Application of xanthine nucleoside as colorectal cancer diagnosis and prognosis biomarker

Abstract

The invention belongs to the field of biological medicine, and relates to application of xanthine nucleoside as a colorectal cancer diagnosis and prognosis biomarker. According to the invention, the intestinal flora metabolite xanthine nucleoside is used as the diagnostic marker of colorectal cancer, the excrement sample required for detection is easy to obtain, the compliance of patients is improved by noninvasive operation, and the intestinal flora metabolite xanthine nucleoside can be used as a biomarker and a therapeutic target for colorectal cancer diagnosis and prognosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of xanthine nucleoside as a biomarker for diagnosis and prognosis of colorectal cancer. Background Art

[0002] The occurrence of colorectal cancer is related to multiple factors such as genetics, environment, and lifestyle. A high-fat, high-protein, and low-fiber diet can increase the risk of colorectal cancer; factors such as lack of exercise and excessive obesity can also increase the risk of colorectal cancer; patients with a family history of colorectal cancer have an increased risk of colorectal cancer, and patients with familial adenomatous polyposis and hereditary non-polyposis colorectal cancer have a lower age of onset; the older the age, the higher the incidence of colorectal cancer: 31% of colorectal cancer worldwide occurs in people over 75 years old; inflammation such as ulcerative colitis and Crohn's disease are also risk factors for colorectal cancer. More and more studies have shown that the imbalance of intestinal flora and changes in the abundance of specific intestinal bacteria have also become risk factors for colorectal cancer. Intestinal flora imbalance can cause inflammatory responses, damage the intestinal microenvironment, and aggravate metabolic disorders. Studies have shown that Fusobacterium nucleatum can adhere to colon cancer cells, and its surface adhesion molecule FadA can bind to VE-cadherin in intestinal epithelial cells. This binding process activates β-catenin, which on the one hand triggers an inflammatory response to change the immune system, and on the other hand stimulates the growth of cancer cells. Enterotoxigenic Bacteroides fragilis reduces the body's immune surveillance and killing ability against tumor cells by inhibiting the activity of natural killer cells and cytotoxic T lymphocytes; it promotes the proliferation and activation of regulatory T cells, creating an immune escape environment for the growth of tumor cells. In the case of intestinal flora imbalance, the metabolism of bile acids, a metabolite of intestinal flora, changes, leading to an increase in the concentration of secondary bile acids. Secondary bile acids can release arachidonic acid and increase the content of reactive oxygen species, causing DNA damage to intestinal epithelial cells, and promoting cancer cell proliferation through a series of signal transductions.

[0003] Currently, the main diagnostic methods for cancer include imaging examinations and tissue biopsies, but these methods have certain disadvantages: imaging examinations have strong radiation, and tissue biopsies are invasive and may cause complications. Detecting changes in fecal metabolites is instructive for cancer diagnosis. Fecal samples are easy to obtain, and the operation method is non-invasive, avoiding the risks of invasive operations. Patients have high acceptance and are expected to serve as biomarkers and therapeutic targets for the diagnosis and prognosis of colorectal cancer. Summary of the invention

[0004] The purpose of the present invention is to provide a biomarker for diagnosing colorectal cancer and its application.

[0005] Another object of the present invention is to provide a reagent for detecting the expression level of xanthine nucleoside in a sample for use in preparing a product for diagnosing colorectal cancer.

[0006] Another object of the present invention is to provide a reagent for detecting the expression level of xanthine nucleoside in a sample for use in preparing a product for diagnosing the prognosis of colorectal cancer treatment.

[0007] The applications include:

[0008] In vitro, the effect of xanthine nucleosides on the proliferation of colorectal cancer cells is concentration-dependent and time-dependent. It can promote the cloning of colorectal cancer cells, significantly increase the proportion of EdU-positive red-stained cells, promote the healing of colorectal cancer cell scratches, and promote the migration of colorectal cancer cells. It has the potential to serve as a biomarker and therapeutic target for the diagnosis and prognosis of colorectal cancer.

[0009] In vivo, xanthine nucleosides can significantly increase the volume and weight of transplanted tumors, significantly increase the cell density within the transplanted tumors, enlarge the cell nuclei, increase the number of cells showing nuclear division, show obvious cell atypia, and place the cells in an active state of proliferation and division. The proportion of cells positive for the proliferation marker Ki67 significantly increases. Xanthine nucleosides have the potential to serve as biomarkers and therapeutic targets for the diagnosis and prognosis of colorectal cancer.

[0010] Another object of the present invention is to provide a kit for diagnosing colorectal cancer and treating the prognosis of colorectal cancer, wherein the kit includes a biomarker: xanthine nucleoside.

[0011] The kit, in addition to the biomarkers, also includes other components: xanthine nucleoside standards and internal standard chlorophenylalanine solution.

