KR20180052109A - A composition for diagnosing colon cancer metastasis or predicting the same, and use thereof - Google Patents

Abstract

According to the present invention, the diagnosis or prognosis prediction of colon cancer metastasis can be performed using a composition for the diagnosis or prognosis prediction of colon cancer metastasis which includes squalene epoxidase and/or an agent for measuring the level of mRNA of p53 gene or protein thereof.

Description

TECHNICAL FIELD The present invention relates to a composition for diagnosing colon cancer metastasis or for predicting prognosis,

The present invention relates to a composition for diagnosing colon cancer metastasis or prediction of prognosis, a kit for diagnosing colorectal cancer metastasis or an information providing method for predicting prognosis, which comprises a preparation for measuring the level of mRNA of squalene epoxidase gene or its protein .

The large intestine is divided into cecum, colon (ascending colon, ascending colon, descending colon, S phase colon) and rectum, and absorbs water and part of vitamin, bile, bilirubin and discharges the debris out of the body It is also home to many different kinds of bacteria. Colorectal cancer refers to a tumor consisting of cancerous cells in the large intestine. Specifically, polyps in the large intestine have been grown from adenomas, and in most cases they have grown to benign tumors, but some are known to develop into malignant tumors.

In addition, colon cancer is a major cause of high fat, meat, processed foods and instant foods. Cholesterol and cholesterol are known to secrete a lot of cholesterol and cholesterol, and cholesterol makes carcinogens in the metabolic process. Cholanic acid is known to play a role in turning colon cells into cancer cells.

Colorectal cancer can be cured if it is detected early, but as the time of discovery is delayed, metastasis to the lung, liver, lymph nodes or peritoneum becomes difficult. The average recurrence rate of colorectal cancer is 12 to 24 months, and it is known that about 70% of recurrence occurs within 24 months, and it is known that 90% recur in 3 to 5 years after surgery.

As the main symptom of the disease, there are no symptoms at the beginning and anemia due to inconspicuous bowel bleeding, loss of appetite and weight loss even in the absence of symptoms. Colon cancer is classified into stages 1 to 4 according to progression. Specifically, stage 1 and stage 2 means that the cancer cells infiltrate into the submucosal layer or muscle layer, and lymph node metastasis occurs in the third or more.

Colorectal cancer usually has no symptoms in the early stages, and symptoms have already progressed considerably. Therefore, it is important to diagnose this early, but it is difficult to diagnose colon cancer metastasis because there are many cases without any special abnormal symptoms.

In addition, Korean Patent Nos. 10-1007573 and 10-0969692 have developed a method for diagnosing colorectal cancer using the overexpressed genes for colon cancer, but it is only for confirming the incidence of colorectal cancer, Therefore, it is still necessary to develop a method for diagnosing the prognosis of colorectal cancer.

Under these circumstances, the inventors of the present invention have made extensive efforts to develop a method for diagnosing colorectal cancer metastasis and prognosis more effectively, and as a result, the expression level of squalene epoxidase has been measured and it has become possible to diagnose colon cancer metastasis and prognosis more effectively The present invention has been completed.

It is an object of the present invention to provide a composition for diagnosing colon cancer metastasis or predicting prognosis, which comprises an agent for measuring the level of mRNA of squalene epoxidase gene or a protein thereof.

Another object of the present invention is to provide a kit for diagnosing colon cancer metastasis or prediction of prognosis, which comprises the above composition.

Yet another object of the present invention is to provide a method for detecting squamous epithelium, comprising the steps of: (a) measuring the mRNA level of a squalene epoxidase gene or a protein level thereof in a sample separated from an individual in which colon cancer has occurred; And (b) comparing the measured mRNA level or a protein level thereof with a mRNA level of the control sample or a protein level thereof in one or two groups of colorectal cancer-diagnosed colon cancer metastases, Providing a method of providing the same.

Yet another object of the present invention is to provide a method for detecting squamous epithelium, comprising the steps of: (a) measuring the mRNA level of a squalene epoxidase gene or a protein level thereof in a sample separated from an individual in which colon cancer has occurred; And (b) comparing the measured mRNA level or a protein level thereof with a mRNA level of the control sample that has died from colon cancer or a protein level thereof, for the prediction of prognosis of colorectal cancer will be.

To achieve the above object, one aspect of the present invention provides a composition for diagnosing colorectal cancer metastasis, which comprises an agent for measuring the level of mRNA of squalene epoxidase gene or a protein thereof.

The term "squalene epoxidase (SqE)" in the present invention is an enzyme which oxidizes squalene to 2,3-oxidosqualene using oxygen and NADPH. The enzyme is known to catalyze the step of oxygenating in the sterol biosynthetic pathway as one of the enzymes that limit the rate in sterol biosynthesis. In addition, the squalene epoxidase is encoded by the SQLE gene.

Squalene epoxidase is also referred to as squalene monooxygenase (SqMO), SQLE, or the like. In the present invention, the squalene epoxidase may be mixed with SqE. Its genetic information can be obtained from known databases, such as, but not limited to, GenBank of the National Center for Biotechnology Information (NCBI). Specifically, squalene epoxidase is commercially available from NCBI GenBank Accession No. < RTI ID = 0.0 > NM_003129.3, but is not limited thereto.

Such a squalene epoxidase of the present invention has not only the amino acid sequence shown in SEQ ID NO: 1 but also a homology of not less than 80%, specifically not less than 90%, more specifically not less than 95%, particularly not less than 97% ≪ / RTI > As the sequence having such homology, any amino acid sequence which represents a protein exhibiting substantially the same or equivalent activity as the respective proteins is included without limitation. It is also apparent that amino acid sequences having deletion, modification, substitution or addition of a part of the sequences are also included within the scope of the present invention, provided that they have such homology.

In addition, the gene encoding each protein of the present invention may contain not only a nucleotide sequence coding for the amino acid sequence shown in the above SEQ ID NOs, but also a sequence having 80% or more, specifically 90% or more, more specifically 95% or more , More specifically not less than 98%, and most particularly not less than 99%, of a protein having substantially the same or equivalent activity as the above-mentioned each protein. Also, it is obvious that base sequences having deletion, modification, substitution or addition of some sequences are also included within the scope of the present invention if they are base sequences having such homology.

As used herein, the term "homology" refers to the percentage of identity between two polynucleotide or polypeptide mimetics. The homology between sequences from one moiety to another can be determined by known techniques. For example, homology can be determined by aligning the sequence information and directly aligning the sequence information between two polynucleotide molecules or two polypeptide molecules using a computer program that is readily available. The computer program may be BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlign (DNASTAR Inc) or the like, but any program capable of determining homology can be used without limitation. Also, homology between polynucleotides can be determined by hybridizing polynucleotides under conditions that form a stable double strand between homologous regions, then resolving to single-strand-specific nucleases to determine the size of the resolved fragment no.

The squalene epoxidase was found to be decreased in the expression of colon cancer metastasis and decreased in survival with decrease in the expression of the squalene epoxidase. The inventors of the present invention have found that the squalene epoxidase can be used for diagnosis of colon cancer metastasis and prediction of prognosis. Respectively.

The squalene epoxidase protein of the present invention or the gene encoding the squalene epoxidase protein of the present invention may be a squalene epoxidase protein derived from various mammals including a human, a gene coding therefor, or a functional gene thereof, as long as it has an activity function of oxidizing squalene to 2,3-oxydosqualene Is a concept that includes equivalent homologous proteins.

The squalene epoxidase protein of the present invention includes not only a protein having a native amino acid sequence thereof but also a homologous protein thereof, within the scope of the present invention. The squalene epoxidase protein or its homologous protein can be extracted or synthesized in nature and can be prepared by a DNA recombinant method based on DNA sequence. In addition, in the present invention, the gene encoding squalene epoxidase protein may be isolated from nature or artificially synthesized.

The term "agent for measuring the level of protein" of the present invention means an agent used in a method for measuring the level of a target protein contained in a sample.

The agent that measures the level of the protein may comprise an antibody specific for the protein, or an aptamer. Specifically, the preparation can be applied to various tissues such as western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, Immunohistochemical staining, Immunoprecipitation Assay, Complement Fixation Assay, Immunofluorescence, Immunochromatography, FACS (fluorescence activated cell sorter (FACS)), immunohistochemical staining, analysis, and protein chip technology assay. However, the present invention is not limited thereto.

The "antibody" means a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. Such an antibody can be produced by a conventional method from the obtained protein by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the marker gene. The form of the antibody is not particularly limited and any polyclonal antibody, monoclonal antibody or antigen-binding antibody thereof may be included in the antibody of the present invention, and all immunoglobulin antibodies may be included. In addition, it may include a special antibody such as a humanized antibody. In addition, the antibody comprises a functional fragment of an antibody molecule as well as a complete form having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and can be Fab, F (ab ') 2, F (ab') 2, Fv and the like.

