Application of lactobacillus johnsonii in colorectal tumor

Abstract

The invention relates to the technical field of biology, in particular to application of lactobacillus johnsonii in colorectal tumor. According to the application, low-abundance lactobacillus johnsonii is used as a biomarker of colorectal tumors, and the abundance ratio of the low-abundance lactobacillus johnsonii to normal-abundance lactobacillus johnsonii is smaller than or equal to 0.9. Based on a large number of experiments in the early stage, the abundance of lactobacillus johnsonii in the intestinal tract of a patient with colorectal tumor shows a significant reduction trend; in addition, the abundance of the lactobacillus johnsonii in the intestinal tract of the patient with colorectal tumor is highly positively correlated with the beneficial bacterium bacteroides simplex in the intestinal tract and is negatively correlated with harmful bacteria in the intestinal tract; in addition, lactobacillus johnsonii is used for intragastric administration, and the number and the volume of tumors of a mouse with colorectal tumors are obviously reduced, so that the lactobacillus johnsonii can be used as a biomarker of the colorectal tumors, and the blank of application of the lactobacillus johnsonii in the aspect of the colorectal tumors in the prior art is filled.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, and in particular to application of Lactobacillus johnsonii in colorectal tumor. BACKGROUND

[0002] Colorectal cancer (CRC) is one of the common malignant tumors of the digestive system, and more than 90% of colorectal cancers occur through three steps: the first step is colon polyp, the second step is surface dysplasia, and the third step is colorectal cancer. Current research shows that the malignant process from polyp to cancer usually takes 5 to 10 years, so timely early treatment can effectively inhibit the development of colorectal tumors. However, the current treatment methods for colorectal tumors include chemotherapy, radiotherapy and surgical treatment, which have serious side effects, including nausea, fatigue, hair loss and digestive problems. Therefore, it is urgent to seek a healthy and drug-resistant treatment for colorectal cancer.

[0003] The occurrence of CRC is closely related to the intestinal microbiota. Current research shows that the composition and abundance of intestinal microorganisms are closely related to the occurrence and development of CRC. For example, certain intestinal beneficial strains can affect the progress of CRC by regulating immune response, affecting tumor microenvironment or producing metabolites. Therefore, it is of great significance to deeply study the mechanism of intestinal microorganisms in the treatment of CRC. Finding new treatment mechanisms from the perspective of intestinal microorganisms can provide new ideas and strategies for solving the problems in the treatment of colorectal cancer.

[0004] Currently, Lactobacillus and Bifidobacterium are two beneficial bacterial genera widely reported in intestinal microorganisms, and L. johnsonii has been used as probiotics to treat various diseases. It has the functions of perfecting the development of the lungs of newborn mice, relieving necrotic enteritis, improving memory disorders, enhancing the morphology and function of mitochondria in the liver and reducing liver fat. In addition, studies have shown that L. johnsonii can improve whole-body glucose metabolism. However, there is currently no application of L. johnsonii in colorectal tumors. SUMMARY

[0005] The present application provides an application of L. johnsonii in colorectal tumors to solve the technical problem of filling the gap in the application of L. johnsonii in colorectal tumors in the prior art.

[0006] In a first aspect, the present application provides an application of L. johnsonii as a biomarker in colorectal tumors, which comprises:

[0007] The low-abundance Lactobacillus johnsonii is used as a biomarker of colorectal tumor, wherein the abundance ratio of the low-abundance Lactobacillus johnsonii to normal-abundance Lactobacillus johnsonii is less than or equal to 0.9.

[0008] Optionally, the abundance of the Lactobacillus johnsonii is negatively correlated with the degree of the colorectal tumor.

[0009] Optionally, the biomarker of the colorectal tumor further comprises intestinal beneficial bacteria, and the abundance of the Lactobacillus johnsonii is positively correlated with the abundance of the intestinal beneficial bacteria.

[0010] Optionally, the intestinal beneficial bacteria comprise at least one of the following:

[0011] Bacteroides uniformis, Lactobacillus rhamnosus and Eubacterium sp. 14-2.

[0012] Optionally, the biomarker of the colorectal tumor further comprises intestinal harmful bacteria, and the abundance of the Lactobacillus johnsonii is negatively correlated with the abundance of the intestinal harmful bacteria.

[0013] Optionally, the intestinal harmful bacteria comprise at least one of the following:

[0014] Anaerotruncus sp. G3 (2012), Turicimonas. Muris and Lleibacterium. Valens.

[0015] In a second aspect, the present application provides a therapeutic drug for colorectal tumor, wherein the therapeutic drug comprises an activator for increasing the abundance of Lactobacillus johnsonii and / or a bacterial preparation containing Lactobacillus johnsonii.

[0016] Optionally, when the therapeutic drug is the bacterial preparation containing Lactobacillus johnsonii, the concentration of Lactobacillus johnsonii in the bacterial preparation is greater than or equal to 1.0 x 10 8 CFU / mL.

