Application of thymidine-5 '-monophosphate as colorectal cancer diagnosis and prognosis biomarker

Abstract

The invention belongs to the field of biological medicine, and relates to application of thymidine-5 '-monophosphate as a colorectal cancer diagnosis and prognosis biomarker. Through comparison of omics data of intestinal flora metabolites of healthy people and colorectal cancer patients and in-vitro and in-vivo level verification, the potential of thymidine-5 '-monophosphate serving as the colorectal cancer diagnostic marker is screened out. Excrement samples needed for detection are easy to obtain, the compliance of patients is improved through non-invasive operation, and the early diagnosis rate of colorectal cancer is expected to be improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of thymidine-5'-monophosphate as a biomarker for the 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 obesity can also increase the risk of colorectal cancer; patients with a family history of colorectal cancer have an increased risk of developing colorectal cancer, and patients with familial adenomatous polyposis and hereditary non-polyposis colorectal cancer have a low onset age; the older the age, the higher the incidence of colorectal cancer: 31% of colorectal cancers globally occur in the elderly over 75 years old; inflammations 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 the gut microbiota and the change in the abundance of specific gut bacteria have also become risk factors for colorectal cancer. The imbalance of the gut microbiota can cause inflammatory responses, disrupt the gut microenvironment, and exacerbate 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 will activate β-catenin, on the one hand, triggering an inflammatory response to change the immune system, and on the other hand, stimulating the growth of cancer cells. Bacteroides fragilis toxin-producing strains can reduce the body's immune surveillance and killing ability of tumor cells by inhibiting the activities of natural killer cells and cytotoxic T lymphocytes; promote the proliferation and activation of regulatory T cells, creating an immune escape environment for the growth of tumor cells. In the case of gut microbiota imbalance, the metabolism of the gut microbiota metabolite bile acid changes, resulting in 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 transduction.

[0003] Currently, the main diagnostic methods for cancer include imaging examinations, tissue biopsies, etc., but these methods have certain disadvantages: imaging examinations have strong radiation, and tissue biopsies are invasive and may bring complications. Detecting changes in fecal metabolites is of guiding significance for cancer diagnosis. Fecal samples are easy to obtain, the operation method is non-invasive, avoiding the risks brought by invasive operations, and patients have a high acceptance, and it is expected to be used as a biomarker and treatment target 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] In view of the fact that the current diagnosis of colorectal cancer is usually invasive, the present invention provides an application of thymidine-5'-monophosphate, a metabolite of the intestinal flora, as a diagnostic, prognostic biomarker and therapeutic target for colorectal cancer.

[0006] By comparing the omics data of metabolites of the intestinal flora between healthy people and colorectal cancer patients and verifying at the in vitro and in vivo levels, the present invention has screened that thymidine-5'-monophosphate has the potential to be a diagnostic biomarker for colorectal cancer. The fecal samples required for detection are easy to obtain, and the non-invasive operation improves the compliance of patients, and it is expected to improve the early diagnosis rate of colorectal cancer.

[0007] Another object of the present invention is to provide an application of a reagent for detecting the expression level of thymidine-5'-monophosphate in a sample in the preparation of a product for diagnosing colorectal cancer.

[0008] Another object of the present invention is to provide an application of a reagent for detecting the expression level of thymidine-5'-monophosphate in a sample in the preparation of a product for diagnosing the prognosis of colorectal cancer treatment.

[0009] The said application includes:

[0010] At the in vitro level, the effect of thymidine-5'-monophosphate on the proliferation of colorectal cancer cells is concentration-dependent and time-dependent. It can promote the colony formation of colorectal cancer cells, significantly increase the proportion of EdU-positive red-stained cells, promote the healing of scratches of colorectal cancer cells, and promote the migration of colorectal cancer cells. It has the potential to be a diagnostic and prognostic biomarker and therapeutic target for colorectal cancer.

[0011] At the in vivo level, thymidine-5'-monophosphate can significantly increase the volume and weight of transplanted tumors. The cell density in the transplanted tumors is significantly increased, the cell nuclei are enlarged, the number of cells with mitotic figures is increased, the cell atypia is obvious, the cells are in an active state of proliferation and division, and the proportion of positive cells of the proliferation marker Ki67 is significantly increased. Thymidine-5'-monophosphate has the potential to be a diagnostic and prognostic biomarker and therapeutic target for colorectal cancer.

[0012] Another object of the present invention is to provide a kit for diagnosing colorectal cancer and treating the prognosis of rectal cancer. The kit includes a biomarker: thymidine-5'-monophosphate.

[0013] In addition to the biomarker, the kit also includes other components: a thymidine-5'-monophosphate standard and an internal standard chlorophenylalanine solution.

