CN115786501B - Enhancer functional site related to colorectal cancer early screening and auxiliary diagnosis and application thereof - Google Patents

Abstract

The present invention relates to an enhancer function site marker related to early screening and auxiliary diagnosis of colorectal cancer and its application, the marker is rs4810856. Large-scale population data and biological functional experiments have confirmed that the rs4810856 site has enhancer function regulatory activity, and individuals carrying the rs4810856[C] genotype are more likely to bind to the transcription factor ZEB1, thereby affecting the relationship between this region and the promoter of the target gene. Chromatin interaction promotes the expression levels of target genes PREX1, CSE1L and STAU1, thereby promoting the proliferation of cancer cells, resulting in an increased risk of individual colorectal cancer. Through the ingenious design of primers and probes for rs4810856, and relying on fluorescent quantitative PCR, the risk sites of rs4810856 can be detected in normal people, so as to identify high-risk groups of colorectal cancer and assist the diagnosis of colorectal cancer patients.

Description

An enhancer functional site associated with early screening and auxiliary diagnosis of colorectal cancer and its application

technical field

The invention relates to the fields of genetic engineering and tumor medicine, in particular to an enhancer functional site related to early screening of colorectal cancer and its application.

Background technique

Colorectal cancer is one of the common malignant tumors of the digestive tract, which seriously threatens human life and health. Its incidence rate ranks third among malignant tumors in my country, and with the development of the country's economic level and the changes in residents' lifestyle and diet structure, the incidence and mortality of colorectal cancer in my country are showing an increasing trend year by year, colorectal cancer It has become a major public health problem to be solved urgently in China and even in the world. Currently, the most effective screening method for colorectal cancer is fecal occult blood test (FIT) combined with colonoscopy. However, this method also has problems such as low detection rate and low patient acceptance. Therefore, if early screening and early diagnosis can be carried out more accurately for high-risk individuals of colorectal cancer, it can make patients more acceptable and save a lot of medical expenses for the country.

In recent years, individualized screening programs based on molecular genetics have become the focus of screening high-risk groups for colorectal cancer. Single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) is the most common genetic variation, and it is a kind of molecular marker that has been confirmed to be associated with the risk of tumor occurrence, which reflects the differences in the genetic background of individuals. So far, GWAS has identified more than 100 colorectal cancer genetic susceptibility SNPs. Among the susceptibility SNPs discovered by GWAS, more than 90% are located in non-coding regions of the genome, which can affect the risk of colorectal cancer by participating in the regulation of gene expression . With the rapid development of GWAS, with the support of genome high-throughput detection technology, researchers found that non-coding regions contain many cis-regulatory elements (Cis-Regulatory Element, CRE), such as promoters, enhancers and insulators, etc. They can play a huge role in the regulation of gene expression, thereby affecting tumorigenesis and susceptibility. With the rise of epigenomics and three-dimensional genomics, researchers have become more and more aware of the characteristics and mechanism of action of CREs. For example, through chromatin transposase accessibility sequencing (Assay for Transposase Accessible Chromatin with high-throughput sequencing, ATAC-seq) technology found that CREs are often enriched in open regions of chromatin, and exposed DNA makes it easier to interact with transcription factors ( Transcription Factor (TF) binding, and then participate in the regulation of gene expression. Through Chromatin Immunoprecipitation and high-throughput sequencing (ChIP-seq) technology, it was found that nucleosomes near CREs often have specific histone modifications, including H3K27ac, H3K27me3 and H3K4me1. These special epigenetic marks can help predict the activity status of CREs and distinguish different CREs. In addition, with the continuous maturity of high-throughput chromatin three-dimensional conformation capture technology (High-Through Chromosome Conformation Capture, Hi-C). The researchers found that chromatin can form a ring structure (ChromatinLooping) through three-dimensional folding, which spatially shortens the distance between the enhancer and the promoter, thereby activating gene transcription.