[0017] In a third aspect, the present application provides a reagent for screening or assisting in screening colorectal cancer, wherein the reagent comprises a medicament for detecting the abundance of Lactobacillus johnsonii.

[0018] In a fourth aspect, the present application provides a reagent for evaluating or assisting in evaluating the prognosis of colorectal cancer, wherein the reagent comprises a medicament for detecting the abundance of Lactobacillus johnsonii.

[0019] Compared with the prior art, the above technical solutions provided by the embodiments of the present application have the following advantages:

[0020] The application provides application of Lactobacillus johnsonii as a biomarker in colorectal tumors, and the application comprises: taking low-abundance Lactobacillus johnsonii as a biomarker of colorectal tumors, wherein the abundance ratio of the low-abundance Lactobacillus johnsonii to normal-abundance Lactobacillus johnsonii is less than or equal to 0.9. Based on a large number of previous experiments, it is found that the abundance of Lactobacillus johnsonii in the intestinal tract of a colorectal tumor patient presents a significant decreasing trend; in addition, the abundance of Lactobacillus johnsonii in the intestinal tract of the colorectal tumor patient is highly positively correlated with the intestinal beneficial bacteria Bacteroides uniformis, and the abundance of Lactobacillus johnsonii in the intestinal tract of the colorectal tumor patient is negatively correlated with intestinal harmful bacteria; in addition, it is found that the number and volume of tumors of a colorectal tumor mouse are significantly reduced by using Lactobacillus johnsonii for gavage treatment, which indicates that Lactobacillus johnsonii has the effect of relieving colorectal tumors, and the abundance of Lactobacillus johnsonii can measure the degree of colorectal tumors, so as to promote Lactobacillus johnsonii to be used as a biomarker of colorectal tumors, so as to fill the blank of application of Lactobacillus johnsonii in colorectal tumors in the prior art. BRIEF DESCRIPTION OF DRAWINGS

[0021] The drawings incorporated into the specification and constituting a part of the specification show embodiments consistent with the application and, together with the specification, serve to explain the principles of the application.

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, those skilled in the art can obtain other drawings according to these drawings without any creative effort.

[0023] Figure 1 The distribution relationship of the abundance of Lactobacillus johnsonii in the intestinal tract of a colorectal patient is provided in the embodiments of the application, wherein, Figure 1 A is a comparative analysis of Lactobacillus johnsonii in the intestinal tract of a colorectal patient and a healthy person, Figure 1 B is a correlation analysis graph of Lactobacillus johnsonii and intestinal beneficial bacteria and intestinal harmful bacteria respectively;

[0024] Figure 2 The experiment graph of Lactobacillus johnsonii inhibiting the colorectal tumor process is provided in the embodiments of the application, wherein, Figure 2 A is an experiment step graph of Lactobacillus johnsonii inhibiting the colorectal tumor process, Figure 2 B is a body weight curve graph of mice subjected to different gavage treatments, Figure 2 C is a statistical graph of stool blood time of mice subjected to different gavage treatments, Figure 2 D is a quantitative schematic diagram of large intestinal tissue tumors, tumor number and tumor volume of mice subjected to different gavage treatments;

[0025] Figure 3Staining section images of liver and kidney of mice with different gavage treatments provided by the embodiments of the present application, wherein, Figure 3 A is a staining section image of liver of mice with different gavage treatments, Figure 3 B is a staining section image of kidney with different gavage treatments;

[0026] Figure 4 Abundance result graphs of intestinal beneficial bacteria and intestinal harmful bacteria of mice with different gavage treatments provided by the embodiments of the present application, wherein, Figure 4 A is an intestinal microorganism α diversity result graph of mice with different gavage treatments, Figure 4 B is an intestinal microorganism β diversity result graph of mice with different gavage treatments, Figure 4 C is a macro-genome heat map of intestinal microorganism of mice with different gavage treatments. DETAILED DESCRIPTION

[0027] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0028] Various embodiments of the present application can exist in the form of a range; it should be understood that the description in the form of a range is merely for the convenience and brevity, and should not be understood as a hard limit on the scope of the present application; therefore, it should be considered that the described range has specifically disclosed all possible sub-ranges and single values in the range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers in the described range, such as 1, 2, 3, 4, 5 and 6, which applies to any range; in addition, whenever a numerical range is indicated in the present application, it refers to any cited number (fraction or integer) in the indicated range.

[0029] In this article, the term includes "includes" and the like means "including but not limited to". The association relationship of "and / or" describing the associated objects means that there can be three kinds of relationships, for example, A and / or B can represent: the case of A alone, the case of A and B existing at the same time, and the case of B alone; Wherein A, B can be singular or plural. "At least one" means one or more, "multiple" means two or more; "At least one", "at least one of the following" or the like means any combination of these items, including single item or any combination of multiple items; For example, "at least one of a, b, or c", or "at least one of a, b, and c", can represent a, b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, where a, b, c can be single or multiple. Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or can be prepared by existing methods.