[0014] The biomarker of the present invention is obtained through a large number of experiments:

[0015] 1. Screening metabolites of the intestinal flora with carcinogenic promotion effects

[0016] By comparing the omics data of gut microbiota metabolites between healthy individuals and colorectal cancer patients, five significantly differentially expressed metabolites were initially screened out. The CCK-8 assay was used to determine the effects of different gut microbiota metabolites on the cell proliferation rates of human colon cancer cell line HCT116 and murine colon adenocarcinoma cell line MC38, so as to identify the metabolites that have a real impact on cancer cell proliferation.

[0017] The CCK-8 assay was used to detect whether the proliferation of colorectal cancer cells by gut microbiota metabolites was concentration-dependent. HCT116 or MC38 cells were seeded in 96-well plates (2×10 3 ), and after incubation with different concentrations of gut microbiota metabolites for 24 h, 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 measured using a microplate reader. Among them, wells with only medium without cells were set as blank wells; wells with cells but without drugs in the medium were set as control wells. Cell viability (%) = (OD value of the drug-treated wells - OD value of the blank wells) / (OD value of the control wells - OD value of the blank wells) × 100%.

[0018] The CCK-8 assay was used to detect whether the proliferation of colorectal cancer cells by gut microbiota metabolites was time-dependent. HCT116 or MC38 cells were seeded in 96-well plates (2×10 3 ), and after incubation with 25 μmol / L and 400 μmol / L thymidine-5′-monophosphate for 24, 48, and 72 h, 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 measured using a microplate reader.

[0019] 2. Promoting cancer effect of gut microbiota metabolite thymidine-5′-monophosphate at the in vitro level

[0020] The colony formation assay and EdU assay were used to further verify the promoting proliferation effect of thymidine-5′-monophosphate on colorectal cancer cell lines HCT116 and MC38.

[0021] HCT116 cells or MC38 cells were seeded in 6-well plates (8×10 2 ), and the medium was changed every 2 days with fresh medium containing 400 μmol / L thymidine-5′-monophosphate. Cultivation was stopped when a large number of clones formed in the well plates. Cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.25% crystal violet for 15 min, then pictures were taken and the clones were counted.

[0022] HCT116 cells or MC38 cells were seeded in 6-well plates (2×10 5) After treatment with 400 μmol / L thymidine-5'-monophosphate for 24 h, the medium was changed to a medium containing 50 μmol / L EdU to label the DNA of proliferating cells, and incubation was continued at 37 °C for 2 h. Fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.3% Triton-X, incubated with the reaction solution for 30 min, stained with Hoechst 33342 for nuclear staining for 10 min, and observed and imaged under a fluorescence microscope.

[0023] Cell scratch assay and Transwell assay were used to detect the effect of thymidine-5'-monophosphate on the migration ability of HCT116 and MC38 cells.

[0024] Colorectal cancer cells were seeded in 24-well plates. When the cells grew to confluence and formed a confluent monolayer, a 10-μl pipette tip was used to scratch the cell monolayer along a straight line to form a scratch. After washing twice with PBS, the cells were incubated in serum-free medium supplemented with 400 μmol / L thymidine-5'-monophosphate or an equal volume of PBS. Images of the cell scratch were taken under a microscope at 0, 24, and 48 h, respectively, and the scratch distance was measured using ImageJ software. Cell migration rate (%) = (scratch distance at 0 h - scratch distance at 24 h or 48 h) / scratch distance at 0 h × 100%.

[0025] An 8-μm pore Transwell chamber was used. HCT116 cells or MC38 cells were seeded in the upper chamber containing serum-free medium with 400 μmol / L thymidine-5'-monophosphate (1.5×10 5 ). 20% fetal bovine serum was added to the lower chamber medium. 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. Observed and imaged under an optical microscope.

[0026] TUNEL assay was used to detect the effect of thymidine-5'-monophosphate on the apoptosis of colorectal cancer cells. HCT116 cells or MC38 cells were seeded in 24-well plates (1×10 5 ). After treatment with 400 μmol / L thymidine-5'-monophosphate for 24 h, fixed with 4% paraformaldehyde at 4 °C for 25 min and permeabilized with 0.3% Triton-X for 5 min. After equilibration with the equilibration buffer for 30 min, the labeling solution was added dropwise to the well plate and incubated at 37 °C in the dark for 1 h. Stained with DAPI for nuclear staining for 5 min, and observed and imaged for green fluorescence under a fluorescence microscope.

[0027] 3. Promoting cancer effect of the gut microbiota metabolite thymidine-5'-monophosphate at in vivo levels

[0028] Five-week-old male C57BL / 6 mice were selected and randomly divided into a PBS control group and a thymidine-5′-monophosphate administration group (400 μmol / L), with 8 mice in each group. After 1 week of feeding adaptation, MC38 cells (8×10 5 ) were subcutaneously injected into the dorsal side of C57BL / 6 mice. When the tumor size reached 50 - 100 mm 3 , PBS or thymidine-5′-monophosphate was intraperitoneally injected every 2 days, with an injection volume of 100 μL / 10 g. The body weight of the mice was measured every 2 days, and the tumor diameter was measured with a caliper. When the tumor size reached 2000 mm 3 , the mice were sacrificed, and the transplanted tumors were completely dissected and weighed and measured for volume.