However, how to effectively integrate these multi-omics data to search for CREs and identify their target genes on a genome-wide scale remains a great challenge. Recently, in order to optimize the prediction of CREs and their target genes, researchers developed the Activity-by-Contact Model (ABC model), by integrating ATAC-seq and H3K27ac ChIP representing the activity status of regulatory elements (Activity) -seq signal peaks and Hi-C data representing chromatin contact frequency (Contact), identify active enhancer elements genome-wide and determine the target genes they regulate. The ABC model is based on the simple biochemical concept that the quantitative impact of a distal candidate regulatory element on target gene expression should depend on its activity as an enhancer, weighted by its chromatin contact frequency with the target gene promoter (Contact) ; while the relative contribution of a candidate regulatory element to the expression of a target gene is determined by dividing the quantitative impact of that element by the total quantitative impact of all candidate regulatory elements within the region of the target gene. The model finally obtains the ABC score corresponding to each enhancer-target gene to evaluate the strength of the enhancer's regulatory effect on gene expression.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and provide an enhancer function SNP site marker for early screening and auxiliary diagnosis of colorectal cancer and its application.

The technical scheme that the present invention solves the problems of the technologies described above is as follows:

An enhancer functional site marker related to early screening and auxiliary diagnosis of colorectal cancer, the marker is rs4810856.

The specific amplification primers for the enhancer function site markers related to the early screening and auxiliary diagnosis of colorectal cancer, the specific amplification primer sequences for rs4810856 are SEQ ID NO: 1 and SEQ ID NO: 2.

The specific probe of the enhancer function site marker related to the early screening and auxiliary diagnosis of colorectal cancer, the probe sequence of rs4810856 is SEQ ID NO: 3 and SEQ ID NO: 4.

The application of the detection reagent of the enhancer function site marker related to early screening and auxiliary diagnosis of colorectal cancer in the preparation of colorectal cancer auxiliary diagnosis kit.

A colorectal cancer early screening and auxiliary diagnostic kit for detecting rs4810856 in peripheral blood DNA.

The kit for early screening and auxiliary diagnosis of colorectal cancer includes the above-mentioned specific amplification primers for SNP markers and specific probes for SNP markers.

The beneficial effects of the invention are: from the molecular biology and gene diagnosis level, it provides a technical method for screening high-risk groups of colorectal cancer. This method is based on our previous integration of multi-omics technology through the ABC model, showing that the rs4810856 locus is associated with the susceptibility of colorectal cancer in the Chinese population. Through the ingenious design of primers and probes for the rs4810856 site, it is possible to detect the risk site of rs4810856 in the normal population by means of fluorescent quantitative PCR, thereby identifying high-risk groups of colorectal cancer, assisting early screening and monitoring of colorectal cancer. Diagnosis, this technical method is ingeniously designed, simple and feasible, and the results are accurate and reliable. It can be promoted in hospitals at all levels. It provides help in assessing the risk of colorectal cancer and is helpful for clinical colorectal cancer screening for this group of people. investigation and early intervention.

Description of drawings

Fig. 1 Flowchart of identification of colorectal cancer susceptibility locus rs4810856 by ABC map method;

Figure 2 Schematic diagram of population screening and potential regulatory effects of enhancer site rs4810856;

Figure 3 AUC curve of colorectal cancer risk prediction model based on SNP rs4810856;

Figure 4 is a schematic diagram of the results of the rs4810856 dual luciferase reporter gene experiment;

Figure 5 is a schematic diagram of the regulation of the expression levels of the target genes PREX1, CSE1L and STAU1 by the rs4810856 genotype;

Fig. 6 Schematic diagram of rs4810856 single nucleotide mutation constructed by CRISPR/Cas9 in SW480 and HCT116 cells;

Figure 7 Schematic diagram of rs4810856 site affecting the proliferation ability of colorectal cancer cells;

Figure 8 Schematic diagram of the effect of overexpression of target genes PREX1, CSE1L and STAU1 on the proliferation ability of colorectal cancer cell lines;

Fig. 9 Schematic diagram of the effect of overexpression of target genes PREX1, CSE1L and STAU1 on subcutaneous tumor formation in nude mice.

