TW201829781A - Method for screening for gene marker of intestinal cancer and/or stomach cancer, gene marker for screening via method, and application of gene marker - Google Patents

Abstract

Provided are a gene marker for detecting stomach cancer, an application of the gene marker, and a method for using the gene marker to detect stomach cancer.

Description

Method for screening gene markers of intestinal cancer, gene markers screened by the method, and uses thereof

The present invention relates to the field of screening for genetic markers by high throughput sequencing. In particular, the present invention relates to a method for screening a genetic marker for intestinal cancer by high-throughput sequencing, and a genetic marker screened by the method and use thereof.

With the changes in living environment and habits, the incidence of cancer has increased year by year in the world, and people's attention has also increased. Gastric cancer and intestinal cancer are the second and third most common cancers in China, respectively. They are malignant tumors with very high morbidity and mortality. Their occurrence and development is a complex process involving multi-factor participation and multi-stage cumulative carcinogenesis. With the existing early treatment of early detection of cancer, the earlier it is discovered, the more likely it is to control or even cure cancer early. How to conduct early screening and timely treatment of gastric cancer and intestinal cancer, and correctly judge the prognosis, has gradually received attention.

Traditional screening methods include gastrointestinal endoscopy and fecal occult blood tests, but all have their own insurmountable shortcomings. Gastrointestinal plus pathological biopsy is considered the gold standard for screening and diagnosis of gastrointestinal cancer, but many patients are reluctant to undergo gastroscopic examination because of the invasiveness of microscopic examination and discomfort in bowel preparation. Recently, studies have shown that colonoscopy is not good for the diagnosis of flat polyps commonly found in the right colon. Colonoscopy does not reduce the incidence of right colon cancer. Fecal occult blood test is another clinically used colorectal cancer screening method. It has the advantages of being completely non-invasive and inexpensive, but its accuracy in detecting tumors is low. Only 50-60% can be detected by immunochemical improvement. Colorectal cancer and about 30% of precancerous adenomas. Moreover, the false positive rate of the detection method is high, so it is difficult to get popularization and popularization.

In addition to the above traditional screening methods, people continue to explore other methods to improve early screening rate, tumor markers are one of them. For example, by THPLC analysis, T1151A was identified as a polymorphic site on the mismatch repair gene hMLH1, which can be used as a candidate for screening gastric and colorectal tumors, especially for high-risk populations of younger stomach and large intestine tumors (see Zhang Xiaomei et al., Oncology). 2005, 25(1): 62-65). The combined detection of tumor markers using CA19-9 (glucose gastrointestinal cancer-associated antigen) and SA (sialic acid) combined detection of colorectal cancer and gastric/cardiac cancer can reach 76.7% and 82.5%, respectively (see Cai Facheng et al. Journal of Practical Medical Technology, 2005, 12 (3B): 722-723). In addition, China has also developed a C12 protein wafer detection system by analyzing serum CA19-9, neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), glycogen 242 (CA242), glycogen 125 (CA125). ), the expression level of these ten tumor markers in glycogen 153 (CA15-3), alpha-fetoprotein (AFP), ferritin, human chorionic gonadotropin (HCG), and human growth hormone (HGH) Early screening and monitoring of tumors. However, most of the tumor markers found remain at the research stage and have not progressed to the clinical screening stage; or have been applied to clinical screening, but the detection accuracy or sensitivity is not high. For example, the study found that the overall screening rate of the above C12 system for patients with gastrointestinal cancer was 39.21%, and the screening rates for patients with stage I, II, III, and IV were 13.73%, 33.33%, 38.30%, and 58.03%, respectively, indicating C12. The detection system has certain value for the screening of advanced gastrointestinal cancer, but it is not sensitive to the early stage (see Yang Xueqin et al., Journal of Nanjing Medical University (Natural Science Edition), 2008 (10): 1285-1289).

Recently, two other in vitro screening methods for intestinal cancer have been developed based on existing conventional genetic markers: Cologuard technology from Exact Sciences, Inc. and Epi proColon technology from Epigenomics. The former mainly detects abnormal DNA changes in feces and occult hemoglobin present in human feces; the latter detects SEPT9 methylation markers in blood free DNA. Large-scale clinical trials have shown that Cologuard's detection rate is better, but it requires manipulation of feces, and the user experience is poor. SEPT9 methylation is a liquid biopsy. Although the user experience is better, the specificity and sensitivity are poor (respectively 80% and 72%).