[0029] HE staining was used to analyze the pathological changes of tumor tissues after thymidine-5′-monophosphate treatment. After the tumor tissues were removed, they were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. The paraffin sections were dewaxed in xylene and then hydrated with gradient ethanol solutions. Hematoxylin was stained for 5 min and rinsed with running water. Eosin was stained for 10 s, and the floating color was washed off with distilled water. The tissue sections were successively immersed in 85%, 95%, and absolute ethanol for 2 min each for dehydration and in xylene for 2 min for clearing. Neutral resin was dropped for mounting and dried for storage. The mounted sections were observed under a microscope.

[0030] Immunohistochemical staining was performed on paraffin-embedded sections of mouse tumor tissues to detect the expression level of Ki67 in tumor tissues, reflecting the effect of thymidine-5′-monophosphate treatment on the proliferation activity of tumor cells in vivo. After the tumor tissue sections were baked in an oven at 60 °C for 1 h, they were successively soaked in xylene I and xylene II for 15 min each, and then successively immersed in absolute ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 min each for dewaxing and rehydration. The sections were placed in a repair box containing EDTA antigen repair solution, boiled at high temperature for 2 min and heated at low temperature for 10 min, and then the tissue sections were taken out and naturally cooled to room temperature for antigen repair. Use 3% H 2 O 2Incubate for 10 min to remove endogenous peroxidase. After the sections are slightly dried, add 50% goat serum and block at room temperature for 30 min. Absorb the surrounding liquid, add Ki67 primary antibody diluent, and incubate overnight at 4°C. The next day, take out the sections and warm them up for 1 h. Incubate with biotin-labeled secondary antibody at 37°C for 20 min. Wipe the liquid on the sections dry, add an appropriate amount of horseradish peroxidase-labeled tertiary antibody and incubate at 37°C for 30 min. Wipe the liquid on the sections dry, add DAB chromogenic solution, let it stand for staining for 3 min, and rinse with tap water to terminate the chromogenesis. Stain with hematoxylin for 10 s for nuclear counterstaining. The tissue sections are successively immersed in 85%, 95%, and absolute ethanol for 2 min each for dehydration and clearing. After air-drying, add neutral resin for mounting and store in a dry place. Observe and collect pictures under a microscope, and quantify them using Image J software.

[0031] 4. Detection of Thymidine-5′-Monophosphate, a Metabolite of Intestinal Flora, in Fecal Samples

[0032] Collect fecal samples from colorectal cancer patients, quickly freeze them in liquid nitrogen, and store them at -80°C. Take the fecal samples stored at -80°C, weigh 50 mg and put them into a 1.5 mL centrifuge tube, add 200 μL of ultrapure water, add zirconia beads, grind them in a tissue grinder, centrifuge at 20,000 rpm for 10 min, aspirate 100 μL of the supernatant into a 1.5 mL centrifuge tube, add 400 μL of methanol (the internal standard is pre-added to the methanol, and the final concentration of the internal standard in methanol is 500 ng / mL), vortex for 1 min, centrifuge at 20,000 rpm for 10 min, take 400 μL of the supernatant, continue to centrifuge at 20,000 rpm for 10 min, take 400 μL of the supernatant into a 1.5 mL centrifuge tube, and evaporate to dryness in a vacuum integrated dryer.

[0033] Weigh 10 mg of thymidine-5′-monophosphate standard product, dissolve it in DMSO to prepare a thymidine-5′-monophosphate stock solution with a concentration of 10 mg / mL; then use methanol as the solvent to dilute to 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 and mix the standard curve working solutions, take 100 μL and put it into a 1.5 mL centrifuge tube, add 400 μL of methanol (the internal standard is added to the methanol, and the final concentration of the internal standard is 500 ng / mL), vortex for 1 min, centrifuge at 20,000 rpm for 10 min, take 400 μL of the supernatant, continue to centrifuge at 20,000 rpm for 10 min, take 400 μL of the supernatant into a 1.5 mL centrifuge tube, and evaporate to dryness in a vacuum integrated dryer.

[0034] Take the dried sample, add 100 μL of 50% methanol to each sample, vortex for 1 min to redissolve, centrifuge twice, centrifuge at 20,000 rpm for 10 min each time, and take the supernatant for injection.

