Tumor metastasis marker, application of protein inhibitor and diagnostic reagent

Abstract

The invention discloses a tumor metastasis marker, application of a protein inhibitor and a diagnostic reagent. The tumor metastasis marker comprises CDC42 protein, and preferably, the CDC42 protein is derived from plasma extracellular vesicles. In addition, the invention also discloses application of the protein inhibitor in preparation of medicines for treating colorectal cancer. According to the technical scheme, the CDC42 protein is found to be obviously higher than normal intestinal cell-derived EVs in CRC-EVs, so that the CDC42 protein can be used as a marker for diagnosing or predicting the metastasis of the colorectal cancer, and has important significance on the treatment of the colorectal cancer.

Description

Technical Field

[0001] The present invention relates to the technical fields of tumor markers and tumor treatment, and particularly to an application of a tumor metastasis marker, a protein inhibitor and a diagnostic reagent. Specifically, it relates to the application of CDC42 as a marker for diagnosing or predicting the metastasis of colorectal cancer, and the application of a CDC42 inhibitor for the treatment of colorectal cancer. Background Art

[0002] Colorectal cancer is one of the common digestive tract malignancies. Among the newly diagnosed cancer types globally, the incidence of colorectal cancer ranks third, with approximately 1.93 million cases, accounting for 10%. Colorectal cancer is also one of the cancer types with a high mortality rate. In 2020, the number of deaths from colorectal cancer was approximately 0.94 million, accounting for 9.4% of the total global cancer deaths, ranking second. According to the development trend of colorectal cancer, it is predicted that the number of new cases will increase to 2.2 million and the number of death cases will reach 1.1 million in 2030, indicating its severity. Tumor metastasis is a key link in tumor evolution and the main cause of death in cancer patients. In colorectal cancer, liver metastasis is the main metastatic site. Clinical data shows that among patients initially diagnosed with colorectal cancer, approximately 20 - 25% of patients are found to have symptoms of liver metastasis; among patients who have undergone radical resection of the primary colorectal cancer focus, the probability of developing liver metastasis within 3 years is as high as 30%. It has been reported in previous studies that the median survival period of patients with colorectal cancer liver metastasis is less than 24 months, and the survival period of patients with untreated colorectal cancer liver metastasis does not exceed 9 - 12 months. All of the above data illustrate that colorectal cancer liver metastasis is one of the important factors affecting the treatment effect and prognosis of patients. Therefore, studying the mechanism of colorectal cancer tumor metastasis is particularly important for the treatment of colorectal cancer.

[0003] Extracellular vesicles (EVs) derived from tumor cells play an important role in tumor metastasis. EVs carry relevant components of tumor cells, transmit information between various cells in the tumor microenvironment, play the function of a "messenger", affect the cells of adjacent tissues and distal tissues, and promote the metastasis of tumor cells.

[0004] Therefore, finding a specific component in extracellular vesicles derived from colorectal cancer cells (CRC-EVs), studying its effect on the metastasis of colorectal cancer, and using it as a marker for diagnosing or predicting liver metastasis of colorectal cancer are of great significance for the treatment of colorectal cancer. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a tumor metastasis marker, the application of a protein inhibitor, and a diagnostic reagent. The present invention uses the CDC42 protein in plasma extracellular vesicles as a marker for diagnosing or predicting colorectal cancer liver metastasis, achieving a more early and accurate diagnosis result of liver metastasis, which is of great significance for the treatment of colorectal cancer.

[0006] In order to achieve the above purpose, the present invention is realized through the following aspects:

[0007] In the first aspect, the present invention proposes an application of a tumor metastasis marker in the preparation of a product for diagnosing colorectal cancer liver metastasis, and the tumor metastasis marker includes the CDC42 protein.

[0008] Preferably, the CDC42 protein is derived from plasma extracellular vesicles. It should be noted that the plasma extracellular vesicles referred to in the present invention are extracellular vesicles derived from plasma.

[0009] It should be pointed out that in the technical solution of the present invention, the CDC42 refers to the cell division cycle 42 protein, which, as a guanine nucleotide enzyme, is a member of the small G protein Rho family (small Rho GTPases), and plays an important role in cell migration and tumor metastasis.