Therefore, there is an urgent need for genetic markers of intestinal cancer with high specificity and high sensitivity for clinical testing, which enables effective screening of intestinal cancer in a non-invasive or minimally invasive and rapid manner to improve the patient's willingness to undergo long-term monitoring.

The inventors unexpectedly discovered multiple high-throughput sequencing of normal samples and intestinal or gastric cancer samples, and analysis of 5-hydroxymethylcytosine (5-hmC) content in each gene. A highly informative genetic marker for detecting intestinal or gastric cancer.

Accordingly, a first aspect of the invention relates to a genetic marker for detecting intestinal cancer comprising one or more genes selected from the group consisting of ADAM metal peptidase domain 20 ( ADAM20 ), F-box and leucine-rich repeat protein 7 ( FBXL7 ), follistatin ( FST ), TP53 apoptotic effector ( PERP ) , Pleek substrate homologous domain family A member 3 ( PHLDA3 ) , Runt - related transcription factor 1 mobile partner 1 ( RUNX1T1 ) Mutulin γ2 ( SNTG2 ), sperm-associated antigen 4 ( SPAG4 ), sulfatase 1 ( SULF1 ) and NME/NM23 nucleoside diphosphate kinase ( NME3 ). Preferably, the genetic marker comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten genes selected from the group consisting of: ADAM20 , FBXL7 , FST , PERP , PHLDA3 , RUNX1T1 , SNTG2 , SPAG4 , SULF1, and NME3 . More preferably, the genetic markers include ADAM20 , FBXL7 , FST , PERP , PHLDA3 , RUNX1T1 , SNTG2 , SPAG4 , SULF1 and NME3 .

A second aspect of the invention relates to a genetic marker for detecting gastric cancer comprising one or more genes selected from the group consisting of Rho GTPase promoter protein 28 ( ARHGAP28 ), BMP binding endothelial regulator ( BMPER ), chromosome 9 open Reading frame 92 ( C9orf92 ), calcium-dependent secretion promoter 2 ( CADPS2 ), cadherin 11 ( CDH11 ), F-box and leucine-rich repeat protein 7 ( FBXL7 ), mesenchymal homeobox 2 ( MEOX2 ), Nitric oxide synthase 1 ( NOS1 ), oncostatin M receptor ( OSMR ), cell membrane regulatory protein paralemmin 2 ( PALM2 ), phosphodiesterase 10A ( PDE10A ), RNA-binding motif single-chain interacting protein 3 (RBMS3) ), sulfatase 1 ( SULF1 ), wntless Wnt ligand secretion regulator ( WLS ), Wilms tumor 1 acting protein ( WTIP ), zinc finger protein 518B ( ZNF518B ), zinc finger protein 714 ( ZNF714 ), and Rad52 motif 1 ( RDM1 ). Preferably, the genetic marker comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17 or 18 genes selected from: ARHGAP28, BMPER, C9orf92, CADPS2 , CDH11, FBXL7, MEOX2, NOS1, OSMR, PALM2 , PDE10A , RBMS3 , SULF1 , WLS , WTIP , ANF518B , ZNF714, and RDM1 . More preferably, the marker gene comprising ARHGAP28, BMPER, C9orf92, CADPS2, CDH11, FBXL7, MEOX2, NOS1, OSMR, PALM2, PDE10A, RBMS3, SULF1, WLS, WTIP, ANF518B, ZNF714 and RDM1.

The invention also relates to the use of the above genetic markers for detecting intestinal or gastric cancer. The present invention also relates to a kit for detecting intestinal cancer or gastric cancer using the above gene marker, which comprises reagents and instructions for determining the 5-hmC content of the above gene marker.

A third aspect of the invention relates to a method for detecting gastric cancer or intestinal cancer, comprising the steps of: (a) determining the content of 5-hmC of the genetic marker of the present invention in a normal sample and a sample of a subject; b) normalizing the 5-hmC content of the corresponding gene marker in the subject sample using the 5-hmC content of the gene marker in the normal sample as reference; (c) on the standardized gene marker The 5-hmC content was mathematically correlated and scored; and (d) the test results were obtained based on the score.

In one embodiment, the sample is a free DNA fragment in a subject or normal human body fluid, or is derived from intact genomic DNA in cells, cells, and tissues. Among them, body fluids are blood, urine, sweat, sputum, feces, cerebrospinal fluid, ascites, pleural effusion, bile, pancreatic juice, and the like.