[0035] Perform liquid chromatography separation and analysis using a Shimadzu liquid chromatography instrument. The chromatographic column is an Amide Bridge HPLC column, the column temperature is 40 °C, the injection volume is 5 μL, the flow rate is 0.4 mL / min, mobile phase A is ultrapure water containing 0.1% formic acid and 5 mM ammonium formate, and mobile phase B is acetonitrile. The solvent gradient is 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 is an AB SCIEX QTRAP 6500 analysis system, the scanning duration is 14 min, the positive ion detection mode is used, the detection ion pair of thymidine-5′-monophosphate is 125.0 / 79.0, and the detection ion pair of chlorophenylalanine (internal standard) is 200.0 / 154.0.

[0036] After the detection of the standard sample, use the analysis software to integrate the peaks of thymidine-5′-monophosphate and the internal standard peak, and statistically analyze the integrated peak area. Use the concentration of thymidine-5′-monophosphate as the abscissa and the ratio of the peak area of thymidine-5′-monophosphate to the internal standard peak as the ordinate to make a standard curve of thymidine-5′-monophosphate, and calculate the concentration of thymidine-5′-monophosphate in the fecal sample from the standard curve.

[0037] Advantages of the present invention:

[0038] Currently, the main diagnostic methods for cancer, such as imaging examinations and invasive tissue biopsies, have strong radiation and invasiveness. This project uses the intestinal flora metabolite thymidine-5′-monophosphate as a diagnostic marker for colorectal cancer. The fecal sample required for detection is easy to obtain, and the non-invasive operation improves the compliance of patients. It can be used as a biomarker and therapeutic target for the diagnosis and prognosis of colorectal cancer.

[0039] For further explanations of the English and terms appearing in the specification:

[0040] HCT116: human colon cancer cells

[0041] MC38: mouse colon adenocarcinoma cells

[0042] CCK-8 reagent: Cell Counting Kit-8 reagent, which contains the water-soluble tetrazolium salt WST-8 and can be used for simple and accurate cell proliferation and toxicity analysis.

[0043] EdU: 5-Ethynyl-2'-deoxyuridine, a thymidine analogue, used for cell proliferation detection.

[0044] Triton X-100: Triton X-100, a non-ionic surfactant, used for permeabilizing cell membranes.

[0045] Transwell assay: Transwell migration or invasion assay experiment, used to study cell migration, chemotaxis and invasion in response to various stimuli (such as growth factors, chemokines or extracellular matrix components).

[0046] PBS: Phosphate Buffered Saline.

[0047] TUNEL assay: TdT-mediated dUTP Nick-End Labeling. When genomic DNA breaks, the exposed 3'-OH can be added with fluorescein (FITC)-labeled dUTP (fluorescein-dUTP) under the catalysis of terminal deoxynucleotidyl transferase (TdT), so that apoptosis can be detected by fluorescence microscopy or flow cytometry.

[0048] DAPI: 4',6-Diamidino-2-phenylindole, a fluorescent dye that can strongly bind to DNA.

[0049] HE staining: Hematoxylin-Eosin staining, a staining method used in histology and pathology.

[0050] EDTA: Ethylenediaminetetraacetic acid.

[0051] Ki67: A cell proliferation marker that can detect the proportion of cells in the cell cycle. The higher the positive rate of Ki67, the larger the proportion of tumor cells in the growth cycle and the faster the growth rate of the tumor.

[0052] DAB chromogenic solution: 3,3'-Diaminobenzidine, the most commonly used chromogenic substrate for horseradish peroxidase. Its chromogenic principle is that it loses electrons under the action of hydrogen peroxide, showing color change and accumulation, forming brown insoluble products. This reaction has high sensitivity and good specificity and is commonly used in immunohistochemistry, in situ hybridization, Western Blot and other membrane chromogenic reactions or for detecting endogenous peroxidase in cells or tissues. Description of the Drawings

[0053] Figure 1In Example 1 of the present invention, the effect of thymidine-5'-monophosphate on the proliferation of colorectal cancer cells was concentration- and time-dependent. A. The promoting effect of thymidine-5'-monophosphate on the proliferation of colorectal cancer cell line HCT116 was concentration-dependent. B. The promoting effect of thymidine-5'-monophosphate on the proliferation of colorectal cancer cell line MC38 was concentration-dependent. C. The promoting effect of xanthosine on the proliferation of colorectal cancer cell line HCT116 was concentration-dependent. D. The promoting effect of xanthosine on the proliferation of colorectal cancer cell line MC38 was concentration-dependent. E. Uric acid had no obvious promoting effect on the proliferation of colorectal cancer cell line HCT116. F. Uric acid had no obvious promoting effect on the proliferation of colorectal cancer cell line MC38. G. The inhibitory effect of citraconic acid on the proliferation of colorectal cancer cell line HCT116 was concentration-dependent. H. The inhibitory effect of citraconic acid on the proliferation of colorectal cancer cell line MC38 was concentration-dependent. I. The inhibitory effect of glutaric acid on the proliferation of colorectal cancer cell line HCT116 was concentration-dependent. J. The inhibitory effect of glutaric acid on the proliferation of colorectal cancer cell line MC38 was concentration-dependent. K. The promoting effect of thymidine-5'-monophosphate on the proliferation of colorectal cancer cell line HCT116 was time-dependent. L. The promoting effect of thymidine-5'-monophosphate on the proliferation of colorectal cancer cell line MC38 was time-dependent. *, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

