KR102350603B1 - Composition for diagnosing or prognosing cancer including exosome based tuba1c proteins - Google Patents

Abstract

According to an embodiment of the present invention, there is provided a marker composition for diagnosing cancer or predicting a prognosis, comprising an exosome overexpressing TUBA1C (Tubulin alpha-1C chain) protein.

Description

Exosome-based cancer diagnosis or prognosis prediction marker composition overexpressing TUBA1C protein {COMPOSITION FOR DIAGNOSING OR PROGNOSING CANCER INCLUDING EXOSOME BASED TUBA1C PROTEINS}

The present invention relates to a marker composition for diagnosing cancer or predicting prognosis, comprising exosomes overexpressing TUBA1C (Tubulin alpha-1C chain) protein.

A tumor is a product of uncontrolled and disordered cell proliferation caused by an excess of abnormal cells, and if such a tumor has destructive proliferation, invasion and metastasis, it is classified as a malignant tumor, that is, cancer. .

Currently, examination means for diagnosing cancer include methods using X-ray imaging, endoscopy, and biopsy. However, in spite of the advantage that the examination process is relatively simple, these methods do not have a high diagnosis success rate, or there is a problem in sanitation and suffering of the patient in the course of the examination. I need this.

In order to treat cancer, it will be important to diagnose cancer with high sensitivity and specificity at the stage prior to treatment.

Therefore, there is a need for the development of a non-invasive, high-sensitivity and high-specification method capable of diagnosing cancer at an early stage. The technology is scarce, and moreover, there is no method specifically applied to a specific cancer.

Republic of Korea Patent Registration No. 10-2080887

Under this background, the present inventors continued research to develop a novel marker for diagnosis or prognosis of exosome-derived cancer. As a result, TUBA1C (Tubulin alpha-1C chain) protein specifically expressed in cancer cell-derived exosomes. The present invention has been completed by confirming that it is possible to accurately and quickly diagnose cancer or predict a prognosis when using

Accordingly, it is an object of the present invention as a marker composition that can improve the accuracy of cancer diagnosis while being used as a non-invasive method. Or to provide a method for providing information necessary for prognosis prediction.

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

According to an embodiment of the present invention, there is provided a marker composition for diagnosing cancer or predicting a prognosis, comprising an exosome overexpressing TUBA1C (Tubulin alpha-1C chain) protein.

According to one side, the exosome may further include a GRIP and coiled-coil domain-containing protein (GCC2) protein.

According to one side, the cancer may be lung cancer, thymus cancer or esophageal cancer.

According to another embodiment of the present invention, a primer or probe that specifically binds to the TUBA1C gene in the exosome; and an antibody that specifically binds to the TUBA1C protein in the exosome; A composition for diagnosing cancer or predicting a prognosis is provided, including any one or more.

According to one side, the composition, a primer or probe that specifically binds to the GCC2 gene in the exosome; and an antibody that specifically binds to the GCC2 protein in the exosome; Any one or more may be further included.

According to one side, the cancer may be lung cancer, thymus cancer or esophageal cancer.

According to another embodiment of the present invention, there is provided a kit for diagnosing or predicting cancer, including the composition.

According to one side, the kit may be at least one selected from the group consisting of an RT-PCR kit, a microarray chip kit, a DNA kit, and a protein chip kit.

According to another embodiment of the present invention, a method of providing information necessary for diagnosis or prognosis of cancer, comprising measuring the expression level of the TUBA1C gene or protein in the exosome isolated from the biological sample this is provided

According to one side, the step of measuring the expression level of the GCC2 gene or protein in the exosome may further comprise.

According to one side, the biological sample may be one or more selected from the group consisting of whole blood, serum, plasma, saliva, urine, sputum, lymph and cells.

Since the marker composition of the present invention includes TUBA1C protein that is specifically and highly expressed in exosomes of cancer patients, it is possible to non-invasively and highly accurately diagnose cancer or predict the prognosis by measuring its expression level.

In addition, the marker composition of the present invention can further improve the sensitivity and accuracy of cancer diagnosis by using TUBA1C and GCC2 overexpressed in exosomes as dual biomarkers.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

1 is an ELISA result comparing the expression levels of GCC2 and TUBA1C proteins from exosomes derived from the blood of a lung cancer patient and a normal group.
2 is an ELISA result comparing the expression level of TUBA1C protein in plasma-derived exosomes of esophageal cancer patients with that of a normal control group.
3 is an ELISA result comparing the expression level of TUBA1C protein in plasma-derived exosomes of a thymic cancer patient with a normal control group.
4 is an ELISA result obtained by dividing the expression levels of TUBA1C and GCC2 proteins in the blood-derived exosomes of lung cancer patients by radix.
5 is an ROC curve confirming the change in the sensitivity of diagnosis of lung cancer when the two markers are used in combination, compared to when TUBA1C and GCC2 are used alone.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. Like reference numerals in each figure indicate like elements.