[0030] It should be noted that the potential mechanisms of intestinal microorganisms in alleviating colorectal tumor progression mainly include the following aspects:

[0031] (1) Regulating immune response: Intestinal microorganisms can affect the host's immune system, regulate the integrity of the intestinal mucosal barrier and immune response. Some probiotics or beneficial bacteria such as lactic acid bacteria and bifidobacterium may enhance the intestinal immune response by promoting the activation of immune cells and the release of cytokines, thereby inhibiting the growth and spread of tumors. (2) Regulating tumor microenvironment: Intestinal microorganisms affect the formation of colorectal cancer tumor microenvironment by regulating the intestinal environment, including pH value, oxygen concentration, nutrient utilization, etc. Normal intestinal flora can inhibit the growth of harmful bacteria and prevent the occurrence of inflammatory response, thereby reducing the risk of tumor occurrence. (3) Producing beneficial metabolites: Intestinal microbial metabolites also play an important role in the development of colorectal cancer. For example, certain beneficial bacteria can metabolize dietary fiber to produce beneficial metabolites such as short-chain fatty acids, which can lower the pH in the intestine, inhibit the growth of harmful bacteria, and also inhibit tumor development by promoting the health of intestinal epithelial cells and enhancing immune function. (4) Affecting host metabolism and nutrient absorption: Intestinal microorganisms can regulate host energy metabolism and nutrient absorption, affecting the development of colorectal cancer. For example, intestinal microorganisms can affect nutrient absorption and fatty acid metabolism in the intestine, thereby affecting the host's intestinal health and immune function, indirectly affecting the development of colorectal cancer. Therefore, it has very promising prospects to use the characteristics of intestinal flora to slow down the incidence of colorectal tumors and enhance the efficacy of chemotherapy or immunotherapy.

[0032] The application provides an application of lactobacillus johnsonii as a biomarker in colorectal tumors, which comprises:

[0033] The low-abundance Lactobacillus johnsonii is used as a biomarker of colorectal tumor, wherein the abundance ratio of the low-abundance Lactobacillus johnsonii to normal-abundance Lactobacillus johnsonii is ≤0.9.

[0034] In some optional embodiments, the abundance of the Lactobacillus johnsonii is negatively correlated with the degree of the colorectal tumor.

[0035] In these embodiments, it is found through preliminary experiments that the smaller the abundance of the Lactobacillus johnsonii in the intestinal tract of the body, the larger the volume and the greater the number of the colorectal tumor in the body, which indicates that the abundance of the Lactobacillus johnsonii is generally negatively correlated with the degree of the colorectal tumor, and indirectly indicates that the abundance of the Lactobacillus johnsonii can be used as a biomarker to judge the colorectal tumor.

[0036] In some optional embodiments, the biomarker of the colorectal tumor further comprises intestinal beneficial bacteria, and the abundance of the Lactobacillus johnsonii is positively correlated with the abundance of the intestinal beneficial bacteria.

[0037] In some optional embodiments, the intestinal beneficial bacteria comprise at least one of the following:

[0038] Bacteroides uniformis, Lactobacillus rhamnosus, and Eubacterium sp. 14-2.

[0039] In these embodiments, the biomarker of the colorectal tumor can further comprise intestinal beneficial bacteria, and the abundance of the Lactobacillus johnsonii is generally positively correlated with the abundance of the intestinal beneficial bacteria. The intestinal beneficial bacteria can comprise at least one of Bacteroides uniformis, Lactobacillus rhamnosus, and Eubacterium sp. 14-2. The specific species of the intestinal beneficial bacteria that are positively correlated with the abundance of the Lactobacillus johnsonii can be determined, so that the abundance of the intestinal beneficial bacteria can be adjusted by adjusting the abundance of the Lactobacillus johnsonii to alleviate the progression of the colorectal tumor.

[0040] In some optional embodiments, the biomarker of the colorectal tumor further comprises intestinal harmful bacteria, and the abundance of the Lactobacillus johnsonii is negatively correlated with the abundance of the intestinal harmful bacteria.

[0041] In some optional embodiments, the intestinal harmful bacteria comprise at least one of the following:

[0042] Anaerotruncus sp. G3 (2012), Turicimonas. Muris, and Lleibacterium. Valens.

[0043] In these embodiments, the biomarker of the colorectal tumor can further include intestinal harmful bacteria, and the abundance of Lactobacillus johnsonii is generally negatively correlated with the abundance of the intestinal harmful bacteria, and the intestinal harmful bacteria can include at least one of Anaerotruncus sp. G3 (2012), Turicimonas. Muris, and Lleibacterium. Valens, and the specific species of the intestinal harmful bacteria that can be negatively correlated with the abundance of Lactobacillus johnsonii can be determined, so that the abundance of the intestinal harmful bacteria can be adjusted by adjusting the abundance of Lactobacillus johnsonii to alleviate the progression of the colorectal tumor.

[0044] Based on one general inventive concept, the embodiments of the present application provide a therapeutic drug for colorectal tumor, which includes an activator for increasing the abundance of Lactobacillus johnsonii and / or a bacterial preparation containing Lactobacillus johnsonii.