[0012] The biomarkers of the present invention are obtained through a large number of experimental screenings:

[0013] 1. Screening of intestinal flora metabolites with cancer-promoting effects

[0014] By comparing the omics data of intestinal flora metabolites in healthy people and colorectal cancer patients, five metabolites with significant differential expression were first screened out. CCK-8 experiments were used to determine the effects of different intestinal flora metabolites on the cell proliferation rate of human colon cancer cells HCT116 and mouse colon adenocarcinoma cells MC38, thereby determining the metabolites that actually affect cancer cell proliferation.

[0015] CCK-8 assay was used to detect whether the intestinal flora metabolites have a concentration-dependent effect on the proliferation of colorectal cancer cells. HCT116 or MC38 cells were seeded in 96-well plates (2×10 3), add different concentrations of intestinal flora metabolites and cultivate for 24 hours, add 10μL CCK-8 reagent to each well, incubate at 37℃ for 1 hour, and use a microplate reader to detect the absorbance at 450nm. Among them, the wells without inoculation of cells and only adding culture medium are set as blank wells; the wells with inoculation of cells but no drug-containing culture medium are set as control wells. Cell survival rate (%) = (OD value of drug-added well-OD value of blank well) / (OD value of control well-OD value of blank well) × 100%.

[0016] CCK-8 assay was used to detect whether the intestinal flora metabolites have a time-dependent effect on the proliferation of colorectal cancer cells. HCT116 or MC38 cells were seeded in 96-well plates (2×10 3 ), 3.125 μmol / L and 50 μmol / L xanthine riboside were added for 24, 48, and 72 h, respectively, and then 10 μL of CCK-8 reagent was added to each well. After incubation at 37°C for 1 h, the absorbance at 450 nm was detected by an enzyme reader.

[0017] 2. Pro-cancer effects of xanthine nucleotides, metabolites of intestinal flora, at the in vitro level

[0018] Plate cloning experiment and EdU experiment were used to further verify the proliferation-promoting effect of xanthine nucleoside on colorectal cancer cells HCT116 and MC38.

[0019] HCT116 cells or MC38 cells were seeded in 6-well plates (8×10 2 ), and the medium was replaced with fresh medium containing 50 μmol / L xanthine nucleoside every 2 days. The culture was stopped when a large number of colonies were formed in the well plate. The cells were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.25% crystal violet for 15 minutes, and pictures were collected and the colonies were counted.

[0020] HCT116 cells or MC38 cells were seeded in 6-well plates (2×10 5 ), after adding 50μmol / L xanthine riboside for 24h, the medium was changed to 50μmol / L EdU to label the DNA of proliferating cells, and incubated at 37℃ for 2h. The cells were fixed with 4% paraformaldehyde for 30min, permeated with 0.3% Triton-X, and then incubated with the reaction solution for 30min. Hoechst33342 was added to stain the nucleus for 10min, and the images were observed and collected under a fluorescence microscope.

[0021] Cell scratch assay and Transwell assay were used to detect the effect of xanthine nucleotides on the migration ability of HCT116 and MC38 cells.

[0022] Colorectal cancer cells were inoculated in a 24-well plate. When the cells filled the well plate and formed a confluent monolayer, the cell monolayer was scraped in a straight line with a 10 μl pipette tip to form a scratch. After washing with PBS twice, 50 μmol / L xanthine nucleoside or an equal volume of PBS was added to the serum-free medium for incubation. Cell scratch images were taken under a microscope at 0, 24, and 48 hours, and the scratch distance was measured using ImageJ software. Cell migration rate (%) = (0h scratch distance - 24h or 48h scratch distance) / 0h scratch distance × 100%.

[0023] HCT116 cells or MC38 cells were inoculated in the upper chamber (1.5×10 5 ), 20% fetal bovine serum was added to the culture medium of the lower chamber. After incubation for 24 h, the cells that migrated to the lower chamber were fixed with 4% paraformaldehyde for 15 min and stained with 0.25% crystal violet for 15 min. The cells were observed and images were collected under an optical microscope.

[0024] The TUNEL assay was used to detect the effect of xanthine nucleosides on apoptosis of colorectal cancer cells. HCT116 cells or MC38 cells were seeded in 24-well plates (1×10 5 ), add 50μmol / L xanthine riboside for 24h, fix with 4% paraformaldehyde at 4℃ for 25min, and permeabilize with 0.3% Triton-X for 5min. After equilibration with equilibration buffer for 30min, add labeling solution to the well plate and incubate at 37℃ in the dark for 1h. Add DAPI to stain the nucleus for 5min, observe the green fluorescence under a fluorescence microscope and collect images.

[0025] 3. The cancer-promoting effect of xanthine nucleotides, metabolites of intestinal flora, at the in vivo level

[0026] Five-week-old male C57BL / 6 mice were randomly divided into a PBS control group and a xanthine nucleoside administration group (50 μmol / L), with 8 mice in each group. After one week of feeding and adaptation, MC38 cells (8×10 5 ) was injected subcutaneously into the dorsal flank of C57BL / 6 mice. When the tumor size reached 50-100 mm 3 Every 2 days, PBS or xanthine nucleoside was injected intraperitoneally at a volume of 100 μL / 10 g. The mice were weighed every 2 days, and the tumor diameter was measured with a caliper. When the tumor size reached 2000 mm 3 The mice were killed at 4 pm and the transplanted tumors were completely removed, weighed and measured.

