KR102401005B1 - A composition for preventing or treating high-risk endometriosis - Google Patents

Abstract

According to the present invention, endometriosis patients belonging to a high-risk group that can progress to ovarian clear cell cancer can be selected early, and furthermore, by inhibiting the transformation of endometriosis into a malignant neoplasm in the high-risk group, the malignancy of high-risk group endometriosis can be reduced. It is expected that it can be used very effectively for prevention or treatment.

Description

A composition for preventing or treating high-risk endometriosis

The present invention relates to a composition for the treatment of high-risk group endometriosis comprising an agent for reducing the expression of TSPAN1 (Tetraspanin 1) protein or a gene encoding the same.

Endometriosis (Em) is a disease in which endometrial tissue attaches to and proliferates in tissues other than the uterus, and corresponds to a common benign gynecological disease occurring in about 10 to 15% of women of childbearing age. Endometriosis can occur at any age, and the main symptoms associated with it include severe menstrual pain, pain in the lower abdomen, and infertility. Although the cause is not clearly known, it is known to be related to genetics, environment, immunology, angiogenesis, and endocrine disorders. Similar to malignant tumors, endometriosis also has the characteristics of heterogeneity, adhesion, invasion and metastasis, and can also be transformed into a malignant tumor. Some of endometriosis can progress to ovarian cancer, and it has been reported that it is closely related to an increased risk of ovarian cancer. A clear study has not been done yet.

Clear cell carcinoma of the ovary can occur in endometriosis, a common benign disease in women, and it has been reported that atypical endometriosis (AtyEm) is a disease in the stage before progression to ovarian clear cell carcinoma. Among ovarian cancers, ovarian clear cell carcinoma (OCCC) accounts for about 10% of epithelial ovarian cancers, but is known as a cancer with high anticancer drug resistance and a very poor treatment prognosis. There is no characteristic tumor marker test, and the current treatment is a treatment similar to that of epithelial ovarian cancer.

As such, when the disease worsens due to malignant transformation from endometriosis to ovarian clear cell carcinoma, the prognosis is poor. Therefore, the molecular mechanism underlying the malignant transformation that can prevent and select high-risk endometriosis patients early is important. research is urgently needed.

One object of the present invention is to provide a preventive or therapeutic agent for high-risk group endometriosis.

Another object of the present invention is to provide a method for screening a cancer progression inhibitor of endometriosis, which is a therapeutic agent for high-risk group endometriosis.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, various specific details are set forth, such as specific forms, compositions and processes, and the like, for a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, references to "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, the particular features, forms, compositions, or properties may be combined in any suitable way in one or more embodiments.

Unless otherwise defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

According to one embodiment of the present invention, it relates to a biomarker for diagnosis of high-risk group endometriosis.

In the present invention, the biomarker may include a TSPAN1 (Tetraspanin 1) protein or a gene encoding the same.

The TSPAN1 (Tetraspanin 1) protein of the present invention is a protein encoded by the TSPAN1 gene and is a member of the transmembrane 4 superfamily, also known as the tetraspanin family. Most of these members correspond to cell surface proteins characterized by the presence of four hydrophobic domains. These proteins are known to mediate signal transduction involved in the regulation of cell development, activation, growth and motility. The amino acid sequence of TSPAN1 and the nucleic acid base sequence encoding the same may be represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively, but are not limited thereto.

In the present invention, the "diagnosis" means confirming the presence or characteristics of a pathological condition, and more specifically, determining the high-risk group endometriosis, determining the possibility of progression from endometriosis to cancer, or malignant cancer It refers to determining whether there is a risk of being transformed. The cancer may be ovarian cancer or uterine cancer as a type of gynecological cancer, preferably ovarian cancer, more preferably ovarian clear cell cancer. Diagnosis is defined as a broad concept that includes providing information on diagnosis.

In the present invention, the "endometriosis" is a disease in which endometrial tissue proliferates in a place other than the surface of the uterine lumen, and is also called heterotopic endometriosis. It is a disease that is difficult to diagnose because it shows various symptoms depending on the location of the lesion, the organ involved, and the degree of the lesion. Clinical tests currently used to diagnose endometriosis include vaginal ultrasound, magnetic resonance imaging (MRI), blood tests, and laparoscopy. Various diagnostic methods are being developed, such as using genetic indicators that can diagnose the disease or predict recurrence after treatment.

In the present invention, the criterion of the "risk group" may be classified according to whether endometriosis patients can be transformed into malignant cancer. Specifically, the high-risk group refers to cases in which endometriosis is likely to undergo transformation from endometriosis to a malignant neoplasm or to progress to cancer. It refers to a case in which there is little or no possibility of transformation or malignant transformation. For the purposes of the present invention, the "high-risk group" means a patient who is likely to progress from endometriosis to cancer, preferably gynecological cancer, to ovarian cancer or uterine cancer, more preferably ovarian cancer, most preferably ovarian clear cell cancer. can

In the present invention, the "ovarian cancer" is a cancer that occurs in the ovaries, which are the gonads located on both sides of the uterus, and is largely classified into epithelial cell carcinoma, germ cell tumor, or sexual stromal tumor depending on the tissue in which the cancer occurs. . Of these, ovarian epithelial cell carcinoma, which arises from epithelial cells on the surface of the ovary, accounts for more than 90% of all ovarian cancers. Ovarian cancer (Mucinous carcinoma), endometrioid carcinoma (Endometroid carcinoma), clear cell carcinoma (Clear cell carcinoma), Malignant brenner tumor (Malignant brenner tumor), undifferentiated cell carcinoma (Undifferentiated carcinoma) or unclassified ovarian cancer (Unclassified Carcinoma) divided

In the present invention, the "ovarian clear cell carcinoma (OCCC)" is a common cancer that accounts for 40 to 50% of types of endometriosis-associated ovarian cancer (EAOC). The histological precancerous lesion of ovarian clear cell carcinoma is known as atypical endometriosis (AtyEm). AtyEm is observed in 12 to 35% of endometriosis cases and is affected by hormone levels, oxidative stress, genetic mutations, and immunomodulatory abnormalities. Transformation of endometriosis into a malignant neoplasm is associated with activation of the oncogenic KRAS (Kirsten rat sarcoma 2 viral oncogene homolog) and PI3K (Phosphoinositide 3-kinases) pathways and the tumor suppressor genes PTEN (Phosphatase and tensin homolog) and ARID1A (AT- There is a study result that it is related to the inactivation of Rich Interaction Domain 1A). In addition, among ovarian cancer, clear cell carcinoma of the ovary accounts for about 10% of epithelial ovarian cancer, but it is a cancer with high anticancer drug resistance and a very poor prognosis for treatment.

According to another embodiment of the present invention, it relates to a composition for diagnosis of high-risk group endometriosis.

The diagnostic composition of the present invention includes an agent for measuring the expression level of TSPAN1 (Tetraspanin 1) protein or a gene encoding the same.

The agent for measuring the level of the TSPAN1 protein of the present invention is at least one selected from the group consisting of an antibody, oligopeptide, ligand, PNA (peptide nucleic acid) and aptamer that specifically binds to TSPAN1 protein. It may include, but is not limited to.

The "antibody" of the present invention refers to a protein molecule capable of specifically binding to the TSPAN1 protein, and the form of the antibody is not particularly limited, but as long as it has polyclonal antibody, monoclonal antibody or antigen-binding property, It may be included even if it is a part of an antibody, and all kinds of immunoglobulin antibodies may be included. Special antibodies such as humanized antibodies may also be included, which include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and may include, but is not limited to, Fab, F(ab'), F(ab') 2, Fv, and the like.

