CN113785200B - Marker composition based on exosomes overexpressing TUBA1C protein - Google Patents
Marker composition based on exosomes overexpressing TUBA1C protein Download PDFInfo
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Abstract
根据本发明的一个实施方案，提供了一种用于诊断癌症或预测预后的标志物组合物，其包含过表达微管蛋白α‑1C链(TUBA1C)蛋白的外泌体，一种试剂盒，以及一种用于提供诊断癌症或预测预后所需信息的方法，其包括测量从生物样品分离的外泌体中TUBA1C基因或蛋白的表达水平的步骤。
According to one embodiment of the present invention, there is provided a marker composition for diagnosing cancer or predicting prognosis, comprising exosomes overexpressing tubulin alpha-1C chain (TUBA1C) protein, a kit, and a method for providing information required for diagnosing cancer or predicting prognosis, comprising the step of measuring the expression level of the TUBA1C gene or protein in exosomes isolated from a biological sample.
Description
Technical Field
The present invention relates to marker compositions comprising exosomes overexpressing alpha-Tubulin-specific 1C chain (Tubulin alpha-1C chain, tuba 1C) proteins for diagnosis of cancer or prognosis prediction.
Background
Tumors (tumor) are the products of uncontrolled and disordered cell proliferation caused by abnormal cell excess, and when such tumors proliferate, spread and metastasize destructively, they will be classified as malignant (MALIGNANT TUMOR), i.e., cancer.
Currently, cancer is diagnosed by methods such as X-ray, endoscopy, pathological examination, and the like. Although the inspection process of the above inspection method is relatively simple, the diagnosis success rate is not high, there is also a sanitary problem, and the patient feels pain during the inspection process, and thus, it is required to develop a cancer diagnosis method that replaces the above method.
In order to treat cancer, a highly sensitive and specific cancer diagnosis is required prior to treatment. It is only by such diagnosis that cancer is found early that the cure rate can be improved.
Therefore, there is a need to develop a method capable of diagnosing cancer at an early stage with high sensitivity and specificity noninvasively, but there is still a lack of molecular diagnostic techniques capable of detecting lesions specifically at an early stage to determine whether the disease is developed or not, and particularly a method specifically applicable to a specific cancer has not yet been developed.
Prior patent document
Patent literature
Korean patent No. 10-2080887
Summary of the invention
Technical problem to be solved
Under such circumstances, the present inventors have continued studies to develop a novel marker for diagnosing cancer or predicting prognosis derived from exosomes, and as a result, have confirmed that when TUBA1C (Tubulin alpha-1C chain) protein specifically expressed in exosomes derived from cancer cells is used, cancer can be accurately and rapidly diagnosed or predicted prognosis, thereby completing the present invention.
It is an object of the present invention to provide a marker composition capable of improving the accuracy of cancer diagnosis by a non-invasive method, the composition comprising an exosome based on over-expression of the TUBA1C protein, and a method for providing information required for diagnosing cancer or predicting prognosis using the composition.
However, the problems to be solved by the present invention are not limited to the above-described technical problems, and other technical problems, which are not mentioned, can be clearly understood by those skilled in the art through the following description.
Technical method for solving the problems
According to one embodiment of the present invention, a marker composition for diagnosing cancer or predicting prognosis is provided, comprising an exosome that overexpresses TUBA1C (Tubulin alpha-1C chain) protein.
According to one aspect, the exosomes further comprise GCC2 (GRIP and coiled-coil domain-containing protein) protein.
According to one aspect, the cancer is lung cancer, thymus cancer or esophageal cancer.
According to another embodiment of the present invention, there is provided a composition for diagnosing cancer or predicting prognosis, comprising a primer or probe that specifically binds to an in-exo-vivo TUBA1C protein, and any one or more of antibodies that specifically bind to an in-exo-vivo TUBA1C protein.
According to one aspect, the composition further comprises a primer or probe that specifically binds to the in-exosome GCC2 gene, and any one or more of antibodies that specifically bind to the in-exosome GCC2 protein.
According to one aspect, the cancer is lung cancer, thymus cancer or esophageal cancer.