[0027] HE staining was used to analyze the pathological changes of tumor tissue after xanthine nucleoside treatment. After the tumor tissue was removed, it was fixed with 4% paraformaldehyde, embedded in paraffin, and sliced. The paraffin sections were dewaxed in xylene and then hydrated with gradient ethanol solutions. Hematoxylin staining for 5 minutes and rinsed with running water. Eosin staining for 10 seconds, and distilled water was used to wash away the floating color. The tissue sections were placed in 85%, 95%, and anhydrous ethanol and soaked for 2 minutes each for dehydration, and soaked in xylene for 2 minutes to make it transparent. Neutral resin was added to seal the sections and dried for storage. The sealed sections were placed under a microscope for observation.

[0028] Immunohistochemical staining was performed on paraffin-embedded sections of mouse tumor tissue to detect the expression level of Ki67 in tumor tissue, reflecting the effect of xanthine nucleoside treatment on tumor cell proliferation activity in vivo. After the tumor tissue sections were baked in a 60°C oven for 1 hour, they were soaked in xylene I and xylene II for 15 minutes each, and then placed in anhydrous ethanol, 95% ethanol, 85% ethanol and 75% ethanol for 5 minutes each for dewaxing and rehydration. The sections were placed in a repair box containing EDTA antigen repair solution, boiled at high temperature for 2 minutes and heated at low temperature for 10 minutes, then the tissue sections were taken out and naturally cooled to room temperature for antigen repair. 3% H2O2 was used for incubation for 10 minutes to remove endogenous peroxidase. After the sections were slightly dry, 50% goat serum was added and blocked at room temperature for 30 minutes. The surrounding liquid was aspirated, and Ki67 primary antibody diluent was added, and it was kept at 4°C overnight. The next day, the sections were taken out and rewarmed for 1 hour. Biotin-labeled secondary antibodies were incubated at 37°C for 20 minutes. Wipe the liquid on the slice dry, add an appropriate amount of horseradish peroxidase-labeled triple antibody and incubate at 37°C for 30 minutes. Wipe the liquid on the slice dry, add DAB colorimetric solution, let it stand for 3 minutes, and rinse with tap water to stop color development. Stain the nucleus with hematoxylin for 10 seconds. Soak the tissue slices in 85%, 95%, and anhydrous ethanol for 2 minutes each to dehydrate and make them transparent. After drying, add neutral resin to seal the slices and store them dry. Observe and collect pictures under a microscope, and quantify them using Image J software.

[0029] 4. Detection of xanthine nucleotides, metabolites of intestinal flora, in fecal samples

[0030] Fecal samples from patients with colorectal cancer were collected and stored at -80°C after rapid freezing in liquid nitrogen. Take the fecal sample stored at -80°C, weigh 50 mg in a 1.5 mL centrifuge tube, add 200 μL ultrapure water, add zirconium beads, grind in a tissue grinder, centrifuge at 20,000 rpm for 10 min, take 100 μL of supernatant in a 1.5 mL centrifuge tube, add 400 μL of methanol (methanol was pre-added with internal standard, and the final concentration of internal standard in methanol was 500 ng / mL), vortex for 1 min, centrifuge at 20,000 rpm for 10 min, take 400 μL of supernatant, continue to centrifuge at 20,000 rpm for 10 min, take 400 μL of supernatant in a 1.5 mL centrifuge tube, and evaporate in a vacuum integrated evaporator.

[0031] Weigh 10 mg of xanthine riboside standard and dissolve it in DMSO to prepare a xanthine riboside stock solution with a concentration of 10 mg / mL; then use methanol as the solvent to dilute and obtain a series of standard curve working solutions with concentrations of 1 μg / mL, 500 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 5 ng / mL, and 1 ng / mL; vortex the standard curve working solution to mix well, take 100 μL in a 1.5 mL centrifuge tube, add 400 μL of methanol (internal standard is added to methanol, and the final concentration of internal standard is 500 ng / mL), vortex for 1 min, centrifuge at 20000 rpm for 10 min, take 400 μL of supernatant, continue to centrifuge at 20000 rpm for 10 min, take 400 μL of supernatant in a 1.5 mL centrifuge tube, and evaporate in a vacuum integrated evaporator.

[0032] Take the evaporated samples, add 100 μL of 50% methanol to each sample, vortex for 1 min to re-dissolve, centrifuge twice, each time at 20,000 rpm for 10 min, and take the supernatant for injection.