[0054] Figure 2 In Example 2 of the present invention, the promoting effect of thymidine-5'-monophosphate on colorectal cancer cell lines HCT116 and MC38. A. Thymidine-5'-monophosphate promoted the formation of colonies of HCT116 and MC38 cells. B. Treatment with thymidine-5'-monophosphate significantly increased the proportion of EdU-positive red-stained cells in HCT116 cells. C. Treatment with thymidine-5'-monophosphate significantly increased the proportion of EdU-positive red-stained cells in MC38 cells. ***, p < 0.001, ****, p < 0.0001.

[0055] Figure 3 In Example 2 of the present invention, the effect of thymidine-5'-monophosphate on the migration ability of colorectal cancer cell lines HCT116 and MC38. A. Treatment with thymidine-5'-monophosphate shortened the distance between the two sides of the scratch of HCT116 cells and promoted the migration of HCT116 cells. B. Treatment with thymidine-5'-monophosphate shortened the distance between the two sides of the scratch of MC38 cells and promoted the migration of MC38 cells. C. Treatment with thymidine-5'-monophosphate 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. Figure 4 In Example 2 of the present invention, the effect of thymidine-5'-monophosphate on the apoptosis ability of colorectal cancer cell lines HCT116 and MC38

[0056] Figure 5This is the carcinogenic promotion effect of thymidine-5'-monophosphate in mice in Example 3 of the present invention. A Photos of transplanted tumors in mice. B Thymidine-5'-monophosphate promotes the growth of the volume of transplanted tumors in mice. C Thymidine-5'-monophosphate promotes the increase in the weight of transplanted tumors in mice. D Thymidine-5'-monophosphate treatment has no obvious effect on the body weight of mice. E HE staining analysis of the pathological changes of transplanted tumors after thymidine-5'-monophosphate treatment. F Thymidine-5'-monophosphate promotes the expression of the proliferation marker Ki67 in tumor tissues. *, p<0.05, ***, p<0.001. Detailed implementation manners

[0057] The present invention will be further described below through examples.

[0058] The products and their preparation methods according to the present invention will be 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.

[0059] Example 1 Screening of gut microbiota metabolites with carcinogenic promotion effects

[0060] By comparing the omics data of gut microbiota metabolites between healthy people and colorectal cancer patients, 5 metabolites with significantly different expressions were first screened out, in order of content: inosine, uric acid, thymidine-5'-monophosphate, citraconic acid, glutaric acid. The CCK-8 assay was used to determine the effects of different gut microbiota metabolites on the cell proliferation rates of human colon cancer cells HCT116 and murine colon adenocarcinoma cells MC38, so as to determine the metabolites that actually affect cancer cell proliferation.

[0061] HCT116 or MC38 cells were inoculated into 96-well plates (2×10 3 ). After incubating with different concentrations of gut microbiota metabolites for 24 h, 10 μL of CCK-8 reagent was added to each well. After incubating at 37 °C for 1 h, the OD value at 450 nm was detected using a microplate reader. At the same time, blank wells and control wells were set up: the blank wells only contained medium without inoculating cells, and the control wells inoculated cells but did not add 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 the gut microbiota metabolite thymidine-5'-monophosphate had an obvious promoting effect on the proliferation of colorectal cancer cells. As the concentration of thymidine-5'-monophosphate increased (0 μmol / L, 12.5 μmol / L, 25 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 400 μmol / L), the proliferation ability of HCT116 and MC38 cells was significantly enhanced, showing a concentration-dependent manner (Figure 1 A, B). The proliferative effect of the gut microbiota metabolite xanthosine on colorectal cancer cells HCT116 and MC38 is concentration-dependent ( Figure 1 C, D). The gut microbiota metabolite uric acid has no obvious effect on the proliferation ability of HCT116 and MC38 cells ( Figure 1 E, F). The gut microbiota metabolites citraconic acid and glutaric acid have an inhibitory effect on the proliferation of colorectal cancer cells HCT116 and MC38, which is concentration-dependent ( Figure 1 G - J). This invention mainly focuses on thymidine - 5′ - monophosphate.

[0062] HCT116 or MC38 cells were seeded in 96 - well plates (2×10 3 ), and 25 μmol / L and 400 μmol / L of thymidine - 5′ - monophosphate were added respectively. After culturing for 24, 48, and 72 h, 10 μL of CCK - 8 reagent was added to each well. After incubating at 37 °C for 1 h, the OD value at 450 nm was measured using an enzyme - linked immunosorbent assay reader. The results showed that both low - concentration and high - concentration thymidine - 5′ - monophosphate had a continuous promoting effect on the proliferation of colorectal cancer cells, which was time - dependent ( Figure 1 K, L).