[0010] Through research, the inventors of this case found that the content of the CDC42 protein in CRC-EVs is significantly higher than that in EVs derived from normal intestinal cells. After CRC-EVs enter macrophages, CDC42 is converted into the activated form GTP-CDC42, which mediates the activation of the NOD1 signaling pathway, and then promotes the secretion of downstream inflammatory factors and chemokines such as IL-6, CCL1, and CCL2, forming an inflammatory microenvironment conducive to the metastasis of colorectal cancer cells and promoting the metastasis of colorectal cancer cells. Therefore, CDC42 can be used as a marker for diagnosing or predicting colorectal cancer metastasis, which is of great significance for the prevention and treatment of colorectal cancer liver metastasis.

[0011] And different from the prior art, what the inventors of this case found is the marker in CRC-EVs, rather than tumor tissues. Thus, it is possible to predict the potential for subsequent metastasis (especially liver metastasis) in colorectal cancer patients through extracellular vesicles in peripheral blood, and then judge the possibility of liver metastasis, providing a basis for preventive metastasis treatment. At the same time, metastasis is prevented and treated by targeting the CDC42 small molecule inhibitor ML141.

[0012] It should be noted that, different from other cancers, colorectal cancer mainly forms through three different pathways, namely: the adenoma carcinogenesis pathway, the serrated adenoma carcinogenesis pathway, and the inflammatory carcinogenesis pathway. And colorectal cancer is a type of cancer that is prone to metastasis. The tissues to which it metastasizes are mainly the liver, lungs, and bones. Among them, the most common metastatic organ is the liver, and 50% of colorectal cancer patients will develop liver metastasis during the course of the disease.

[0013] For other cancers, such as gastric cancer, its pathogenesis is mainly related to exogenous factors such as environmental diet or Helicobacter pylori infection.

[0014] Preferably, the preparation is to design a reagent based on the tumor metastasis marker, and the reagent contains biomolecules that specifically hybridize with the tumor metastasis marker.

[0015] Preferably, the biomolecules include one or more selected from primers, probes, and antibodies.

[0016] In a second aspect, the present invention proposes an application of a protein inhibitor in the preparation of a drug for treating colorectal cancer, and the protein inhibitor is an inhibitor that inhibits Rho protein.

[0017] Preferably, the Rho protein is CDC42 protein.

[0018] Preferably, the protein inhibitor is ML141, and the CAS NO of ML141 is: 71203-35-5, and its chemical formula is as follows:

[0019]

[0020] It should be noted that there is currently no experimental evidence that ML141 can be used to inhibit the metastasis of tumor cells. The relevant animal experiments are about treating anxiety and hypertension (see the literature: Competing targets of microRNA-608 affect anxiety and hypertension, vol 23, pg 4569, 2014, Geifman-Shochat.et).

[0021] In a third aspect, the present invention proposes a diagnostic reagent related to colorectal cancer, which includes a detection reagent and a sample to be detected; wherein, the sample to be detected is extracellular vesicles derived from plasma, and the detection reagent is used to detect the content of CDC42 protein in the sample to be detected.

[0022] In a fourth aspect, the present invention proposes a system for detecting tumor metastasis markers, and the system includes:

[0023] A data processing module for calculating the received or input data of tumor metastasis markers; and

[0024] A judgment and output module for judging whether the calculation result meets a preset judgment condition and outputting a prediction result;

[0025] Optionally, in the judgment and output module, when the calculation result meets the judgment condition, the detection result is output as "normal", and when the calculation result does not meet the judgment condition, the detection result is output as "at risk";

[0026] Optionally, the judgment condition is that when the content of CDC42 in the plasma extracellular vesicles of colorectal cancer patients is less than 1.3 times that of normal people's plasma extracellular vesicles, it is judged to meet the condition; when the content of CDC42 in the plasma extracellular vesicles of colorectal cancer patients is greater than or equal to 1.3 times that of normal people's plasma extracellular vesicles, it is judged not to meet the condition;

[0027] In a fifth aspect, the present invention proposes a method for computer-aided detection of tumor metastasis markers, including the following steps:

[0028] (1) Calculate the received or input data of tumor metastasis markers to obtain a calculation result;

[0029] (2) Judge whether the calculation result meets a preset judgment condition and output a detection result;