[0033] Shimadzu liquid chromatography instrument was used for liquid chromatography separation and analysis. The chromatographic column was Amide Bridge HPLC column, the column temperature was 40 °C, the injection volume was 5 μL, the flow rate was 0.4 mL / min, the mobile phase A was ultrapure water containing 0.1% formic acid and 5 mM ammonium formate, and the mobile phase B was acetonitrile. The solvent gradient was as follows: 0–2 min, 85% mobile phase B; 2–5 min, 85–20% mobile phase B; 5–8 min, 20% mobile phase B; 8–10 min, 20–85% mobile phase B; 10–14 min, 85% mobile phase B. The mass spectrometry detection and analysis system was AB SCIEX QTRAP 6500 analysis system, the scan duration was 14 min, and the positive ion detection mode was used. The detection ion pair of xanthine nucleoside was 153.1 / 110.0, and the detection ion pair of chlorophenylalanine (internal standard) was 200.0 / 154.0.

[0034] After the standard sample test is completed, the xanthosine peak and the internal standard peak are integrated using analysis software, and the integrated peak area is calculated. A xanthosine standard curve is drawn with the xanthosine concentration as the horizontal axis and the ratio of the xanthosine to internal standard peak areas as the vertical axis. The standard curve is used to calculate the xanthosine concentration in the fecal sample.

[0035] Beneficial effects of the present invention:

[0036] At present, the main diagnostic methods for cancer, such as imaging examinations and invasive tissue biopsies, are highly radiation- and invasive. This project uses xanthine nucleosides, metabolites of intestinal flora, as diagnostic markers for colorectal cancer. The stool samples required for the test are easy to obtain, and the non-invasive operation improves patient compliance. It can be used as a biomarker and therapeutic target for the diagnosis and prognosis of colorectal cancer.

[0037] Further explanation of the English and terms appearing in the manual:

[0038] HCT116: human colon cancer cells

[0039] MC38: mouse colon adenocarcinoma cells

[0040] CCK-8 reagent: Cell Counting Kit-8 reagent, which contains water-soluble tetrazolium salt WST-8 and can be used for simple and accurate cell proliferation and toxicity analysis.

[0041] EdU: 5-ethynyl-2'-deoxyuridine, a thymidine analog, used for cell proliferation detection.

[0042] Triton X-100: Triton X-100, a non-ionic surfactant, is used to permeabilize cell membranes.

[0043] Transwell assay: Transwell migration or invasion assay is used to study the migration, chemotaxis and invasion of cells in response to various stimuli such as growth factors, chemokines or extracellular matrix components.

[0044] PBS: phosphate buffered saline.

[0045] TUNEL assay: TdT-mediated dUTP Nick-End Labeling. When genomic DNA breaks, the exposed 3'-OH can be catalyzed by terminal deoxynucleotidyl transferase (TdT) to add fluorescein (FITC)-labeled dUTP (fluorescein-dUTP), thereby detecting cell apoptosis by fluorescence microscopy or flow cytometry.

[0046] DAPI: 4',6-diamidino-2-phenylindole, a fluorescent dye that binds strongly to DNA.

[0047] HE staining: Hematoxylin-eosin staining, a staining method used in histology and pathology.

[0048] EDTA: ethylenediaminetetraacetic acid.

[0049] Ki67: A cell proliferation marker that can detect the proportion of cells in the cell cycle. The higher the Ki67 positive rate, the greater the proportion of tumor cells in the growth cycle and the faster the tumor grows.

[0050] DAB color developing solution: diaminobenzidine, the most commonly used chromogenic substrate of horseradish peroxidase. Its color development principle is that it loses electrons under the action of hydrogen peroxide and shows color change and accumulation, forming a brown insoluble product. The reaction has high sensitivity and good specificity. It is often used for membrane color development such as immunohistochemistry, in situ hybridization, Western Blot, or to detect endogenous peroxidase in cells or tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The effect of xanthine nucleosides on the proliferation of colorectal cancer cells in Example 1 of the present invention is concentration- and time-dependent. A. The promoting effect of xanthine nucleosides on the proliferation of colorectal cancer cells HCT116 is concentration-dependent. B. The promoting effect of xanthine nucleosides on the proliferation of colorectal cancer cells MC38 is concentration-dependent. C. Uric acid has no obvious promoting effect on the proliferation of colorectal cancer cells HCT116. D. Uric acid has no obvious promoting effect on the proliferation of colorectal cancer cells MC38. E. The promoting effect of thymidine-5′-monophosphate on the proliferation of colorectal cancer cells HCT116 is concentration-dependent. F. The promoting effect of thymidine-5′-monophosphate on the proliferation of colorectal cancer cells MC38 is concentration-dependent. G The inhibitory effect of citraconic acid on the proliferation of colorectal cancer cells HCT116 was concentration-dependent H The inhibitory effect of citraconic acid on the proliferation of colorectal cancer cells MC38 was concentration-dependent I The inhibitory effect of glutaric acid on the proliferation of colorectal cancer cells HCT116 was concentration-dependent J The inhibitory effect of glutaric acid on the proliferation of colorectal cancer cells MC38 was concentration-dependent K The promoting effect of xanthine nucleoside on the proliferation of colorectal cancer cells HCT116 was time-dependent L The promoting effect of xanthine nucleoside on the proliferation of colorectal cancer cells MC38 was time-dependent. **, p<0.01, ***, p<0.001, ****, p<0.0001.