[0045] The therapeutic drug is based on the application of Lactobacillus johnsonii as a biomarker in colorectal tumor, and the specific principle of the application of Lactobacillus johnsonii as a biomarker in colorectal tumor can refer to the above embodiments. Since the therapeutic drug uses part or all of the technical solutions of the above embodiments, it at least has all the beneficial effects brought by the technical solutions of the above embodiments, which will not be repeated here.

[0046] In some optional embodiments, when the therapeutic drug is a bacterial preparation containing Lactobacillus johnsonii, the concentration of Lactobacillus johnsonii in the bacterial preparation is ≥1.0×10 8 CFU / mL;

[0047] In these embodiments, under the premise that the therapeutic drug is a bacterial preparation containing Lactobacillus johnsonii, the concentration of Lactobacillus johnsonii in the bacterial preparation is ≥1.0×10 8 CFU, which can cause the bacterial preparation to have sufficient Lactobacillus johnsonii to effectively increase the abundance of Lactobacillus johnsonii in the body, so as to increase the abundance of intestinal beneficial bacteria and reduce the abundance of intestinal harmful bacteria in the body, thereby alleviating the progression of the colorectal tumor.

[0048] Based on one general inventive concept, the embodiments of the present application provide a reagent for screening or assisting in screening colorectal cancer, which includes a medicament for detecting the abundance of Lactobacillus johnsonii.

[0049] The reagent is based on the application of Lactobacillus johnsonii as a biomarker in colorectal tumor, and the specific principle of the application of Lactobacillus johnsonii as a biomarker in colorectal tumor can refer to the above embodiments. Since the reagent uses part or all of the technical solutions of the above embodiments, it at least has all the beneficial effects brought by the technical solutions of the above embodiments, which will not be repeated here.

[0050] Based on a general inventive concept, the embodiments of the present application provide a reagent for evaluating or assisting in evaluating the prognosis effect of colorectal cancer, which comprises a reagent for detecting the abundance of Lactobacillus johnsonii.

[0051] The reagent is realized based on the application of Lactobacillus johnsonii as a biomarker in colorectal tumors, and the specific principles of the application of Lactobacillus johnsonii as a biomarker in colorectal tumors can be referred to the above embodiments. Since the reagent adopts part or all of the technical solutions of the above embodiments, it at least has all the beneficial effects brought by the technical solutions of the above embodiments, which will not be repeated here.

[0052] The present application will be further described below in conjunction with specific embodiments. The experimental methods not specified in the following embodiments are generally determined according to industry standards; if there is no corresponding industry standard, the general international standard, conventional conditions, or the conditions suggested by the manufacturer are followed.

[0053] Example 1

[0054] I. Test materials:

[0055] 1. Test animals

[0056] Apc Min / + mice of 4 weeks old and C57BL / 6J wild-type mice of 4 weeks old were purchased from the Animal Experimental Center of the Army Medical University, and all animal operations followed the relevant regulations of the Animal Research of the Army Medical University. Min / + Mice and 4-week-old C57BL / 6J wild-type mice, all animal operations followed the relevant regulations of the Animal Research of the Army Medical University.

[0057] II. Test methods:

[0058] 1. Mouse feeding

[0059] The mice in each group were adaptively fed with standard diet and drinking water for one week. The feed used for feeding the mice needs to be strictly sterilized by radiation, and the drinking water bottles, cages and bedding are sterilized by high-pressure steam before use. The breeding environment is SPF level, and the environmental temperature is set to 23-25°C. The automatic lighting device is adjusted according to the daylight time. All procedures follow the guidelines approved by the Animal Experiment Ethics Committee of Huazhong Agricultural University.

[0060] 2. Modeling and feeding of conventional colorectal cancer model mice

[0061] In the azoxymethane / dextran sodium sulfate (AOM / DSS) model, 4-week-old C57BL / 6 mice (purchased from Hunan SJA Experimental Animal Co., Ltd.) were first adaptively fed for one week, then randomly divided into two groups according to body weight, and fed with black rice feed (China Shenzhen Readydietech Co., Ltd.) and control feed, respectively, with 45 mice in each group.

[0062] At 8 weeks of age, mice were injected intraperitoneally with 10 mg / kg of AOM (Merck, Darmstadt, Germany) and then administered 3 cycles of DSS (MP Biomedicals, Solon, OH) to mimic CRC associated with colitis. Each cycle was 7 days with 2.0% DSS added to the drinking water, followed by 14 days of regular drinking water. Mice were housed in a temperature-controlled, specific pathogen-free environment and subjected to a 12-h light / dark cycle; no voluntary culling of mice was performed throughout the experiment. All procedures were in accordance with the guidelines approved by the Animal Experimental Ethics Committee of Huazhong Agricultural University.

