Colorectal cancer treatment target and serum marker and application thereof

Abstract

The invention provides a colorectal cancer treatment target, a colorectal cancer diagnosis serum marker and application of the colorectal cancer treatment target and the colorectal cancer diagnosis serum marker. The screened drug target capable of being used for precise treatment of colorectal cancer and the screened biomarker capable of being used for early screening are one or more of the following proteins: ADH1B, ARRDC1, ANGPTL2, GATM, GTF2H4, MGME1 or LILRB2. A new thought is provided for clinical diagnosis and treatment of colorectal cancer, and improvement of prognosis of patients with colorectal cancer is facilitated. The diagnosis serum marker shows high sensitivity, specificity, accuracy and diagnosis efficiency, the effect of the diagnosis serum marker is superior to that of traditional serum markers CEA and CA19-9, and early screening and diagnosis of colorectal cancer and precancerous lesions are promoted.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of September 5, 2023, application number 2023111344685, and invention name “A colorectal cancer therapeutic target and serum marker and its application”. Technical Field

[0002] The present invention relates to the field of medical biotechnology, and in particular to a colorectal cancer treatment target and diagnostic serum marker and applications thereof. Background Art

[0003] Although the diagnosis and treatment of colorectal cancer have improved significantly compared to the past, the overall survival rate is still not ideal, and the 5-year survival rate of patients with advanced colorectal cancer is less than 20%, which has brought serious medical and economic burdens to patients' families and society. When diagnosed based on clinical symptoms such as abdominal discomfort, changes in bowel habits, or blood in the stool, colorectal cancer has often progressed to an irreversible stage. Therefore, early screening for colorectal cancer and precancerous lesions and precision treatment of specific targets are of great significance to improving patient prognosis and reducing the social medical burden.

[0004] There are various methods for early screening of colorectal cancer, including colonoscopy, fecal occult blood test, serological tumor markers, etc. The ideal screening method should have high compliance, high sensitivity and specificity, be minimally invasive and cost-effective. Colonoscopy combined with endoscopic tissue biopsy is the gold standard for diagnosing colorectal cancer, but because it is an invasive examination, there is a certain risk of bleeding and perforation, which requires endoscopists to have rich experience in endoscopic operation, limiting the popularization and promotion of colonoscopy in clinical screening. Fecal occult blood test is recommended as one of the non-invasive screening methods for colorectal cancer. It is non-invasive, inexpensive and easy to be accepted by patients. However, due to its limitations such as low sensitivity and specificity, the need for repeated screening, and the presence of many false positives, patients' compliance with fecal occult blood test is greatly reduced. Serological screening is a non-invasive screening method generally accepted by the public, and is widely used in gastroenterology outpatient clinics, physical examination centers, etc. Commonly used serological screening indicators for gastrointestinal diseases include CEA, CA19-9, etc. These commonly used serological screening indicators have been used clinically for nearly decades and have been unanimously recognized by clinical physicians and patients. Currently, there is still no new serum screening indicator that can surpass them. However, in actual clinical work, it can be observed that some patients with colorectal cancer and precancerous lesions do not have abnormal increases in the above indicators, that is, the sensitivity is limited. Therefore, a new, non-invasive, low-cost, highly sensitive and specific diagnostic serum marker for colorectal cancer is urgently needed in the clinic to predict colorectal cancer or precancerous lesions and achieve early diagnosis and precise treatment. Summary of the invention

[0005] The purpose of the present invention is to provide a colorectal cancer treatment target and diagnostic serum marker and its application, so as to solve the technical problems of limited sensitivity and low specificity of commonly used serum screening indicators in the prior art.

[0006] To solve the above technical problems, the present invention provides a colorectal cancer therapeutic target and diagnostic serum marker, which is one or more selected from the following proteins: ADH1B (alcohol dehydrogenase), ARRDC1 (capture domain-containing protein 1), ANGPTL2 (angiopoietin-like protein 2), GATM (glycine amidinotransferase), GTF2H4 (general transcription factor IIH subunit 4), MGME1 (mitochondrial genome maintenance exonuclease 1) and LILRB2 (leukocyte immunoglobulin-like receptor B2).