According to yet another embodiment of the present invention, a kit for diagnosing cancer or predicting prognosis comprising the composition is provided.
According to one aspect, the kit is one or more selected from the group consisting of an RT-PCR kit, a microarray chip kit, a DNA kit and a protein chip kit.
According to yet another embodiment of the present invention, there is provided a method for providing information required for diagnosing cancer or predicting prognosis, including the step of measuring the expression level of TUBA1C gene or protein in an exosome isolated from a biological sample.
According to one aspect, the method further comprises the step of measuring the expression level of the GCC2 gene or protein in said exosome.
According to one aspect, the biological sample is one or more selected from the group consisting of whole blood, serum, plasma, saliva, urine, sputum, lymph fluid, and cells.
ADVANTAGEOUS EFFECTS OF INVENTION
The marker compositions of the invention include TUBA1C protein that is overexpressed in the exosomes of cancer patients, enabling a non-invasive accurate diagnosis of cancer or prediction prognosis by measuring its expression level.
In addition, the marker composition of the invention takes TUBA1C and GCC2 which are over expressed in exosomes as dual biomarkers, so that the sensitivity and accuracy of cancer diagnosis can be further improved.
However, the effects to be attained by the present invention are not limited to the above-described effects, but include all effects that can be derived based on the description of the embodiments of the present invention or the claims.
Drawings
FIG. 1 shows ELISA results comparing the expression levels of GCC2 and TUBA1C proteins in blood-derived exosomes of lung cancer patients with those of the normal group.
FIG. 2 shows ELISA results comparing the expression level of TUBA1C protein in plasma derived exosomes of esophageal cancer patients with normal control.
FIG. 3 shows ELISA results comparing the expression level of TUBA1C protein in the plasma-derived exosomes of patients with thymus cancer with that of the normal control group.
FIG. 4 shows ELISA results of measurement of TUBA1C and GCC2 expression levels in plasma-derived exosomes of lung cancer patients according to the number of phases.
Fig. 5 is a ROC curve confirming changes in lung cancer diagnostic sensitivity when TUBA1C and GCC2 are used alone and when both markers are used.
Detailed Description
The embodiments are described in detail below with reference to the accompanying drawings. Like reference symbols in the various drawings indicate like elements.
Various modifications can be made to the embodiments described below. The implementation forms of the present invention are not limited to the following embodiments, but are intended to include all modifications of the following embodiments, their equivalents, and their substitutes.
The terminology used in the description of the embodiments is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of protection. Where not specifically stated in the context, singular expressions include plural meanings. In this specification, the terms "comprises" and "comprising" are used to specify the presence of stated features, integers, steps, operations, elements, components, or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or groups thereof.
All terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art without other definitions. Terms commonly used as dictionary defined should be understood as meaning in the related art, and should not be interpreted as idealized or excessively formalized meaning without being explicitly defined in the specification.
In the description with reference to the drawings, the same reference numerals are used for the same components irrespective of the reference numerals, and duplicate descriptions are omitted. In describing the embodiments, when it is judged that detailed description of the related art will unnecessarily obscure the embodiments, detailed description thereof will be omitted.
According to one embodiment of the present invention, a marker composition is provided that includes an exosome that overexpresses the TUBA1C (Tubulin alpha-1C chain) protein for diagnosis of cancer or prognosis prediction. Furthermore, the exosomes may also include GRIP and coiled-coil domain 2 (GRIP and coiled-coil domain-containing protein, GCC 2) proteins.
The terms "an exosome overexpressing the TUBA1C protein" and "an exosome overexpressing the GCC2 protein" as used in this specification refer to an exosome capable of achieving higher levels of GCC2 or TUBA1C protein expression than an exosome present in a normal cell.
Exosomes (exosomes) are small vesicles of nanometric size (30-150 nm) secreted by most cells. It is known that various proteins derived from cells, genetic material (DNA, mRNA, miRNA), lipids, and the like are included in the inside of the exosomes and in the phospholipid bilayer membrane. Furthermore, the tissue-derived exosomes can reflect the state of the tissue from which they are secreted, and can therefore be used to diagnose diseases.