In one embodiment, the 5-hmC content of the genetic markers of the invention can be determined by any method known to those skilled in the art, including, for example, but not limited to, glucosylation, restriction endonucleases Method, chemical labeling method, precipitation method combined with high-throughput sequencing method, single molecule immediate sequencing method (SMRT), oxidized bisulfite sequencing method (OxBS-Seq), and the like. The principle of the glucosylation method is to transfer glucose to a hydroxyl group in the presence of a glucose donor substrate, urinary nucleoside diphosphate glucose (UDP-Glu), using T4 bacteriophage β -glucose transferase ( β- GT). Position, thereby producing β -glucosyl-5-hydroxymethylcytosine (5-ghmC). At the same time, isotopically labeled receptors can be used for quantification The restriction endonuclease method and the chemical labeling method were further developed on the basis of the glucosylation method. The principle of the restriction endonuclease method is that the glucosylation reaction changes the enzymatic cleavage properties of some restriction enzymes. The methylation-dependent restriction enzymes MspI and HpaII recognize the same sequence (CCGG), but their sensitivity to methylation status is different: MspI recognizes and cleaves 5-methylcytosine (5-mC) And 5-hmC, but not 5-ghmC; HpaII only cleaves completely unmodified sites, and any modification on cytosine (5-mC, 5-hmC, 5-ghmC) blocks cleavage. If the CpG locus contains 5-hmC, the band can be detected after glycosylation and enzymatic hydrolysis, and there is no band in the unglycosylated control reaction; qPCR can be used for quantitative analysis. In addition, other restriction enzymes also have a hindrance to 5-ghmC digestion, and can be applied to 5-hmC detection (eg, GmrSD, MspJI, PvuRts1I, Taq I, etc.). The principle of chemical labeling is to chemically modify the glucose of the enzyme reaction to UDP-6-N3-glucose, and transfer 6-N3-glucose to the position of hydroxymethyl to form N3-5ghmC. Subsequently, a single molecule of biotin was added to each 5-hmC by click chemistry, combined with next-generation high-throughput DNA sequencing technology or single-molecule sequencing technology to analyze the distribution of 5-hmC in genomic DNA. The precipitation method is to modify 5-hmC in a special way and then specifically capture it from genomic DNA and perform sequence analysis. The oxidized bisulfite sequencing method is the first method to quantitatively sequence 5-hmC with a single base resolution. First, 5-hmC was subjected to KRuO4 oxidation treatment to produce 5-mercaptocytosine (5fC), which was then subjected to bisulfite sequencing. In this process, 5-hmC is first oxidized to 5fC, and then deaminated to form U. Usually, quantitative detection of 5-hmC is performed simultaneously using a variety of detection methods.