[0063] 3. HE staining

[0064] Isolation of Apc Min / + The small and large intestinal segments of mice and WT mice were fixed with 4% paraformaldehyde, paraffin sections were made for HE staining, and the histological morphology of the small and large intestines was observed. Image J software was used to analyze the Apc Min / Differences in intestinal tissue structure integrity. The specific steps for the preparation process of HE staining paraffin sections are as follows:

[0065] (1) Sampling and fixation: Fresh tissue blocks (generally not more than 0.5 cm thick) were taken from animals and placed in pre-prepared fixatives (10% formalin, Bouin's fixative) to denature and coagulate the proteins of the tissues and cells, preventing autolysis or bacterial decomposition after cell death, thereby maintaining the original morphological structure of the cells.

[0066] (2) Dehydration and transparency: Generally, low-concentration to high-concentration alcohol is used as a dehydrating agent to gradually remove water from the tissue blocks. Then, the tissue blocks are placed in dimethylbenzene, which is soluble in alcohol and wax, to make them transparent. Dimethylbenzene is used to replace the alcohol in the tissue blocks, and then the tissue blocks are immersed in wax for embedding.

[0067] (3) Wax immersion and embedding: Place the transparent tissue blocks in melted wax and store them in a wax melting box for preservation. After the wax completely penetrates the tissue blocks, embedding is performed: prepare a container (such as a small folded paper box), pour the melted wax into it, quickly pick up the tissue blocks that have been soaked in wax, and place them in the container. After cooling and solidification, the embedded tissue blocks become hard, allowing them to be cut into thin sections on a microtome.

[0068] (4) Sectioning and mounting: Fix the embedded wax blocks on a microtome and cut them into thin sections, typically 5-8 μm thick. The cut sections are often wrinkled and need to be flattened by heating in water, then mounted on glass slides and placed in a 45°C constant temperature oven for drying.

[0069] (5) Dewaxing: HE staining is commonly used to increase the color difference of each part of the tissue cell structure, which is conducive to observation; hematoxylin (H) is an alkaline dye that can dye the cell nucleus and intracellular ribosomes into blue-violet, and the structure dyed by the alkaline dye has alkaliphilia; eosin (E) is an acid dye that can dye the cytoplasm into red or light red, and the structure dyed by the acid dye has acidophilia; before staining, xylene is used to remove paraffin in the section, and then high-concentration to low-concentration alcohol is used, and finally distilled water is used for staining.

[0070] (6) Staining:

[0071] ① Put the section into the hematoxylin aqueous solution after entering the distilled water for several minutes;

[0072] ② Color separation in acid water and ammonia water, each for several seconds;

[0073] ③ After washing with running water for 1 hour, enter the distilled water for a moment;

[0074] ④ Dehydrate in 70% and 90% alcohol for 10 min each;

[0075] ⑤ Stain in alcohol eosin staining solution for 2-3 min.

[0076] (7) Dehydration and transparency: after staining, the section is dehydrated by pure alcohol, and then made transparent by xylene.

[0077] (8) Fixation: drop the transparent section with Canada balsam, cover with a cover glass for fixation, and after the balsam is slightly dry, paste the label, and the section specimen can be used.

[0078] Result analysis standard: HE is a method for checking tumor cells, which is observed under a microscope, and the metabolite nucleus presents purple blue, the cytoplasm presents light magenta red, and the red blood cells present light red.

[0079] III. Bioinformatics analysis

[0080] 1. Programming language and main software as shown in Table 1 and Table 2.

[0081] Table 1 Main programming languages

[0082] Name Version URL Linux 4.15.0 http: / / www.gnu.org / / software / bash / Python 3.6.3 https: / / ww.python.org / R 4.2.1 https: / / www.r-project.org /

[0083] Table 2 Main bioinformatics software

[0084]

[0085] The obtained data is analyzed on a local workstation.

[0086] 2. Analysis of metagenomic data:

[0087] (1) Library construction and sequencing

[0088] The collected mouse fecal samples were mailed to Huada Gene (Wuhan, China) for PE library construction. The sequencing process and instruments were provided by Huada Gene.

[0089] According to the conventional metagenomic analysis process, including high-quality reads for data quality control, host genome alignment, host contamination cleaning, and then downstream abundance construction and species / function difference analysis, the specific analysis process is as follows:

[0090] Sequence quality control: After the data is downloaded, the KneadData process (Beghini et al. 2021) is called on the Linux server for metagenomic data quality control. Trimmomatic (Bolger et al. 2014) is used to cut the adapter sequence of reads, and reads with length less than 50 bp, average base quality value less than 20 bp and containing N base are removed, to obtain better quality sequences for subsequent analysis.

[0091] Remove host contamination: Call the KneadData process to align the host genome sequence to the corresponding database and remove the host sequence. Bowtie2 (Langmead and Salzberg 2012) is used to align the genome reads with the human reference genome (hg19), and after removing the host contamination reads on the alignment, the double-end reads are combined for subsequent microbial species classification.