Thus, the present inventors have found that cancer can be accurately and rapidly diagnosed or prognosis can be predicted by using TUBA1C or GCC2 protein specifically expressed in the exosomes of cancer patients, and thus completed the present invention.
The cancers herein include all cancers, non-limiting examples of which include lung cancer, esophageal cancer, thymus cancer, breast cancer, liver cancer, stomach cancer, rectal 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, with lung cancer, esophageal cancer or thymus cancer being preferred.
The term "diagnosis" as used in this specification refers to the confirmation of the presence or character of a pathological condition, i.e. the confirmation of the presence or character of a cancer. Also, "prognosis" refers to the determination of whether or not a corresponding individual has developed relapse, metastasis, drug response, resistance, etc. after cancer treatment. I.e., by measuring the TUBA1C or GCC2 expression levels in the exosomes isolated from a sample of the individual, thereby determining whether the respective individual has cancer and predicting whether the prognosis of the respective individual is good after survival.
Thus, by measuring the expression level of the TUBA1C or GCC2 protein derived from exosomes to be able to diagnose cancer or predict prognosis, primers or probes specifically binding to the gene, or antibodies specifically binding to the protein, can be used in a composition for diagnosing cancer or predicting prognosis.
Furthermore, the present invention can provide a kit for diagnosing cancer or predicting prognosis by adapting any one or more of a primer, a probe, or an antibody specifically binding to a TUBA1C or GCC2 gene, or an antibody specifically binding to a TUBA1C or GCC2 protein to a kit.
Non-limiting examples of such kits may include RT-PCR kits, microarray chip kits, DNA kits, protein chip kits, and the like. The kit can confirm and detect the expression level of TUBA1C, GCC2 protein corresponding to a marker in the exosome, thereby diagnosing cancer or predicting prognosis.
For diagnosing cancer or predicting prognosis, the kit includes primers, probes or antibodies that selectively recognize markers, and further includes compositions, solutions or devices suitable for use in one or more other components of the analytical method.
For example, for immunological detection of antibodies, the kit may comprise a substrate, a suitable buffer solution, a secondary antibody labeled with chromogenic enzyme or fluorescent substance, a chromogenic substrate, and the like. Further, as the substrate, a nitrocellulose membrane, a 96-well plate synthesized from polyethylene resin, a 96-well plate synthesized from polystyrene resin, and a glass slide, a peroxidase (peroxidase), an alkaline phosphatase (alkaline phosphatase) or the like may be used as the chromogenic enzyme, a Fluorescein Isothiocyanate (FITC), rhodamine Isothiocyanate (RITC) or the like may be used as the fluorescent substance, and ABTS (2, 2-diaza-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) or OPD (o-phenylenediamine), TMB (tetramethylbenzidine) may be used as the chromogenic substrate, but not limited thereto.
According to another embodiment of the present invention, there is provided a method for providing information required for diagnosing cancer or predicting cancer, including the step of measuring the expression level of TUBA1C gene or protein in exosomes (exosomes) isolated from a biological sample.
In order to increase the sensitivity and accuracy of diagnosing cancer, the method of the present invention may further comprise the step of measuring the expression level of GCC2 gene or protein in the exosome.
The biological sample may be one or more selected from the group consisting of whole blood, serum, plasma, saliva, urine, sputum, lymph fluid, and cells, and preferably may be whole blood or cells, but is not limited thereto.
The measurement of the gene expression level is a process of confirming the presence or absence of mRNA of the TUBA1C, GCC2 gene and the degree of expression thereof from a biological sample for the purpose of diagnosing cancer or predicting prognosis, i.e., measuring the expression amount of mRNA.
Non-limiting examples of analytical methods used for this purpose include reverse transcription-polymerase chain reaction (RT-PCR), competitive RT-PCR (Competitive RT-PCR), real-time RT-PCR (Real-time RT-PCR) and RNase Protection Assay (RPA), northern blotting (Northern blotting), DNA chips, etc.
And, measuring the expression level of the protein is a process of confirming whether the TUBA1C, GCC2 protein and the expression level thereof are present in the biological sample for diagnosis of cancer or prediction prognosis.