[0007] Preferably, the protein that is the therapeutic target and diagnostic serum marker is ANGPTL2 protein.

[0008] Preferably, the protein that is the therapeutic target and diagnostic serum marker is LILRB2 protein.

[0009] The present invention also provides an application of a colorectal cancer therapeutic target and a diagnostic serum marker, characterized in that ADH1B, ARRDC1, ANGPTL2, GATM, GTF2H4, MGME1 and / or LILRB2 proteins are used in drugs for screening, diagnosing and treating colorectal cancer pathological conditions.

[0010] The pathological conditions of colorectal cancer include colorectal cancer, polyps, and adenomas.

[0011] Furthermore, the protein screening tools include proteomics technology based on ultra-high performance liquid chromatography-mass spectrometry, bioinformatics analysis, immunohistochemical staining and enzyme-linked immunosorbent assay.

[0012] Preferably, immunohistochemical staining is used to quantitatively detect the expression of ANGPTL2 and LILRB2 proteins.

[0013] Preferably, the serum concentration of LILRB2 protein is quantitatively detected by enzyme-linked immunosorbent assay.

[0014] Furthermore, the ANGPTL2 protein is upregulated in colorectal cancer tissues, which is 4.55 times the protein amount in normal control tissues.

[0015] Furthermore, the LILRB2 protein was upregulated in colorectal tissue, which was 5.5 times the protein amount in normal control tissue.

[0016] Furthermore, the application of the serum concentration of the LILRB2 protein in the early screening of colorectal cancer: when the serum concentration of LILRB2 is ≥4557pg / ml, it indicates the possible presence of colorectal precancerous lesions such as adenoma ≥1cm, villous or tubulovillous adenoma, severe atypical hyperplasia, high-grade intraepithelial neoplasia, etc.; when the serum concentration of LILRB2 is ≥5256pg / ml, it indicates that it may have progressed to colorectal cancer.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects:

[0018] The present invention provides a colorectal cancer therapeutic target and diagnostic serum marker and its application, on the one hand, to provide more options for the future treatment of colorectal cancer. ANGPTL2 protein and LILRB2 protein can be used as precise therapeutic targets to improve the treatment effect of colorectal cancer and improve drug resistance; on the other hand, compared with traditional serum markers, LILRB2 protein serum concentration has high sensitivity and specificity in screening colorectal cancer and precancerous lesions, and has good diagnostic efficacy. Under the currently advocated hierarchical diagnosis and treatment model, it is more conducive to the early screening and early diagnosis of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1a is the overall survival curve of ADH1B protein;

[0021] Figure 1b is the overall survival curve of ARRDC1 protein;

[0022] Figure 1c is the overall survival curve of GATM protein;

[0023] Figure 1d is the overall survival curve of GTF2H4 protein;

[0024] Figure 1e is the overall survival curve of MGME1 protein;

[0025] Figure 1f is the overall survival curve of LILRB2 protein;

[0026] Figure 1g is the overall survival curve of ANGPTL2 protein;

[0027] Figure 2 is the expression level of LILRB2 protein;

[0028] Figure 3a for the correlation between LILRB2 protein expression and nerve involvement;

[0029] Figure 3b for the correlation between LILRB2 protein expression and vascular involvement;

[0030] Figure 3c is the correlation between LILRB2 protein expression and differentiation degree;

[0031] Figure 3d is the correlation between LILRB2 protein expression and invasion depth;

[0032] Figure 3e is the correlation between LILRB2 protein expression and lymph node metastasis;

[0033] Figure 3f for the correlation between LILRB2 protein expression and distant metastasis;

[0034] Figure 3g The correlation between LILRB2 protein expression and TNM stage;

[0035] Figure 4 is the expression level of ANGPTL2 protein;