[0030] Optionally, in step (2), when the calculation result meets the judgment condition, the detection result is output as "normal", and when the calculation result does not meet the judgment condition, the detection result is output as "at risk". The judgment condition is that when the content of CDC42 in the plasma extracellular vesicles of colorectal cancer patients is less than 1.3 times that of normal people's plasma extracellular vesicles, it is judged to meet the condition; when the content of CDC42 in the plasma extracellular vesicles of colorectal cancer patients is greater than or equal to 1.3 times that of normal people's plasma extracellular vesicles, it is judged not to meet the condition; Preferably, the data of the tumor metastasis marker is the ratio of the expression level of CDC42 protein in the plasma extracellular vesicles of colon cancer patients to the expression level in normal people's plasma extracellular vesicles;

[0031] More preferably, the CDC42 protein is derived from plasma extracellular vesicles.

[0032] In a sixth aspect, the present invention proposes a computer-readable storage medium storing a computer program, which when executed by a processor, can implement the functions of the above system or the above method.

[0033] In a seventh aspect, the present invention provides a computer device, which includes a memory and a processor. The memory stores a computer program, and the processor is configured to execute the computer program to implement the steps of the above-described method.

[0034] In an eighth aspect, the present invention provides the use of CDC42 protein as a target for screening drugs for reducing or preventing colorectal cancer or liver metastasis of colorectal cancer. Preferably, the CDC42 protein is derived from plasma extracellular vesicles.

[0035] Based on common general knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0036] The reagents and raw materials used in the present invention are all commercially available.

[0037] Compared with the prior art, the present invention has at least the following advantages and beneficial effects:

[0038] The present invention provides a biomarker for diagnosing or predicting liver metastasis of colorectal cancer, as well as a targeted small molecule inhibitor for the treatment of colorectal cancer, and it has been confirmed in a series of in vitro experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention. Therefore, it should not be regarded as a limitation of the scope. For those of ordinary skill in the art, other corresponding drawings can be obtained based on these drawings without creative efforts.

[0040] Figure 1 The content of CDC42 protein in CRC-EVs is significantly higher than that in EVs derived from normal colon cells; among them, Figure 1 A is the process of extracting EVs from cell culture supernatant by ultracentrifugation; Figure 1 B is to quantitatively analyze the differences in protein components carried by EVs (EV-HT29) derived from colon cancer cell HT29 and EVs (EV-CCD18Co) derived from normal colon cell CCD18Co by mass spectrometry; Figure 1 C is to analyze the expression levels of CDC42 protein in EV-CCD18Co and EV-HT29 by quantitative mass spectrometry; Figure 1 D is to detect the protein level of CDC42 in 10 μg of EV-CCD18Co and 10 μg of EV-HT29 by Western Blotting, and qualitatively identify EVs with the classical marker protein Flotillin 1 of EVs; Figure 1E was to detect the protein levels of CDC42 in CCD18Co, HT29, and MC38 cells by Western Blotting, with β-actin as the internal reference;

[0041] Figure 2 To obtain EVs with low expression of CDC42 protein; among them, Figure 2 A was to construct CDC42 knockdown cell lines sh-CDC42-HT29 and corresponding control cells sh-NC-HT29 using shRNA technology, and detect the protein expression of CDC42 in different cells by Western Blotting; Figure 2 B was to detect the gene expression of CDC42 in different types of cells by qPCR; Figure 2 C was to detect the protein levels of CDC42 in EVs (EV-HT29, EV-sh-CDC42-HT29, EV-sh-NC-HT29) derived from HT29, sh-CDC42-HT29, and sh-NC-HT29 cells by Western Blotting, and qualitatively identify EVs with the classical marker protein Flotillin 1 of EVs;

[0042] Figure 3 It was the flow chart of the experimental protocol for CRC-EVs stimulating macrophages to promote tumor cell metastasis;