Various modifications may be made to the embodiments described below. It should be understood that the embodiments described below are not intended to limit the embodiments, and include all modifications, equivalents, and substitutes thereto.

Terms used in the examples are used only to describe specific examples, and are not intended to limit the examples. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present specification, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It should be understood that this does not preclude the possibility of the presence or addition of numbers, steps, operations, components, parts, or combinations thereof.

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

In addition, in the description with reference to the accompanying drawings, the same components are given the same reference numerals regardless of the reference numerals, and the overlapping description thereof will be omitted. In describing the embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.

According to an embodiment of the present invention, there is provided a marker composition for diagnosing cancer or predicting a prognosis, comprising an exosome overexpressing TUBA1C (Tubulin alpha-1C chain) protein. In addition, the exosome may further include a GRIP and coiled-coil domain-containing protein (GCC2) protein.

As used herein, the terms "TUBA1C protein overexpressing exosome" and "GCC2 protein overexpressing exosome" refer to exosomes expressing GCC2 or TUBA1C protein at a higher level than exosomes present in normal cells.

Exosomes are nano-sized (30-150 nm) small endoplasmic reticulum secreted by most cells. It is known that various types of proteins, genetic materials (DNA, mRNA, miRNA), lipids, etc. derived from cells are contained in the exosome inner and phospholipid double membranes. In addition, it has been reported that tissue-derived exosomes can be used for diagnosis of diseases because they reflect the state of the tissue that secreted them.

Accordingly, the present inventors have completed the present invention by confirming that when using TUBA1C or GCC2 protein specifically expressed in exosomes of cancer patients, it is possible to accurately and quickly diagnose cancer or predict the prognosis.

Here, cancer includes all cancers, and includes lung cancer, esophageal cancer, thymus cancer, breast cancer, liver cancer, stomach cancer, colorectal cancer, pancreatic cancer, cervical cancer, skin cancer, prostate cancer, ovarian cancer, thyroid cancer, bladder cancer, head and neck cancer, bone marrow cancer, biliary tract cancer Cancer may be exemplified, but is not limited thereto, and preferably lung cancer, esophageal cancer or thymus cancer.

As used herein, the term “diagnosis” refers to determining the presence or characteristics of a pathological condition, ie, whether or not cancer has developed. In addition, "prognosis" refers to determining whether the subject relapses, metastases, drug reactivity, or resistance after cancer treatment. This may include the concept of predicting whether or not the subject has a good survival prognosis in the future as well as whether the subject has cancer by measuring the expression level of TUBA1C or GCC2 in the exosomes isolated from the sample of the subject.

As such, it is possible to diagnose cancer or predict the prognosis by measuring the expression level of TUBA1C or GCC2 protein derived from exosomes, so a primer or probe that specifically binds to its gene, or an antibody that specifically binds to a protein It can be used as a composition for cancer diagnosis or prognosis prediction.

In addition, the present invention provides a kit for diagnosing cancer or predicting prognosis to which any one or more of a primer, a probe, and an antibody that specifically binds to a TUBA1C or GCC2 gene is applied. .

The kit may include, but is not limited to, an RT-PCR kit, a microarray chip kit, a DNA kit, a protein chip kit, and the like. The kit can determine the expression level in the exosome of TUBA1C, GCC2 gene or protein corresponding to the marker and detect it to diagnose lung cancer or predict the prognosis.

The kit may include one or more other component compositions, solutions, or devices suitable for analysis methods, in addition to primers, probes, or antibodies that selectively recognize markers for diagnosis or prognosis of cancer.

For example, the kit may include a substrate, a suitable buffer, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, and a chromogenic substrate for immunological detection of the antibody. In addition, as the substrate, a nitrocellulose membrane, a 96-well plate synthesized from a polyvinyl resin, a 96-well plate synthesized from a polystyrene resin, and a glass slide glass may be used, and the chromogenic enzyme is peroxidase, alkaline Phosphatase (alkaline phosphatase), etc. may be used, FITC, RITC, etc. may be used as a fluorescent material, and ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-) sulfonic acid)) or OPD (O-phenylenediamine), or TMB (tetramethyl benzidine), but is not limited thereto.

According to another embodiment of the present invention, a method of providing information necessary for diagnosis or prognosis of cancer, comprising measuring the expression level of the TUBA1C gene or protein in the exosome isolated from the biological sample this is provided

The method of the present invention may further include measuring the expression level of the GCC2 gene or protein in the exosome in order to improve the sensitivity and accuracy of cancer diagnosis.