In one embodiment of the invention, the 5-hmC content of the genetic markers of the invention is determined by chemical labeling in combination with high throughput sequencing. In this particular embodiment, the method of determining the 5-hmC content of a genetic marker of the present invention comprises the steps of: fragmenting DNA from a sample of a patient with a bowel or gastric cancer and a normal human; the fragmented DNA The ends are repaired and end-filled; the end-filled DNA is ligated to the sequencing linker to obtain a ligation product; the 5-hydroxymethylcytosine in the ligation product is labeled by a labeling reaction; enrichment or enrichment is collected. To enrich the DNA fragment containing 5-hydroxymethylcytosine labeling to obtain an enriched product; PCR amplification of the enriched product to obtain a sequenced gene pool; high-throughput sequencing of the sequenced gene pool Sequencing results; the genetic content of 5-hydroxymethylcytosine was determined based on the sequencing results. Wherein the labeling reaction comprises: i) covalent attachment of a sugar having a modifying group to a methylol group of 5-hydroxymethylcytosine using a glycosyltransferase, and ii) direct or indirect attachment of biotin Click on the chemical acceptor to react with 5-hydroxymethylcytosine with a modifying group. Wherein step i) and step ii) may be carried out sequentially or simultaneously in one reaction. This labeling method reduces the amount of sample required for sequencing, and the biotin tag on 5-hydroxymethylcytosine gives it a higher kinetic signal in sequencing, which improves the accuracy of nucleotide recognition. Sex. In this embodiment, the glycosyltransferases include, but are not limited to, T4 phage β-glucosyltransferase (β-GT), T4 phage α-glucosyltransferase (α-GT), and the same Or a similarly active derivative, analog, or recombinase; the saccharide with a modifying group includes, but is not limited to, a saccharide with an azide modification (eg, 6-N3-glucose) or with other chemical modifications ( a saccharide such as a carbonyl group, a thiol group, a hydroxyl group, a carboxyl group, a carbon-carbon double bond, a carbon-carbon triple bond, a disulfide bond, an amine group, a decylamino group, a diene or the like, wherein a saccharide having an azide modification is preferred; Chemical groups for indirect attachment of biotin and click chemical acceptors include, but are not limited to, carbonyl, sulfhydryl, hydroxy, carboxy, carbon-carbon double bonds, carbon-carbon triple bonds, disulfide bonds, amine groups, guanamine Base, diene. In this embodiment, the concentrated DNA fragment containing the 5-hmC label is preferably collected by a solid phase material. Specifically, a DNA fragment containing a 5-hydroxymethylcytosine label can be bound to a solid phase material by a solid phase affinity reaction or other specific binding reaction, and then the unbound DNA fragment can be removed by multiple washings. Solid phase materials include, but are not limited to, bracts or other wafers with surface modifications, such as artificial polymer beads (preferably 1 nm-100 mm in diameter), magnetic beads (preferably 1 nm-100 mm in diameter), agar Sugar globules, etc. (preferably 1 nm to 100 mm in diameter). The washing liquid used for solid phase enrichment is a buffer well known to those skilled in the art, including but not limited to: containing Tris-HCl, MOPS, HEPES (pH=6.0-10.0, concentration between 1 mM and 1 M), NaCl (0-2M) or a buffer such as Tween20 (0.01%-5%). In this embodiment, PCR amplification is preferably performed directly on the solid phase to prepare a sequencing gene pool. If necessary, after performing PCR amplification on the solid phase, the amplification product can be recovered and subjected to a second round of PCR amplification to prepare a sequencing gene pool. The second round of PCR amplification can be performed using conventional methods known to those skilled in the art. Optionally, one or more purification steps may be further included in the process of preparing the sequencing gene pool. Any purification kit known or commercially available to those skilled in the art can be used in the present invention. Purification methods include, but are not limited to, gel electrophoresis gel extraction, silicone film spin column method, magnetic bead method, ethanol or isopropanol precipitation method, or a combination thereof. Optionally, a quality check is performed on the sequencing gene pool prior to high throughput sequencing. For example, the library is subjected to fragment size analysis and the concentration of the library is absolutely quantified using the qPCR method. A sequenced gene pool that passes quality checks can be used for high-throughput sequencing. A certain number (1-96) of libraries containing different barcodes were then mixed at the same concentration and sequenced according to the standard method of the second generation sequencer to obtain sequencing results. Various second generation sequencing platforms and related reagents known in the art can be used in the present invention.

In one embodiment of the invention, the sequencing results are preferably aligned with a standard human genome reference sequence, and sequences aligned to the gene markers of the invention are selected, ie, aligned bit positions and gene features are selected ( The number of reads of the coincident region, such as histone modification site, transcription factor binding site, gene exon intron region, and gene promoter, etc., to represent the modification level of 5-hmC on the gene, thereby determining 5 -hmC content on the gene marker. Preferably, prior to performing the alignment, the sequencing result is first cleared of low quality sequencing sites, wherein factors determining the quality of the sequencing sites include, but are not limited to, base quality, reads quality, GC content, repeat sequences, and number of Overrepresented sequences. Wait. The various alignment software and analytical methods involved in this step are known in the art.

In one embodiment of the invention, determining the 5-hmC content of the genetic marker means determining the 5-hmC content of the full length of the genetic marker or determining the 5-hmC content of a fragment of the genetic marker or a combination thereof .

According to the present invention, after determining the 5-hmC content of each gene marker, the 5-hmC content of the corresponding gene marker in the sample of the subject is used as a reference with the 5-hmC content of the gene marker in the normal sample. standardization. For example, the 5-hmC content of the same gene marker in the normal sample and the subject sample is X and Y, respectively, and the normalized 5-hmC content of the gene marker in the subject sample is Y/X.