[0092] Species taxonomy annotation: Call MetaPhlAn2 (Truong et al. 2015) to calculate species abundance. Bowtie2 is used for comparison with microbial genome data. Then the species abundance data obtained by analysis is combined, classified according to kingdom, phylum, class, order, family, genus and species, and in addition, only bacterial species are analyzed for difference.

[0093] Microbial diversity analysis: For the study of microbial community diversity or ecological diversity, diversity index is usually used for evaluation and comparison. Commonly used diversity indexes include alpha diversity analysis and beta diversity analysis. Alpha diversity refers to the diversity within a specific environmental area or ecosystem, mainly reflecting the richness of species and the uniformity of individual distribution in the community. There are usually four indicators to describe alpha diversity: Observed species, Chao1, Shannon and Simpson. Among them, Observed species refers to the number of OTUs (species) actually contained in the sample, and Chao1 refers to the estimated number of OTUs in the sample. Both indexes can reflect the number of species in the sample. The higher the two indexes, the higher the species richness in the sample. Shannon and Simpson indexes can reflect the abundance distribution of each species in the sample. The more OTUs in the sample, the more uniform the abundance distribution, and the higher the alpha diversity index. The Shannon index and Simpson index were calculated using the diversity function of the vegan package (version 2.6-4) (Dixon 2003). Beta diversity analysis: The relative abundance table of species, genus and door classification was used to calculate the beta diversity. The vegdist function of the vegan package (version 2.6-4) was used to calculate the distance between species, and the Bray-Curtis-based distance was selected. The pcoa function in the ape package (version 5.6.2) was used for principal coordinates analysis (PCoA). The difference between different groups in PCoA analysis was analyzed by permutational multivariate analysis of variation (PERMANOVA), and the significance of the analysis was calculated by the adonis2 function of the pairwiseAdonis package (version 0.4). P<0.05 was statistically different.

[0094] Differential analysis: Differential analysis was performed on each group of mice. The analysis method was wilcoxon rank sum test. The P value obtained was corrected to obtain FDR value. FDR value and Log2 fold change (log2FC) were used for screening. FDR<0.05 and log2FC≥1 were up-regulated, and FDR<0.05 and log2FC≤-1 were down-regulated.

[0095] Microbial genome and pathway annotation: Humann3 (Beghini et al. 2021) is a computational tool for functional profiling analysis, which is used to analyze metagenomic sequencing data, especially for gene function analysis of microbial communities. The specific process includes:

[0096] (1) Bowtie2 is used to accelerate nucleic acid level search;

[0097] (2) Diamond is used to accelerate translational protein level search;

[0098] (3) Known and unknown biological analysis of population function: MetaPhlAn2 and ChocoPhlAn pan-genome database can obtain functional spectrum more quickly and accurately;

[0099] (5) Obtain results at the levels of genome, gene and pathway: UniRef database provides the definition of gene family; MetaCyc pathway gene pathway definition; MinPath provides a defined minimum path set.

[0100] 3. Metabolome data analysis:

[0101] (1) Non-targeted metabolome detection

[0102] The collected fecal samples of mice were mailed to Metware Biotechnology Co., Ltd. (Wuhan, China) for metabolome sequencing using liquid chromatography mass spectrometry (LC-MS). The instrument platform used for LC-MS analysis is the ultra-high performance liquid chromatography tandem time-of-flight mass spectrometry UPLC-TripleTOF system of AB SCIEX Company. The sequencing process and instrument are provided by Metware Biotechnology Co., Ltd.

[0103] (2) TM broad-target metabolome processing analysis

[0104] Data preprocessing: The metabolome data obtained by TM broad-target has some null values, and the null values in the original data are grouped and filled. For each metabolite in each group, if the number of missing values is greater than 70% of the total sample size, the metabolite is removed, and if the number of missing values is less than 70% of the total sample size, the missing values are filled with half of the minimum value of the metabolite. The data matrix is normalized by sum. Then remove the variables with relative standard deviation (RSD) greater than 20% of the QC control sample, and then perform log10 standardization.

[0105] Differential metabolite analysis: Principal component analysis (PCA) was performed using the R package PCAtools (version 2.0.0), and Variable Importance in Projection (VIP) values were obtained using the R package ropls (version 1.30.0) for orthogonal partial least squares discriminant analysis (OPLS-DA). Wilcoxon rank-sum test was used for differential analysis of ApcMin / + male and female mice, and the obtained P values were corrected to obtain FDR values. FDR < 0.05 and VIP ≥ 1 threshold were used to screen differential metabolites. MetOrigin (Yu et al. 2022) (http: / / metorigin.met-bioinformatics.cn / home / ) was used for differential metabolite source analysis and pathway enrichment analysis of metabolites from different sources (Metabolite Pathway Enrichment Analysis, MPEA). Finally, differential metabolites and enriched pathways of microorganisms and microorganisms and host co-metabolism were selected.