In the present invention, the "oligopeptide" is a peptide consisting of 2 to 20 amino acids and may include a dipeptide, a tripeptide, a tetrapeptide, and a pentapeptide, but is not limited thereto.

In the present invention, the "PNA (Peptide Nucleic Acid)" refers to an artificially synthesized polymer similar to DNA or RNA, and was first introduced in 1991 by Professors Nielsen, Egholm, Berg and Buchardt of the University of Copenhagen, Denmark. Whereas DNA has a phosphate-ribose sugar backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which greatly increases binding strength and stability to DNA or RNA, resulting in molecular biology , diagnostic assays and antisense therapy. PNA is described in Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254 (5037): 1497-1500.

The "aptamer" of the present invention refers to a single-stranded oligonucleotide, and refers to a nucleic acid molecule having binding activity to the TSPAN1 protein. The aptamer may have various three-dimensional structures according to its nucleotide sequence, and may have high affinity for a specific substance, such as an antigen-antibody reaction. The aptamer may inhibit the activity of a given target molecule by binding to the given target molecule. The aptamer may be RNA, DNA, modified nucleic acid, or a mixture thereof, and the form may be linear or cyclic.

The agent for measuring the expression level of the gene encoding the TSPAN1 protein of the present invention may include one or more selected from the group consisting of primers, probes and antisense nucleotides that specifically bind to the gene encoding the TSPAN1 protein. , but is not limited thereto.

In the present invention, the "primer" is a fragment recognizing a target gene sequence, including a pair of forward and reverse primers, but preferably, a primer pair that provides analysis results having specificity and sensitivity. High specificity can be conferred when the primer's nucleic acid sequence is a sequence that is inconsistent with a non-target sequence present in the sample, thus amplifying only the target gene sequence containing the complementary primer binding site and not causing non-specific amplification. .

In the present invention, the "probe" refers to a substance capable of specifically binding to a target substance to be detected in a sample, and refers to a substance capable of specifically confirming the presence of a target substance in a sample through the binding. The type of probe is not limited as a material commonly used in the art, but preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, and most preferably It is PNA. More specifically, the probe is a biomaterial derived from or similar thereto, or manufactured in vitro, and includes, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, neurons, DNA, and It may be RNA, and DNA includes cDNA, genomic DNA, and oligonucleotides, RNA includes genomic RNA, mRNA, and oligonucleotides, and examples of proteins include antibodies, antigens, enzymes, peptides, and the like.

In the present invention, the "LNA (Locked nucleic acids)" means a nucleic acid analog including a 2'-O, 4'-C methylene bridge [J Weiler, J Hunziker and J Hall Gene Therapy (2006) 13, 496.502) ]. LNA nucleosides include common nucleic acid bases in DNA and RNA, and can form base pairs according to Watson-Crick base pairing rules. However, due to the 'locking' of the molecule due to the methylene bridge, the LNA does not form the ideal shape in the Watson-Crick bond. When LNA is incorporated into DNA or RNA oligonucleotides, LNA can pair with complementary nucleotide chains more rapidly, increasing the stability of the double helix.

In the present invention, the "antisense" means that the antisense oligomer is hybridized with a target sequence in RNA by Watson-Crick base pairing, and typically mRNA and RNA in the target sequence: A sequence of nucleotide bases allowing the formation of an oligomeric heteroduplex and oligomers having an inter-subunit backbone. An oligomer may have exact sequence complementarity or approximate complementarity to a target sequence.

Since information on the TSPAN1 protein according to the present invention or genes encoding them is known, those skilled in the art can easily design primers, probes or antisense nucleotides that specifically bind to the gene encoding the protein based on this. .

The diagnostic composition of the present invention compares the expression level of the TSPAN1 protein or the gene encoding it measured in a biological sample isolated from a target individual with that of the control group to determine whether the expression level is increased, thereby preventing high-risk group endometriosis. can be diagnosed

In the present invention, the "control" is a group of individuals suffering from endometriosis and histologically free of atypical endometriosis, or a group of individuals suffering from endometriosis and known for good survival, suffering from endometriosis and not developing cancer. It may be obtained from a sample derived from a group of individuals known to not have endometriosis, or from a group of individuals known to have endometriosis and not produced malignant neoplasms, or from a group of individuals known to have endometriosis and not undergone malignant transformation. A group of individuals known to have endometriosis and have good survival outcomes, a group of individuals known to have endometriosis and not progressed to cancer, or a group of individuals known to have endometriosis and no malignant neoplasms or uterus A group of individuals known to have endometriosis and not undergone malignant transformation may be a low-risk group of patients with endometriosis.

According to another embodiment of the present invention, it relates to a kit for diagnosis of high-risk group endometriosis comprising the composition of the present invention.

The kit of the present invention can be predicted to correspond to a high-risk group of endometriosis when the TSPAN1 protein or a gene encoding it is present at a higher level in the target individual using the composition of the present invention compared to the control group.

In the diagnostic kit of the present invention, descriptions of the TSPAN1 protein or a gene encoding it, endometriosis, etc. are the same as those described in the diagnostic composition, and thus are omitted to avoid excessive complexity of the present specification.

The kit of the present invention may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a Rapid kit, or a Multiple reaction monitoring (MRM) kit, but is not limited thereto.

The kit of the present invention may further include one or more other component compositions, solutions or devices suitable for the assay method. For example, in the present invention, the kit may further include essential elements necessary for performing the reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes a pair of primers specific for a gene encoding a marker protein. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably, a length of about 10 bp to 30 bp. It may also include a primer specific for the nucleic acid sequence of the control gene. Other reverse transcription polymerase reaction kits include test tubes or other suitable containers, reaction buffers (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC -Water (DEPC-water), sterile water, etc. may be included.

In addition, the diagnostic kit of the present invention may include essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, etc. for preparing a fluorescently-labeled probe. The substrate may also contain cDNA or oligonucleotides corresponding to control genes or fragments thereof.

In addition, the diagnostic kit of the present invention may include essential elements necessary for performing ELISA. The ELISA kit contains an antibody specific for this protein. Antibodies are antibodies with high specificity and affinity for a marker protein and little cross-reactivity with other proteins, and are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. The ELISA kit may also include an antibody specific for a control protein. Other ELISA kits include reagents capable of detecting bound antibody, for example, labeled secondary antibodies, chromophores, enzymes (eg, conjugated with an antibody) and their substrates or capable of binding the antibody. other materials and the like.

In the diagnostic kit of the present invention, as a fixture for antigen-antibody binding reaction, a well plate synthesized from a nitrocellulose membrane, a PVDF membrane, a polyvinyl resin or polystyrene resin, and a glass slide glass and the like may be used, but is not limited thereto.

In addition, in the diagnostic kit of the present invention, the label of the secondary antibody is preferably a conventional color developer that reacts with color, and HRP (horseradish peroxidase), basic dephosphorylation enzyme (alkaline phosphatase), colloidal gold (colloid gold), FITC ( Poly L-lysine-fluorescein isothiocyanate) and fluorescein such as RITC (rhodamine-B-isothiocyanate) and labels such as dyes may be used, but the present invention is not limited thereto. .

In addition, in the diagnostic kit of the present invention, it is preferable to use a chromogenic substrate for inducing color development according to a marker that undergoes a color reaction, TMB (3,3',5,5'-tetramethyl bezidine), ABTS[ 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], OPD(o-phenylenediamine), etc. can be used. In this case, it is more preferable that the chromogenic substrate be provided in a dissolved state in a buffer solution (0.1 M NaAc, pH 5.5). A chromogenic substrate such as TMB is decomposed by HRP used as a label of the secondary antibody conjugate to generate chromogenic deposits, and the presence or absence of the marker proteins is detected by visually confirming the degree of deposition of the chromogenic deposits.