[0043] Figure 4 To reduce the content of CDC42 in CRC-EVs and inhibit macrophage activation to promote tumor metastasis; among them, Figure 4 A was the change in the scratch area in the scratch experiment after culturing HT29 cells for 24 hours with different conditioned media (CM), namely CM-Ctrl, CM-EV-sh-NC-HT29, and CM-EV-sh-CDC42-HT29; Figure 4 B was the statistical analysis of the migration rates of cells in each group in the scratch experiment; Figure 4 C was the change in the number of HT29 cells transferred from the upper layer to the lower layer of the Transwell membrane in the Transwell experiment after culturing HT29 cells for 24 hours with different CM, namely CM-Ctrl, CM-EV-sh-NC-HT29, and CM-EV-sh-CDC42-HT29; Figure 4 D was the statistical analysis of the migration rates of cells in each group in the Transwell experiment;

[0044] Figure 5 To inhibit CDC42 in EVs and weaken the promotion of tumor cell metastasis by EV-HT29 stimulating THP-1; Figure 5A are different CMs, namely CM-Ctrl, CM-EV-HT29, and CM-EV-HT29+ML141. After culturing HT29 cells for 24 hours, the change in the scratch area in the scratch assay; Figure 5 B is the statistical analysis of the migration rate of each group of cells in the scratch assay; Figure 5 C are different CMs, namely CM-Ctrl, CM-EV-HT29, and CM-EV-HT29+ML141. After culturing HT29 cells for 24 hours, the change in the number of HT29 cells transferred from the upper layer to the lower layer of the Transwell filter membrane in the Transwell assay; Figure 5 D is the statistical analysis of the migration rate of each group of cells in the Transwell assay;

[0045] Figure 6 The content of CDC42 protein is significantly higher in plasma EVs of patients with colorectal cancer liver metastasis than in plasma EVs of normal people; among them, Figure 6 A is the protocol for extracting and purifying EVs from plasma by size exclusion chromatography; Figure 6 B is the determination of the particle size and its distribution of plasma-derived EVs by nanoparticle tracking analysis; Figure 6 C is the expression of CDC42 protein in plasma EVs (EV-CRC-LM) of patients with colorectal cancer liver metastasis (CRC-LM) and plasma EVs (EV-HD) of normal people (healthy donor, HD); Figure 6 D is the gray scale statistics of the expression level of CDC42 protein in different EV-CRC-LM and EV-HD. The average gray scale of CDC42 protein in different EV-HD is set to 1, and EV-CRC-LM is compared with this average value. Detailed implementation mode

[0046] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as a limitation of the present invention. For those not specified in the embodiments, the techniques or conditions described in the literature in the art or according to the product specifications are followed. Those reagents or instruments not indicated by the manufacturer can be obtained through the market as conventional products.

[0047] Example 1

[0048] Culture the human normal colon cell line CCD18Co and the human colorectal cancer cell line HT29.

[0049] The human normal colon cell line CCD18Co and the human colorectal cancer cell line HT29 are commercially available products, and their culture methods adopt conventional techniques in the art, so they will not be elaborated here.

[0050] After culturing for 24 h, the cell culture supernatant was collected, and according to Figure 1 the experimental procedure shown in A of

[0051] , EVs were extracted by ultracentrifugation. The specific steps were as follows: after taking the cell culture supernatant, centrifuge at 300×g for 5 min, collect the supernatant, then centrifuge at 2,000×g for 15 min, filter with a 0.22 μm filter, centrifuge the filtrate at 100,000×g for 2 h, discard the supernatant, wash the precipitate with PBS, and then centrifuge at 100,000×g for 2 h. Add PBS to resuspend to obtain the required EVs. All centrifugation processes were carried out at 4 °C. Figure 1 The process for determining the target protein can refer to

[0052] B of Figure 1 . Specifically: EVs were lysed with a protein lysis buffer to extract proteins. After measuring the protein concentration with a BCA kit, each sample was labeled with TMT. The spectra of tandem mass spectrometry were separated in high-performance liquid chromatography-mass spectrometry (HPLC), and compared with the theoretical spectra in the protein database UniprotKB using an automatic data alignment program to obtain peptide sequences and perform protein identification, and obtain the relative content of proteins in different types of EVs. Through mass spectrometry data analysis and combined with existing literature reports, it was found that there were significant differences in the CDC42 proteins carried by EV-HT29 and EV-CCD18Co. Figure 1 D of