The term "aptamer " refers to a single-stranded oligonucleotide, which refers to a nucleic acid molecule having binding activity to a given target molecule. The aptamer may have various three-dimensional structures depending on its nucleotide sequence, and may have a high affinity for a specific substance such as an antigen-antibody reaction. An aptamer can inhibit the activity of a given target molecule by binding to a predetermined target molecule.

The aptamer of the present invention may be RNA, DNA, modified nucleic acid, or a mixture thereof, and the form thereof may be linear or cyclic, but is not limited thereto. The aptamers of the present invention can be used for squalene epoxidase. The aptamers having a binding activity to the squalene epoxidase can be easily produced according to a known method having a person skilled in the art with reference to each base sequence.

The term " agent for measuring mRNA level "of the present invention means a preparation used in a method for measuring the level of mRNA transcribed from the target gene in order to confirm the expression of the target gene contained in the sample. Specifically, the preparation can be used for RT-PCR, quantitative real time PCR, competitive RT-PCR, real time quantitative RT-PCR, RNase protection assay (RPA; a primer or a probe capable of specifically binding to a target gene used in a method such as protection assay, Northern blotting and DNA chip analysis, but is not particularly limited thereto.

The "primer" can form base pairs with a complementary template with a nucleic acid sequence having a short free 3 'hydroxyl group and serves as a starting point for template strand copying Quot; means a short nucleic acid sequence. Primers can be used to initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures.

On the other hand, the "probe" may be a probe capable of complementarily binding to the squalene epoxidase gene, and the nucleotide sequence of the probe is not limited as long as it can complementarily bind to each gene.

In the present invention, the composition may further include an agent for measuring the mRNA level of the p53 gene or the level of the protein expressed therefrom.

The above-mentioned "agent for measuring mRNA level" and "agent for measuring protein level" are as described above.

The term "p53" of the present invention is a tumor suppressor gene, and plays a crucial role in regulating the responses such as cell damage, cell death, DNA repair and cell senescence caused by various stresses such as DNA damage, hypoxia and abnormal expression of tumor genes (Vousden, Cell, 103: 691-694, 2000; Vogelstein et al ., Nature, 408: 307-310, 2000). In addition, activation of p53 following DNA damage is known to regulate cell cycle stasis such as p21, Bax, PUMA and MDM2, DNA damage repair, and the expression of genes involved in apoptosis (Juven et al ., Oncogene, 8: 3411 -3416, 1993; Barak et al ., EMBO J., 12: 461-468, 1993).

In the present invention, the expression of p53 in colon cancer metastasis is decreased, and thus it can be used to diagnose colon cancer metastasis more accurately with squalene epoxidase.

In a specific example of the present invention, the amount of protein expression of SqE and p53 was significantly reduced in cholesterol-treated colon cancer cells (FIG. 4). In addition, in one embodiment, in order to confirm the decrease of p53 expression due to the decrease of SqE expression, Western blotting revealed a decrease in the expression amount of p53 protein by induction of increase of hdm2 expression by siSqE treatment (Fig. 10).

This suggests that the reduction of p53 expression by inhibiting the expression of squalene epoxidase could be a target for the production of a diagnostic kit for colon cancer metastasis.

The term " colorectal cancer metastasis "of the present invention means that tumor cells of colon cancer migrate and settle and proliferate in different places. Specifically, colon cancer cells grow in a manner invading surrounding tissues unlike normal cells, so that when they come into contact with tubular structures such as blood vessels or lymph vessels, they can travel to other places by taking blood or lymph. When the colon cancer cells are moved to another organs or tissues, they grow again with cell division, which is called colon cancer.

The term "diagnosis" of the present invention means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to ascertain whether metastatic or metastatic colorectal cancer is metastatic.

According to one embodiment of the present invention, it was confirmed that the survival of colorectal cancer cells was increased by lowering the expression of squalene epoxidase (Fig. 7), and the expression of p53 was suppressed and the metastatic potential of colon cancer cells was increased (Figs. 10 and 11). Therefore, the inventors of the present invention have determined that degradation of squalene epoxidase may be a target for diagnosing colon cancer metastasis, and thus the present invention has been developed. This is the opposite of what is known in the field of colon cancer diagnosing colon cancer through overexpression of squalene epoxidase in the prior art. In order to confirm that the expression levels of SqE and p53 in the present invention are biomarkers superior to those of CAF and CA19-9 used in conventional clinical studies, ROC curve analysis, optimal cut-off value analysis, and maximum As a result of the optimal cut-off value analysis predicted by the maximum Youden index, SqE proved to be more effective than CEA and CA19-9 in clinical use. This indicates that SqE is a biomarker showing high efficiency in the diagnosis of colon cancer metastasis (Fig. 20).

Another aspect of the present invention provides a kit for colon cancer metastasis diagnosis comprising a composition for colon cancer metastasis diagnosis. Specifically, the kit may be a reverse transcription polymerase chain reaction kit (RT-PCR), a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, But is not limited thereto.

Specifically, the kit for measuring the mRNA expression level of the squalene epoxidase or the p53 gene of the present invention may be a kit containing essential elements necessary for performing RT-PCR. The RT-PCR kit contains a test tube or other appropriate container, reaction buffer (pH and magnesium concentration varying), deoxynucleotides (dNTPs), Taq-polymerase (dNTPs) in addition to the respective primer pairs specific for squalene epoxidase or p53 gene Enzymes such as enzyme and reverse transcriptase, DNase, RNAse inhibitor, DEPC-water, sterile water, and the like. In addition, it may contain a primer pair specific to a gene used as a quantitative control.

In addition, the kit of the present invention may contain essential elements necessary for performing DNA chip analysis. The kit for DNA chip analysis may include a substrate on which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and a reagent, a preparation, and an enzyme for producing a fluorescent-labeled probe. In addition, the substrate may contain a cDNA corresponding to a quantitative control gene or a fragment thereof.

In addition, the kit of the present invention may be a kit for analyzing a protein chip for measuring the level of a protein expressed from a squalene epoxidase gene. The kit may be, but not limited to, a kit for immunologically detecting an antibody, A 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, but a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, and a glass slide glass can be used, and the coloring enzyme is not particularly limited The fluorescent substance may be FITC, RITC or the like, and the coloring substrate liquid is not particularly limited, but ABTS (2, 3, 4, 5, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) or OPD (o-phenylenediamine), TMB (tetramethylbenzidine).

Another aspect of the present invention is a method for screening a squalene epoxidase gene, comprising: (a) measuring an mRNA level of a squalene epoxidase gene or a protein level thereof in a sample separated from an individual in which colon cancer has occurred; And (b) comparing the measured mRNA level or a protein level thereof with a mRNA level of the control sample or a protein level thereof in one or two groups of colorectal cancer-diagnosed colon cancer metastases, Providing a method of providing the same.

In addition, the information providing method may further comprise the step of (a) measuring the mRNA level of the p53 gene or the protein level thereof in a sample isolated from a subject suffering from colon cancer.

The above "squalene epoxidase "," p53 "and" colorectal cancer metastasis "

The term "individual" of the present invention may refer to all animals, including humans, who have metastasized or metastasized to colon cancer, assuming colon cancer has developed. The animal may be, but is not limited to, a mammal such as a cow, a horse, a sheep, a pig, a goat, a camel, a nutrient, a dog, a cat,

MRNA levels of the squalene epoxidase gene may be determined by RT-PCR, competitive RT-PCR, real-time quantitative RT-PCR ), RNase protection method, Northern blotting, or DNA chip technology assay. However, the present invention is not limited thereto.

The protein level of squalene epoxidase and p53 may be determined by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Immunoassay, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography but are not limited to, immunochromatography, fluorescence activated cell sorter analysis, and protein chip technology assay.

The term "control group " of the present invention means an individual in which colon cancer has developed, specifically, a colon cancer cell in which the colon cancer cells have not been transferred to other organs or tissues such as lymph node metastasis or blood , More specifically, individuals having one or two colon cancer cancers. Colorectal cancer can be divided into stages 1 to 4 based on the Astler-Coller staging system or TNM classification. Specifically, according to the TNM classification, when the cancer cells are confined to the submucosal layer or the muscle layer, it is classified into the first stage of colon cancer. When the cancer cells infiltrate into the subserosal layer or infiltrate into adjacent adjacent organs without lymph node metastasis, (National Cancer Information Center http://www.cancer.go.kr).

The term "sample" of the present invention means a direct object of measuring the expression level of squalene epoxidase gene from a patient suffering from colon cancer, and specifically, the sample may be a colon cancer cell or a colon cancer tissue.

The information providing method may further include a step of judging that the expression level of the squalene epoxidase or p53 protein measured by the sample is lower than the level of the protein of interest in the normal control sample to determine that it is a colon cancer metastasis.

Another aspect of the present invention is a method for screening a squalene epoxidase gene, comprising: (a) measuring an mRNA level of a squalene epoxidase gene or a protein level thereof in a sample separated from an individual in which colon cancer has occurred; And (b) comparing the measured mRNA level or a protein level thereof with a mRNA level of the control sample that has died from colon cancer or a protein level thereof, for the prediction of prognosis of colorectal cancer will be.