In the diagnostic kit of the present invention, the washing solution preferably includes a phosphate buffer solution, NaCl, and Tween 20, and a buffer solution (PBST) composed of 0.02 M phosphate buffer, 0.13 M NaCl, and 0.05% Tween 20. more preferably. After the antigen-antibody binding reaction, the secondary antibody is reacted with the antigen-antibody conjugate, and an appropriate amount is added to the immobilizer and washed 3 to 6 times. As the reaction stop solution, a sulfuric acid solution (H2SO4) may be preferably used.

According to another embodiment of the present invention, there is provided a method for providing information for the diagnosis of high-risk group endometriosis.

The method of the present invention includes measuring the level of TSPAN1 protein present in a biological sample isolated from a subject of interest.

In the present invention, the "target subject" refers to mammals including humans, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cattle, horses, pigs, sheep and goats. It may be selected from the group consisting of, and preferably may be a human, but is not limited thereto.

In the present invention, the term “human” refers to a patient who has or is suspected of having developed endometriosis, and may mean a patient in need or expected to treat endometriosis, but is not limited thereto.

The "biological sample" of the present invention refers to any material, biological fluid, tissue or cell obtained from, or derived from, an endometriosis patient or suspected high-risk group endometriosis disease, for example, whole blood ( whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, blood including plasma and serum, sputum, tears , mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, intrapelvic pelvic fluids, cystic fluids, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid , nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cell, cell extract or It may include, but is not limited to, cerebrospinal fluid.

In the method of providing information for diagnosis of the present invention, the description of TSPAN1 protein or a gene encoding it, endometriosis, high-risk group, agents for measuring expression levels, etc. are the same as those described in the diagnostic composition, omitted to avoid complexity.

The step of measuring the level of the protein of the present invention is a protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) using the composition of the present invention. Analysis, SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion method, octeroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, 2D by electrophoretic analysis, liquid chromatography-Mass Spectrometry (LC-MS), liquid chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS), Western blotting or ELISA (enzyme linked immunosorbent assay) It may be performed, but is not limited thereto.

The step of measuring the expression level of the gene encoding the protein of the present invention is reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerization using the composition of the present invention. It may be performed by an enzyme reaction (Real-time RT-PCR), an RNase protection assay (RPA), Northern blotting, or a DNA chip, but is not limited thereto.

The method of the present invention can predict that the target subject will have high-risk endometriosis disease when the level of TSPAN1 protein measured in the biological sample isolated from the subject is higher than that of the control group.

According to another embodiment of the present invention, it relates to a composition for preventing or treating high-risk group endometriosis.

The composition of the present invention may act as a mechanism to inhibit the progression of endometriosis to cancer in individuals diagnosed with a high-risk group, and accordingly, the composition and terms for inhibiting the progression of endometriosis to cancer in the present specification may be used interchangeably. .

The composition of the present invention may include an agent that inhibits the activity of TSPAN1 protein or an agent that reduces the expression level of a gene encoding the same.

The composition of the present invention has a function of preventing cancer by inhibiting the progression to cancer, and the cancer may be uterine cancer or ovarian cancer as a type of gynecological cancer, preferably ovarian cancer, more preferably ovarian transparent cells It could be cancer. In addition, the pharmaceutical composition can inhibit the transformation of endometriosis into malignant cancer, more preferably, it can inhibit transformation into ovarian cancer, and most preferably, it can inhibit transformation into ovarian clear cell carcinoma. Through this, by delaying or suppressing the exacerbation of endometriosis, it can be applied to the prevention or treatment of endometriosis diseases, such as high-risk group endometriosis, in which endometrial cells are ultimately predicted to become cancerous.

In the present invention, the "prevention" means any action capable of blocking symptoms caused by endometriosis disease or inhibiting or delaying the progression of endometriosis to cancer using the composition of the present invention without limitation. there is.

In the present invention, the term "treatment" refers to a series of activities performed for alleviation and/or improvement of a desired disease. Treatment for purposes of the present invention includes the action of inhibiting or delaying the progression of endometriosis to cancer.

In the composition for inhibiting malignant transformation of the present invention, descriptions of the TSPAN1 protein or its encoding gene, endometriosis, clear cell cancer, risk group, etc. are the same as those described in the diagnostic composition, and thus are omitted to avoid excessive complexity of the present specification. .

The agent for inhibiting the activity of the TSPAN1 protein of the present invention may include any one or more selected from the group consisting of a compound that specifically binds to TSPAN1, a peptide, a peptide mimetics, an aptamer, an antibody, and a natural product. , The agent for inhibiting the expression of the TSPAN1 gene is a TSPAN1 gene, preferably antisense nucleotides complementary to the mRNA of the gene, short interfering RNA (siRNA), short hairpin RNA (short hairpin RNA) and It may include any one or more selected from the group consisting of ribozyme, but it acts directly or indirectly on TSPAN1, a target protein or gene, and corresponds to a means for deriving an effect that inhibits its activity or expression. It is not limited thereto and may include any one that can be easily derived by a known technique in a method commonly used in the present invention.

In the present invention, the "peptide mimetics (Peptide Minetics)" is a peptide or non-peptide that inhibits the binding domain of the TSPAN1 protein leading to the inhibition of TSPAN1 activity. Major residues of non-hydrolyzable peptide analogs include the β-turn dipeptide core (Nagai et al. Tetrahedron Lett 26:647, 1985), keto-methylene pseudopeptides (Ewenson et al. J Med chem 29:295, 1986; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th AmeriCan Peptide Symposium) Pierce chemiCal co. Rockland, IL, 1985), azepine (Huffman et al. in Peptides: chemistry and Biology, G.R. Marshall ed., EScom Publisher: Leiden, Netherlands, 1988), benzodiazepines (Freidinger et al. in Peptides; chemistry and Biology, G.R. Marshall ed., EScom Publisher: Leiden, Netherlands, 1988), β-aminoalcohols (Gordon et al. Biochem Biophys Res) commun 126:419 1985) and substituted gamma lactam rings (Garvey et al. in Peptides: chemistry and Biology, G.R. Marshell ed., EScOM Publisher: Leiden, Netherlands, 1988).

In the present invention, the "aptamer (Aptamer)" is a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure by itself and having a characteristic capable of binding to a target molecule with high affinity and specificity. After the first development of an aptamer discovery technology called SELEX (Systematic Evolution of Ligands by EXponential enrichment) (Ellington, AD and Szostak, JW., Nature 346:818-822, 1990), Many aptamers capable of binding to various target molecules have been continuously discovered. Aptamers are comparable to single antibodies because of their inherent high affinity (usually at pM level) and specificity to bind to a target molecule, and have high potential as an alternative antibody, especially as a “chemical antibody”.

In the present invention, the "antibody" prepared by injection of TSPAN1 protein or commercially purchased can be used. In addition, the antibody includes polyclonal antibodies, monoclonal antibodies, and fragments capable of binding to an epitope.

Here, the polyclonal antibody can be produced by a conventional method of injecting the TSPAN1 protein into an animal and collecting blood from the animal to obtain a serum containing the antibody. Such polyclonal antibodies may be purified by any method known in the art, and may be made from a host of any animal species, such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, and the like.

In addition, the monoclonal antibody can be prepared using any technique that provides for the production of the antibody molecule through culture of a continuous cell line. Such techniques include, but are not limited to, hybridoma technology, human B-cell line hybridoma technology, and EBV-hybridoma technology.

In addition, an antibody fragment containing a specific binding site for the TSPAN1 protein can be prepared. For example, but not limited thereto, the F(ab')2 fragment may be prepared by digesting an antibody molecule with pepsin, and the Fab fragment may be prepared by reducing the disulfide bridge of the F(ab')2 fragment. Alternatively, by making the Fab expression library small, monoclonal Fab fragments having a desired specificity can be quickly and conveniently identified.