[0036] Figure 5a for the correlation between ANGPTL2 protein expression and tumor size;

[0037] Figure 5b for the correlation between ANGPTL2 protein expression and vascular involvement;

[0038] Figure 5c is the correlation between ANGPTL2 protein expression and differentiation degree;

[0039] Figure 5d is the correlation between ANGPTL2 protein expression and invasion depth;

[0040] Figure 5e for the correlation between ANGPTL2 protein expression and lymph node metastasis;

[0041] Figure 5f for the correlation between ANGPTL2 protein expression and distant metastasis;

[0042] Figure 5g The correlation between ANGPTL2 protein expression and TNM stage;

[0043] Figure 6is the serum concentration of LILRB2 protein in healthy controls, colorectal adenoma, and colorectal cancer patients;

[0044] Figure 7 To determine the diagnostic performance of serum LILRB2 protein concentration in the diagnosis of advanced adenomas (precursor lesions of colorectal cancer);

[0045] Figure 8a To compare the sensitivity, specificity and accuracy of serum concentration of LILRB2 protein with traditional serum markers CEA and CA19-9 in diagnosing colorectal cancer;

[0046] Figure 8b This is a comparison of the receiver operating characteristic curve and area under the curve between the serum concentration of LILRB2 protein and the traditional serum markers CEA and CA19-9 in the diagnosis of colorectal cancer. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] The present invention is further explained below in conjunction with specific implementation modes.

[0049] Example 1

[0050] like Figure 1a-1g As shown, candidate therapeutic targets and diagnostic serum markers for colorectal cancer were screened out. The specific experimental steps are as follows:

[0051] Colorectal cancer tissue and paired para-cancer tissue samples were collected, and 30 μL of solvent A (A: 0.1% formic acid aqueous solution) was added to each sample to make a suspension. 9 μL was taken out and added with 1 μL of 10×iRT peptides. After mixing, it was separated by nano-LC and analyzed by online electrospray tandem mass spectrometry. The whole system was an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific, MA, USA) connected to EASY-nano-LC 1200. A total of 4 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm), and the sample was separated by a gradient of 90 min. The column flow was controlled at 250 nL / min, the column temperature was 55 °C, the gradient started from 4% B phase, equilibrated for 3 minutes, increased to 50% by nonlinear gradient at 82 minutes, increased to 95% within 1 minute, and maintained for 7 minutes. The mass spectrometer was operated in data-independent acquisition mode, and the mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 350–1200; resolution: 120,000; Normalized AGC target: 250%; maximum injection time: 100 ms. (2) HCD-MS / MS: resolution: 50,000; Normalized AGC target: 200%; maximum injection time: 86 ms; collision energy: 33. (3) Variable window acquisition, 60 windows were set, overlapping serial ports were set, and each window overlapped 1 m / z. The data were analyzed using Spectronaut15.0 default parameters (BGS Factory Settings (default)). The sequence database was the uniprot-homo sapiens (version 201907, 20428 entries) database. Among all the identified proteins, the screening criteria for differentially expressed proteins were: proteins with a P value of <0.05 and an absolute value of the difference fold >2 after t-test analysis.

[0052] The CPTAC protein database was used for survival analysis to screen for proteins with differential prognosis in colorectal cancer. The protein quantitative detection values, clinical outcomes, overall survival, and progression-free survival data of colorectal cancer patients in the CPTAC database were downloaded, and the patients were divided into survival group and death group according to clinical outcomes. The R language code was used to identify prognosis-related proteins, and the differentially expressed proteins were intersected with the prognosis-related proteins to obtain proteins with differential prognosis in colorectal cancer. The R language code is as follows:

[0053]

[0054]

[0055]

[0056] Based on the above-mentioned ultra-performance liquid chromatography-mass spectrometry-coupled proteomics technology and bioinformatics analysis, candidate therapeutic targets and diagnostic serum markers for colorectal cancer were screened out, selected from one or more of the following proteins: ADH1B (alcohol dehydrogenase), ARRDC1 (capture domain-containing protein 1), ANGPTL2 (angiopoietin-like protein 2), GATM (glycine amidinotransferase), GTF2H4 (general transcription factor IIH subunit 4), MGME1 (mitochondrial genome maintenance exonuclease 1) and LILRB2 (leukocyte immunoglobulin-like receptor B2).