The biological sample may be at least one selected from the group consisting of whole blood, serum, plasma, saliva, urine, sputum, lymph and cells, and preferably whole blood or cells, but is not limited thereto.

The gene expression level measurement is a process of determining the presence and expression level of mRNA of TUBA1C and GCC2 genes from a biological sample for diagnosis or prognosis of cancer, and means measuring the mRNA expression level.

Analysis methods for this include reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection) assay), Northern blotting, and a DNA chip, but is not limited thereto.

In addition, the protein expression level measurement refers to a process of confirming the presence and expression level of TUBA1C and GCC2 proteins from a biological sample for cancer diagnosis or prognosis prediction.

The protein expression level measurement or comparative analysis method includes protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) 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, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (liquid chromatography-Mass Spectrometry, LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry), Western blotting, and ELISA (enzyme-linked immunosorbent assay), but are not limited thereto.

After measuring the expression level of the TUBA1C, GCC2 gene or protein, when the expression level is higher than that of the normal control compared to the normal control, it can be determined that cancer has occurred or the incidence is high.

In addition, according to an embodiment of the present invention, (a) processing a cancer drug candidate in a biological sample collected from a cancer patient; (b) separating the exosomes from the biological sample; And (c) measuring the expression level of the TUBA1C gene or protein in the exosome; A method for screening a cancer therapeutic agent is provided, comprising a.

As an extension of the method for providing information necessary for cancer diagnosis or prognosis prediction, the screening for therapeutic candidates may be applied. That is, when a cancer treatment candidate is treated with a biological sample isolated from a cancer patient and the expression level of the TUBA1C gene or protein is decreased in the exosomes present therein, the candidate material effectively functions as a cancer treatment. can check that

Hereinafter, the present invention will be described in more detail through examples. The following examples are described for the purpose of illustrating the present invention, but the scope of the present invention is not limited thereto.

Example One: exosomes separation and proteomic Assay Preparation

Five lung cancer cell lines (H522, A549, H1650, PC9, H1299), thymic cancer cell lines, and esophageal cancer cell lines were each cultured in a 150 mm diameter dish. At this time, the supernatant of FBS (Fetal Bovine Serum) from which exosomes were removed by centrifugation at 120,000 g for 4 hours using an ultra-high speed centrifuge was used as a culture medium. Using the culture medium, cells were continuously cultured for 2-3 days to reach 70-80% confluency.

The obtained culture medium was centrifuged at 10,000 g for 30 minutes to remove cell debris, and passed sequentially through 0.45 μm and 0.22 μm filters to preferentially remove relatively bulky substances. Thereafter, the filtered cell culture solution was concentrated using an Amicon tube 100K (Millipore, USA), leaving only particles of the desired size.

Next, only the particles of the exosome size (50-100 nm) were separated from the concentrated cell culture using the column liquid chromatography method, and concentrated again using an Amicon tube 100K.

Proteins were obtained from the concentrated exosomes using RIPA lysis buffer (Thermo Fisher Scientific, USA), and the result of proteomic analysis was obtained by requesting the Korea Basic Science Institute (KBSI).

Based on this, TUBA1C (Tubulin alpha-1C chain) and GCC2 (GRIP and coiled-coil domain-containing protein 2), which are overexpressed in exosomes of cancer cell lines, were finally selected.

Example 2: Lung cancer patients exosomes my GCC2 and TUBA1C Expression level measurement

In order to confirm whether the exosomes containing the GCC2 and TUBA1C proteins selected in Example 1 can be used as a marker for diagnosis or prognosis of lung cancer, blood of the normal group (n = 3) and the lung cancer patient group (n = 5) Expression levels of GCC2 and TUBA1C from exosomes extracted from ELISA (Enzyme linked immunoassay) were confirmed through analysis.

As a result, as shown in Figure 1, it was confirmed that the expression of GCC2 and TUBA1C increased in the lung cancer patient group compared to the normal group (control).

Next, in order to confirm the characteristics of exosomes isolated from the plasma of lung cancer patients, blood was collected from 20 patients with stage 1 to 3 lung cancer visiting the hospital, and exo from plasma using Exoquick (Systembio, USA) moth was isolated. Expression levels of GCC2 and TUBA1C derived from isolated blood-derived exosomes were confirmed through ELISA analysis (GCC2: Mybiosource's GRIP and coiled-coil domain containing proten 2 ELISA KIT (Cat No. MBS9330667), 2) TUBA1C: Mybiosource TUBA1C ELISA KIT (Cat No. MBS9336377) from the company. As a result, as shown in FIG. 4 , it was confirmed that the expression levels of GCC2 and TUBA1C significantly increased in all stages of lung cancer compared to the normal group, and the expression levels of GCC2 and TUBA1C also increased as the stage of lung cancer increased.