In the present invention, the antibody may be bound to a solid substrate to facilitate subsequent steps such as washing or separation of the complex. Solid substrates include, for example, synthetic resins, nitrocellulose, glass substrates, metal substrates, glass fibers, microspheres and microbeads. In addition, the synthetic resin includes polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF and nylon.

The agent for inhibiting the activity in the present invention may be one that specifically binds to the epitope represented by SEQ ID NO: 1 of the TSPAN1 protein, and preferably, the pharmaceutical composition of the present invention may include an antibody specific to the TSPAN1 protein. Here, the antibody may be one that specifically binds to the epitope represented by SEQ ID NO: 1, but is not limited thereto.

In the present invention, the "antisense nucleotide" binds (hybridizes) to the complementary nucleotide sequence of DNA, immature-mRNA, or mature mRNA as defined in Watson-Click base pairing to disrupt the flow of genetic information from DNA to protein will be. The nature of antisense nucleotides specific to a target sequence makes them exceptionally versatile. Since antisense nucleotides are long chains of monomeric units, they can be readily synthesized for the target RNA sequence. Many recent studies have demonstrated the usefulness of antisense nucleotides as biochemical means to study target proteins. Due to recent advances in oligonucleotide chemistry and nucleotide synthesis with improved cell line adsorption, target binding affinity and nuclease resistance, the use of antisense nucleotides can be considered as a new type of inhibitor.

In the present invention, the "siRNA" and "shRNA" are nucleic acid molecules capable of mediating RNA interference or gene silencing, and because they can inhibit the expression of a target gene, an efficient gene knockdown method or gene therapy used in a way shRNA is a single-stranded oligonucleotide that forms a hairpin structure by bonding between complementary sequences. In vivo, the shRNA is cut by a dicer and a small RNA fragment of 21 to 25 nucleotides in size. siRNA, which is a double-stranded oligonucleotide, and can specifically bind to mRNA having a complementary sequence to inhibit expression. Therefore, which means of shRNA and siRNA to use may be determined by a person skilled in the art, and if the mRNA sequences targeted by them are the same, a similar expression reduction effect can be expected. For the purpose of the present invention, by acting specifically on the gene encoding TSPAN1 to cut the gene (eg, mRNA molecule) of TSPAN1 to induce RNA interference (RNAi, RNA interference) phenomenon, the expression of TSPAN1 can be suppressed there is. siRNA can be synthesized chemically or enzymatically. The method for preparing siRNA is not particularly limited, and methods known in the art may be used. For example, a method for synthesizing siRNA directly chemically, a method for synthesizing siRNA using in vitro transcription, a method for cleaving long double-stranded RNA synthesized by in vitro transcription using an enzyme; Expression method through intracellular delivery of an shRNA expression plasmid or viral vector and expression method through intracellular delivery of a PCR (polymerase chain reaction)-induced siRNA expression cassette (cassette), but are not limited thereto.

In the present invention, the "ribozyme" refers to an RNA molecule having catalytic activity. Ribozymes having various activities are known, and ribozymes of the TSPAN1 gene include known or artificially generated ribozymes, and ribozymes having a target-specific RNA cleavage activity are selectively prepared by standard known techniques. can be

As an example of the present invention, the agent for reducing the expression may be an siRNA that knocked down the TSPAN1 gene, and the siRNA may include a forward siRNA represented by SEQ ID NO: 3 and a reverse siRNA represented by SEQ ID NO: 4, However, the present invention is not limited thereto.

The composition provided in the present invention may be used in the form of a pharmaceutical composition or a food composition, but is not limited thereto.

The pharmaceutical composition of the present invention may be characterized in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be characterized in that it is targeted to humans.

The pharmaceutical composition of the present invention is not limited thereto, but each is formulated in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods to be used. can The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, fragrances, etc., for oral administration, and in the case of injections, buffers, preservatives, pain-freezing agents Agents, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used, and in the case of topical administration, bases, excipients, lubricants, preservatives, etc. may be used. The dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injection, it can be prepared in the form of unit dosage ampoules or multiple dosage forms there is. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained release formulation, and the like.

Meanwhile, examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used. In addition, fillers, anti-agglomeration agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives and the like may be further included.

The route of administration of the pharmaceutical composition of the present invention is, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal. Oral or parenteral administration is preferred.

The "parenteral" of the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. Preferably, the pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration, but is not limited thereto.

The pharmaceutical composition of the present invention depends on several factors including the activity of the specific compound used, age, weight, general health, sex, formula, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, disease severity, drug form, administration route and period, but may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg/day per day kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.

The food composition of the present invention may be prepared in the form of various foods, for example, beverages, gums, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, and the like.

When the active ingredient of the present invention is included in the food composition, the amount may be added in a proportion of 0.1 to 50% of the total weight, but is not limited thereto.

When the food composition of the present invention is prepared in the form of a beverage, there is no particular limitation except for including the food composition in the indicated ratio, and it may contain various flavoring agents or natural carbohydrates as additional ingredients, like a conventional beverage. . Specifically, as natural carbohydrates, monosaccharides such as glucose, disaccharides such as fructose, and polysaccharides such as sucrose, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol are used as natural carbohydrates. may include The flavoring agent may be a natural flavoring agent (taumartin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agent (saccharin, aspartame, etc.).

The food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, A pH adjuster, a stabilizer, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like may be further included.

The ingredients included in the food composition of the present invention may be used independently or in combination. The proportion of the additive is not a key element of the present invention, but may be selected from 0.1 to about 50 parts by weight per 100 parts by weight of the food composition of the present invention, but is not limited thereto.

According to another embodiment of the present invention, it relates to a method for screening a therapeutic agent for high-risk group endometriosis.

The therapeutic agent for the high-risk group of endometriosis of the present invention may include any drug having a mechanism for inhibiting the progression of endometriosis to cancer, and the term may be used interchangeably with the inhibitor of the progression of endometriosis to cancer in the present specification. .

The screening method of the present invention comprises the steps of: treating a target candidate with a biological sample isolated from a high-risk group of endometriosis patients; and measuring the expression level of the TSPAN1 protein or a gene encoding the same in the biological sample.

In the screening method of the present invention, descriptions of TSPAN1 protein or a gene encoding it, endometriosis and ovarian clear cell carcinoma are the same as those described in the diagnostic composition, and thus are omitted to avoid excessive complexity of the present specification.

The candidate substance of the present invention has inhibitory activity for overcoming high-risk group endometriosis, that is, the expression level of TSPAN1 protein or a gene encoding the same, equal to, or similar to, the expression level in a normal person without low-risk group endometriosis or endometriosis. It refers to a drug for testing for the presence of an activity capable of reducing to a certain level, and includes any molecule such as proteins, oligopeptides, organic molecules, polysaccharides, polynucleotides and a wide range of compounds. Such candidate materials may include not only natural materials but also synthetic materials.

In the screening method of the present invention, the expression level of the TSPAN1 protein or the gene encoding it measured after the treatment of the candidate material is reduced compared to the expression level of the TSPAN1 protein or the gene encoding it measured before the treatment of the candidate material In this case, the method may further include selecting the candidate substance as an inhibitor of the progression of endometriosis to cancer.

In the present invention, by measuring the expression level of TSPAN1, it is possible to select high-risk endometriosis patients predicted to be transformed into malignant cancer. In addition, by inhibiting the proliferation of endometrial cells by suppressing the expression of TSPAN1, and furthermore, by inhibiting the malignant transformation into ovarian clear cell carcinoma in the high-risk group, it can be very effectively used for the prevention or treatment of malignancy of high-risk group endometriosis. .