[0063] Example 2 Promoting cancer effect of the gut microbiota metabolite thymidine - 5′ - monophosphate at the in vitro level

[0064] The plate colony formation assay and EdU assay were used to further verify the proliferative effect of thymidine - 5′ - monophosphate on colorectal cancer cells HCT116 and MC38.

[0065] HCT116 cells or MC38 cells were seeded in 6 - well plates (8×10 2 ), and the medium was changed every 2 days with fresh medium containing 400 μmol / L thymidine - 5′ - monophosphate. Culturing was stopped when a large number of clones formed in the well plates. The cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.25% crystal violet for 15 min, then pictures were taken and the clones were counted. The results showed that the number of clone formations of HCT116 cells and MC38 cells treated with thymidine - 5′ - monophosphate was significantly more than that of the control group, indicating that thymidine - 5′ - monophosphate has a significant ability to promote the proliferation of tumor cells ( Figure 2 A).

[0066] HCT116 cells or MC38 cells were seeded in 6 - well plates (2×10 5) After treatment with 400 μmol / L thymidine-5'-monophosphate for 24 h, the medium was replaced with a medium containing 50 μmol / L EdU to label the DNA of proliferating cells, and incubation was continued at 37 °C for 2 h. Fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.3% Triton-X, incubated with the reaction solution for 30 min, stained with Hoechst 33342 for nuclear staining for 10 min, and observed and images were collected under a fluorescence microscope. The results showed that the proportion of EdU-positive red-stained cells in the thymidine-5'-monophosphate treatment group was significantly higher than that in the control group, indicating that the cells entered the active proliferation phase, further confirming the promoting effect of thymidine-5'-monophosphate on the proliferation of colorectal cancer cells( Figure 2 B).

[0067] The effects of thymidine-5'-monophosphate on the migration ability of HCT116 and MC38 cells were detected by cell scratch assay and Transwell assay.

[0068] Colorectal cancer cells HCT116 or MC38 were seeded in 24-well plates. When the cells grew to confluence and formed a confluent monolayer, the cell monolayer was scratched in a straight line with a 10 μl pipette tip to form a scratch. After washing twice with PBS, incubation was carried out in serum-free medium supplemented with 400 μmol / L thymidine-5'-monophosphate or an equal volume of PBS. Images of the cell scratch were taken under a microscope at 0, 24, and 48 h, and the scratch distance was measured using ImageJ software. Cell migration rate (%) = (scratch distance at 0 h - scratch distance at 24 h or 48 h) / scratch distance at 0 h × 100%. The results showed that after continued incubation of HCT116 cells in serum-free medium for 24 h and 48 h, compared with the control group, the cell spacing on both sides of the scratch in the thymidine-5'-monophosphate treatment group was significantly shortened, and cell migration was active. After continued incubation of MC38 cells in serum-free medium for 24 h, the cell spacing on both sides of the scratch was significantly shortened. After incubation for 48 h, the scratch in the thymidine-5'-monophosphate treatment group was basically healed, and the distribution of the scratch and surrounding cells tended to be uniform. Thymidine-5'-monophosphate has a promoting effect on the migration of colorectal cancer cells( Figure 3 A, B).

[0069] Using an 8 μm pore Transwell chamber, HCT116 cells or MC38 cells were seeded in the upper chamber of serum-free medium containing 40 μmol / L thymidine-5'-monophosphate (1.5×10 5), 20% fetal bovine serum was added to the lower chamber medium. 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. Images were observed and collected under an optical microscope. The results showed that compared with the control group, the number of migrated cells of HCT116 and MC38 cells treated with thymidine-5′-monophosphate was significantly increased, further indicating that thymidine-5′-monophosphate has a promoting effect on the migration of colorectal cancer cells( Figure 3 C).

[0070] The effect of thymidine-5′-monophosphate on the apoptosis of colorectal cancer cells was detected by TUNEL assay. HCT116 cells or MC38 cells were seeded in 24-well plates (1×10 5 ), after treatment with 400 μmol / L thymidine-5′-monophosphate for 24 h, they were fixed with 4% paraformaldehyde at 4 °C for 25 min and permeabilized with 0.3% Triton-X for 5 min. After equilibration with the equilibration buffer for 30 min, the labeling solution was added dropwise to the wells and incubated at 37 °C in the dark for 1 h. DAPI was added to stain the nuclei for 5 min, and green fluorescence was observed and images were collected under a fluorescence microscope. The results showed that there was no significant change in the proportion of green-stained cells in the thymidine-5′-monophosphate treatment group compared with the control group, indicating that thymidine-5′-monophosphate could not inhibit the apoptosis of tumor cells( Figure 4 ).