[0052] Figure 2The following are the effects of xanthocyanin on the proliferation of colorectal cancer cells HCT116 and MC38 in Example 2 of the present invention. A. Xanthocyanin promotes the formation of HCT116 and MC38 cell clones. B. Xanthocyanin treatment significantly increases the proportion of EdU-positive red-stained cells in HCT116 cells. C. Xanthocyanin treatment significantly increases the proportion of EdU-positive red-stained cells in MC38 cells. ***, p<0.001, ****, p<0.0001.

[0053] Figure 3 The effect of xanthocyanine nucleosides on the migration ability of colorectal cancer cells HCT116 and MC38 in Example 2 of the present invention. A. Treatment with xanthocyanine nucleosides shortened the distance between the two sides of the scratch of HCT116 cells and promoted the migration of HCT116 cells. B. Treatment with xanthocyanine nucleosides shortened the distance between the two sides of the scratch of MC38 cells and promoted the migration of MC38 cells. C. Treatment with xanthocyanine nucleosides significantly increased the number of migrating cells of HCT116 and MC38 cells passing through the Transwell chamber. **, p<0.01, ***, p<0.001, ****, p<0.0001.

[0054] Figure 4 The effect of xanthine nucleotides on the apoptosis of colorectal cancer cells HCT116 and MC38 in Example 2 of the present invention

[0055] Figure 5 The carcinogenic effect of xanthine nucleosides in mice in Example 3 of the present invention. A Photo of mouse transplanted tumor B Xanthine nucleosides promote the growth of mouse transplanted tumor volume C Xanthine nucleosides promote the increase of mouse transplanted tumor weight D Xanthine nucleosides treatment has no significant effect on mouse weight E HE staining analysis of pathological changes in transplanted tumors after xanthine nucleosides treatment F Xanthine nucleosides promote the expression of Ki67, a proliferation marker in tumor tissue. *, p<0.05, ***, p<0.001. DETAILED DESCRIPTION

[0056] The present invention is further illustrated by the following examples.

[0057] The product and preparation method of the present invention are further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The raw materials can be obtained from public commercial channels unless otherwise specified.

[0058] Example 1 Screening of intestinal flora metabolites with cancer-promoting effects

[0059] By comparing the omics data of intestinal flora metabolites of healthy people and colorectal cancer patients, five significantly differentially expressed metabolites were first screened out, in order of content: xanthine nucleoside, uric acid, thymidine-5′-monophosphate, citraconic acid, and glutaric acid. CCK-8 experiments were used to determine the effects of different intestinal flora metabolites on the cell proliferation rate of human colon cancer cells HCT116 and mouse colon adenocarcinoma cells MC38, thereby determining the metabolites that actually affect cancer cell proliferation.

[0060] HCT116 or MC38 cells were seeded in 96-well plates (2×10 3 ), add different concentrations of intestinal flora metabolites and cultivate for 24 hours, add 10μL CCK-8 reagent to each well, incubate at 37℃ for 1 hour, and use a microplate reader to detect the OD value at 450nm. At the same time, set up blank wells and control wells: only culture medium is added to the blank well without inoculating cells, and cells are inoculated in the control well without adding drugs. Substituting the OD value into the equation can calculate the cell survival rate, cell survival rate (%) = (OD value of the drug-added well-OD value of the blank well) / (OD value of the control well-OD value of the blank well) × 100%. The results showed that xanthine nucleosides, metabolites of intestinal flora, significantly promoted the proliferation of colorectal cancer cells. With the increase of xanthine nucleosides concentration (0 μmol / L, 1.5625 μmol / L, 3.125 μmol / L, 6.25 μmol / L, 12.5 μmol / L, 25 μmol / L, 50 μmol / L), the proliferation ability of HCT116 and MC38 cells was significantly enhanced, and MC38 cells were more susceptible to this metabolite. The effect of xanthine nucleosides on the proliferation of colorectal cancer cells was concentration-dependent ( Figure 1 A, B). Uric acid, a metabolite of intestinal flora, has no significant effect on the proliferation ability of HCT116 and MC38 cells ( Figure 1 C, D). The intestinal flora metabolite thymidine-5′-monophosphate promotes the proliferation of colorectal cancer cells HCT116 and MC38 in a concentration-dependent manner ( Figure 1 E, F). Citraconic acid and glutaric acid, metabolites of intestinal flora, inhibit the proliferation of colorectal cancer cells HCT116 and MC38 in a concentration-dependent manner ( Figure 1 GJ). The present invention mainly focuses on xanthine ribosides.