[0053] As Figure 1 shown in C of

[0054] , in the quantitative proteomics, the protein content of CDC42 in EV-HT29 was 1.6 times that in EV-CCD18Co, with a significant difference statistically (p < 0.001). Figure 1 Using BCA to measure the protein concentration after EVs lysis, Western Blotting was used to detect the expression of CDC42 in EV-HT29 and EV-CCD18Co with the same protein content (10 μg), and the classical marker protein Flotillin 1 of EVs was used for qualitative identification of EVs. The results were as Figure 1 shown in D of

[0055] The protein levels of CDC42 in CCD18Co, HT29, and MC38 cells were further detected, with β-actin as the internal reference. MC38 cells were commercially available products. We found that the expression level of CDC42 in CCD18Co was decreased compared with HT29 and MC38 (see Figure 1 E). It can be seen that CDC42 is highly expressed in both colorectal cancer cells and EVs secreted by colorectal cancer cells.

[0056] Example 2

[0057] Using the shRNA technique, stable cell lines sh-CDC4-HT29 with knocked-down CDC42 and the corresponding empty vector (negative control, NC) were constructed to establish a control cell line sh-NC-HT29, and EVs with low expression of CDC42 protein were obtained. The specific results are shown in Figure 2 , where, as Figure 2 A in Figure 2 shows the construction of the cell line sh-CDC42-HT29 with knocked-down CDC42 and the corresponding control cell sh-NC-HT29 using the shRNA technique, and the protein expression of CDC42 in different cells was detected by Western Blotting. Blank is HT29 cells without transfection treatment; the experimental results showed that the protein expression level of CDC42 was significantly decreased in sh-CDC4-HT29 cells; Figure 1 B in Figure 2 shows the detection of the gene expression of CDC42 in sh-CDC42-HT29 and sh-NC-HT29 cells by qPCR. Blank is HT29 cells without transfection treatment. The experimental results showed that the gene expression level of CDC42 was significantly decreased in sh-CDC4-HT29 cells; further, using the method in

[0058] Example 3

[0059] Figure 3 It is a flow chart of the experimental protocol for CRC-EVs stimulating macrophages to promote tumor cell metastasis. According to Figure 3The experimental protocol shown is as follows: CRC-EVs are diluted in serum-free medium, and then the diluted CRC-EVs (20 μg / mL) are added to differentiated and mature macrophages 1, and co-incubated with the macrophages for 24 hours. After 24 hours, the cell culture supernatant is collected and centrifuged at 1,000×g for 5 minutes to remove detached cells. Subsequently, the macrophage culture supernatant and the complete medium of colorectal cancer cells are prepared as conditional medium 2 (conditional medium, CM) in a ratio of 1:1, and scratch assay and Transwell assay are performed respectively.

[0060] For the scratch assay, colorectal cancer cells 4 are seeded in a 6-well plate and cultured in serum-free medium for 24 hours. "Scratches" are made in each well, and the medium is replaced with 2, and the culture is continued. The healing ability of the "scratches" of colorectal cancer cells under the action of the conditional medium is observed under a microscope. Through the scratch assay, it is analyzed whether the inflammatory microenvironment formed after CRC-EVs stimulate macrophages 1 promotes the healing ability of the "scratches" of colorectal cancer cells 4.

[0061] For the Transwell assay, colorectal cancer cells 4 are cultured in serum-free medium 3 for 24 hours, then seeded in a Transwell chamber, and serum-free basal medium 3 is added thereto. At the same time, 2 is added to the lower chamber of the Transwell device. The number of colorectal cancer cells 4 transferred from the upper layer to the lower layer of the filter membrane of the Transwell chamber is observed and counted by the Transwell assay. Through the Transwell assay, it is analyzed whether the inflammatory microenvironment formed after CRC-EVs stimulate macrophages promotes the migration of colorectal cancer cells.

[0062] In this example, stable cell lines sh-CDC4-HT29 with low expression of CDC42 protein are constructed using shRNA technology, and control cell lines sh-NC-HT29 are established using empty vectors (negative control). EVs secreted by the cells are extracted to obtain EV-sh-CDC42-HT29 with low expression of CDC42 and the corresponding control EV-sh-NC-HT29. 20 μg / mL of EV-sh-NC-HT29 or EV-sh-CDC42-HT29 are respectively added to differentiated and mature THP-1 cells and co-incubated with the cells for 24 hours. The culture supernatant of THP-1 cells is collected and mixed with the medium of HT29 cells in a ratio of 1:1 to prepare conditional medium CM, which are: CM-Ctrl (blank control, without EVs stimulation), CM-EV-sh-NC-HT29, and CM-EV-sh-CDC42-HT29. The obtained CM is according to Figure 3The experimental protocol shown in the figure was co-incubated with colorectal cancer cells HT29 for 24 hours, and scratch assay and Transwell migration assay were performed.