In addition, the information providing method may further comprise the step of (a) measuring the mRNA level of the p53 gene or the protein level thereof in a sample isolated from a subject suffering from colon cancer.

The above "squalene epoxidase "," p53 "and" colorectal cancer metastasis "

The term "control group " of the present invention means an individual who has died from the onset of colorectal cancer, and specifically, a colon cancer cell can be an individual or a subject that has died from metastasis to other organs or tissues such as lymph node metastasis or blood But is not limited thereto. The term "sample" of the present invention means a direct object to measure the expression level of squalene epoxidase gene from a patient who has died from the onset of colorectal cancer. Specifically, the sample may be a colon cancer cell or a colon cancer tissue have.

The term "prognosis" of the present invention means an act of predicting the progress of the disease and the result of death or survival. More specifically, the prognosis or prognosis prediction means that the progression of the disease may vary depending on the physiological or environmental condition of the patient, and it refers to all the actions that predicting the progress of the pre- . ≪ / RTI > For the purpose of the present invention, the prognosis can be interpreted as predicting the disease-free survival rate or survival rate of patients with colorectal cancer by anticipating the progression and cure of disease before and after treatment of colon cancer. For example, predicting a "good prognosis" indicates a high level of disease-free survival or survival rate in patients with colorectal cancer, regardless of treatment, indicating that patients with colorectal cancer are more likely to be treated, and "poor prognosis" Suggesting that the disease-free survival rate or survival rate of patients after colorectal cancer treatment is low, indicating that cancer is likely to be recurred from patients with colorectal cancer or death due to colorectal cancer. For example, the diagnosis of squamous cell carcinoma of the squamous cell carcinoma at the reference point was confirmed to be 64.43%, confirming that the diagnosis rate of mortality was higher than that of CEA.

The term " disease free survival rate "of the present invention means the possibility that a patient can survive without recurrence of cancer regardless of treatment or not.

The term "survival rate" of the present invention means the possibility that a patient can survive, regardless of whether or not the cancer recurs, whether treated or not.

The information providing method may be that the expression level of the squalene epoxidase protein measured by the sample is lower than the level of the protein expression level of the control sample of the surviving colon cancer patient to determine that the colorectal cancer prognosis is bad. Specifically, when the expression level of the squalene epoxidase protein is lower than the level of the protein expression of the corresponding protein in the survival colon cancer patient control sample, it is preferable that the cancer patient (for example, a patient suffering from colon cancer, a patient with colon cancer metastasis, Patients, etc.) may die. For example, the survival probability of patients with colorectal cancer whose expression level of squalene epoxidase is 0.01438 ± 0.0071 (optimal cut-off value) is -0.002650 ± 0.0062. But is not limited thereto. On the contrary, the possibility of death of colorectal cancer patients whose expression level of squalene epoxidase belongs to -0.002650 +/- 0.0062 is significantly higher than that of patients with colorectal cancer belonging to 0.001438 0.0071, but not limited thereto.

In one embodiment of the present invention, the utility of SqE and p53 as prognostic markers in colorectal cancer patients is analyzed by ROC curve analysis, SQE, p53 and combinations thereof, optimal cut-off value analysis, , And the maximum cut-off value predicted by the maximum Youden index. As a result, the prognosis of patients with colorectal cancer could be predicted by the combination of SqE, p53 or SqE and p53.

The present invention provides a composition comprising an agent for measuring the level of mRNA of squalene epoxidase gene or a protein thereof, to diagnose colon cancer metastasis or prognosis.

FIG. 1 shows the accumulation of intracellular cholesterol by cholesterol treatment at different concentrations.
FIG. 2 shows the ability of colon cancer cells to migrate by treatment with cholesterol.
FIG. 3 shows the induction of epithelial-mesenchymal transition and the survival of colon cancer cells in colorectal cancer cells by treatment with cholesterol.
Figure 4 shows the decrease in SqE and p53 expression by cholesterol treatment.
FIG. 5 shows the acceleration of degradation of SqE protein by cholesterol.
FIG. 6 shows colon cancer cell migration by siSqE treatment.
FIG. 7 shows the induction of epithelial-mesenchymal transition in colorectal cancer cells due to a decrease in expression of SqE.
FIG. 8 shows the increase in survival of colon cancer cells by treatment with cholesterol using an adhesion-inhibiting cell culture dish.
FIG. 9 shows statistical treatment of increase in survival of colon cancer cells by treatment with cholesterol using an adhesion-inhibiting cell culture dish.
Fig. 10 shows the decrease in p53 expression by siSqE treatment.
11 shows the increase in metastatic potential of colon cancer cells due to a decrease in SqE expression.
Figures 12, 13 and 14 show the degree of metastasis of shSqE colon cancer cells using the in vivo luciferase method.
Fig. 15 shows the promotion of colon cancer metastasis by high cholesterol ingestion and decreased SqE expression in a mouse model.
Figure 16 shows the malignancy of cancer caused by high cholesterol intake and decreased SqE expression in a mouse model.
FIG. 17 shows changes in expression of SqE, p53 and GSK3? (Serine 9) according to the progression of colorectal cancer in human colorectal cancer tissues.
FIG. 18 shows the sensitivity and specificity analysis of SqE and p53 for colorectal cancer diagnosis using ROC (Receiver Operating Characteristic) curve.
FIG. 19 shows the results of SqE sensitivity and specificity analysis for colorectal cancer patients using ROC (Receiver Operating Characteristic) curves.
FIG. 20 shows the results of comparative analysis of diagnosis efficiency of colon cancer metastasis of each biomarker based on the ROC curve.
FIG. 21 shows the results of comparing the diagnostic efficiency of each biomarker based on the ROC curve for the diagnosis of colon cancer.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  1: Colon cancer due to cholesterol treatment Intracellular  Cholesterol accumulation

1-1. Cell culture

The specific expression of squalene epoxidase (SqE) and related proteins by cholesterol was confirmed and HCT116 and HT29, colon cancer cell lines, were cultured to confirm the function of the gene. HCT116 was cultured in DMEM-high glucose (Corning, USA) and HT29 was cultured in RPMI-high glucose (Corning, USA), and the cell lines were cultured in DMEM supplemented with 10% fetal bovine serum, 100 mg / ml streptomycin and 100 IU / ml ampicillin And cultured in the included medium.

1-2. Intracellular  Measurement of cholesterol

Measurement of intracellular cholesterol was investigated using the following method using filin (Cayman, UK) binding to cholesterol. Cholesterol was treated with colorectal cancer cells at different concentrations for 3 days, fixed with methanol, and reacted with filin (filip III). After incubation for 60 min at 37 ° C, images were obtained using ultra-precision confocal microscopy and quantitative computations were performed using the program (Zen lite, Carl Zeiss, Germany).

As a result, cholesterol accumulation inside the cells by cholesterol treatment by concentration was confirmed (Fig. 1).

Example  2: Cholesterol-Treated Colon Cancer Cells Mobile ability / Invasiveness  increase

Cell migration experiments using transwell were performed to observe the colon cancer cell migration by cholesterol treatment. First, the lower surface of a pore size 8 micrometer Transwell (Corning Incorporated Costar, USA) was coated with 2% gelatin (Sigma-Aldrich, USA) and cell suspension of serum-free medium was added to the inside of the transwell insert. At this time, the outer container was filled with a medium containing 20 μM cholesterol and 20% serum. The cell suspension was incubated for 72 hours. When the incubation was completed, the inner surface of the insert was wiped with a cotton swab to remove the non-migrated endothelial cells. The number of cells that migrated downward was counted in a 200-fold microscope field after staining with a crystal violet solution and then plotted and compared (FIG. 2).

As a result, it was confirmed that 20μM cholesterol treatment significantly affected invasiveness and migration ability of colon cancer cells.

Example  3: Epithelialization of colorectal cancer cells by cholesterol treatment - Intermediate lobe  Induction of transfusion and increased survival of colon cancer cells

In order to confirm epithelial-mesenchymal transition and SqE protein degradation of colon cancer cells, the following method was used. Cells were seeded in 6-well plates and 20 μM cholesterol was treated for 3 days each day. After collecting intracellular proteins using RIPA buffer (Invitrogen, USA), E-cahderin, N-cadherin, Viment And Western blotting using an antibody against GAPDH. The antibodies used are shown in Table 1.

Antibody Catalog No. company E-cadherin sc-21791 Santa Cruz N-cadherin 4061s Cell Signaling Technology Vimentin 5741s Cell Signaling Technology GAPDH LF PA-0212 AbFrontier

As a result, epithelial-mesenchymal transition (decrease in expression of E-cadherin, increase in expression of N-cadherin, and vimentin) and degradation of SqE protein were observed in colorectal cancer cells by treatment with 20 μM cholesterol (FIG.