1 is a view showing specific details regarding clinical information of a tissue sample according to an embodiment of the present invention.
2 is a diagram showing the results of checking differentially expressed genes (DEG) from various FFPE tissue blocks according to an embodiment of the present invention through DESeq2 and edgeR methods.
3A is a diagram illustrating a result of creating a Venn diagram by selecting a common DEG for each tissue comparison group according to an embodiment of the present invention.
3B is a diagram showing the final 14 common gene heatmaps according to an embodiment of the present invention using Cluster3.0 and Java Treeview.
3c and 3d are diagrams showing the results of comparing and confirming the expression levels of 14 genes for each tissue according to an embodiment of the present invention.
Figure 4a shows immortalized endometrial cell lines (6595 and 6866_SV40), immortalized endometriosis cells; EM cells; 6045_SV40 and 9585_SV40) and ovarian clear cell carcinoma cells according to an embodiment of the present invention; It is a diagram showing the results of confirming the expression levels of TSPAN1 mRNA and protein in cell carcinoma cells; OCCC cells; ES-2, TOV-21G, OVTOKO and OVISE).
Figure 4b is a diagram comparing the mRNA expression level of TSPAN1 in normal cells and ovarian clear cell carcinoma cells (OCCC) according to an embodiment of the present invention.
4C is a diagram showing the results of immunohistochemistry (IHC) scoring in order to confirm the expression level of TSPAN1 for each patient tissue sample type according to an embodiment of the present invention.
5A and 5B are diagrams showing the results of comparing and confirming the TSPAN1 protein level by IHC scoring for each ovarian cancer subtype according to an embodiment of the present invention.
6A and 6B are diagrams confirming the transfection results for each cell line prepared by TSPAN1 overexpression or TSPAN1 knockdown according to an embodiment of the present invention through Western blotting and immunofluorescence staining analysis.
7A and 7B show short-term and long-term effects on cell proliferation according to the expression of TSPAN1 for each cell line according to an embodiment of the present invention using a WST-1 Cell Proliferation Assay System. It is a figure showing the measurement result.
8A and 8B show the results of staining with crystal violet and WST-1 Cell Proliferation Assay System to investigate cell proliferation activity according to the expression of TSPAN1 for each cell line according to an embodiment of the present invention. It is a diagram showing the measurement result using .
9A and 9B are diagrams comparing and analyzing the degree of cell invasion according to the expression of TSPAN1 for each cell line according to an embodiment of the present invention.
10 is a diagram illustrating the measurement of phosphorylation levels of major kinases related to cell growth and survival according to the expression of TSPAN1 for each cell line according to an embodiment of the present invention.
11A shows the phosphorylation level of each kinase and the degree of AMPK, ERK and AKT activity upon treatment with the AKT upstream kinase PI3K inhibitor (Wortmannin), the AMPK inhibitor (Compound C; Dorsomorphin) and the ERK inhibitor (U0126) according to an embodiment of the present invention. is a confirmation of
Figure 11b is a diagram confirming the effect on cell growth and invasion when treated with an AKT upstream kinase PI3K inhibitor (Wortmannin), an AMPK inhibitor (Compound C) and an ERK inhibitor (U0126) according to an embodiment of the present invention.
12 is a diagram showing the degree of cell invasion upon treatment with an AKT upstream kinase PI3K inhibitor (Wortmannin), an AMPK inhibitor (Compound C), and an ERK inhibitor (U0126) according to an embodiment of the present invention.
13 is a diagram showing the results of crystal violet analysis and WST-1 cell proliferation analysis in order to confirm the AMPK inhibitor (Compound C) action effect according to an embodiment of the present invention.
14 is a diagram comparing the degree of cell growth in AMPK alpha1 knockdown and the siControl control group according to an embodiment of the present invention.
Figure 15a is a diagram showing the expression level of TSPAN1 in TOV-21G and OVTOKO according to an embodiment of the present invention.
15b and 15c show the degree of cell growth in TOV-21G and OVTOKO cell lines according to an embodiment of the present invention stained with crystal violet and confirmed using a WST-1 Cell Proliferation Assay System. A diagram showing the results.
16 is a diagram confirming the degree of phosphorylation change according to siTSPAN1 treatment in TOV-21G and OVTOKO cell lines according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Preparation Example 1: Recruiting patients and obtaining tumor tissue samples

All formalin-fixed, paraffin-embedded (FFPE) tissue samples were provided by the Korean Gynecological Cancer Bank through the Biomedical Technology Development Program of the Korean Ministry of Education, Science and Technology. FFPE tissue blocks for RNA sequencing were selected for endometriosis (Em) (n = 9), atypical endometriosis (AtyEm) (n = 18), and adjacent endometriosis to OCCC. : AdjEm) (n = 7), ovarian clear cell carcinoma (OCCC) (n = 17) and ovarian endometrioid carcinoma (OEC) (n = 12). For tissue microarray (TMA), FFPE tissue blocks were Em (n = 83), AtyEm (n = 13), AdjEm (n = 4), OCCC (n = 51) and OEC (n = 53) was included Tumor staging was determined according to the International Federation of Gynecology and Obstetrics (FIGO) classification. Pelvic and aortic lymphadenectomy were performed in all FIGO Stage I/II ovarian cancer patients in accordance with the National Comprehensive Cancer Network (NCCN) clinical practice guidelines, and medical records were performed to collect data including age, surgical procedure, duration of survival, and survival status. was reviewed. Pathology reports were reviewed to obtain data on tumor grade and cell type. The inventors of the present invention performed all experiments with the approval of the Yonsei University School of Medicine Evaluation Committee (Institutional Review Board, IRB), and performed with prior consent from each patient before sample collection.

Endometrial tissue samples were obtained from two patients with histologically confirmed endometriosis, whereas endometrial tissue samples were obtained from two patients without endometriosis. The biopsy material was stored in sterile saline at 4 °C and transferred to the laboratory. Fresh endometrial biopsy specimens were washed with PBS, minced into small pieces, and centrifuged at 1000 rpm for 5 minutes. After discarding the supernatant, the tissue was incubated in 1 mg/mL collagenase type IV at 37°C for 1 hour. Dispersed cells were filtered through a 50-μm cell strainer and centrifuged at 1000 rpm for 5 minutes. Collected cells were suspended in DMEM containing 1% DMEM/F12 or 10% FBS. Cells were cultured in an incubator at 37° C., 5% CO ₂ .

Preparation Example 2: Cell culture

For the experiment, TOV-21G, ES-2, NIH3T3 and HS-5 cell lines were obtained from the American Type Culture Collection (ATCC), and OVISE from the Japanese Collection of Research Bioresources Cell Bank (JCRB). and OVTOKO cell line, HEK293T cell line from System Biosciences, SBI Inc., and SNU-251 cell line from Korean Cell Line Bank (KCLB). Following the guidelines of the cell line bank, the TOV-21G, OVISE, OVTOKO and SNU-251 cell lines were in RPMI 1640 medium supplemented with 1% penicillin-streptomycin, and the ES-2 cell line was prepared in 10% FBS with 1% penicillin-streptomycin. In McCoy's 5A medium containing It was cultured in a 5% CO ₂ incubator (HERAcell 150i, Thermo Scientific, Waltham, MA, USA) and used for the experiment.

Preparation Example 3: Lentivirus production and infection

pLenti CMV/TO SV40 small + Large T vector and human TERT were obtained from Addgene (Cambridge, MA). HEK293T cells (1Х 106)) were co-transfected with 2 ug lentiviral vector and 2 ug pPACKH1 lenti vector packaging kit (System Biosciences, Palo Alto, CA). Viral supernatants (10 mL of total harvested viral medium) were obtained at 48 and 72 hours post-transfection. Cells were infected with 500 uL of the obtained crude virus medium per well, and the medium was changed to fresh after 24 hours.