[0106] (3) Targeted metabolomics detection:

[0107] 1) Sample pretreatment:

[0108] ① Take out the sample and thaw on ice (all subsequent operations are performed on ice);

[0109] ② Mix the thawed sample and transfer 50 μL to a centrifuge tube;

[0110] ③ Add 250 μL of methanol solution (containing 10 μL of internal standard working solution with a concentration of 250 ng / mL) to the centrifuge tube, vortex for 3 min, mix well, and then place in a -20°C refrigerator for 30 min;

[0111] ④ Centrifuge at 12000 r / min for 10 min at 4°C, and transfer 150 μL of supernatant to a new centrifuge tube;

[0112] ⑤ After centrifugation again, transfer 100 μL of supernatant to a sample bottle and store in a -20°C refrigerator for LC-MS / MS analysis;

[0113] 2) Chromatography mass spectrometry collection conditions:

[0114] The data collection instrument system mainly includes ultra performance liquid chromatography (UPLC) (ExionLC TM AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (MS / MS) (Triple QuadTM 6500+, https: / / sciex.com.cn / ). The main liquid phase conditions include:

[0115] ① Column: Waters HSS T3 C18 column (1.8 μm, 100 mm x 2.1 mm i.d.);

[0116] ② Mobile phase: A phase, ultrapure water (containing 0.1% formic acid); B phase, acetonitrile (containing 0.1% formic acid);

[0117] ③ Flow rate 0.35 mL / min; column temperature 40℃; injection volume 5 μL;

[0118] ④ Mobile phase gradient: 0 min, A / B 90:10 (V / V); 1 min, A / B 90:10 (V / V); 8 min, A / B 5:95 (V / V); 9.5 min, A / B 5:95 (V / V); 9.6 min, A / B 90:10 (V / V); 12 min, A / B 90:10 (V / V).

[0119] The main mass spectrometry conditions include: Electrospray Ionization (ESI) temperature 550℃, mass spectrometry voltage 5500V in positive ion mode, mass spectrometry voltage -4500V in negative ion mode, curtain gas (CUR) 35 psi. In Q-Trap 6500+, each ion pair is scanned and detected according to the optimized declustering potential (DP) and collision energy (CE).

[0120] 3) Qualitative and quantitative principles:

[0121] Based on the standard product, the MWDB (Metware Database) database is constructed for qualitative analysis of mass spectrometry data.

[0122] Quantitative analysis is completed by using the Multiple Reaction Monitoring (MRM) mode of triple quadrupole mass spectrometry. In the MRM mode, the quadrupole first selects the precursor ion (parent ion) of the target substance to exclude other molecular weight substances to preliminarily exclude interference; the precursor ion is induced to ionize in the collision chamber to form multiple fragment ions, and the characteristic fragment ions required are selected by the triple quadrupole to exclude non-target ion interference, so that the quantification is more accurate and the repeatability is better. After obtaining the mass spectrometry data of different samples, the chromatographic peaks of all target substances are integrated, and the quantitative analysis is performed by the standard curve.

[0123] 4) Standard curve:

[0124] Prepare standard solution with different concentrations of 0.001 ng / mL, 0.002 ng / mL, 0.005 ng / mL, 0.01 ng / mL, 0.02 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, respectively, obtain the chromatographic peak intensity data of the corresponding quantitative signal of each concentration standard; take the external standard and internal standard concentration ratio or external standard concentration (Concentration Ratio or Concentration) as the abscissa, and the external standard and internal standard peak area ratio or external standard peak area (Area Ratio or Area) as the ordinate, draw the standard curve of different substances.

[0125] 5) Sample content:

[0126] Substitute the integral peak area of all detected samples into the standard curve linear equation for calculation, and further substitute into the calculation formula after calculation, to finally obtain the content data of the substance in the actual sample. The calculation formula used has been unit converted, and the corresponding numerical value can be directly substituted to obtain the sample content. The specific calculation formula includes:

[0127] The content of tryptophan in liquid sample (ng / mL) = c*V1 / 1000 / V2,

[0128] In the formula, c is the concentration value (ng / mL) obtained by substituting the integral peak area in the sample into the standard curve;

[0129] V1 is the total volume of the extract (μL);

[0130] V2 is the amount of sample transferred (mL).

[0131] (4) Targeted metabolomics analysis:

[0132] According to the total concentration ranking of different groups, select the top 5 compounds for visualization.

[0133] Related experiments and effect data:

[0134] (1) The abundance of Lactobacillus johnsonii decreases in colorectal patients:

[0135] Twelve published fecal metagenomic datasets of colorectal cancer patients and healthy people were collected for Meta-analysis, which showed that L. johnsonii was significantly decreased in colorectal cancer patients (as shown in Figure 1 A). And through correlation analysis, the results showed that L. johnsonii was highly positively correlated with the intestinal probiotic B. uniformis, and negatively correlated with harmful bacteria in the intestine such as pathogenic E. coli (as shown in Figure 1 B).