Non-limiting examples of the method for measuring the expression level of the protein or the comparative analysis method include a protein chip analysis method, an immunoassay method, a ligand binding analysis method, a Matrix assisted laser Desorption-ionization time of flight (MALDI-TOF) analysis, a surface enhanced laser Desorption-ionization time of flight (Sulface ENHANCED LASER Desorption/Ionization Time of FLIGHT MASS Spectrometry, SELDI-TOF) analysis, a radioimmunoassay, an Oldham immunodiffusion method, rocket immunoelectrophoresis, an immunohistochemical staining method, a complement fixation method, a two-dimensional electrophoresis analysis, a liquid chromatography-mass Spectrometry (liquid chromatography-Mass Spectrometry, LC-MS), a liquid chromatography-mass Spectrometry/mass Spectrometry (liquid chromatography-Mass Spectrometry/Mass Spectrometry, LC-MS/MS), a Western ink dot method, an enzyme-linked immunosorbent assay (ELISA), and the like.
After measuring the expression level of the gene or protein of TUBA1C, GCC, the expression level is compared with the normal control group, and when the expression level is higher than that of the normal control group, the cancer onset or onset possibility can be judged to be higher.
Also, according to an embodiment of the present invention, there is provided a cancer therapeutic screening method comprising the steps of (a) applying a cancer therapeutic candidate substance to a collected biological sample of a cancer patient, (b) isolating exosomes (exosomes) from the biological sample, and (C) measuring the expression level of the TUBA1C gene or protein in the exosomes.
Extending the methods of providing information necessary to diagnose cancer or predict prognosis may be used as screening for such therapeutic candidate substances. That is, after a cancer therapeutic candidate substance is applied to a biological sample isolated from a cancer patient, when it is confirmed that the expression level of the TUBA1C gene or protein in the exosomes present therein is reduced, it is confirmed that the corresponding candidate substance has an effective function as a cancer therapeutic.
The present invention will be described in more detail with reference to examples. The following examples are intended to illustrate the invention, but the scope of the invention is not limited thereto.
Example 1 preparation of isolation of exosomes and analysis of proteomes
The 5 kinds of cancer cell lines (H522, A549, H1650, PC9, H1299), thymus cancer cell line and esophageal cancer cell line were each cultured in a culture dish having a diameter of 150 mm. At this time, the supernatant from which the exosomes had been removed of fetal bovine serum (FBS, fetal Bovine Serum) was used as a culture broth by centrifugation at 120000g for 4 hours using a super-high speed centrifuge. And (3) continuously culturing for 2-3 days by using the culture solution until the cell fusion degree reaches 70-80%.
The resulting culture broth was centrifuged at 10000g for 30 minutes to remove cell debris (cell debris), and then passed through a 0.45 μm and 0.22 μm filter in order to preferentially remove substances having a larger volume. Thereafter, for the filtered cell culture fluid, only particles of a desired size were retained by Amicon tube 100K (Amicon tube 100K) (Millipore Co., U.S.A.), and then concentrated.
Then, the concentrated cell culture broth was subjected to column liquid chromatography (column liquid chromatography) to separate only exosome-sized (50-100 nm) particles, and was concentrated again using Amicon tube 100K.
Protein was obtained from the concentrated exosomes using RIPA lysate (RIPA LYSIS buffer, thermo FISHER SCIENTIFIC, usa), entrusted to Korean Basic Science Institute (KBSI) to obtain proteome analysis results.
Based on this, TUBA1C (Tubulin alpha-1C chain) and GCC2 (GRIP and coiled-coil domain-containing protein 2) which were overexpressed in exosomes of the cancer cell line were finally selected.
Example 2 measurement of expression level of GCC2 and TUBA1C protein in exosomes of Lung cancer patients
To confirm whether the exosomes comprising GCC2 and TUBA1C proteins screened in example 1 were viable as markers for diagnosing lung cancer or predicting prognosis, GCC2 and TUBA1C expression levels in exosomes extracted from blood of the normal group (n=3) and the lung cancer patient group (n=5) were analyzed using ELISA (Enzyme linked immunoassay).