[0057] Example 2

[0058] like Figure 2 , Figure 3a-3g , Figure 4 , Figure 5a-5g As shown, immunohistochemical staining was used to quantitatively detect ANGPTL2 protein and LILRB2 protein. The specific steps are as follows:

[0059] 1) Preparation of paraffin sections and dewaxing and hydration: Collect colorectal cancer tissue and paired adjacent cancer tissue, fix the tissue in 10% formalin solution for 3-4 hours, then place the tissue in an embedding box for dehydration, and sequentially enter 75% alcohol for 1.5 hours, 95% alcohol for 1.5 hours, 95% alcohol for 1 hour, anhydrous ethanol for 1.5 hours, anhydrous ethanol for 1 hour, and xylene for 1 hour. Turn on the embedding machine, turn on the cold stage and tissue slot to work, put the dehydrated tissue together with the embedding box into the machine tissue slot, drip liquid paraffin, and then put it on the cold stage to cool. After fixation, dehydration, wax immersion and embedding, the tissue wax block is prepared. Turn on the slicer, adjust the slice thickness, and cut into 4μm paraffin slices for use. Before staining, the paraffin sections need to be dewaxed and hydrated. Place the sections in xylene for 15 minutes - xylene for 15 minutes - xylene for 15 minutes - anhydrous ethanol for 15 minutes - 90% ethanol for 5 minutes - 80% ethanol for 5 minutes - 70% ethanol for 5 minutes - distilled water for 3 minutes * 3 times.

[0060] 2) Heat antigen repair: Place the tissue sections in a repair box filled with Tris-EDTA antigen repair buffer (pH 9.0) for antigen repair, heat and boil in a microwave or pot, control the temperature at 96-98°C, keep warm for 15 minutes, and finally cool naturally at room temperature. During this process, excessive evaporation of the buffer should be prevented and the slides should not be dried. After natural cooling, place the slides in a phosphate buffered saline solution (PBS) at pH 7.4 and shake and wash on a decolorizing shaker 3 times, 5 minutes each time.

[0061] 3) Blocking endogenous peroxidase: Place the sections in a 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 minutes. Place the slides in PBS and wash them three times on a decolorizing shaker, each time for 5 minutes.

[0062] 4) Serum blocking of non-specific sites: Add 3% bovine serum albumin in the tissue circle to evenly cover the tissue, block at room temperature for 30 minutes to block non-specific antigen-antibody reactions.

[0063] 5) Add primary antibody: shake off the blocking solution, dry the surface water droplets with absorbent paper, add 20μl of primary antibody on the slice (the volume ratio of LILRB2 antibody to PBS solution is 1:50 or the volume ratio of ANGPTL2 antibody to PBS solution is 1:200), and lay the slice flat in an immunohistochemical humidified box and incubate overnight at 4℃. Use PBS solution for antibody dilution.

[0064] 6) Add secondary antibody: Rewarm at room temperature for 30 minutes, place the slide in PBS and wash on a decolorizing shaker for 3 times, 5 minutes each time. Dried the slices, blot the surface water droplets with absorbent paper, then add HRP-labeled anti-rabbit secondary antibody in the circle to cover the tissue, and incubate at room temperature for 50 minutes.

[0065] 7) Color development: Place the slide in PBS and shake it on a decolorizing shaker for 3 times, 5 minutes each time, and use absorbent paper to dry the surface water droplets. 10mM Tris-HCl (pH7.6) 9mL + 3-diaminobenzidine + 0.3% (W / V) NiCl2 or CoCl2 1mL, add 5-10mL 30% H2O2 and mix well during color development, and 0.05% DAB color development solution is ready. Add 100μL of freshly prepared DAB color development solution in the circle, control the color development under a microscope for about 3 minutes, the positive color is brown-yellow, and rinse the slices with tap water to terminate the color development reaction.