[0061] HCT116 or MC38 cells were seeded in 96-well plates (2×10 3), 3.125μmol / L and 50μmol / L xanthine nucleoside were added for 24, 48, and 72h, respectively, and 10μL CCK-8 reagent was added to each well. After incubation at 37℃ for 1h, the OD value at 450nm was detected by microplate reader. The results showed that both low and high concentrations of xanthine nucleoside had a continuous promoting effect on the proliferation of colorectal cancer cells in a time-dependent manner ( Figure 1 K, L).

[0062] Example 2 Promoting cancer effects of xanthosine nucleotides, metabolites of intestinal flora, at the in vitro level

[0063] Plate cloning experiment and EdU experiment were used to further verify the proliferation-promoting effect of xanthine nucleoside on colorectal cancer cells HCT116 and MC38.

[0064] HCT116 cells or MC38 cells were seeded in 6-well plates (8×10 2 ), and the medium was replaced with fresh culture medium containing 50 μmol / L xanthine nucleoside every 2 days. The culture was stopped when a large number of clones were formed in the well plate. The cells were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.25% crystal violet for 15 minutes, and then pictures were collected and the clones were counted. The results showed that the number of clones formed by HCT116 cells and MC38 cells treated with xanthine nucleoside was significantly higher than that of the control group, indicating that xanthine nucleoside has a significant ability to promote tumor cell proliferation ( Figure 2 A).

[0065] HCT116 cells or MC38 cells were seeded in 6-well plates (2×10 5 ), after adding 50μmol / L xanthine nucleoside for 24h, the medium was changed to 50μmol / L EdU to mark the DNA of proliferating cells, and incubated at 37℃ for 2h. Fix with 4% paraformaldehyde for 30min, permeate with 0.3% Triton-X, add reaction solution and incubate for 30min, add Hoechst33342 to stain the nucleus for 10min, observe and collect images under a fluorescence microscope. The results showed that the proportion of EdU-positive red-stained cells in the xanthine nucleoside-treated group was significantly higher than that in the control group, indicating that the cells entered an active proliferation period, further confirming the promoting effect of xanthine nucleoside on the proliferation of colorectal cancer cells ( Figure 2 B).

[0066] Cell scratch assay and Transwell assay were used to detect the effect of xanthine nucleotides on the migration ability of HCT116 and MC38 cells.

[0067] Colorectal cancer cells HCT116 or MC38 were inoculated in 24-well plates. When the cells grew all over the wells and formed a confluent monolayer, the cell monolayer was scraped in a straight line with a 10μl pipette tip to form a scratch. After washing with PBS twice, 50μmol / L xanthine nucleoside or an equal volume of PBS was added to the serum-free medium for incubation. Cell scratch images were taken under a microscope at 0, 24, and 48h, and the scratch distance was measured using ImageJ software. Cell migration rate (%) = (0h scratch distance - 24h or 48h scratch distance) / 0h scratch distance × 100%. The results showed that after HCT116 cells were incubated in serum-free medium for 24h and 48h, the cell spacing on both sides of the scratch in the xanthine nucleoside-treated group was significantly shortened compared with the control group, and the cell migration was active. After MC38 cells were incubated in serum-free medium for 24 hours, the distance between cells on both sides of the scratch was significantly shortened. After 48 hours of incubation, the scratch in the xanthine nucleoside treatment group was basically healed, and the distribution of cells between the scratch and the surrounding area tended to be uniform. Xanthine nucleoside has a promoting effect on the migration of colorectal cancer cells ( Figure 3 A, B).

[0068] HCT116 cells or MC38 cells were inoculated in the upper chamber (1.5×10 5 ), 20% fetal bovine serum was added to the culture medium in the lower chamber. After incubation for 24 hours, the cells that migrated to the lower chamber were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.25% crystal violet for 15 minutes. The images were observed and collected under an optical microscope. The results showed that compared with the control group, the number of migrating cells of HCT116 and MC38 cells treated with xanthine nucleosides increased significantly, further indicating that xanthine nucleosides have a promoting effect on the migration of colorectal cancer cells ( Figure 3 C).

[0069] The TUNEL assay was used to detect the effect of xanthine nucleosides on apoptosis of colorectal cancer cells. HCT116 cells or MC38 cells were seeded in 24-well plates (1×10 5 ), after adding 50μmol / L xanthine nucleoside for 24h, fix with 4% paraformaldehyde at 4℃ for 25min, and permeabilize with 0.3% Triton-X for 5min. After equilibration with equilibrium buffer for 30min, add labeling solution to the well plate and incubate at 37℃ in the dark for 1h. Add DAPI to stain the nucleus for 5min, observe the green fluorescence under a fluorescence microscope and collect images. The results showed that the proportion of green-stained cells in the xanthine nucleoside treatment group did not change significantly compared with the control group, indicating that xanthine nucleoside cannot inhibit the apoptosis of tumor cells ( Figure 4 ).