For generation of stable TSPAN1-expressing cell lines, the cDNA encoding human TSPAN1 was amplified using primer sets 5'-AAGCTAGCATGCAGTGCTTCAGCTTC-3' (forward) and 5'-TTGGATCCTTATTGTAGATTGCAGTA-3' (reverse). The amplified cDNA was cloned into the NheI / BamHI restriction site of the pCDH-promoter-MCS-EF1 lentivector (System Biosciences, Mountain View, CA) without the GFP sequence. The GFP deletion was amplified by the PCR product using the primer sets 5'-CCTACGCTAGACGCCACCATGACCGAGTACAAGCCC-3' (forward) and 5'-GGGCTTGTACTCGGTCATGGTGGCGTCTAGCGTAGG-3' (reverse) and the pCDH-promoter MCS-EF1-lentivector as a template was the restriction enzyme DpnI was selected by and the empty lenti vector was used as a control for a stable cell line. A viral packaging plasmid composed of pCMV delta and pMDG was used to generate a stable cell line expressing TSPAN1. Viral particles were obtained 48 and 72 hours after transfection, and positively infected cells were selected with 2 ug/mL puromycin (Sigma-Aldrich, St. Louis, MO).

Preparation Example 4: Preparation of siRNA

Knockdown of TSPAN1 (#1157352), AMPK (#5562-1) and negative control (#SN-1003) was performed by purchasing a pre-designed siRNA sequence from Bioneer. Transfection of siTSPAN1 was performed using Lipofectamine RNAiMax (Thermo Scientific, MA, Waltham, MA) according to the manufacturer's instructions. Transfection of siAMPK was performed using G-Fectin (Genolution Pharmaceuticals Inc., Seoul, Korea) according to the manufacturer's instructions.

Example 1: Gene expression profiling through DESeq2 and edgeR analysis

1.1 Identification of differentially expressed genes

To discover differentially expressed genes that can explain the oncogenic transition from endometriosis (Em) to ovarian clear cell carcinoma (OCCC), Laser Capture Microdissection (LCM) RNA sequencing was performed using the mRNA extracted from the lesion excised from the FFPE tissue obtained in Preparation Example 1. Specific details regarding the clinical information of the tissue sample are shown in FIG. 1 . After primary selection by DESeq2 and edgeR methods, gene expression data heatmaps were generated through RNA sequencing. At this time, RNA sequencing reads were mapped to the human genome (hg19), and read counts of each gene were calculated. Each gene was normalized using read counts, and differentially expressed genes (DEG) were analyzed using DESeq2 and edgeR.

Differentially expressed genes (DEGs) in six control groups (Em vs. AtyEm, Em vs. AdjEm, Em vs. OCCC, AtyEm vs. AdjEm, AtyEm vs. AdjEm and AdjEm vs. OCCC) were analyzed. To increase the accuracy of DEG analysis, both DESeq2 and edgeR methods were applied, the number of DEGs was greater in edgeR than DESeq2 in all comparisons, and 113 genes (DESeq2) or 586 genes (edgeR) when AtyEm and AdjEm were compared. ) showed the lowest total number of DEGs, whereas between Em and OCCC, 3,305 genes (DESeq2) or 5,288 genes (edgeR) were confirmed to be the highest (see FIG. 2 ). Genes were selected based on fold change of 2 fold or more and p-value < 0.05. The result of selecting a common DEG from 5 comparisons except AtyEm and AdjEm and creating a Venn diagram is shown in Fig. 3a. At this time, 14 genes in DESeq2 analysis and 34 genes in edgeR analysis were found, and the final 14 common The gene heat map is shown in Figure 3b using Cluster3.0 and Java Treeview. The 14 genes discovered through the above process were identified as TSPAN1, EPCAM, TMEM84A, PKP3, ERBB3, MUC20, B4GALNT3, B3GNT3, EPS8L1, KRT19, BSPRY, SYTL1, SGK2 and CDK2P2 (see FIGS. 3c and 3d). As a result of comparison through normalized counts (log2), the expression levels of 14 genes were higher than Em in AtyEm and AdjEm and higher than that of AtyEm and AdjEm in OCCC (see Fig. 3c). All results were confirmed to have statistically significant values (see FIG. 3D ). In particular, it can be confirmed that the TSPAN1 gene has the most significant increase in OCCC compared to Em among a total of 14 genes.

Example 2: Excavation of TSPAN1 markers

2.1 Assessment of TSPAN1 Expression in Cells

In order to confirm the expression level of TSPAN1 and evaluate the efficacy of TSPAN1, the cells of Em obtained in Preparation Example were first cultured, and the hTERT and SV40 T antigens were expressed using a lentivirus to obtain an immortalized cell line and used for the experiment. did endometrial cell lines (6595 and 6866_SV40), endometriosis cells (EM cells; 6045_SV40 and 9585_SV40) and ovarian clear cell carcinoma cells (OCCC cells; ES-2, TOV-21G; OVTOKO and OVISE) were used to confirm the mRNA and protein expression levels of TSPAN1. It was confirmed that the expression of TSPAN1 was higher in ovarian clear cell carcinoma cells (TOV-21G, OVTOKO and OVISE except for ES-2) than in endometrial and endometriosis cells (see FIG. 4a ). According to the analysis of the Gene Expression Omnibus (GEO) data set, it was confirmed that the mRNA expression of TSPAN1 was higher in OCCC than in normal cells (see FIG. 4b ).

2.2 Evaluation of TSPAN1 Expression in Patient Tissues

In addition, to evaluate TSPAN1 expression in patient tissues, immunohistochemistry (IHC) was performed. Endogenous peroxidase was blocked by deparaffinization from paraffinic tissue sections with xylene, rehydration with ethanol, and treatment with 3% H ₂ O ₂ solution in methanol for 30 min. After blocking for 30 min, sections were incubated with mouse monoclonal anti-human TSPAN1 antibody (Santa Cruz Biotechnology, Cat #sc-376551) for 1 h at RT for detection using a Dako LSAB+ (Dako, Glostrup, Denmark). did. The reaction product was detected by DAB (3,3'-diaminobenzidine) chromogen solution reaction. Tissue sections were stained with hematoxylin and fixed with Paramount aqueous mounting medium (Dako). Staining for TSPAN1 was scored as positive when tumors or epithelial cells showed immunoreactivity in the cytoplasm or membrane.

As a result, TMA (tissue microarray) was performed with Em (n = 83), AtyEm (n = 13), AdjEm (n = 4) and OCCC (n = 51) among patient tissue samples. As a result, TSPAN1 protein expression was low. It was confirmed that AtyEm and AdjEm, which are endometriosis, which are more likely to develop into OCCC, than Em, which is a risk group endometriosis, showed significantly higher in OCCC (see FIG. 4c ).

The RNA sequencing results of ovarian endometrial cancer were obtained, and it was confirmed that the TSPAN1 mRNA level was higher in the ovarian clear cell carcinoma type but not in the ovarian endometrial cancer. was confirmed to represent (see Fig. 5a). These results are shown in Figure 5b according to the GEO data set analysis (GSE6008, GSE655986 and GSE29175). As such, TPSAN1 is expressed differently depending on the ovarian cancer subtype.