[0066] 8) Re-staining of cell nuclei: Re-stain with hematoxylin for 3 minutes, wash with tap water, differentiate with 1% hydrochloric acid alcohol for 3 seconds, rinse with tap water, blue with 0.5% ammonia water, and rinse with running water.

[0067] 9) Dehydration and sealing: Put the slices into 75% alcohol for 5 min-85% alcohol for 5 min-anhydrous ethanol for 5 min-anhydrous ethanol for 5 min-n-butanol for 5 min-xylene for 5 min to dehydrate and make them transparent. Take the slices out of xylene and air dry for half a day, then seal the slices with sealing glue.

[0068] 10) Observation and analysis under an optical microscope.

[0069] The experimental results showed that both proteins were significantly overexpressed in colorectal cancer tissues compared with normal control tissues. The expression of LILRB2 protein was 5.5 times that of normal control tissues, and was significantly correlated with colorectal cancer poor differentiation (P=0.03), vascular involvement (P<0.001), nerve involvement (P=0.002), lymph node metastasis (P<0.001), distant metastasis (P=0.009), later clinical stage (P<0.001) and poor prognosis (P=0.045); while the expression of ANGPTL2 protein was 4.55 times that of normal control tissues, and was significantly correlated with colorectal cancer size (P=0.013), poor differentiation (P=0.003), vascular involvement (P=0.002), lymph node metastasis (P<0.001), distant metastasis (P=0.001), later clinical stage (P<0.001) and poor prognosis (P=0.004). It has been verified that both can serve as potential precision treatment targets for colorectal cancer.

[0070] Example 3

[0071] like Figure 6 , Figure 7 , Figure 8a and Figure 8b As shown, the serum concentration of LILRB2 protein was quantitatively detected by enzyme-linked immunosorbent assay, and the operation was performed according to the instructions of the Human LILRB2 ELISA Kit. The specific steps are as follows:

[0072] 1) Prepare all reagents: human CD85d capture antibody 10X 600μL, human CD85d detection antibody 10X 600μL, 2 bottles of human CD85d lyophilized recombinant protein, antibody diluent CPI 6mL, sample diluent 12mL, wash buffer PT 10X 20mL, TMB substrate solution 12mL, stop solution 12mL, 96-well microplate and sealing film.

[0073] 2) Sample addition: prepare blank wells and standard wells with serial dilutions (according to the instructions, the concentration gradient of the standard in this kit is 0pg / ml, 62.5pg / ml, 125pg / ml, 250pg / ml, 500pg / ml, 1000pg / ml, 2000pg / ml, 4000pg / ml) at the same time, and add 100μl of the serum sample to be tested diluted 3.3 times to the coated reaction wells to be tested.

[0074] 3) Incubation and washing: Seal the plate with a sealing film and incubate at 37°C for 1-2 hours. Discard the liquid, add 300 μL of 10X wash buffer PT to each well, soak for 1-2 minutes, pat dry on absorbent paper, and repeat 3-5 times.

[0075] 4) Add enzyme-labeled antibody: Mix 300 μL 10X human CD85d capture antibody and 300 μL 10X human CD85d detection antibody with 2.4 mL antibody diluent CPI, stir evenly, and prepare 3 ml enzyme-labeled antibody working solution. Add 100 μL of 1:5 diluted enzyme-labeled antibody working solution to each well.

[0076] 5) Incubation and washing: Seal the plate with a sealing film and incubate at 37°C for 1 hour. Discard the liquid and wash as in step 3.