[0063] The final results are shown in Figure 4 .

[0064] The results of the scratch assay are shown in Figure 4 Figure A of Figure 4 . Compared with the CM-Ctrl treatment group, when HT29 cells were cultured with the conditioned medium (CM-EV-sh-NC-HT29) prepared from the supernatant collected after THP-1 cells were stimulated with EV-sh-NC-HT29 for 24 hours, the "scratch" healing ability of HT29 cells could be significantly promoted (see Figure A of Figure 4 for details). However, in the CM-EV-sh-CDC42-HT29 treatment group, the "scratch" healing ability of HT29 cells was relatively low and there was no significant change compared with the CM-Ctrl group (see Figure A of Figure 4 for details). By statistically analyzing the migration rate of HT29 cells in the scratch assay, it was found that compared with the CM-EV-sh-NC-HT29 treatment group, the "scratch" healing ability of HT29 cells was significantly decreased in the CM-EV-sh-CDC42-HT29 treatment group, with a statistical difference (p < 0.05) (see Figure B of Figure 4 for details). This indicates that reducing the content of CDC42 in EV-HT29 can effectively reduce the promoting effect of the microenvironment formed by macrophages on the "scratch" healing ability of HT29 cells.

[0065] The results of the Transwell assay are shown in Figure 4 Figure C of Figure 4 . Compared with the CM-Ctrl group, the CM-EV-sh-NC-HT29 treatment group could significantly promote the transfer of HT29 cells from the upper layer to the lower layer of the Transwell chamber filter membrane (see Figure C of Figure 4 for details). While compared with the CM-Ctrl group, the number of HT29 cells transferred from the upper layer to the lower layer of the Transwell chamber filter membrane was not significantly affected in the CM-EV-sh-CDC42-HT29 treatment group (see Figure C of Figure 4 for details). By statistically analyzing the migration rate of HT29 cells in the Transwell assay, it was found that compared with the CM-EV-sh-NC-HT29 treatment group, the migration ability of HT29 cells was significantly decreased in the CM-EV-sh-CDC42-HT29 treatment group, with a statistical difference (p < 0.05) (see Figure D of Figure 4 for details). This indicates that reducing the content of CDC42 in EV-HT29 can effectively reduce the promoting effect of the microenvironment formed by macrophages on the migration ability of HT29 cells.

[0066] Example 4

[0067] Mix 20 μg / mL EV-HT29 with the CDC42 inhibitor ML141 (20 μM) and incubate at 37 °C for 1 hour, or directly incubate EV-HT29 at 37 °C for 1 hour. Then, add them to differentiated and mature THP-1 cells and let them act for 24 hours. After that, collect the culture supernatant of THP-1 cells and prepare the conditioned medium CM by mixing it with the medium for culturing HT29 cells at a ratio of 1:1, obtaining 3 different CMs, namely: CM-Ctrl (blank control without EVs stimulation), CM-EV-HT29, and CM-EV-HT29+ML141. According to Figure 3 the experimental protocol shown, co-incubate the obtained CM with HT29 cells for 24 hours, and perform the scratch assay and Transwell migration assay.

[0068] The results are shown in Figure 5 :

[0069] The results of the scratch assay are as shown in Figure 5 A of. Compared with the CM-Ctrl treatment group, the healing ability of the "scratch" of HT29 cells in the CM-EV-HT29 treatment group was significantly enhanced. At the same time, compared with the CM-EV-HT29 treatment group, the healing ability of the "scratch" of HT29 cells in the CM-EV-HT29+ML141 treatment group was significantly decreased. By statistically analyzing the migration rate of HT29 cells in the scratch assay, it was found that compared with the CM-EV-HT29 treatment group, the healing ability of the "scratch" of HT29 cells was significantly decreased in the CM-EV-HT29+ML141 treatment group, with a statistical difference (p<0.05) (see Figure 5 B of for details). This indicates that inhibiting CDC42 in CRC-EVs can effectively reduce the promoting effect of the microenvironment formed by macrophages on the healing ability of the "scratch" of colorectal cancer cells.