According to the present invention, after the data is standardized, the standardized 5-hmC content of each gene marker is mathematically correlated to obtain a score, thereby obtaining a detection result based on the score. As used herein, "mathematical association" refers to any computational or machine learning method that correlates the 5-hmC content of a genetic marker from a biological sample with an intestinal cancer or gastric cancer screening result. One of ordinary skill in the art will appreciate that different computing methods or tools may be selected to provide the mathematical associations of the present invention, such as elastic network regularization, decision trees, generalized linear models, logistic regression, highest score pairs, neural networks, Linear and quadratic discriminant analysis (LQA and QDA), naive Bayes, random forests, and support vector machines.

In one embodiment of the present invention, the specific steps for mathematically correlating the standardized 5-hmC content of each gene marker and obtaining a score are as follows: multiplying the normalized 5-hmC content of each gene marker by a weighting coefficient to obtain the gene The predictor of the marker t; the predictor t of each gene marker is added to obtain a total predictor T; the total predictor T is subjected to Logistic conversion to obtain a score P; if P>0.5, the subject sample suffers Intestinal or gastric cancer; if P < 0.5, the subject sample is normal. The weighting factors described herein refer to various advanced statistics in consideration of factors that may affect the 5-hmC content (eg, subject area, age, gender, below, smoking history, drinking history, family history, etc.). The coefficients obtained by the analytical method.

The method for detecting intestinal cancer and/or gastric cancer in the present invention is based on the 5-hmC content on the gene marker as compared with the prior art, and thus a wider source of DNA samples can be used. Therefore, the method for detecting intestinal cancer and/or gastric cancer in the present invention has the following advantages: (1) safe and non-invasive, and high acceptance of the test even in asymptomatic people; (2) wide source of DNA, no image In-school detection blind zone; (3) high accuracy, high sensitivity and specificity; (4) easy to operate, user experience is good, easy to monitor disease dynamics.

The invention will be described in detail below with reference to the drawings, in order to provide a better understanding of the invention. It should be understood that the drawings and the embodiments of the present invention are intended to be illustrative only and not restrictive. The embodiments of the present application and the features of the embodiments may be combined with each other without contradiction.

Example 1. Screening of colon cancer gene markers

(1) Extraction of plasma DNA:

10 ng of plasma DNA was extracted from samples from 15 colon cancer patients and 18 normal subjects, respectively. This step can be carried out using any method and reagent suitable for extracting plasma DNA well known to those skilled in the art.

(2) The plasma DNA is end-filled, suspended A and connected to the sequencing linker:

Prepare a reaction mixture containing 50 mL of plasma DNA, 7 mL of End Repair & A-Tailing Buffer and 3 mL of End Repair & A-Tailing Enzyme mix according to the Kapa Hyper Perp Kit instructions (total volume 60 mL), at 20 °C in a warm bath 30 Minutes, then warm at 65oC for 30 minutes. The following ligation reaction mixture was placed in a 1.5 mL low adsorption EP tube: 5 mL Nuclease free water, 30 mL Ligation Buffer and 10 mL DNA Ligase. Add 5 mL of the sequencing link to the 45 mL ligation reaction mixture, mix, heat at 20 °C for 20 minutes, then hold at 4 °C. The reaction product was purified using Ampure XP beads and eluted with 20 mL of a buffer containing Tris-HCl (10 mM, pH = 8.0) and EDTA (0.1 mM) to obtain a final DNA-ligated sample.

(3) Labeling 5-hydroxymethylcytosine:

Prepare a total of 26 mL of labeled reaction mixture: azide-modified uridine diphosphate glucose (ie UDP-N3-Glu, final concentration 50 mM), β-GT (final concentration 1 mM), Mg ²⁺ (final concentration 25 mM), HEPES (pH = 8.0, final concentration 50 mM) and 20 mL of DNA from the above procedure. The mixture was incubated at 37 ° C for 1 hour. The mixture was taken out and purified with Ampure XP beads to obtain purified 20 mL of DNA.

Then, 1 mL of biotin-containing diphenylcyclooctyne (DBCO-Biotin) was added to the purified 20 mL of the above DNA, and reacted at 37 ° C for 2 hours, followed by purification with Ampure XP beads to obtain a purified labeled product.

(4) Solid phase collection of concentrated DNA fragments containing labeled 5-hydroxymethylcytosine:

First, prepare the magnetic beads as follows: Remove 0.5 mL of C1 streptadvin beads (life technology) and add 100 mL of buffer (5 mM Tris, pH=7.5, 1 M NaCl, 0.02% Tween 20), vortex for 30 seconds, then use 100 The magnetic beads were washed 3 times with mL Wash (5 mM Tris, pH = 7.5, 1 M NaCl, 0.02% Tween 20), and finally 25 mL of binding buffer (10 mM Tris, pH = 7.5, 2 M NaCl, 0.04% Tween 20 or other surfactant) was added. ), and mix evenly.