[0070] Through the above experiments, it is comprehensively proved that xanthine nucleosides have a significant promoting effect on the proliferation and migration of colorectal cancer in vitro, and have no inhibitory effect on cell apoptosis. Xanthine nucleosides, metabolites of intestinal flora, have the potential to be used as biomarkers and therapeutic targets for the diagnosis and prognosis of colorectal cancer.

[0071] Example 3 Cancer-promoting effects of xanthosine, a metabolite of intestinal flora, at the in vivo level

[0072] Five-week-old male C57BL / 6 mice were randomly divided into a PBS control group and a xanthine nucleoside administration group (50 μmol / L), with 8 mice in each group. After one week of feeding and adaptation, MC38 cells (8×10 5 ) was injected subcutaneously into the dorsal flank of C57BL / 6 mice. When the tumor size reached 50-100mm3, PBS or xanthine nucleoside was injected intraperitoneally every 2 days with an injection volume of 100μL / 10g. The mice were weighed every 2 days, and the tumor diameter was measured with a caliper. When the tumor size reached 2000mm3, the mice were killed, the transplanted tumor was completely removed, weighed, and the volume was measured. The results showed that over time, the tumor volume of the control group mice gradually increased, but the growth rate was relatively slow, while the tumor volume of the xanthine nucleoside-treated mice increased significantly faster than that of the control group, and the tumor weight was also significantly higher than that of the control group ( Figure 5 A, B, C). At the same time, the treatment with xanthine nucleotides had no significant effect on the body weight of mice ( Figure 5 D).

[0073] HE staining was used to analyze the pathological changes of tumor tissue after xanthine nucleoside treatment. After the tumor tissue was removed, it was fixed with 4% paraformaldehyde, embedded in paraffin, and sliced. The paraffin sections were dewaxed in xylene and then hydrated with gradient ethanol solutions. Hematoxylin staining for 5 minutes and rinsed with running water. Eosin staining for 10 seconds, and distilled water was used to wash away the floating color. The tissue sections were placed in 85%, 95%, and anhydrous ethanol and soaked for 2 minutes each for dehydration, and soaked in xylene for 2 minutes to make them transparent. Neutral resin was added to seal the sections and dried for storage. The sealed sections were placed under a microscope for observation. The results showed that compared with the colorectal cancer model group, the density of transplanted tumor cells in the xanthine nucleoside-treated group increased significantly, the cell nucleus enlarged, the number of cells showing nuclear division increased, and the cell atypia was obvious, indicating that the cells were in an active state of proliferation and division, indicating that xanthine nucleoside has a significant promoting effect on the proliferation of tumor cells ( Figure 5 E).

[0074] Immunohistochemical staining was performed on paraffin-embedded sections of mouse tumor tissue to detect the expression level of Ki67 in tumor tissue, reflecting the effect of xanthine nucleoside treatment on tumor cell proliferation activity in vivo. After the tumor tissue sections were baked in a 60°C oven for 1 hour, they were soaked in xylene I and xylene II for 15 minutes each, and then placed in anhydrous ethanol, 95% ethanol, 85% ethanol and 75% ethanol for 5 minutes each for dewaxing and rehydration. The sections were placed in a repair box containing EDTA antigen repair solution, boiled at high temperature for 2 minutes and heated at low temperature for 10 minutes, then the tissue sections were taken out and naturally cooled to room temperature for antigen repair. 3% H2O2 was used for incubation for 10 minutes to remove endogenous peroxidase. After the sections were slightly dry, 50% goat serum was added and blocked at room temperature for 30 minutes. The surrounding liquid was aspirated, and Ki67 primary antibody diluent was added, and it was kept at 4°C overnight. The next day, the sections were taken out and rewarmed for 1 hour. Biotin-labeled secondary antibodies were incubated at 37°C for 20 minutes. Wipe the liquid on the slice dry, add an appropriate amount of horseradish peroxidase-labeled triple antibody and incubate at 37°C for 30 minutes. Wipe the liquid on the slice dry, add DAB colorimetric solution, let it stand for 3 minutes, and rinse with tap water to terminate the color development. Stain the nucleus with hematoxylin for 10 seconds. Soak the tissue sections in 85%, 95%, and anhydrous ethanol for 2 minutes each to dehydrate and make them transparent. After drying, add neutral resin to seal the sections and store them dry. Observe and collect pictures under a microscope, and quantify them using Image J software. The results showed that compared with the model group, the proportion of positive cells for the proliferation marker Ki67 in the xanthine nucleoside-administered group increased significantly, indicating that xanthine nucleoside has a significant effect in promoting the proliferation of colorectal cancer cells ( Figure 5 F).

[0075] The results of in vivo studies have shown that xanthine nucleosides, a metabolite of intestinal flora, have a significant effect in promoting the proliferation of colorectal cancer cells and have the potential to serve as a biomarker and therapeutic target for the diagnosis and prognosis of colorectal cancer.