Example 4: Decreased expression of SqE and p53 by cholesterol

4-1 Confirmation of reduction of SqE and p53 expression by cholesterol treatment

(HCT116) and 20 (HT29) μM cholesterol were treated with time (0, 4, 12, 24, 48 hours) after seeding the cells in the same amount in 6 well plates to confirm the decrease of SqE and p53 expression by cholesterol treatment After the intracellular proteins were collected using RIPA buffer (Invitrogen, USA), western blotting was performed using antibodies against each. The antibodies used are shown in Table 2.

Antibody Catalog No. company SQE sc-21791 Santa Cruz E-cadherin sc-21791 Santa Cruz GSK3 sc-377213 Santa Cruz GAPDH LF PA-0212 AbFrontier b-catein sc-7963 Santa Cruz pGSK3b 9336s Cell Signaling Technology pGSK3b sc-135653 Santa Cruz hdM2 OP-46 Calbiochem p53 sc-56180 Santa Cruz p-p53 (S315) 2528s Cell Signaling Technology p-p53 (S376) ab60021 Abcam

As a result, it was confirmed that the expression level of SqE and p53 was significantly decreased in 10 and 20 μM cholesterol colon cancer cells. E - cadherin, which is an indicator of epithelial - mesenchymal transition, was also inactivated and GS -catenin significantly reduced the expression of the epithelial-mesenchymal transition of colon cancer cells by cholesterol (Fig. 4).

4-2. Confirmation of decomposition of SqE protein by cholesterol using Western blot

The degree of decomposition of SqE protein by cholesterol in colorectal cancer cells was confirmed by Western blot based on the following method. Colorectal cancer cell lines were inoculated on 6-well plates, and 24 hours later, cells were treated with 200 nM siRNAs (siCont, siSqE) established in Example 5-1 under DMEM-high glucose medium conditions and treated with 5% lipoprotein deficient serum ) And 20 μg / ml cyclodextrin (LPDS / CD) medium. After the cells were ruptured with RIPA buffer (Invitrogen, USA), proteins were collected and used for antibodies against SqE and GAPDH Western blot was performed.

4-3. Total RNA isolation and Reverse transcription  Reactor (cDNA) generation

Total RNA was extracted from colon cancer cells using a commercially available system, the RNeasy Total RNA kit (Nanohelix co., Korea). Only the total RNA was purified by the phenol-chloroform method. Thereafter, reverse transcription was performed using 1 mg of RNA as a template and oligo dT as a primer (Nanohelix, South Korea).

4-4. Semi-quantitative Reverse transcription  Polymerase chain reaction Semiquantative  RT- PCR )

The semi-quantitative RT-PCR (10 μl) of the semi-quantitative RT-PCR performed with the reverse transcriptase (cDNA) performed in Example 4-3 and the primer shown in Table 3 was as follows: 2X premix PCR mixture (Nanohelix, Korea), 0.5 μM of each primer, 1 μl of template cDNA. The semi-quantitative reverse transcription polymerase chain reaction was performed at 95 ° C for 2 minutes and 30 seconds, followed by denaturation at 95 ° C for 20 seconds, at 58 ° C for 45 seconds, and at 72 ° C for 30 seconds. . The resulting PCR products were confirmed using 1% agarose gel.

designation Accession Primer base sequence SEQ ID NO: E-cadherin XM_011525788 Forward 5'-CAACGACCCAACCCAAGAAT-3 ' 4 Reverse 5'-TCCGCCTCCTTCTTCATCAT-3 ' 5 N-cadherin NM_004360 Forward 5'-ACAGTGGCAGCTGGACTTGA-3 ' 6 Reverse 5'-TCCCTTGGCTAATGGCACTT-3 ' 7 Vimentin XM_011519649 Forward 5'-CCTTGAACGCAAAGTGGAAT-3 ' 8 Reverse 5'-GCTTCAACGGCAAAGTTCTC-3 ' 9 TP53 NM_001276698.1 Forward 5'-CCTCACCATCATCACACTGC-3 ' 10 Reverse 5'-CCTCATTCAGCTCTCGGAAC-3 ' 11 Squalene epoxidase NM_003129 Forward 5'-GCTTCCTTCCTCCTTCATCA-3 ' 12 Reverse 5'-ACACATTCGCCACCAAGTTT-3 ' 13 GAPDH NM_001289746 Forward 5'-ACCATCTTCCAGGAGCGAGA-3 ' 14 Reverse 5'-AGTGATGGCATGGACTGTGG-3 ' 15

As a result, it was confirmed that decomposition of SqE protein and slight decrease of mRNA expression were observed by 20 μM cholesterol treatment (FIG. 5).

Example  5: SqE  Of colorectal cancer cells due to decreased expression Mobile ability / Invasiveness  increase

5-1. SqE siRNA  Produce

In order to investigate the intracellular function by suppression of SqE gene expression, siRNAs (SEQ ID NO: 2 and SEQ ID NO: 3) consisting of 20 nucleotides in the base sequence of SqE (SEQ ID NO: 1) were prepared. The amino acid sequence of SqE is shown in SEQ ID NO: 16.

5-2. SqE  Experiment of colon cancer cell migration by decreased expression

Cell migration experiments using transwell were performed to observe the colon cancer cell migration by siSqE treatment. The lower surface of a pore size 8 micrometer Transwell (Corning Incorporated Costar, USA) was coated with 2% gelatin (Sigma-Aldrich, USA) and 200 nM siCont or siSqE Each of the cell suspension solutions treated for two days was added. At this time, a medium containing 20% serum was added to the outer container. The cell suspension was incubated for 48 hours. When the incubation was completed, the inner surface of the insert was wiped with a cotton swab to remove the non-migrated endothelial cells. The number of cells migrated downward was counted in a 200-fold microscope field after staining with a crystal violet solution and then plotted and compared (FIG. 6).

As a result, it was confirmed that siSqE treatment significantly inhibited invasion and migration of colon cancer cells.

Example  6: SqE  The epithelium of colon cancer cells due to decreased expression Intermediate lobe  Induction of migration and increased survival of colon cancer cells

6-1. Western Blut method  Semi-quantitative Reverse transcription  Using polymerase chain reaction SqE  The expression of the epithelial- Intermediate lobe  Confirmation of transition

Cells were seeded in 6-well plates and treated with 200 nM siSqE for 3 days to collect the intracellular proteins using RIPA buffer (Invitrogen, USA) Semi-quantitative RT-PCR was performed based on the Western blot and the method described in Example 4 (4-2) using antibodies against the mesenchymal indicators E-cahderin, N-cadherin, Viment and GAPDH .

As a result, it was confirmed that E-cadherin protein expression and mRNA expression decrease and expression of protein and mRNA of N-cadherin and viment were increased in colorectal cancer cells by inhibition of SqE expression (Fig. 7).

6-2 Confirmation of Increased Survival of Colon Cancer Cells by Treatment of Cholesterol Using an Inhibitory Cell Culture Plate

Anoikis is a kind of apoptosis. It is a kind of cell death that occurs when the cell does not attach to the cell substrate or is attached to a place other than its original position. If this anoikis is not made well, Cells can survive or grow in a place other than the original organ. As a result, cancer metastasis is induced.

Therefore, in order to confirm the survival of colon cancer cells in the floating state by cholesterol treatment, colon cancer cells were cultured in an adhesion-inhibitory cell culture dish (Corning, USA) and 20 μM cholesterol was added every day for 3 days And the cells were photographed with a microscope to confirm cell viability. At the same time, 200 nM siRNAs prepared in Example 5 (5-1) were treated with colon cancer cells as a comparative group, and then cultured under the same conditions to confirm cell viability (FIG. 8).

Statistical Analysis of Increased Survival of Colon Cancer Cells by Cholesterol Treatment Using 6-3 Attachment Inhibitory Cell Culture Plate

In order to statistically confirm the increase of survival of colon cancer cells by siRNA against cholesterol and SqE identified in the previous 6-2, MTT method was used to perform the following method. After 3 days of 20 μM cholesterol or siRNA for 200 nM of SqE established in Example 5 (5-1), the cells were collected and transferred to a 96-well plate and treated with 2 mg / ml MTT After incubation for 2 hours at 37, the reaction was stopped with DMSO. After reading the absorbance with a microplate reader (570 nm), the cell death / viability was determined as a percentage. In addition, 50 μM Terbinafine, which is an activity inhibitor of SqE, was treated as a control, and the death / survival of colon cancer cells was confirmed by the same method (FIG. 9).

As a result, it was confirmed that the survival of colon cancer cells was significantly increased by treatment with 20 μM cholesterol (FIG. 9A) or 200 nM siSqE (FIG. 9B) compared to the control group in which almost all the cells were killed. However, significant survival of colon cancer cells by SqE inhibition (Fig. 9C) was not confirmed.

Example  7: SqE  Reduction of p53 expression by inhibition of expression

In order to confirm the decrease of p53 expression by SqE expression reduction, the cells were seeded in the same amount in 6-well plate and treated with 200 nM siCont and / or siSqE for 3 days, and then the intracellular proteins were treated with RIPA buffer (Invitrogen, USA) After collection, Western blotting was performed using antibodies against each.