Example 3: Confirmation of TSPAN1 action effect in endometriosis cell line

3.1 Preparation of Transfected Cells for Confirmation of TSPAN1 Function in Em

In order to confirm the TSPAN1 function in Em, TSPAN1 overexpressing cell lines were prepared using the 6045_SV40 and 9585_SV40 cells obtained in the above preparation example. Each cell line was infected with a TSPAN1-overexpressing lentivirus and a control lentivirus (empty lentivirus). Thereafter, it was confirmed that the TSPAN1 overexpressing cell line had higher TSPAN1 mRNA and protein levels compared to the control, and it was confirmed that the TSPAN1 mRNA and protein expression levels of the TSPAN1 overexpressing cell line were reduced through siRNA (small interfering RNA) ( FIG. 6a ). reference). The previously designed siRNA sequence was purchased from Bioneer and the above experiment was performed, and the transfection of siTSPAN1 was performed using Lipofectamine RNAiMax (Thermo Scientific, MA, Waltham, MA) according to the manufacturer's instructions.

In order to confirm the expression of TSPAN1 in the transfected 6045_SV40 and 9585_SV40 cells, immunofluorescence staining was performed on the cytoplasm. First, the 6045_SV40 and 9585_SV40 cells were cultured in a 24-well cell culture plate until the confluence of the cells reached an appropriate 30 to 40%, the cells were washed with PBS, and fixed with cold methanol at room temperature for 1 hour with PBS. Washed 3 times. It was then incubated with TSPAN1 (1:100) with 1% bovine serum albumin in Tris-buffered saline containing 0% 1% Tween 20 (TBS-T) solution overnight at 4°C. After washing 3 times with TBS-T, cells were incubated with anti-mouse IgG (cell signal) conjugated to Alexa Fluor 488 dye for 2 h at room temperature. Cells were washed 3 times with TBS-T, and nuclei were labeled with 1 g/mL Hoechst 33342 (Sigma-Aldrich) for 10 minutes at room temperature. After washing the cells three times with PBS for 5 minutes at room temperature, the images of the cells were observed with a microscope (Life Technologies, EVOS®FL Cell Imaging System), the results are shown in FIG. 6b. Referring to the results, the expression level of TSPAN1 in the nucleus can be confirmed.

3.2 Effect of TSPAN1 Expression on Cell Growth Rate

To determine the effect of TSPAN1 on the growth rate of cells, the short-term and long-term effects on cell proliferation in 6045_SV40 and 9585_SV40 stable cell lines were analyzed. The degree of cell proliferation in each of the 6045_SV40 and 9585_SV40 stable cell lines overexpressing or knocking down TSPAN1 was measured using a WST-1 Cell Proliferation Assay System. More specifically, the TSPAN1 overexpressing 6045_SV40 and 9585_SV40 stable cell lines were inoculated at 0.1 Х 10 ⁴ cells/well on a 96-well plate to make a final volume of 100 uL/well. In order to investigate cell proliferation activity, premix WST-1 (Dail Lab, Korea) was added and incubated for 2 hours at 37 °C, and OD (optical density) value at 450 nm wavelength was measured by VERSA maxTM (Bio-Rad Laboratories, Inc.) was measured and recorded every 0, 3, and 5 days, and is shown in FIGS. 7A and 7B. In addition, in order to obtain TSPAN1 knockdown 6045_SV40 and 9585_SV40 stable cell lines, 0.5 to 2 Х 10 ⁴ cells/well were inoculated on a 24-well plate and cells were adhered, and then, 20 to 50 nM of siTSPAN1 or siControl was transfected into the cells. After transfection, cells were fixed using 10% acetic acid solution with 10% methanol, stained with 0-5% crystal violet for 1 hour, photographed, and extracted using 1% SDS solution. The absorbance at 595 nm wavelength of VERSA maxTM from the cells was measured and shown in FIGS. 8A and 8B, and all of the above experiments were repeated three times.

7A and 8A, it was confirmed that the endometriosis cell line, TSPAN1 overexpressing 6045_SV40, proliferated faster than the control group, and when TSPAN1 was knocked down, it was confirmed that the growth rate of the TSPAN1 overexpressing cell line was reduced. In addition, the 9585_SV40 cell line showed faster growth only when tested for a long period of time compared to the 6045_SV40, and it was confirmed that the growth rate of the TSPAN1 overexpressing cell line was reduced when TSPAN1 was knocked down (see FIGS. 7b and 8b).

3.3 Effect of TSPAN1 Expression on Cell Invasion

Cell invasion assays were performed in an invasion chamber (Neuuro Probe 48-well Micro Chemotaxis Chamber, Neuro Probe, Inc., Gaithersburg, MD) according to the manufacturer's instructions. 0.3-2 x 10 ⁴ cells were resuspended in serum-free medium (56 uL) and plated in an upper chamber coated with Matrigel (BD Transduction Lab, San Jose, CA). The lower chamber was filled with a medium containing 0.1 to 10% FBS (27 uL), and after 24 hours of incubation, the cells that migrated through the membrane were stained with a Differential Quik Stain Kit (Triangle Biomedical Sciences, Inc., Durham, NC). Infiltrating cells in six randomly selected fields were counted using a microscope Axio Imager.M2 (Carl Zeiss, Thornwood, NY, Magnification x200).

As a result of the Matrigel invasion assay as described above, it was confirmed that the increase or decrease of cell invasion in the 6045_SV40 stable cell line was confirmed in response to TSPAN1, whereas the 9585_SV40 stable cell showed no change (see FIGS. 9a and 9b). This confirms that TSPAN1 increases the growth rate of Em cells and affects cell invasion.

3.4 Mechanism of cell growth and invasion of TSPAN1 in Em cells

To determine the mechanism of cell growth and invasion according to the TSPAN1 response in Em cells, we measured the phosphorylation level of key kinases related to cell growth and survival in Em stable cell lines. In the 6045_SV40 and 9585_SV40 stable cell lines, overexpression of TSPAN1 increased AMPK-Thr172 phosphorylation, which was decreased in response to siTSPAN1. However, TSPAN1 expression did not induce changes in pAKT-Ser473 and pERK-Thr201/Thr204 (see FIG. 10 ). The present inventors treated the 6045_SV40 stable cell line with an AKT upstream kinase PI3K inhibitor (Wortmannin), an AMPK inhibitor (Compound C; Dorsomorphin) and an ERK inhibitor (U0126) to confirm the effect on the growth and invasion of TSPAN1 overexpressing cells. As a result, it was confirmed that treatment with wortmannin and compound C delayed the growth of the TSPAN1 overexpressing cell line, whereas treatment with U0126 did not change (see FIG. 11b ). In the invasion assay, wortmannin significantly reduced cell invasion regardless of TSPAN1, but treatment with U0126 and compound C did not reduce cell invasion of TSPAN1-overexpressing cells (see FIG. 12 ). As described above, the activity of kinases after each inhibitor treatment was confirmed by the phosphorylation level of each kinase. Although the AMPK and ERK activities were attenuated by the inhibitor, it can be seen that the AKT activity was maintained regardless of the treatment with the inhibitor (see FIG. 11a ). Although AKT-Ser473 phosphorylation was not reduced by treatment with wortmannin, TSPAN1-induced cell growth was reduced thereby, confirming that the effect on cell growth was due to other PI3K pathways instead of PI3K/AKT signaling. .

Here, it is noteworthy that Compound C, a well-known AMPK inhibitor, reduced AMPK-Thr172 phosphorylation and TSPAN1-induced cell growth. To confirm the effect of compound C once again, crystal violet analysis was performed. Cell growth in response to TSPAN1 decreased with the concentration of Compound C (see FIGS. 12 and 13 ). Finally, cell growth was assessed via AMPK alpha1 knockdown. Expression of AMPK alpha1 and pAMPK-Thr172 was attenuated by siAMPK. Cell growth in response to TSPAN1 was accelerated in siControl, whereas AMPK alpha1 knockdown cells showed no difference in growth (see Fig. 14). These results suggest that TSPAN1 induces cell growth through AMPK phosphorylation in Em cells.