[0136] (2) Lactobacillus johnsonii alleviates the tumor progression of colorectal cancer model mice:

[0137] To study the effect of L. johnsonii on the progression of colorectal tumors, L. johnsonii (1.0 x 10 8 CFU per mouse), non-tumorigenic E. coli strain (as a bacterial control group) or PBS (control group) were gavaged into colorectal cancer model Apc Min / + Min / + mice once a day for eight consecutive weeks (as shown in Figure 2 A). The body weight of mice was counted every week during gavage, and the results showed that there was no significant difference in the body weight of mice (as shown in Figure 2 B). After 8 weeks of gavage, the incidence of blood stool in the L. johnsonii group was lower than that in the PBS group (as shown in Figure 2 C). After sampling, it was found that the number and volume of tumors in the L. johnsonii group were significantly lower than those in the E. coli group and the PBS control group (as shown in Figure 2 D). The above results preliminarily showed that L. johnsonii alleviated the progression of colorectal tumors to some extent.

[0138] (3) In vivo toxicity of Lactobacillus johnsonii:

[0139] To explore whether gavaging L. johnsonii has toxic effects on mouse organs, liver and kidney tissues of mice in different groups were taken at the end of gavage to prepare paraffin sections for HE staining. The results of HE staining showed that different gavage groups had no significant damage to the structure and function of mouse liver and kidney (as shown in Figure 3 A and Figure 3 B). The results showed that gavaging L. johnsonii did not have damage to the main organs of mice, such as liver and kidney, which laid a solid foundation for realizing clinical application.

[0140] (4) Lactobacillus johnsonii up-regulates the abundance of Lactobacillus rhamnosus in the intestine:

[0141] Current studies have shown that beneficial bacteria can inhibit the progression of colorectal cancer by increasing the abundance of beneficial bacteria in the gut, reducing the abundance of harmful bacteria, regulating intestinal microbial homeostasis, and other methods. After the above gavage was completed, the feces of mice in different groups were collected for metagenomic detection, and the results showed that the intestinal microbial alpha and beta diversity of mice in the L. johnsonii group was significantly increased (as shown in Figure 4 A and Figure 4 B). The abundance of beneficial bacteria B. uniformis, L. rhamnosus and Eubacterium sp. 14-2 in the feces of mice in the L. johnsonii group was significantly increased, while the abundance of harmful bacteria Anaerotruncus sp. G3 (2012), Turicimonas. Muris, Lleibacterium. Valens was significantly reduced (as shown in Figure 4 C). The above results show that L. johnsonii can maintain intestinal homeostasis by increasing the abundance of beneficial bacteria in the gut and reducing the abundance of harmful bacteria in the gut.

[0142] In summary, the application provides a L. johnsonii as a biomarker in colorectal tumors. Through a large number of experiments, it is found that L. johnsonii can be used as a biomarker for colorectal tumors, thereby filling the gap in the prior art for the application of L. johnsonii in colorectal tumors.

[0143] The above description is merely a specific implementation of the present application, which enables those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the present application, but will conform to the widest scope consistent with the principles and novel features of the present application.

Claims

1. Use of Lactobacillus johnsonii as a biomarker in colorectal tumours, characterised in that, The application comprises: The low abundance of Lactobacillus johnsonii is used as a biomarker of colorectal tumor, wherein the abundance ratio of the low abundance of Lactobacillus johnsonii to normal abundance of Lactobacillus johnsonii is ≤0.

9.

2. Use according to claim 1, characterized in that, The abundance of Lactobacillus johnsonii is negatively correlated with the degree of colorectal tumor.

3. Use according to claim 1, characterized in that, The biomarker of colorectal tumor also includes intestinal beneficial bacteria, and the abundance of Lactobacillus johnsonii is positively correlated with the abundance of intestinal beneficial bacteria.

4. Use according to claim 3, characterized in that, The intestinal beneficial bacteria include at least one of the following: Bacteroides uniformis, Lactobacillus rhamnosus and Eubacterium sp. 14-2.

5. The use according to claim 1, characterized in that, The biomarker of colorectal tumor also includes intestinal harmful bacteria, and the abundance of Lactobacillus johnsonii is negatively correlated with the abundance of intestinal harmful bacteria.

6. Use according to claim 5, characterized in that, The intestinal harmful bacteria include at least one of the following: Anaerotruncus sp. G3 (2012), Turicimonas. Muris and Lleibacterium. Valens.

7. A therapeutic agent for colorectal tumor, characterized by, The therapeutic drug includes an activator for increasing the abundance of Lactobacillus johnsonii and / or a bacterial preparation containing Lactobacillus johnsonii.

8. The therapeutic medicament according to claim 7, characterized in that, When the therapeutic drug is a bacterial preparation containing Lactobacillus johnsonii, the concentration of Lactobacillus johnsonii in the bacterial preparation is > 1.0 x 10 8 CFU / mL.

9. An agent for screening or aiding in the screening of colorectal cancer, characterized in that, The reagent includes a medicament for detecting the abundance of Lactobacillus johnsonii.

10. An agent for evaluating or aiding in the evaluation of the prognosis of colorectal cancer, characterized by, The reagent includes a medicament for detecting the abundance of Lactobacillus johnsonii.

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