As a result, it was confirmed that the expression of p53 protein was decreased by induction of hdm2 expression induced by 200 nM siSqE treatment, and E-cadherin, which is an indicator of epithelial-mesen transition, The expression of phospho-ser9 GSK3β and the activation of β-catenin were significantly decreased, thereby confirming the induction of epithelial-mesenchymal transition of colorectal cancer cells by cholesterol (FIG. 10).

Example  8: SqE  Promoting colon cancer metastasis by decreased expression

In order to confirm the increase of metastatic ability of colorectal cancer cells by reduction of SqE expression, colon cancer cells (shSqE) stably inhibited the expression of SqE were constructed by the following method. HCT11 and HT29 colon cancer cells were infected with SqE Mission shRNA Lentiviral Transduction Particles (Sigma: TRCN0000046155) and Mission pLKO.1-puro Non-Target shRNA Control Transduction Particles (Sigma: SHC016V) in order to inhibit stable expression of SqE. Then, 4 μg of Luciferase vector, PGL4.17, was treated with lipofectamine 3000 (Invitrogen, USA) to observe the in vivo migration of human colorectal cancer cells. Thereafter, colonies were selected and cells were proliferated for about 3 weeks with 10 μg / ml puromycin and 0.3 mg / ml G418. Firstly, the migration and invasion of colon cancer cells were confirmed by SqE expression inhibition using the Transwell method. Secondly, semi-quantitative RT-PCR was used to determine the expression level of SqE Was inhibited. Finally, the expression level of PGL4.17 inserted into colon cancer cells and the degree of luciferase activity were confirmed using quantitative RT-PCR (Fig. 11).

As a result, the inhibition of stable expression of SqE and the corresponding increase in migration and invasiveness of colon cancer cells were confirmed in both HCT116 (FIG. 11A) and HT29 (FIG. 11B) cells, Lt; 17 > and luciferase activity.

Example  9: In animal models SqE  Promoting colon cancer metastasis by decreased expression

9-1. in vivo How to make Lucifera  Used shSqE  Confirm the degree of metastasis of colon cancer cells

The HCT116-based shCont / shSqE cells established in Example 8 above were injected into the subcutaneously and the orthotopic animals of the experimental animals and compared with the control cells (shCont) for 100 days. The degree of metastasis of shSqE colon cancer cells was measured in vivo Luciferase assay (IVIS Lumina II, Caliper Life Science). After 100 days, lung and liver were removed and the degree of cancer progression was confirmed by hematoxylin / eosin staining. In addition, in order to confirm the in vivo anoikis resistance of cancer cells, immunohistochemical staining and immunoaffinity staining using an antibody against caspase-3 (cell death marker) and MCM-7 (cancer cell proliferation marker) Focus fluorescence microscopy was used (Figs. 12, 13, 14).

As a result, it was confirmed that the metastasis of colorectal cancer cells (shSqE) stably inhibited the expression of SqE was significantly increased compared with the metastasis of the control colorectal cancer cells (shCont). Also, in the liver and lungs of the shSqE- Significant increase of cancer cells was confirmed. In addition, by staining lung tissue of shSqE cell-injected experimental animals, significant increase of cancer cells and decrease of cell death were confirmed, and significant increase of in vivo cell survival of colon cancer cells by inhibition of SqE expression could be confirmed .

9-2: Promoting colon cancer metastasis by high cholesterol intake and decreased SqE expression in mouse models constructed using HT29 cells

After the mice were fed with high cholesterol diet and control diet for one month and their blood cholesterol levels were measured immediately before the mouse model, blood cholesterol levels in hypercholesterol-supplemented rats were significantly increased (200 mg / dl, Fig. 15) . In addition, the HT29-based shCont / shSqE cells established in Example 8 were injected into the mouse's oropharynx and the high cholesterol diet and the control diet were consumed for 120 days. Then, shSqE colon cancer cells (IVIS Lumina II, Caliper Life Science). As a result, significant transfer of cancer cells was confirmed (Fig. 15B-LA).

After 120 days, the lungs and liver were removed and the degree of cancer progression was examined using hematoxylin / eosin staining (Fig. 15 (d)). As a result, the formation of cancer was confirmed.

In addition, the survival of each group was confirmed, and the survival rate of the remaining groups was significantly reduced in comparison with rats fed with the control feed and shCont (FIG. 15 bar). This means that a decrease in SqE expression due to high cholesterol intake promotes metastasis of colorectal cancer (Fig. 15).

9-3: Cancer Malignancy and Increased Blood Cancer Cells by High Cholesterol Intake and SqE Expression Decrease in Mouse Model Constructed with HT29 Cells

The lung tissue established in Example 9-2 was extracted and the degree of cancer progression was confirmed by hematoxylin / eosin staining. As a result, the cholesterol intake or SqE expression decreased significantly Cancer production was confirmed (Fig. 16A). In addition, a significant increase in Vimentin indicating invasion of cancer cells was confirmed (FIG. 16B). In addition, increased survival of cancer cells due to increase of Minichromosome maintenance complex component 7 (MCM7) and decrease of Histone H2A.X and caspase-3 (C3), a cancer cell marker in high cholesterol intake or SqE expression reduction group, Inhibition of the death was confirmed (Fig. 16A-D). In addition, the presence of CUB domain-containing protein 1 (CDCP1), a significant colon cancer cell marker, was confirmed in the high cholesterol intake group and the SqE expression reduction group (FIG.

This means that the cancer is aggravated by the increase of blood cancer cells by the decrease of SqE expression by high cholesterol intake (Fig. 16).

Example  10: Follow-up of colorectal cancer SqE and  Confirming significant inhibition of p53 expression

Human colon cancer microarray slides (CO702, CO803, US Biomax, USA) were purchased and tissue specimens were taken using immunofluorescence staining and ultra-precision confocal microscopy in order to confirm changes in SqE and p53 expression following colon cancer progression The changes in SqE and p53 expression and co-localization were investigated using the post-analysis program (Zen lite, Germany) according to the extent of colorectal cancer progression in patients (Table 4 and Table 5).

The tumor margin is not recognized as an independent prognostic factor for colorectal cancer, but less than 5 tumor buddies in the invasive aspect of invasive growth are observed in more than 10 , It is reported that prognosis is significantly worse.

Analysis of the expression level of SqE and p53 in each tissue using fluorescence immunoassay and ultrafine confocal fluorescence microscopy revealed that high tumor budding, ie, progression of colorectal cancer to SqE (Fig. 17A) and p53 (Fig. 17B) expression was decreased. In addition, it was found that the degree of Ser9 phosphorylation (pSer9-GSK3?) Of GSK3? Increases as the degree of dissociation of SqE and p53 increases (Figs. 17C and 17D).

These results indicate that the expression of SqE and p53 is significantly decreased according to the progression of colorectal cancer to malignancy, that is, the degree of colon cancer metastasis.

Example  11: Tissue microarray (Tissue microarray ; TMA Analysis of expression of SqE and p53 by image analysis

TMA slides composed of colorectal cancer, metastatic colorectal cancer, and normal tissue were purchased from US Biomax, stained with fluorescent antibodies using SqE and p53 antibodies, and examined by fluorescence microscopy (x200) The expression patterns of SqE and p53 were analyzed. Quantitative expression analysis of each protein was performed using ZEN (blue edition) provided by Carl Zeiss. The obtained values were converted by global equalization and then analyzed by GraphPad Prism (Idikio et al., International Journal of Clinical Experimental Pathology 2011; 4: 505-512) (Table 6).

Example  12: SqE  And p53 in Colorectal Cancer Metastasis Diagnosis Sensitivity and Specificity Analysis

12-1: Using Receiver Operating Characteristic (ROC) curve SqE  And p53 in Colorectal Cancer Metastasis Diagnosis Sensitivity and Specificity Analysis

In order to investigate the utility of SqE and p53 as metastatic diagnostic markers in colorectal cancer patients, ROC curve analysis was performed from Table 6 data (original cancer vs. metastatic cancer) of Example 11 using GraphPad Prism.

As a result, the Area Under the Curve (AUC) of SqE was 0.7101 (95% CI: 0.6174-0.8029) higher than 0.5827 (95% CI: 0.4619-0.7035) of p53. The AUC value of the combination of two proteins (SqE + p53) was 0.6530 (95% CI: 0.5783-0.7276). This means that SqE alone or combination of SqE and p53, p53 alone can diagnose the metastasis of colorectal cancer (Fig. 18).

12-2: SqE  And < RTI ID = 0.0 > p53 & off  Colorectal cancer metastasis based on cut-off value

Based on the optimal cut-off value of SqE and p53, the optimal cut-off value of SqE and p53 was calculated from the data of Table 6 of Example 11 (primary cancer vs. metastatic cancer) The diagnosis of colorectal cancer metastasis was confirmed.