[0070] The results of the Transwell assay are as shown in Figure 5As shown in C of [reference], compared with the CM-Ctrl treatment group, the CM-EV-HT29 treatment group could significantly promote the transfer of HT29 cells from the upper layer to the lower layer of the Transwell chamber filter membrane. Moreover, compared with the CM-EV-HT29 treatment group, the promoting effect of the CM-EV-HT29+ML141 treatment group on the transfer of HT29 cells from the upper layer to the lower layer of the Transwell chamber filter membrane was significantly reduced. By statistically analyzing the migration rate of HT29 cells in the Transwell experiment, it was found that compared with the CM-EV-HT29 treatment group, the migration ability of HT29 cells was significantly decreased in the CM-EV-HT29+ML141 treatment group, with a statistical difference (p<0.05) (see Figure 5 D of [reference]). This indicates that inhibiting CDC42 in CRC-EVs can effectively reduce the promoting effect of the microenvironment formed by macrophages on the migration ability of colorectal cancer cells.

[0071] Example 5

[0072] EVs were extracted from plasma samples of healthy donors (HD) and patients with colorectal liver metastasis (CRC-LM) using size exclusion chromatography. The specific experimental protocol is as Figure 6 shown in A: First, the collected plasma was centrifuged at 6,000×g for 15 minutes, and then the supernatant was filtered through a 0.22μm filter to remove residual cells and large particulate matter in the plasma. Then, the filtered plasma was added to a size exclusion chromatography column, and 0.5 mL of PBS was added each time for elution and the eluate was collected. The collected eluates were numbered in sequence, and the eluates numbered #7 to #12 (a total of 3 mL) were combined. Finally, it was concentrated in an ultrafiltration concentration device and centrifuged at 3,000×g for 4 - 10 minutes to obtain a solution containing EVs of approximately 500 μL.

[0073] The particle size in the EVs solution extracted from plasma of different sources was measured using a nanoparticle tracking analyzer. The experimental results showed that the particle sizes of EVs derived from plasma of normal people and CRC-LM patients separated by the SEC method were mainly distributed in the range of 50 - 200 nm and concentrated around 100 nm (see Figure 6 B of [reference]).

[0074] The content of CDC42 in 10 μg of EVs derived from HD plasma and CRC-LM patient plasma was detected by Western Blotting. The results are as Figure 6C display showed that in the detection of plasma EVs from normal individuals (n = 7) (EVs-HD) and plasma EVs from CRC-LM patients (n = 7) (EVs-P), we found that the content of CDC42 in EVs-P was generally higher than that in EVs-HD. The gray value statistics of the expression level of CDC42 protein in different EV-CRC-LM and EV-HD were performed using ImageJ software, and it was found that the protein level of CDC42 in EVs derived from the plasma of CRC-LM patients was 1.32 times that in EVs derived from the plasma of normal individuals (see specifically Figure 6 D), which was consistent with the data of Figure 1 C in the proteomics, indicating that the protein level of CDC42 increased in patients with liver metastasis of colorectal cancer.

[0075] Example 6

[0076] In this example, a system for detecting tumor metastasis markers is provided. The system includes: a data processing module for calculating the data of the received or input tumor metastasis markers; and a judgment and output module for judging whether the calculation result meets the preset judgment conditions and outputting the detection result.

[0077] The specific operation of the system is as follows:

[0078] (1) Taking CDC42 as the tumor metastasis marker, inputting the sample data. Among them, the sample data can be derived from the content of CDC42 protein detected in plasma EVs. Preferably, CDC42 is derived from extracellular vesicles of plasma. Of course, the sample data can also be data from the cloud platform database in some other embodiments.

[0079] (2) After performing data calculation on the sample data, obtaining the calculation result.

[0080] (3) The judgment and output module outputs the detection result for the calculation result. The detection result includes "normal" or "at risk". When the calculation result meets the judgment conditions, the output detection result is "normal", and when the calculation result does not meet the judgment conditions, the output detection result is "at risk".