[0077] 6) Add color substrate: Add 100μl of TMB substrate solution to each well, react at 37℃ in the dark for 10-30min, until a clear color gradient appears in the standard wells diluted according to the instructions. The results can be observed directly with the naked eye on a white background: the darker the color in the reaction well, the stronger the positive degree, depending on the depth of the color. The optical density (OD) value can also be measured: at 450nm on the ELISA detector, adjust the blank control well to zero and measure the OD value of each well. If it is greater than 3 times the negative control OD value specified in the instructions, it is positive.

[0078] 7) Stop reaction: Add 100 μl of stop solution to each reaction well, and the color will change from blue to yellow.

[0079] 8) Interpretation of results: Measure the OD value of each well on the microplate reader at 450nm after adjusting the blank control well to zero. Make a standard curve based on the concentration and OD value of the standard, then calculate the sample concentration based on the standard curve equation, and multiply the result by the sample dilution factor.

[0080] The experimental results showed that the cutoff value of LILRB2 serum concentration for diagnosing colorectal precancerous lesions was 4557pg / ml, with a sensitivity of 78.95%, a specificity of 77.78%, an accuracy of 78.38%, an AUC value of 0.84 (0.71, 0.96), and P=0.0005.

[0081] The cutoff value of LILRB2 serum concentration for diagnosing colorectal cancer was 5256pg / ml, with a sensitivity of 89.74%, a specificity of 88.89%, an accuracy of 89.63%, and an AUC value of 0.95 (0.89, 1.00), P < 0.0001. The sensitivity, specificity, accuracy, and AUC values ​​of the traditional indicator CEA were 42.2%, 94.4%, 49.6%, and 0.79 (0.73, 0.85), respectively, and the sensitivity, specificity, accuracy, and AUC values ​​of CA19-9 were 22.2%, 100%, 33.3%, and 0.89 (0.84, 0.94), respectively. It can be seen that the serum concentration of LILRB2 protein has high sensitivity, specificity, accuracy, and diagnostic efficacy in diagnosing colorectal cancer and precancerous lesions, which is superior to the traditional serum markers CEA and CA19-9. It has been validated that LILRB2 can serve as a potential new, non-invasive, highly sensitive and specific screening marker for colorectal cancer.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An application of a colorectal cancer therapeutic target and diagnostic serum marker, characterized in that: The therapeutic targets and diagnostic serum markers are one or more of alcohol dehydrogenase ADH1B, capture domain-containing protein 1 ARRDC1, angiopoietin-like protein 2 ANGPTL2, glycine amidinotransferase GATM, general transcription factor IIH subunit 4 GTF2H4, mitochondrial genome maintenance exonuclease 1 MGME1, and leukocyte immunoglobulin-like receptor B2 LILRB2.

2. The use of colorectal cancer therapeutic targets and diagnostic serum markers according to claim 1, characterized in that: ADH1B, ARRDC1, ANGPTL2, GATM, GTF2H4, MGME1 and / or LILRB2 proteins are used in drugs for screening, diagnosis and treatment of colorectal cancer pathological conditions.

3. The use of colorectal cancer therapeutic targets and diagnostic serum markers according to claim 2, characterized in that: The pathological conditions of colorectal cancer include colorectal cancer, polyps and / or adenomas.

4. The use of colorectal cancer therapeutic targets and diagnostic serum markers according to claim 2, characterized in that: Immunohistochemical staining was used to quantitatively detect the expression of ANGPTL2 and LILRB2 proteins.

5. The use of colorectal cancer therapeutic targets and diagnostic serum markers according to claim 2, characterized in that: The serum concentration of LILRB2 protein was quantitatively detected by enzyme-linked immunosorbent assay.

6. The use of colorectal cancer therapeutic targets and diagnostic serum markers according to claim 2, characterized in that: The content of LILRB2 protein in colorectal cancer tissue is 5.5 times that of normal control tissue.

7. The use of colorectal cancer therapeutic targets and diagnostic serum markers according to claim 2, characterized in that: The content of the ANGPTL2 protein in colorectal tissue is 4.55 times that of normal control tissue.

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