As a result, as shown in Table 7, high sensitivity of SqE in colon cancer was confirmed (86.96%). In contrast, high specificity was confirmed in p53 (94.38%) and 72.1% in SqE + p53. These results suggest that SqE or p53 alone is more specific for prognostic markers of colorectal cancer in sensitivity and specificity, respectively.

Colon cancer SqE SqE / p53 p53 Sensitivity (%) 86.96
(0.7668-0.9386) 72.07
(0.6276-0.8017) 7.143
(0.01498-0.1948) Specificity
(specificity,%) 12.36
(0.0634-0.2104) 28.09
(0.2162-0.3530) 94.38
(0.8737-0.9815)

12-3: maximum Yuden  Index (maximum Youden  index) SqE  And p53 in Colorectal Cancer Metastasis Diagnosis Sensitivity and Specificity Analysis

In order to evaluate the accuracy of SqE, p53 and SqE + p53 as diagnostic indexes for colorectal cancer metastasis, the optimal cut-off predicted by the maximum Youden index from the data of Table 6 of Example 11 (primary cancer vs. metastatic cancer) The sensitivity and specificity (%) of SqE, p53, and SqE + p53 for colorectal cancer metastasis according to off values were confirmed.

As a result, as shown in Table 8 below, the sensitivity of SqE was 59.4% and the specificity was 91%. In contrast, the sensitivity of p53 was 64.3% and the specificity was 71.9%. The combination of SqE and p53 showed a sensitivity of 64% and a specificity of 79.2%. This indicates that the combination of SqE or SqE + p53 is more specific for colon cancer metastasis than p53 alone.

Colon cancer SqE SqE / p53 p53 Y-index 0.5043 0.4374 0.3732 Sensitivity (%) 59.42
(0.4837-0.7240) 63.96
(0.5430-0.7286) 64.29
(0.4803-0.7845) Specificity
(specificity,%) 91.01
(0.8305-0.9604) 79.21
(0.7251-0.8492) 71.91
(0.6138-0.8093)

Example  13: SqE's  Sensitivity and specificity of colorectal cancer prognosis

13-1: Using ROC (Receiver Operating Characteristic) curve SqE's  Sensitivity and specificity of colorectal cancer prognosis

To investigate the utility of SqE and p53 as prognostic markers in colorectal cancer patients, ROC curve analysis was performed using GraphPad Prism from the data of Table 6 of Example 11 (Survival versus Death Patients in Colorectal Cancer Patients).

As a result, the AUC value of SqE was 0.6443 (95% CI (confidence interval): 0.5030-0.7856) higher than the AUC value of p53, 0.6131 (95% CI: 0.4291-0.7971). The AUC value of the combination of two proteins (SqE + p53) was 0.6261 (95% CI: 0.5178-0.7344). It was confirmed that the prognosis of patients with colorectal cancer can be predicted by SqE alone or a combination of SqE and p53 (Fig. 19).

13-2: SqE  And < RTI ID = 0.0 > p53 & off  Diagnosis of colorectal cancer prognosis based on cut-off value

The optimal cut-off value of SqE and p53 was calculated from the data of Table 6 of Example 11 (survival patients versus death patients in colorectal cancer patients) and the optimal cut-off value of SqE and p53 value) were evaluated for sensitivity and specificity of colorectal cancer prognosis.

As a result, as shown in Table 9 below, a high sensitivity of SqE was confirmed in the diagnosis of colorectal cancer prognosis (64.3%). In contrast, the specificity of p53 for diagnosis was 100%, and for SqE + p53, the specificity of diagnosis was 96.88%. These results suggest that SqE or p53 alone is more specific for colorectal cancer prognostic markers in sensitivity and specificity, respectively.

Colorectal cancer prognosis SqE SqE / p53 p53 responsiveness
(95% Cl,%) 64.29
(0.4803-0.7845) 3.571
(0.007427-0.1008) 2.381
(0.0006026-0.1257) Specificity
(95% Cl,%) 31.25
(0.1102-0.5866) 96.88
(0.8378-0.9992) 100.0
(0.7941-1.000)

13-3: maximum Yuden  Index (maximum Youden  index) SqE  And p53 in colorectal cancer patient prognosis sensitivity and specificity analysis

In order to evaluate the accuracy of SqE, p53 and SqE + p53 as prognostic indicators of colorectal cancer patient prognosis, it was predicted by the maximum Youden index from the data of Table 6 of Example 11 (surviving patients vs. death patients in colorectal cancer patients) The prognostic sensitivity and specificity (%) of SqE, p53 and SqE + p53 in colorectal cancer patients were investigated according to the optimal cut - off value.

As a result, as shown in Table 10 below, the sensitivity of SqE was 52.4% and the specificity was 93.8%. In contrast, the sensitivity of p53 was 97.6% and the specificity was 43.8%. The combination of SqE and p53 showed a sensitivity of 45.2% and a specificity of 81.3%. This means that the combination of SqE alone or SqE + p53 is sensitive to the prognosis of colorectal cancer patients and that p53 alone is sensitive.

Colorectal cancer prognosis SqE SqE / p53 p53 Y-index 0.461 0.265 0.414 responsiveness
(95% Cl,%) 52.38
(0.3642-0.6800) 45.24
(0.3434-0.5648) 97.62
(0.8743-0.9994) Specificity
(95% Cl,%) 93.75
(0.6977-0.9984) 81.25
(0.6356-0.9279) 43.75
(0.1975-0.7012)

Example  14: Tissue Microarray microarray ; TMA ) Image analysis is used in clinical clinics CAF , CA19-9 and SqE , p53 expression pattern analysis

Carbohydrate antigen (CA19-9), SqE (CA19-9), and carbohydrate antigens were purchased from US Biomax using the method presented in Example 11 after purchasing a TMA slide (CO953) consisting of various types of colon cancer, , p53 antibody was used to react under the same conditions and the expression pattern was analyzed. The obtained values were transformed through global equalization and then statistical analysis was performed using GraphPad Prism (Table 11).

Example  15: CEA , CA-19-9 and SqE , p53 in colorectal cancer metastasis diagnosis sensitivity and specificity comparison analysis

15-1: Using ROC (Receiver Operating Characteristic) curve CEA , CA-19-9 and SqE , p53 in colorectal cancer metastasis diagnosis sensitivity and specificity comparison analysis

In order to investigate the utility of SqE and p53 as colon cancer metastasis diagnostic markers, ROC curve analysis was performed using the data of Table 11 of Example 14 (primary cancer vs. metastatic cancer) and the correlation between CEA and CA19-9 The metastatic diagnostic efficiency was compared.

As a result, the AUC value of CEA was 0.8867 (95% confidence intervals: 0.8144-0.9590), the AUC value of CA19-9 was 0.8075 (95% CI: 0.7140-0.9010) and the AUC value of SqE was 0.9983 % CI (confidence intervals): 0.9938-1.003), and the AUC value of p53 was 0.5208 (95% CI: 0.3734-0.6682). In other words, SqE showed a significantly better diagnosis of colorectal cancer metastasis than the CEA and CA19-9 used in clinical practice. This indicates that SqE is a biomarker showing high efficiency in the diagnosis of colon cancer metastasis (Fig. 20).

15-2: CEA , CA19-9 and SqE , the optimal cut of p53- off  Comparison of colorectal cancer metastasis diagnostic efficiency based on cut-off value

The optimal cut-off values of CEA, CA19-9, SqE and p53 were calculated from the data of Table 11 of Example 14 (primary cancer vs. metastatic cancer), and the optimal cut-off values of CEA, CA19-9, SqE and p53 The diagnostic efficiency of colorectal cancer metastasis based on cut-off value was confirmed.

As shown in Table 12, high sensitivity (80%, 70%, 100%) and high specificity (83.3%, 80%, 88.3%) were observed in CEA, CA19-9 and SqE, (40%) and high specificity (68.3%) in p53, respectively. In other words, SqE showed significantly higher sensitivity and specificity than the biomarkers used in clinical practice. This suggests that SqE is a biomarker with high diagnostic efficiency in the diagnosis of colon cancer (Table 12).

Colon cancer CEA Sensitivity (95% CI,%) 80.0 (0.5634-0.9427) Specificity (95% CI,%) 83.33 (0.7148-0.9171) CA19-9 Sensitivity (95% CI,%) 70.0 (0.4572-0.8811) Specificity (95% CI,%) 80.0 (0.6767-0.8922) SqE Sensitivity (95% CI,%) 100 (0.8316-1.000) Specificity (95% CI,%) 88.33 (0.7743-0.9518) p53 Sensitivity (95% CI,%) 40.0 (0.1912-0.6395) Specificity (95% CI,%) 68.33 (0.5504-0.7974)

15-3: maximum Yuden  Index (maximum Youden  index) CEA , CA19-9 and SqE, of p53  Colorectal cancer metastasis sensitivity and specificity analysis

To evaluate the accuracy of CEA, CA19-9, SqE, and p53 as diagnostic indexes for colorectal cancer metastasis, the optimal predicted by the maximum Youden index from the data of Table 11 of Example 14 (primary cancer vs. metastatic cancer) Sensitivity and specificity (%) of colon cancer metastasis of CEA, CA19-9 and SqE, p53 according to cut-off value were confirmed.