[0081] Whether the conditions are met can be set according to the specific situation of each embodiment. In this embodiment, the judgment condition is that when the content of CDC42 in plasma EVs of colorectal cancer patients is less than 1.3 times that in plasma EVs of normal individuals, it is judged to meet the conditions, and the output detection result is "normal"; when the content of CDC42 in plasma EVs of colorectal cancer patients is greater than or equal to 1.3 times that in plasma EVs of normal individuals, it is judged to not meet the conditions, and the output detection result is "at risk".

[0082] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that this is only an example, and the protection scope of the present invention is defined by the appended claims. Without departing from the principle and essence of the present invention, those skilled in the art can make various changes or modifications to these embodiments, but these changes and modifications all fall within the protection scope of the present invention.

Claims

1. Use of a tumor metastasis marker in the preparation of a product for diagnosing liver metastasis of colorectal cancer, characterized in that, The tumor metastasis marker includes CDC42 protein.

2. The application according to claim 1, wherein The CDC42 protein is derived from plasma extracellular vesicles.

3. The application according to claim 1, characterized in that, The preparation is to design a reagent based on the tumor metastasis marker, and the reagent contains biomolecules that specifically hybridize with the tumor metastasis marker; Preferably, the biomolecules include one or more selected from primers, probes, and antibodies.

4. Use of a protein inhibitor in the preparation of a drug for treating colorectal cancer, characterized in that, The protein inhibitor is an inhibitor that inhibits Rho protein; preferably, the Rho protein is CDC42 protein.

5. The application according to claim 4, wherein The protein inhibitor is ML141.

6. A diagnostic reagent related to colorectal cancer, characterized in that, It includes a detection reagent and a sample to be detected; wherein, the sample to be detected is plasma-derived extracellular vesicles, and the detection reagent is used to detect the content of CDC42 protein in the sample to be detected.

7. A system for detecting tumor metastasis markers, characterized in that, The system includes: A data processing module for calculating the data of the tumor metastasis marker received or input; and A judgment and output module for judging whether the calculation result meets a preset judgment condition and outputting a detection result; Optionally, in the judgment and output module, when the calculation result meets the judgment condition, the detection result output is "normal", and when the calculation result does not meet the judgment condition, the detection result output is "at risk"; Optionally, the judgment condition is that when the CDC42 content in the plasma extracellular vesicles of colorectal cancer patients is less than 1.3 times that in the plasma extracellular vesicles of normal people, it is judged to meet the condition, and the detection result output is "normal"; when the CDC42 content in the plasma extracellular vesicles of colorectal cancer patients is greater than or equal to 1.3 times that in the plasma extracellular vesicles of normal people, it is judged to not meet the condition, and the detection result output is "at risk"; Preferably, the tumor metastasis marker is CDC42 protein; More preferably, the CDC42 protein is derived from plasma extracellular vesicles.

8. A computer-aided method for detecting tumor metastasis markers, characterized in that, It includes the following steps: (1) Calculate the data of the tumor metastasis marker received or input to obtain a calculation result; (2) Judge whether the calculation result meets a preset judgment condition and output a detection result; Optionally, in step (2), when the calculation result meets the judgment condition, the detection result output is "normal", and when the calculation result does not meet the judgment condition, the detection result output is "at risk", and the judgment condition is that when the CDC42 content in the plasma extracellular vesicles of colorectal cancer patients is less than 1.3 times that in the plasma extracellular vesicles of normal people, it is judged to meet the condition; when the CDC42 content in the plasma extracellular vesicles of colorectal cancer patients is greater than or equal to 1.3 times that in the plasma extracellular vesicles of normal people, it is judged to not meet the condition; Preferably, the tumor metastasis marker is CDC42 protein; More preferably, the CDC42 protein is derived from plasma extracellular vesicles.

9. A computer-readable storage medium storing a computer program, characterized in that, When the computer program is executed by a processor, it can implement the functions of the system as claimed in claim 7, or implement the method as claimed in claim 8.

10. A computer device, which includes a memory and a processor, wherein the memory stores a computer program, and is characterized in that, The processor is used to execute the computer program to implement the steps of the method as claimed in claim 8.

11. Use of CDC42 protein as a target in screening drugs for reducing or preventing colorectal cancer or metastasis of colorectal cancer; Preferably, the CDC42 protein is derived from plasma extracellular vesicles.

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