Example 3: Esophageal cancer and thymus cancer patient's exosomes my TUBA1C Expression level measurement

First, exosomes were extracted from plasma samples of 5 normal people and 5 esophageal cancer patients, and then the TUBA1C protein concentration in the sample was confirmed using the TUBA1C ELISA KIT (TUBA1C ELISA KIT (Cat No. MBS9336377, Mybiosource)).

As a result, as shown in FIG. 2 , the average concentration of exosome-derived TUBA1C protein in normal individuals was 939.306 ng/ml, whereas in esophageal cancer patients, the average concentration of exosome-derived TUBA1C protein was high as 1236.764 ng/ml. . The concentration of exosome-derived TUBA1C protein in esophageal cancer patients was increased 1.36 times compared to that of normal people, and the p value was 0.021, confirming that it was statistically significant.

Next, after extracting the exosomes from the plasma samples of 5 normal people and 5 thymic cancer patients, the TUBA1C protein concentration in the sample was checked using the TUBA1C ELISA KIT (Mybiosource's TUBA1C ELISA KIT (Cat No. MBS9336377)). did

As a result, as shown in FIG. 3 , the average concentration of exosome-derived TUBA1C protein in normal individuals was 909.306 ng/ml, whereas in esophageal cancer patients, the average concentration of exosome-derived TUBA1C protein was very high as 3503.15 ng/ml. became The concentration of exosome-derived TUBA1C protein in patients with esophageal cancer was increased 3.85 times compared to that of normal people, and the p value was 0.005, confirming that it was statistically significant.

Example 4: TUBA1C and GCC2's double as a biomarker Diagnostic usefulness evaluation

In order to evaluate the usefulness of using TUBA1C and GCC2 as dual biomarkers for cancer diagnosis, changes in diagnostic sensitivity when TUBA1C and GCC2 are used alone or when both markers are used in combination were confirmed with an ROC curve. Specifically, after extracting exosomes from the plasma of 7 normal people and 21 lung cancer patients, the concentrations of GCC2 and TUBA1C proteins in the exosomes were obtained using GCC2 ELISA KIT and TUBA1C ELISA KIT, and then statistically using the ROC curve. The AUC value was confirmed, and it is shown in FIG. 5 .

Referring to FIG. 5 , when using the GCC2 antibody alone, the AUC value was 0.905 (p = 0.002), and when the TUBA1C antibody was used alone, the AUC value was 0.8787 (p = 0.003). On the other hand, when GCC2 and TUBA1C antibodies were treated simultaneously, AUC was confirmed to be 1 (P 0.0000963), indicating that more precise diagnosis is possible when TUBA1C and GCC2 are used as dual biomarkers than when TUBA1C or GCC2 is used alone. could check

As described above, although the embodiments have been described with reference to the limited embodiments and drawings, various modifications and variations are possible from the above description by those skilled in the art. For example, even if the described techniques are performed in an order different from the described method, and/or the described components are combined or combined in a different form from the described method, or replaced or substituted by other components or equivalents Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (11)

A marker composition for cancer diagnosis or prognosis prediction, comprising an exosome overexpressing TUBA1C (Tubulin alpha-1C chain) protein and GCC2 (GRIP and coiled-coil domain-containing protein) protein.
delete The method of claim 1,
The cancer is lung cancer, thymus cancer, or esophageal cancer, a marker composition for diagnosing cancer or predicting prognosis.
a primer or probe that specifically binds to the TUBA1C gene in the exosome; and an antibody that specifically binds to the TUBA1C protein in the exosome; any one or more of and
a primer or probe that specifically binds to the GCC2 gene in the exosome; and an antibody that specifically binds to the GCC2 protein in the exosome; A composition for diagnosing cancer or predicting a prognosis, comprising any one or more.
delete 5. The method of claim 4,
The cancer is lung cancer, thymus cancer or esophageal cancer, cancer diagnosis or prognosis prediction composition.
A kit for diagnosing cancer or predicting a prognosis, comprising the composition of claim 4 .
8. The method of claim 7,
The kit is at least one selected from the group consisting of an RT-PCR kit, a microarray chip kit, a DNA kit, and a protein chip kit, a kit for diagnosing cancer or predicting a prognosis.
Measuring the expression level of the TUBA1C gene or protein in the exosome isolated from the biological sample; And measuring the expression level of the GCC2 gene or protein in the exosome; A method of providing information necessary for diagnosis or prognosis of cancer, including a.
delete 10. The method of claim 9,
The biological sample is at least one selected from the group consisting of whole blood, serum, plasma, saliva, urine, sputum, lymph and cells, a method of providing information necessary for diagnosis or prognosis of cancer.

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