As a result, as shown in Table 13 below, SqE showed significantly higher Y-index, sensitivity, and specificity than CEA and CA19-9 used in clinical practice. In contrast, p53 has a lower Y-index and sensitivity and specificity than other biomarkers. In other words, SqE showed significantly higher Y-index, sensitivity and specificity than the biomarkers used in clinical practice. This implies that SqE is a biomarker with clinically high colorectal cancer metastasis diagnostic efficiency (Table 13).

Colon cancer CEA Sensitivity (95% CI,%) 95.0 (0.7513-0.9987) Specificity (95% CI,%) 75 (0.6214-0.8528) Y-index 70 CA19-9 Sensitivity (95% CI,%) 90.0 (0.6830-0.9877) Specificity (95% CI,%) 73.33 (0.6034-0.8393) Y-index 63.33 SqE Sensitivity (95% CI,%) 100 (0.8316-1.000) Specificity (95% CI,%) 76.67 (0.6396-0.8662) Y-index 76.67 p53 Sensitivity (95% CI,%) 45.0 (0.2306-0.6847) Specificity (95% CI,%) 58.33 (0.4488-0.7093) Y-index 3.33

Example  16: CEA , CA-19-9 and SqE , p53 in prognosis of colorectal cancer patients

16-1: Using ROC (Receiver Operating Characteristic) curve CEA , CA-19-9 and SqE Comparison of p53 diagnosis of colorectal cancer mortality

To investigate the utility of SqE and p53 as prognostic markers for colorectal cancer patients, we performed ROC curve analysis using the data from Table 11 (survival versus death) of Example 14 and compared the prognosis with CEA and CA19-9 Respectively.

As a result, the AUC value of CEA was 0.6220 (95% confidence intervals: 0.4653-0.7788), the AUC value of CA19-9 was 0.5818 (95% CI: 0.4099-0.7538) and the AUC value of SqE was 0.6443 % CI (confidence intervals): 0.5030-0.7856), and the AUC value of p53 was 0.6414 (95% CI: 0.4903-0.7924). In other words, SqE and p53 were found to be predictive of the prognosis of patients with colorectal cancer higher than that of CEA and CA19-9 used in clinical practice. This implies that SqE and p53 are highly efficient biomarkers in predicting the prognosis of colon cancer patients (Fig. 21).

16-2: CEA , CA19-9 and SqE , the optimal cut of p53- off  Comparison of diagnosis of colorectal cancer prognosis based on cut-off value

The optimal cut-off values of CEA, CA19-9 and SqE, p53 from the data of Table 11 of Example 14 (survival versus death) were calculated to determine the optimal cut-off value of CEA, CA19-9 and SqE, The sensitivity and specificity of colorectal cancer prognosis were evaluated based on the cut-off value.

As a result, the diagnostic efficiency of CEA, CA19-9, SqE, and p53 was confirmed in patients with colorectal cancer. As a result, the sensitivity (69%, 64%, 52%, 50%) of CEA, CA19-9, SqE, and p53 which are used clinically was confirmed as shown in Table 14 below. In addition, high specificity (88%, 81%) of SqE and p53 was confirmed compared with CEA and CA19-9. These results indicate that SqE has a significantly higher specificity than the biomarkers used in clinical practice and has a significant diagnostic efficiency in predicting the prognosis of colorectal cancer patients (Table 14).

Prognosis of colorectal cancer patients CEA Sensitivity (95% CI,%) 69.05 (0.5291-0.8238) Specificity (95% CI,%) 68.75 (0.4134-0.8898) CA19-9 Sensitivity (95% CI,%) 64.29 (0.4803-0.7845) Specificity (95% CI,%) 56.25 (0.2988-0.8025) SqE Sensitivity (95% CI,%) 52.38 (0.3642-0.6800) Specificity (95% CI,%) 87.50 (0.6165-0.9845) p53 Sensitivity (95% CI,%) 50.0 (0.3419-0.6581) Specificity (95% CI,%) 81.25 (0.5435-0.9595)

16-3: maximum Yuden  Index (maximum Youden  index) CEA , CA19-9, and SqE, p53 in patients with colorectal cancer were analyzed for sensitivity and specificity

In order to evaluate the accuracy of CEA, CA19-9, SqE, and p53 as diagnostic indicators of mortality in colorectal cancer patients, the optimal cut estimated by the maximum Youden index from the data of Table 11 of Example 14 (survival versus death) The sensitivity and specificity of CEA, CA19-9 and SqE, p53 according to the off-value were examined in colorectal cancer patients.

As a result, as shown in Table 15 below, SqE showed a Y-index similar to that of CEA and CA19-9 used in clinical practice, but the sensitivity was found to be significantly higher. In contrast, p53 showed lower Y-index and specificity than other biomarkers. This indicates that SqE is a biomarker with clinically high diagnostic efficiency in predicting the mortality of colon cancer patients (Table 15).

Prognosis of colorectal cancer patients CEA Sensitivity (95% CI,%) 45.24 (0.2985-0.6133) Specificity (95% CI,%) 68.75 (0.4134-0.8898) Y-index 13.99 CA19-9 Sensitivity (95% CI,%) 45.24 (0.2985-0.6133) Specificity (95% CI,%) 68.75 (0.4134-0.8898) Y-index 13.99 SqE Sensitivity (95% CI,%) 61.9 (0.4564-0.7643) Specificity (95% CI,%) 50.0 (0.2465-0.7535) Y-index 11.9 p53 Sensitivity (95% CI,%) 57.14 (0.4096-0.7228) Specificity (95% CI,%) 37.50 (0.1520-0.6457) Y-index -0.0536