[0076] Example 4 Detection of xanthosine nucleosides, metabolites of intestinal flora, in fecal samples

[0077] Fecal samples from patients with colorectal cancer were collected and stored at -80°C after rapid freezing in liquid nitrogen. Take the fecal sample stored at -80°C, weigh 50 mg in a 1.5 mL centrifuge tube, add 200 μL ultrapure water, add zirconium beads, grind in a tissue grinder, centrifuge at 20,000 rpm for 10 min, take 100 μL of supernatant in a 1.5 mL centrifuge tube, add 400 μL of methanol (methanol was pre-added with internal standard, and the final concentration of internal standard in methanol was 500 ng / mL), vortex for 1 min, centrifuge at 20,000 rpm for 10 min, take 400 μL of supernatant, continue to centrifuge at 20,000 rpm for 10 min, take 400 μL of supernatant in a 1.5 mL centrifuge tube, and evaporate in a vacuum integrated evaporator.

[0078] Weigh 10 mg of xanthine riboside standard and dissolve it in DMSO to prepare a xanthine riboside stock solution with a concentration of 10 mg / mL; then use methanol as the solvent to dilute and obtain a series of standard curve working solutions with concentrations of 1 μg / mL, 500 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 5 ng / mL, and 1 ng / mL; vortex the standard curve working solution to mix well, take 100 μL in a 1.5 mL centrifuge tube, add 400 μL of methanol (internal standard is added to methanol, and the final concentration of internal standard is 500 ng / mL), vortex for 1 min, centrifuge at 20000 rpm for 10 min, take 400 μL of supernatant, continue to centrifuge at 20000 rpm for 10 min, take 400 μL of supernatant in a 1.5 mL centrifuge tube, and evaporate in a vacuum integrated evaporator.

[0079] Take the evaporated samples, add 100 μL of 50% methanol to each sample, vortex for 1 min to re-dissolve, centrifuge twice, each time at 20,000 rpm for 10 min, and take the supernatant for injection.

[0080] Shimadzu liquid chromatography instrument was used for liquid chromatography separation and analysis. The chromatographic column was Amide Bridge HPLC column, the column temperature was 40 °C, the injection volume was 5 μL, the flow rate was 0.4 mL / min, the mobile phase A was ultrapure water containing 0.1% formic acid and 5 mM ammonium formate, and the mobile phase B was acetonitrile. The solvent gradient was as follows: 0–2 min, 85% mobile phase B; 2–5 min, 85–20% mobile phase B; 5–8 min, 20% mobile phase B; 8–10 min, 20–85% mobile phase B; 10–14 min, 85% mobile phase B. The mass spectrometry detection and analysis system was AB SCIEX QTRAP 6500 analysis system, the scan duration was 14 min, and the positive ion detection mode was used. The detection ion pair of xanthine nucleoside was 153.1 / 110.0, and the detection ion pair of chlorophenylalanine (internal standard) was 200.0 / 154.0.

[0081] After the standard sample test is completed, the xanthosine peak and the internal standard peak are integrated using analysis software, and the integrated peak area is calculated. The xanthosine standard curve is drawn with the xanthosine concentration as the horizontal axis and the ratio of the xanthosine to internal standard peak areas as the vertical axis: y=0.0282x+0.0692, r=0.9968. The xanthosine concentration in the fecal sample is calculated based on the standard curve.

[0082] The above description is only a preferred embodiment of the present invention. It should be noted that the embodiments of the present invention are not limited to the embodiments. Without departing from the principle of the present invention, several changes, modifications, substitutions, combinations, and simplifications can be made, all of which should be equivalent replacement methods, and these should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting the expression level of xanthine nucleoside in a sample in the preparation of a product for diagnosing colorectal cancer.

2. Use of reagents for detecting xanthine nucleoside expression levels in samples in the preparation of products for diagnosing the prognosis of colorectal cancer treatment.

3. The use according to claim 1 or 2, characterized in that: The effects of xanthine nucleosides on the proliferation of colorectal cancer cells are concentration-dependent and time-dependent.

4. The use according to claim 1 or 2, characterized in that: Xanthine nucleosides can promote the colony formation of colorectal cancer cells and significantly increase the proportion of EdU-positive red-stained cells.

5. The use according to claim 1 or 2, characterized in that: It can promote the healing of scratches on colorectal cancer cells and the migration of colorectal cancer cells, and has the potential to serve as a biomarker and therapeutic target for the diagnosis and prognosis of colorectal cancer.

6. The use according to claim 1 or 2, characterized in that: Xanthine nucleoside can significantly increase the volume and weight of transplanted tumors, significantly increase the cell density in transplanted tumors, enlarge the cell nuclei, increase the number of cells showing nuclear division, show obvious cell atypia, and the cells are in an active state of proliferation and division. The proportion of positive cells for the proliferation marker Ki67 increases significantly.

7. A kit for diagnosing colorectal cancer and treating rectal cancer prognosis, the kit comprising a biomarker: xanthine nucleoside.

8. The kit according to claim 7, characterized in that Kit includes: Xanthine nucleoside standard and internal standard chlorophenylalanine solution.

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