[0071] Through the above experiments, it was comprehensively proved that thymidine-5′-monophosphate has an obvious promoting effect on the proliferation and migration of colorectal cancer at the in vitro level, and at the same time has no inhibitory effect on cell apoptosis. The intestinal flora metabolite thymidine-5′-monophosphate has the potential to be used as a biomarker and therapeutic target for the diagnosis and prognosis of colorectal cancer.

[0072] Example 3 Promoting cancer effect of intestinal flora metabolite thymidine-5′-monophosphate at the in vivo level

[0073] Five-week-old male C57BL / 6 mice were selected and randomly divided into a PBS control group and a thymidine-5′-monophosphate administration group (400 μmol / L), with 8 mice in each group. After 1 week of adaptation to feeding, MC38 cells (8×10 5) Subcutaneously inject into the dorsal side of C57BL / 6 mice. When the tumor size reaches 50 - 100 mm3, intraperitoneally inject PBS or thymidine-5′-monophosphate every 2 days with an injection volume of 100 μL / 10 g. Weigh the mice every 2 days and measure the tumor diameter with calipers. When the tumor size reaches 2000 mm3, sacrifice the mice, completely dissect the transplanted tumors and weigh and measure the volume. The results show that over time, the tumor volume of the control group mice gradually increases, but the growth rate is relatively slow, while the tumor volume of the mice in the thymidine-5′-monophosphate administration group increases significantly faster than that of the control group, and the tumor weight is also significantly higher than that of the control group( Figure 5 A, B, C). At the same time, the treatment with thymidine-5′-monophosphate has no obvious effect on the body weight of the mice( Figure 5 D).

[0074] Use HE staining to analyze the pathological changes of the tumor tissue after treatment with thymidine-5′-monophosphate. After the tumor tissue is taken out, it is fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. The paraffin sections are dewaxed in xylene and then hydrated with a gradient ethanol solution. Stain with hematoxylin for 5 min and rinse with running water. Stain with eosin for 10 s and wash off the floating color with distilled water. The tissue sections are successively immersed in 85%, 95%, and absolute ethanol for 2 min each for dehydration and in xylene for 2 min for clearing. Drop neutral resin for mounting and store after drying. Place the mounted sections under a microscope for observation. The results show that compared with the colorectal cancer model group, the density of transplanted tumor cells in the thymidine-5′-monophosphate treatment group is significantly increased, the cell nuclei are enlarged, the number of cells with mitotic figures increases, and the cell atypia is obvious, indicating that the cells are in an active proliferation and division state, suggesting that thymidine-5′-monophosphate has an obvious promoting effect on the proliferation of tumor cells( Figure 5 E).

[0075] Perform immunohistochemical staining on paraffin-embedded sections of mouse tumor tissue to detect the expression level of Ki67 in the tumor tissue, reflecting the effect of thymidine-5′-monophosphate treatment on the proliferation activity of tumor cells in vivo. After baking the tumor tissue sections in an oven at 60 °C for 1 h, soak them in xylene I and xylene II successively for 15 min each, and then successively immerse them in absolute ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 min each for dewaxing and rehydration. Place the sections in a repair box containing EDTA antigen retrieval solution, boil at high temperature for 2 min and heat at low temperature for 10 min, then take out the tissue sections and let them cool naturally to room temperature for antigen retrieval. Use 3% H 2 O 2Incubate for 10 min to remove endogenous peroxidase. After the sections are slightly dried, add 50% goat serum and block at room temperature for 30 min. Absorb the surrounding liquid, add the Ki67 primary antibody dilution, and incubate overnight at 4°C. The next day, take out the sections and warm them up for 1 h. Incubate with biotin-labeled secondary antibody at 37°C for 20 min. Wipe the liquid on the sections dry, add an appropriate amount of horseradish peroxidase-labeled tertiary antibody and incubate at 37°C for 30 min. Wipe the liquid on the sections dry, add DAB chromogenic solution, let it stand for 3 min for staining, and rinse with tap water to terminate the chromogenesis. Stain with hematoxylin for 10 s for nuclear counterstaining. The tissue sections are successively immersed in 85%, 95%, and absolute ethanol for 2 min each for dehydration and clearing. After drying, add neutral resin for mounting and store in a dry place. Observe and collect pictures under a microscope, and quantify them using Image J software. The results show that compared with the model group, the proportion of positive cells of the proliferation marker Ki67 is significantly increased in the thymidine-5′-monophosphate administration group, indicating that thymidine-5′-monophosphate has a significant effect on promoting the proliferation of colorectal cancer cells( Figure 5 F).

[0076] The results of in vivo studies show that the gut microbiota metabolite thymidine-5′-monophosphate has a significant effect on promoting the proliferation of colorectal cancer cells and has the potential to be used as a biomarker and therapeutic target for the diagnosis and prognosis of colorectal cancer.

