, we investigated the effect on adherent and floating cells after NRP2 knockdown, and the number of dividing cells decreased after NRP2 siRNA treatment for both adherent and floating cells (Figure 4C and 4D), and the invasion ability of floating cells was confirmed. . In addition, in this case, it was confirmed that floating cells had significantly increased invasion ability than adherent cells (Fig. 4E). In addition, the invasive ability of floating cells significantly decreased after NRP2 knockdown (Fig. 4F), and the migration of adherent cells after NRP2 siRNA treatment was also inhibited (Fig. 4G). From these results, it was confirmed that regulation of NRP2 expression can be utilized as one of the treatment strategies for breast cancer patients.

From the results of Experimental Example 4, it was confirmed that knockdown of NRP2 inhibits the proliferation, migration and invasion of MDA-MB-468 cells.

[Experimental Example 5] Confirmation of the role of MiR-146a in circulating tumor cells

An experiment was conducted to confirm the specific role of miR-146a, a regulator of NRP2 expression, in the exacerbation of breast cancer.

The results of this are shown in FIG. 6 .

Referring to Fig. 6, after miR-146a overexpression, the number of infiltrating cells was significantly reduced in MDA-MB-468 floating cells (Fig. 5A), and the migration of adherent cells was inhibited after miR-146a analogue transfection (Fig. 5B ).

In Experimental Example 5, the effect of miR-146a on the invasion induced by SEMA3C was examined in detail.

As a result, the increased invasion capacity by SEMA3C in floating cells was reduced by overexpression of miR-146a (Fig. 5C), and miR-146a also inhibited the proliferation induced by SEMA3C (Fig. 5D), in which case 10% Fetal bovine serum was used as a positive control factor. From these results, it was confirmed that miR-146a plays a role in suppressing SEMA3C-induced invasion and proliferation in floating cells, and the possibility of miR-146a as an important factor in suppressing circulating breast cancer cells was confirmed.

From the results of Experimental Example 5, it was confirmed that miR-146a suppresses the metastasis-promoting trait induced by SEMA3C in MDA-MB-468 floating cells.

[Experimental Example 6] Confirmation of correlation between NRP2 overexpression and breast cancer

To investigate the role of NRP2 related to the survival of breast cancer patients, the data of breast cancer patients were analyzed in The Cancer Genome Atlas (TCGA).

The results for this are shown in FIG. 7 .

Although there was no significant difference in overall survival related to NRP2 expression, it was confirmed that breast cancer patients with high NRP2 expression patterns had a shorter survival period without distant metastasis (Fig. 6A and 6B). From this, it was confirmed that NRP2 can serve as a biomarker to predict the recurrence of breast cancer patients, and these analysis results indicate that miR-146a and NRP2 have the potential to be used as a treatment strategy.

From the results of Experimental Example 6, it was confirmed that the increased expression of NRP2 is correlated with poor prognosis of breast cancer patients.

The above description of the present application is for illustrative purposes, and those skilled in the art will understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present application. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present application is indicated by the following claims rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts thereof should be construed as being included in the scope of the present application.

<110> SOOKMYUNG WOMEN'S UNIVERSITY Research & Business Development Foundation <120> COMPOSITION FOR DIAGNISING CANCER, KIT FOR DIAGNOSING, PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING AND METHOD FOR MEASURING OF CANCER USING THE SAME <130> DP200373 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> microRNA 146a <400> 1 ugagaacuga auuccauggg uu 22 <210> 2 <211> 6659 <212> PRT <213> Artificial Sequence <220> <223> Neuropilin2 <400> 2 Ala Gly Ala Gly Ala Thr Cys Gly Cys Gly Ala Gly Cys Gly Ala Gly 1 5 10 15 Gly Cys Ala Cys Cys Ala Gly Cys Cys Thr Gly Cys Ala Gly Cys Cys 20 25 30 Gly Gly Cys Cys Cys Cys Cys Ala Gly Cys Ala Cys Ala Thr Cys Cys 35 40 45 Thr Cys Ala Gly Cys Cys Gly Cys Ala Cys Ala Gly Ala Cys Ala Cys 50 55 60 Thr Cys Gly Gly Cys Gly Ala Gly Gly Thr Gly Gly Ala Gly Gly Thr 65 70 75 80 Gly Ala Gly Gly Gly Cys Gly Gly Gly Cys Gly Cys Cys Ala Gly Cys 85 90 95 Gly Ala Ala Cys Thr Cys Gly Gly Ala Gly Ala Gly Gly Gly Gly Cys 100 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Claims (5)

A method for determining whether cancer occurs, comprising measuring the level of NRP2 protein in a biological sample.
According to claim 1,
Wherein the biological sample is a circulating-tumor cell (CTC) or a circulating-tumor cell-mimicking suspension cell thereof.
According to claim 1,
The method for determining whether cancer occurs, further comprising the step of measuring the mRNA level of the NRP2 before expression of the NRP2 protein.
According to claim 1,
The method for determining whether cancer occurs, further comprising the step of injecting an expression inhibitor of the NRP2 protein into the biological sample.
According to claim 1,
The expression inhibitor of the NRP2 protein, characterized in that miR-146a, a method for determining whether cancer occurs.

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