Then, the purified labeled product obtained in the above procedure was added to the magnetic bead mixture, and mixed for 15 min in a rotary mixer to sufficiently bind.

Finally, the magnetic beads were washed 3 times with 100 mL of washing solution (5 mM Tris, pH = 7.5, 1 M NaCl, 0.02% Tween 20), the supernatant was removed by centrifugation, and 23.75 mL of nuclease-free water was added.

(5) PCR amplification:

Add 25 mL of 2 X PCR master mix and 1.25 mL PCR primer (total volume 50 mL) to the final system of the above procedure and amplify according to the temperature and conditions of the PCR reaction cycle described below:

The amplified product was purified using Ampure XP beads to obtain a final sequencing gene pool.

(6) High-throughput sequencing after quality inspection of the sequencing gene bank:

The obtained sequencing gene pool was subjected to concentration determination by qPCR, and the DNA fragment size content in the library was determined using Agilent 2100. The sequencing gene pools passed through the QC were mixed at the same concentration and sequenced with an Illumina Hiseq 4000.

(7) Determine the 5-hmC content and weighting coefficient of each gene marker

The obtained sequencing results were subjected to preliminary quality control evaluation, and after the low-quality sequencing sites were removed, the readings that met the sequencing quality criteria were compared with the human standard genome reference sequence using the Bowtie 2 tool. The feature counts and HtSeq-Count tools were then used to count the number of reads to determine the 5-hmC content of each gene marker. At the same time, using high-throughput sequencing results, the factors that may affect the 5-hmC content were used as covariates, and the weighting coefficients of each gene marker were obtained by logistic regression and elastic network regularization. The results are shown in Table 1. Table 1: Average normalized 5-hmC content and weighting coefficient of intestinal cancer gene markers of the present invention

As described above, the average normalized 5-hmC content refers to the ratio of the average 5-hmC content of the gene marker in the intestinal cancer sample to the average 5-hmC content of the same gene marker in the normal sample. As can be seen from Table 1, the 5-hmC content of the intestinal cancer gene marker of the present invention is significantly different between the normal sample and the intestinal cancer sample, and the 5-hmC content of the remaining genetic markers except for NME3 is relative to Normal people have increased significantly.

Example 2. Effectiveness of gut cancer gene markers

This example demonstrates the effectiveness of the intestinal cancer gene marker of the present invention for detecting intestinal cancer.

The 5-hmC content of the 10 gut cancer gene markers of the present invention in the first batch of 59 samples (24 intestinal cancers and 35 controls) was determined according to the method of Example 1, and the weighting of each gene marker was determined. coefficient.

Multiplying the normalized 5-hmC content of each gene marker by its corresponding weighting coefficient to obtain the predictive factor t of the gene marker, adding the predictor t of each gene marker to obtain the total predictor T, and then The total predictor T is scored by Logistic conversion according to the following formula:

If P > 0.5, the subject sample has intestinal cancer; if P < 0.5, the subject sample is normal.

Figure 1 shows the results of distinguishing the batch of samples in accordance with the method of the present invention. As shown in Figure 1, the method of the invention is capable of achieving a sensitivity of 83% and a specificity of 94%. The inventors further distinguished the second batch of 69 samples (32 intestinal cancers and 37 controls) using the method of the present invention, and the results showed that the method was able to achieve 88% sensitivity and 89% specificity (Fig. 2).

These results indicate that the method according to the present invention is capable of detecting intestinal cancer with higher sensitivity and specificity than the prior art.

Example 3. Screening of gastric cancer gene markers

The genetic markers of gastric cancer were screened according to the method described in Example 1, the only difference being that the samples used were plasma free DNA from 7 gastric cancer patients and 18 normal humans. The gastric cancer markers screened are shown in Table 2. Table 2: Average normalized 5-hmC content and weighting coefficient of gastric cancer gene markers of the present invention

As described above, the average normalized 5-hmC content refers to the ratio of the average 5-hmC content of the gene marker in the gastric cancer sample to the average 5-hmC content of the same gene marker in the normal sample. As can be seen from Table 2, the 5-hmC content of the gastric cancer gene marker of the present invention is significantly different between the normal sample and the intestinal cancer sample, wherein among the gastric cancer samples, the three gene markers show a 5-hmC content decrease: RDM1, ZNF714 and ZNF518B, 15 marker genes show elevated levels of 5-hmC: ARHGAP28, BMPER, C9orf92, CADPS2 , CDH11, FBXL7, MEOX2, NOS1, OSMR, PALM2, PDE10A, RBMS3, SULF1, WLS and WTIP.