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> A composition for diagnosing colon cancer metastasis or predicting the same,          and use thereof <130> KPA161140-KR-P1 <150> KR 10-2016-0148930 <151> 2016-11-09 <160> 16 <170> KoPatentin 3.0 <210> 1 <211> 1725 <212> DNA <213> Homo sapiens <400> 1 atgtggactt ttctgggcat tgccactttc acctattttt ataagaagtt cggggacttc 60 atcactttgg ccaacaggga ggtcctgttg tgcgtgctgg tgttcctctc gctgggcctg 120 gtgctctcct accgctgtcg ccaccgaaac gggggtctcc tcgggcgcca gcagagcggc 180 tcccagttcg ccctcttctc ggatattctc tcaggcctgc ctttcattgg cttcttctgg 240 gccaaatccc cccctgaatc agaaaataag gagcagctcg aggccaggag gcgcagaaaa 300 ggaaccaata tttcagaaac aagcttaata ggaacagctg cctgtacatc aacatcttct 360 cagaatgacc cagaagttat catcgtggga gctggcgtgc ttggctctgc tttggcagct 420 gtgctttcca gagatggaag aaaggtgaca gtcattgaga gagacttaaa agagcctgac 480 agaatagttg gagaattcct gcagccgggt ggttatcatg ttctcaaaga ccttggtctt 540 ggagatacag tggaaggtct tgatgcccag gttgtaaatg gttacatgat tcatgatcag 600 gaaagcaaat cagaggttca gattccttac cctctgtcag aaaacaatca agtgcagagt 660 ggaagagctt tccatcacgg aagattcatc atgagtctcc ggaaagcagc tatggcagag 720 cccaatgcaa agtttattga aggtgttgtg ttacagttat tagaggaaga tgatgttgtg 780 atgggagttc agtacaagga taaagagact ggagatatca aggaactcca tgctccactg 840 actgttgttg cagatgggct tttctccaag ttcaggaaaa gcctggtctc caataaagtt 900 tctgtatcat ctcattttgt tggctttctt atgaagaatg caccacagtt taaagcaaat 960 catgctgaac ttattttagc taacccgagt ccagttctca tctaccagat ttcatccagt 1020 gaaactcgag tacttgttga cattagagga gaaatgccaa ggaatttaag agaatacatg 1080 gttgaaaaaa tttacccaca aatacctgat cacctgaaag aaccattctt agaagccact 1140 gacaattctc atctgaggtc catgccagca agcttccttc ctccttcatc agtgaagaaa 1200 cgaggtgttc ttcttttggg agacgcatat aatatgaggc atccacttac tggtggagga 1260 atgactgttg cttttaaaga tataaaacta tggagaaaac tgctaaaggg tatccctgac 1320 ctttatgatg atgcagctat tttcgaggcc aaaaaatcat tttactgggc aagaaaaaca 1380 tctcattcct ttgtcgtgaa tatccttgct caggctcttt atgaattatt ttctgccaca 1440 gatgattccc tgcatcaact aagaaaagcc tgttttcttt atttcaaact tggtggcgaa 1500 tgtgttgcgg gtcctgttgg gctgctttct gtattgtctc ctaaccctct agttttaatt 1560 ggacacttct ttgctgttgc aatctatgcc gtgtattttt gctttaagtc agaaccttgg 1620 attacaaaac ctcgagccct tctcagtagt ggtgctgtat tgtacaaagc gtgttctgta 1680 atatttcctc taatttactc agaaatgaag tatatggttc attaa 1725 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siCont <400> 2 augaacguga auugcucaat t 21 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> siSqE <400> 3 gcagagccca atgcaaagtt tdtdt 25 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for E-cadherin <400> 4 caacgaccca acccaagaat 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for E-cadherin <400> 5 tccgcctcct tcttcatcat 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for N-cadherin <400> 6 acagtggcag ctggacttga 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for N-cadherin <400> 7 tcccttggct aatggcactt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Vimentin <400> 8 ccttgaacgc aaagtggaat 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Vimentin <400> 9 gcttcaacgg caaagttctc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for TP53 <400> 10 cctcaccatc atcacactgc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for TP53 <400> 11 cctcattcag ctctcggaac 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Squalene epoxidase <400> 12 gcttccttcc tccttcatca 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Squalene epoxidase <400> 13 acacattcgc caccaagttt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for GAPDH <400> 14 accatcttcc aggagcgaga 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for GAPDH <400> 15 agtgatggca tggactgtgg 20 <210> 16 <211> 574 <212> PRT <213> Homo sapiens <400> 16 Met Trp Thr Phe Leu Gly Ile Ala Thr Phe Thr Tyr Phe Tyr Lys Lys   1 5 10 15 Phe Gly Asp Phe Ile Thr Leu Ala Asn Arg Glu Val Leu Leu Cys Val              20 25 30 Leu Val Phe Leu Ser Leu Gly Leu Val Leu Ser Tyr Arg Cys Arg His          35 40 45 Arg Asn Gly Gly Leu Leu Gly Arg Gln Gln Ser Ser Ser Ser Gln Phe Ala      50 55 60 Leu Phe Ser Asp Ile Leu Ser Gly Leu Pro Phe Ile Gly Phe Phe Trp  65 70 75 80 Ala Lys Ser Pro Pro Glu Ser Glu Asn Lys Glu Gln Leu Glu Ala Arg                  85 90 95 Arg Arg Arg Lys Gly Thr Asn Ile Ser Glu Thr Ser Leu Ile Gly Thr             100 105 110 Ala Ala Cys Thr Ser Thr Ser Ser Gln Asn Asp Pro Glu Val Ile Ile         115 120 125 Val Gly Ala Gly Val Leu Gly Ser Ala Leu Ala Ala Val Leu Ser Arg     130 135 140 Asp Gly Arg Lys Val Thr Val Ile Glu Arg Asp Leu Lys Glu Pro Asp 145 150 155 160 Arg Ile Val Gly Glu Phe Leu Gln Pro Gly Gly Tyr His Val Leu Lys                 165 170 175 Asp Leu Gly Leu Gly Asp Thr Val Glu Gly Leu Asp Ala Gln Val Val             180 185 190 Asn Gly Tyr Met Ile His Asp Gln Glu Ser Ser Ser Ser Val Val Gln Ile         195 200 205 Pro Tyr Pro Leu Ser Glu Asn Asn Gln Val Gln Ser Gly Arg Ala Phe     210 215 220 His His Gly Arg Phe Ile Met Ser Leu Arg Lys Ala Ala Met Ala Glu 225 230 235 240 Pro Asn Ala Lys Phe Ile Glu Gly Val Val Leu Gln Leu Leu Glu Glu                 245 250 255 Asp Val Val Met Gly Val Gln Tyr Lys Asp Lys Glu Thr Gly Asp             260 265 270 Ile Lys Glu Leu His Ala Pro Leu Thr Val Val Ala Asp Gly Leu Phe         275 280 285 Ser Lys Phe Arg Lys Ser Leu Val Ser Asn Lys Val Ser Val Ser Ser     290 295 300 His Phe Val Gly Phe Leu Met Lys Asn Ala Pro Gln Phe Lys Ala Asn 305 310 315 320 His Ala Glu Leu Ile Leu Ala Asn Pro Ser Ser Val Leu Ile Tyr Gln                 325 330 335 Ile Ser Ser Ser Glu Thr Arg Val Leu Val Asp Ile Arg Gly Glu Met             340 345 350 Pro Arg Asn Leu Arg Glu Tyr Met Val Glu Lys Ile Tyr Pro Gln Ile         355 360 365 Pro Asp His Leu Lys Glu Pro Phe Leu Glu Ala Thr Asp Asn Ser His     370 375 380 Leu Arg Ser Met Pro Ala Ser Phe Leu Pro Pro Ser Ser Val Lys Lys 385 390 395 400 Arg Gly Val Leu Leu Leu Gly Asp Ala Tyr Asn Met Arg His Pro Leu                 405 410 415 Thr Gly Gly Gly Met Thr Val Ala Phe Lys Asp Ile Lys Leu Trp Arg             420 425 430 Lys Leu Leu Lys Gly Ile Pro Asp Leu Tyr Asp Asp Ala Ala Ile Phe         435 440 445 Glu Ala Lys Lys Ser Phe Tyr Trp Ala Arg Lys Thr Ser His Ser Phe     450 455 460 Val Val Asn Ile Leu By Ala Gln Ala Leu By Tyr Glu Leu Phe Ser Ala Thr 465 470 475 480 Asp Asp Ser Leu His Gln Leu Arg Lys Ala Cys Phe Leu Tyr Phe Lys                 485 490 495 Leu Gly Gly Glu Cys Val Ala Gly Pro Val Gly Leu Leu Ser Val Leu             500 505 510 Ser Pro Asn Pro Leu Val Leu Ile Gly His Phe Phe Ala Val Ala Ile         515 520 525 Tyr Ala Val Tyr Phe Cys Phe Lys Ser Glu Pro Trp Ile Thr Lys Pro     530 535 540 Arg Ala Leu Leu Ser Ser Gly Ala Val Leu Tyr Lys Ala Cys Ser Val 545 550 555 560 Ile Phe Pro Leu Ile Tyr Ser Glu Met Lys Tyr Met Val His                 565 570

Claims (14)

A composition for diagnosing colon cancer metastasis diagnosis or prediction of prognosis, which comprises an agent for measuring the level of mRNA of squalene epoxidase gene or a protein thereof.
The method according to claim 1,
Wherein the agent for measuring the level of the protein comprises an antibody specific for the protein or an aptamer.
The method according to claim 1,
Wherein the prognosis is a mortality prognosis.
The method according to claim 1,
Wherein the agent for measuring the level of the mRNA is a primer or a probe that specifically binds to the gene.
The method according to claim 1,
Wherein the composition further comprises an agent for measuring the mRNA level of the p53 gene or the level of the protein expressed therefrom.
A kit for diagnosing colon cancer metastasis or prognosis, comprising the composition of any one of claims 1 to 5.
The method according to claim 6,
Wherein the kit is a reverse transcription polymerase chain reaction kit (RT-PCR), a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, Kits for colorectal metastasis diagnosis or prognosis prediction.
(a) measuring the mRNA level of a squalene epoxidase gene or a protein level thereof in a sample separated from an individual having colon cancer; And
(b) comparing the measured mRNA level or a protein level thereof with a mRNA level or a protein level thereof in a control sample in which one or two colon cancer incidences are provided, for diagnosis of colon cancer metastasis Way.
9. The method of claim 8,
The method further comprises the step of measuring the mRNA level of the p53 gene or the protein level thereof in a sample isolated from the individual in which colon cancer has occurred in step (a).
10. The method according to claim 8 or 9,
The mRNA level of the gene may be determined by RT-PCR, competitive RT-PCR, real-time quantitative RT-PCR, RNase protection assay (RNase protection method, northern blotting, or DNA chip technology assay. &lt; Desc / Clms Page number 19 &gt;
10. The method according to claim 8 or 9,
The level of the protein may be determined by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, Immunohistochemical staining, immunoprecipitation assays, complement fixation assays, immunofluorescence, immunochromatography, FACS (Immunohistochemical staining), immunofluorescence (immunohistochemical staining), immunoprecipitation assays a fluorescence activated cell sorter analysis, and a protein chip technology assay. The present invention also provides a method for diagnosing colon cancer metastasis.
The method according to claim 1,
Wherein the colorectal cancer metastasis is induced by cholesterol.
(a) measuring the mRNA level of a squalene epoxidase gene or a protein level thereof in a sample separated from an individual having colon cancer; And
(b) comparing the measured mRNA level or a protein level thereof with a mRNA level of a control sample that has died from colon cancer or a protein level thereof.
14. The method of claim 13,
The method further comprises the step of measuring the mRNA level of the p53 gene or the protein level thereof in a sample isolated from the individual in which colorectal cancer has occurred in step (a).

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