As a result, as shown in fig. 1, the expression of GCC2 and TUBA1C was increased in the lung cancer patient group compared to the normal group (control).
Thereafter, in order to confirm the characteristics of the exosomes isolated from the plasma of lung cancer patients, 20 lung cancer patients at stage 1 to stage 3 were collected from the hospital, and exosomes were isolated from the plasma using Exoquick (Systembio company, usa). Expression levels of GCC2 and TUBA1C derived from the isolated blood exosomes were confirmed by ELISA (GCC 2: mybiosourc Co. GRIP and coiled-coil domain containing proten; ELISA KIT (Cat No. MBS 933067), 2) TUBA1C: mybiosource Co. TUBA1C ELISA KIT (Cat No. MBS 9336377)). As a result, as shown in fig. 4, the expression levels of GCC2 and TUBA1C increased significantly in all lung cancer stage numbers, and the expression levels of GCC2 and TUBA1C increased together with the increase in lung cancer stage numbers, as compared with the normal group.
Example 3 measurement of TUBA1C expression levels in the exosomes of patients with esophageal cancer and thymus cancer
First, exosomes were extracted from plasma (plasma) samples of normal 5 humans and esophageal cancer patients 5 humans, and then the TUBA1C protein concentration in the samples was confirmed by TUBA1C ELISA KIT (TUBA 1C ELISA KIT (Cat No. MBS 9336377) from Mybiosource).
As a result, as shown in FIG. 2, the average concentration of TUBA1C protein derived from the exosome of normal persons was 939.306ng/ml, whereas the average concentration of TUBA1C protein derived from the exosome of patients with esophageal cancer was 1236.764ng/ml higher. The concentration of TUBA1C protein from the exosome of the patients with esophageal cancer is increased by 1.36 times compared with that of normal people, and the p value (value p value) is 0.021, which is statistically significant.
Then, exosomes were extracted from plasma (plasma) samples of normal 5 humans and thymus cancer patient 5 humans, and then the TUBA1C protein concentration in the samples was confirmed by TUBA1C ELISA KIT (TUBA 1C ELISA KIT (Cat No. MBS 9336377) from Mybiosource).
As a result, as shown in FIG. 3, the average concentration of TUBA1C protein derived from the exosome of normal persons was 939.306ng/ml, whereas the average concentration of TUBA1C protein derived from the exosome of patients with esophageal cancer was extremely high, 3503.15ng/ml. The concentration of TUBA1C protein of the exosome source of the esophageal cancer patients is increased by 3.85 times compared with that of normal patients, and the p value is 0.005, which is statistically significant.
Example 4 evaluation of diagnostic effectiveness Using TUBA1C and GCC2 as Dual biomarkers
In order to evaluate the effectiveness of TUBA1C and GCC 2as dual biomarkers in diagnosing cancer, the diagnosis sensitivity change in the case of TUBA1C and GCC2 used alone and in the case of using both markers at the same time was confirmed by ROC curve. Specifically, after extracting exosomes from plasma (plasma) of 7 normal persons and 21 lung cancer patients, the concentrations of GCC2 and TUBA1C proteins in the exosomes were obtained by GCC2ELISA KIT and TUBA1C ELISA KIT, and AUC values were confirmed by using a statistically ROC curve and are shown in fig. 5.
Referring to fig. 5, AUC was 0.905 (p=0.002) when GCC2 antibody alone and 0.8787 (p=0.003) when TUBA1C antibody alone. In contrast, when the antibodies of GCC2 and TUBA1C are used simultaneously, AUC is 1 (P0.0000963), and diagnosis can be performed more precisely when TUBA1C and GCC2 are used as dual biomarkers than when TUBA1C or GCC2 are used alone.
In summary, the embodiments are described with limited figures, and a person skilled in the art can make various technical modifications and variations based on the description. For example, the described techniques may be performed in a different order than the described methods, and/or the described components may be combined or combined in a different manner than the described methods, or substituted or replaced with other components or equivalents, as appropriate.
Accordingly, other implementations, other embodiments, and equivalents of the claims are within the scope of the following claims.
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