Example 4. Determination of the effectiveness of gastric cancer gene markers

This example demonstrates the effectiveness of the gastric cancer gene marker of the present invention for detecting gastric cancer.

The 5-hmC content of the 18 gastric cancer gene markers of the present invention in the third batch of 60 samples (25 gastric cancers and 35 controls) was determined according to the method of Example 1, and the weighting coefficients of the respective gene markers were determined.

Multiplying the normalized 5-hmC content of each gene marker by its corresponding weighting coefficient to obtain the predictive factor t of the gene marker, adding the predictor t of each gene marker to obtain the total predictor T, and then The total predictor T is scored by Logistic conversion according to the following formula:

If P > 0.5, the subject sample has gastric cancer; if P < 0.5, the subject sample is normal.

Figure 3 shows the results of distinguishing the batch of samples in accordance with the method of the present invention. As shown in Figure 3, the method of the invention is capable of achieving a sensitivity of 92% and a specificity of 91%. The inventors further distinguished the fourth batch of 63 samples (29 gastric cancers and 37 controls) by the method of the present invention, and the results showed that the method was able to achieve 90% sensitivity and 97% specificity (Fig. 4).

These results indicate that the method according to the present invention is capable of detecting intestinal cancer with higher sensitivity and specificity than the prior art.

It is apparent to those skilled in the art that various modifications and variations can be made without departing from the scope and spirit of the invention. Although the present invention has been described in terms of specific preferred embodiments, it should be understood that the invention should not be In fact, the various modifications that are apparent to those skilled in the art are also contemplated by the scope of the invention.

Figure 1: Results of distinguishing the first batch of samples using the intestinal cancer gene markers of the present invention. Figure 2: Results of distinguishing a second batch of samples using the intestinal cancer gene markers of the present invention. Figure 3: Results of distinguishing a third batch of samples using the gastric cancer gene markers of the present invention. Figure 4: Results of distinguishing the fourth batch of samples using the gastric cancer gene markers of the present invention.

Claims (8)

A genetic marker for detecting intestinal cancer, comprising one or more genes selected from the group consisting of ADAM metal peptidase domain 20 ( ADAM20 ), F-box and leucine-rich repeat protein 7 ( FBXL7 ), follistatin ( FST ), TP53 apoptotic effector ( PERP ), pleckin protein homologous domain family A member 3 ( PHLDA3 ), Runt-related transcription factor 1 mobile partner 1 ( RUNX1T1 ), mutual protein γ2 ( SNTG2 ), sperm associated antigen 4 (SPAG4), sulfatase 1 (SULF1) and NME / NM23 nucleoside diphosphate kinase (NME3). The gene markers according to claim 1 include ADAM20 , FBXL7 , FST , PERP , PHLDA3 , RUNX1T1 , SNTG2 , SPAG4 , SULF1 and NME3 . Use of the genetic marker of claim 1 or 2 in a method for detecting intestinal cancer. A method for detecting intestinal cancer comprising the steps of: (a) determining 5-hydroxymethylcytosine (5-hmC) of the gene marker according to claim 1 or 2 in a normal sample and a subject sample. (b) normalize the 5-hmC content of the corresponding gene marker in the sample of the subject with the 5-hmC content of the gene marker as described in the normal product; (c) the step (b) The 5-hmC content of the standardized gene marker is mathematically correlated and scored; and (d) the test results are obtained according to the score. The method of claim 4, wherein the step (a) is to determine the content of 5-hmC on the entire length of the gene marker or a fragment thereof. The method of claim 4, wherein the sample is a free DNA fragment from a normal human or a subject's body fluid, or a complete genomic DNA derived from a cell, a cell, and a tissue. The method of claim 4, wherein the body fluid is blood, urine, sweat, sputum, feces, cerebrospinal fluid, ascites, pleural effusion, bile or pancreatic juice. A kit for detecting intestinal cancer, comprising: (a) an agent for determining a 5-hmC content of the gene marker according to claim 1 or 2; and (b) a specification.