Example 4: Confirmation of association between the expression of TSPAN1 and the growth regulation response of OCCC

To determine whether TSPAN1 regulates the growth of OCCC in Em cells, siTSPAN1 consisting of the nucleotide sequences shown in SEQ ID NOs: 3 and 4 was transiently transfected into TOV-21G and OVTOKO, and then the degree of cell growth was measured. In response to siTSPAN1, the expression level of TSPAN1 decreased in TOV-21G and OVTOKO (see FIG. 15a ), and it was confirmed that the cell growth rate was delayed accordingly (see FIGS. 15b and 15c ). Unlike Em cells, siTSPAN1 treatment of TOV-21G and OVTOKO did not induce changes in AMPK-Thr172 phosphorylation compared to siControl (see Fig. 16). This means that when cells are transformed into a malignant type such as OCCC, TSPAN1 regulates cell growth through other mechanisms instead of AMPK. By using this mechanism, it is expected to delay and further suppress the progression of high-risk endometriosis to ovarian clear cell carcinoma (OCCC).

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> A composition for preventing or treating high-risk endometriosis <130> PDPB204064d01 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 241 <212> PRT <213> Homo sapiens <400> 1 Met Gln Cys Phe Ser Phe Ile Lys Thr Met Met Ile Leu Phe Asn Leu 1 5 10 15 Leu Ile Phe Leu Cys Gly Ala Ala Leu Leu Ala Val Gly Ile Trp Val 20 25 30 Ser Ile Asp Gly Ala Ser Phe Leu Lys Ile Phe Gly Pro Leu Ser Ser 35 40 45 Ser Ala Met Gln Phe Val Asn Val Gly Tyr Phe Leu Ile Ala Ala Gly 50 55 60 Val Val Val Phe Ala Leu Gly Phe Leu Gly Cys Tyr Gly Ala Lys Thr 65 70 75 80 Glu Ser Lys Cys Ala Leu Val Thr Phe Phe Phe Ile Leu Leu Leu Ile 85 90 95 Phe Ile Ala Glu Val Ala Ala Ala Val Val Ala Leu Val Tyr Thr Thr 100 105 110 Met Ala Glu His Phe Leu Thr Leu Leu Val Val Pro Ala Ile Lys Lys 115 120 125 Asp Tyr Gly Ser Gln Glu Asp Phe Thr Gln Val Trp Asn Thr Thr Met 130 135 140 Lys Gly Leu Lys Cys Cys Gly Phe Thr Asn Tyr Thr Asp Phe Glu Asp 145 150 155 160 Ser Pro Tyr Phe Lys Glu Asn Ser Ala Phe Pro Pro Phe Cys Cys Asn 165 170 175 Asp Asn Val Thr Asn Thr Ala Asn Glu Thr Cys Thr Lys Gln Lys Ala 180 185 190 His Asp Gln Lys Val Glu Gly Cys Phe Asn Gln Leu Leu Tyr Asp Ile 195 200 205 Arg Thr Asn Ala Val Thr Val Gly Gly Val Ala Ala Gly Ile Gly Gly 210 215 220 Leu Glu Leu Ala Ala Met Ile Val Ser Met Tyr Leu Tyr Cys Asn Leu 225 230 235 240 Gln <210> 2 <211> 1619 <212> DNA <213> Homo sapiens <400> 2 agacacacac accagcagct acacctacac gctgaccatc acaggcacac agaggcacat 60 ccacctcaca tccacctcat acttgtgtac tctcagggtt cagtctttca tcctatccct 120 ctctgatctg tgcctcccaa taccttccaa gatgtttaca gagacccttc tccctgtgca 180 gttaggagtg taaggcaaga gagcccctac ttcatggggc agatcaagag ctgagaccaa 240 agatggtcta tgttgctgac cttgtcctgt cctcctgctg tcttaaacta tgatccctgc 300 tgcggtcact gaagcctttc cctgtgagca gtggtgtgtg agagccaggc gtccctctgc 360 ctgcccactc agtggcaaca cccgggagct gttttgtcct ttgtggagcc tcagcagttc 420 cctctttcag aactcactgc caagagccct gaacaggagc caccatgcag tgcttcagct 480 tcattaagac catgatgatc ctcttcaatt tgctcatctt tctgtgtggt gcagccctgt 540 tggcagtggg catctgggtg tcaatcgatg gggcatcctt tctgaagatc ttcgggccac 600 tgtcgtccag tgccatgcag tttgtcaacg tgggctactt cctcatcgca gccggcgttg 660 tggtctttgc tcttggtttc ctgggctgct atggtgctaa gactgagagc aagtgtgccc 720 tcgtgacgtt cttcttcatc ctcctcctca tcttcattgc tgaggttgca gctgctgtgg 780 tcgccttggt gtacaccaca atggctgagc acttcctgac gttgctggta gtgcctgcca 840 tcaagaaaga ttatggttcc caggaagact tcactcaagt gtggaacacc accatgaaag 900 ggctcaagtg ctgtggcttc accaactata cggattttga ggactcaccc tacttcaaag 960 agaacagtgc ctttccccca ttctgttgca atgacaacgt caccaacaca gccaatgaaa 1020 cctgcaccaa gcaaaaggct cacgaccaaa aagtagaggg ttgcttcaat cagcttttgt 1080 atgacatccg aactaatgca gtcaccgtgg gtggtgtggc agctggaatt gggggcctcg 1140 agctggctgc catgattgtg tccatgtatc tgtactgcaa tctacaataa gtccacttct 1200 gcctctgcca ctactgctgc cacatgggaa ctgtgaagag gcaccctggc aagcagcagt 1260 gattggggga ggggacagga tctaacaatg tcacttgggc cagaatggac ctgccctttc 1320 tgctccagac ttggggctag atagggacca ctccttttag gcgatgcctg actttccttc 1380 cattggtggg tggatgggtg gggggcattc cagagcctct aaggtagcca gttctgttgc 1440 ccattccccc agtctattaa acccttgata tgccccctag gcctagtggt gatcccagtg 1500 ctctactggg ggatgagaga aaggcatttt atagcctggg cataagtgaa atcagcagag 1560 cctctgggtg gatgtgtaga aggcacttca aaatgcataa acctgttaca atgttgcca 1619 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> TSPAN1 siRNA sense <400> 3 gcuucaccaa cuauacgga 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> TSPAN1 siRNA antisense <400> 4 uccguauacu uggugaagc 19

Claims (9)

A pharmaceutical composition for inhibiting the progression of endometriosis to ovarian clear cell carcinoma, comprising an agent for inhibiting the activity of TSPAN1 (Tetraspanin 1) protein or an agent for reducing the expression level of a gene encoding the protein. The method of claim 1,
The agent for inhibiting the activity is any one or more selected from the group consisting of compounds that specifically bind to TSPAN1 protein, peptides, peptide mimetics, aptamers, antibodies, and natural products, a pharmaceutical composition. The method of claim 1,
The agent for reducing the expression level is selected from the group consisting of antisense nucleotides complementary to TSPAN1 gene, short interfering RNA (siRNA), short hairpin RNA and ribozyme. One or more pharmaceutical compositions. delete delete processing a candidate material on a biological sample isolated from a subject of interest; and
A method of screening an inhibitor of progression of endometriosis to ovarian clear cell carcinoma, comprising: measuring the expression level of a TSPAN1 (Tetraspanin 1) protein or a gene encoding the same in the biological sample. 7. The method of claim 6,
In the screening method, the expression level of the TSPAN1 protein or the gene encoding it measured after the treatment of the candidate substance is reduced compared to the expression level of the TSPAN1 protein or the gene encoding the TSPAN1 protein measured before the treatment of the candidate substance. The method further comprising the step of selecting the candidate as an inhibitor of the progression of endometriosis to cancer. delete delete

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