[0077] Example 4 Detection of gut microbiota metabolite thymidine-5′-monophosphate in fecal samples

[0078] Collect fecal samples from colorectal cancer patients, quickly freeze them in liquid nitrogen, and store them at -80°C. Take the fecal samples stored at -80°C, weigh 50 mg into a 1.5 mL centrifuge tube, add 200 μL of ultrapure water, add zirconia beads, grind them in a tissue grinder, centrifuge at 20,000 rpm for 10 min, pipette 100 μL of the supernatant into a 1.5 mL centrifuge tube, add 400 μL of methanol (the internal standard is pre-added to the methanol, and the final concentration of the internal standard in methanol is 500 ng / mL), vortex for 1 min, centrifuge at 20,000 rpm for 10 min, take 400 μL of the supernatant, continue to centrifuge at 20,000 rpm for 10 min, take 400 μL of the supernatant into a 1.5 mL centrifuge tube, and dry it in a vacuum integrated dryer.

[0079] Weigh 10 mg of thymidine-5′-monophosphate standard, dissolve it with DMSO to prepare a thymidine-5′-monophosphate stock solution with a concentration of 10 mg / mL; then use methanol as the solvent to dilute it to 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 and mix the standard curve working solutions evenly, take 100 μL and put it into a 1.5 mL centrifuge tube, add 400 μL of methanol (the internal standard is added to the methanol, and the final concentration of the internal standard is 500 ng / mL), vortex and shake for 1 min, centrifuge at 20,000 rpm for 10 min, take 400 μL of the supernatant, continue to centrifuge at 20,000 rpm for 10 min, take 400 μL of the supernatant and put it into a 1.5 mL centrifuge tube, and dry it in a vacuum integrated dryer.

[0080] Take the dried sample, add 100 μL of 50% methanol to each sample, vortex for 1 min to redissolve, centrifuge twice, each time centrifuge at 20,000 rpm for 10 min, and take the supernatant for injection.

[0081] Use a Shimadzu liquid chromatography instrument for liquid chromatography separation and analysis. The chromatographic column is an Amide Bridge HPLC column, the column temperature is 40 °C, the injection volume is 5 μL, the flow rate is 0.4 mL / min, mobile phase A is ultrapure water containing 0.1% formic acid and 5 mM ammonium formate, and mobile phase B is acetonitrile. The solvent gradient is 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 is an AB SCIEX QTRAP 6500 analysis system, the scanning duration is 14 min, the positive ion detection mode is used, the detection ion pair of thymidine-5′-monophosphate is 125.0 / 79.0, and the detection ion pair of chlorophenylalanine (internal standard) is 200.0 / 154.0.

[0082] After the detection of the standard sample, use the analysis software to integrate the thymidine-5′-monophosphate peak and the internal standard peak, count the integrated peak area, use the thymidine-5′-monophosphate concentration as the abscissa, and the ratio of the thymidine-5′-monophosphate peak area to the internal standard peak area as the ordinate to make a thymidine-5′-monophosphate standard curve: y = 0.0172x + 0.00877, r = 0.9956, and calculate the thymidine-5′-monophosphate concentration in the fecal sample according to the thymidine-5′-monophosphate standard curve.

[0083] The above is only the preferred embodiment of the present invention. It should be noted that the embodiments of the present invention are not limited by the described examples. 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 protection scope of the present invention.

Claims

1. Use of a reagent for detecting the expression level of thymidine-5′-monophosphate in a sample in the preparation of a product for diagnosing colorectal cancer.

2. Use of a reagent for detecting the expression level of thymidine-5′-monophosphate in a sample in the preparation of a product for diagnosing the prognosis of colorectal cancer treatment.

3. The use according to claim 1 or 2, characterized in that: The effect of thymidine-5′-monophosphate on the proliferation of colorectal cancer cells is concentration-dependent and time-dependent.

4. The use according to claim 1 or 2, characterized in that: Thymidine-5′-monophosphate 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: Thymidine-5′-monophosphate can promote the healing of scratches in colorectal cancer cells.

6. The use according to claim 1 or 2, characterized in that: Thymidine-5′-monophosphate can promote the migration of colorectal cancer cells.

7. The use according to claim 1 or 2, characterized in that: Thymidine-5′-monophosphate can significantly increase the volume and weight of transplanted tumors. The cell density in the transplanted tumors is significantly increased, the cell nuclei are enlarged, the number of cells showing nuclear division increases, the cell atypia is obvious, the cells are in an active state of proliferation and division, and the proportion of positive cells for the proliferation marker Ki67 is significantly increased.

8. A kit for diagnosing colorectal cancer and treating colorectal cancer prognosis, the kit comprising a biomarker: thymidine-5′-monophosphate.

9. The kit according to claim 8, characterized in that Kit includes: Thymidine-5′-monophosphate standard and internal standard chlorophenylalanine solution.

Images