Detailed ways

The principles and features of the present invention are described below, and the examples given are only used to explain the present invention, and are not intended to limit the scope of the present invention.

The technical scheme that the present invention solves the problems of the technologies described above is as follows:

An enhancer functional site marker related to early screening and auxiliary diagnosis of colorectal cancer, the marker is rs4810856.

The specific amplification primer sequences of rs4810856 are SEQ ID NO: 1 and SEQ ID NO: 2.

The specific probe of the enhancer function site marker related to early screening and auxiliary diagnosis of colorectal cancer, the probe sequence of rs4810856 is SEQ ID NO: 3 and SEQ ID NO: 4.

The application of the detection reagent of the enhancer functional site marker related to early screening and auxiliary diagnosis of colorectal cancer in the preparation of colorectal cancer auxiliary diagnosis kit.

A kit for early screening and auxiliary diagnosis of colorectal cancer, which is used to detect rs4810856 in peripheral blood DNA.

The kit includes specific amplification primers for the above-mentioned SNP markers and specific probes for the above-mentioned SNP markers.

Specifically, the technical solution to the problem of the present invention includes: (1) establishing a unified standard specimen library and database: collecting standard operating procedures (SOPs) to collect standard blood samples from subjects, systematically collecting complete demographic data and clinical information. (2) Genotype detection: Select colorectal cancer cases and healthy controls, and use the ABC model combined with ATAC-seq, CHIP-seq, RNA-se, Hi-C and other multi-omics to more accurately find the correlation with the incidence of colorectal cancer The SNP marker rs4810856(3) of the screened positive association markers was verified in an independent large population sample to judge the stability of the association. (4) Development of auxiliary diagnostic kits for colorectal cancer: develop early screening and auxiliary diagnostic kits based on genetic markers with significant differences in genotype distribution frequencies between colorectal cancer cases and healthy controls.

Specifically the experimental method of the research of the present invention mainly comprises the following contents:

First, we collected 10 fresh colorectal cancer tissue samples from Tongji Hospital affiliated to Huazhong University of Science and Technology, which were resected by colorectal cancer surgery. All 10 samples were subjected to ATAC-seq, H3K27ac ChIP-seq and RNA-seq experiments. We use the ABC model system to integrate ATAC-seq, H3K27acChIP-seq, RNA-seq and Hi-C omics data. The Hi-C experimental data is downloaded from the ENCODE database, considering enhancer activity and chromatin with the target gene promoter The effect of interaction on gene expression, and then identify enhancer elements and their regulated target genes on a genome-wide scale, and draw an enhancer-target gene mapping map. With the ABC score ≥ 0.02 as the significance threshold, a total of 26,877 significant ABC enhancer SNPs were identified. The specific process is shown in Figure 1.

Next, we downloaded three colorectal cancer GWAS datasets for European populations from the dbGaP database, and the combined data contained a total of 17,789 colorectal cancer cases and 19,951 control samples. The basic demographic characteristics are shown in Table 1. Based on the unconditional Logistic regression model, and after adjusting the confounding factors of age and sex, we calculated the association between the identified ABC enhancer SNPs and the susceptibility to colorectal cancer, and then drew the Manhattan plot. The results are shown in panel A of Figure 2 Show. In the calculation process, we identified a total of 4,847 ABC enhancer SNPs associated with colorectal cancer risk using P-value <0.05 as the significance threshold. Among them, we found that the enhancer region where rs4810856 is located: chr20:48682097-48683408, has the strongest gene expression regulation effect, and can simultaneously regulate three target genes: PREX1, CSE1L and STAU1, the results are shown in Figure 2 B . The results of the case-control study showed that individuals carrying the rs4810856 risk genotype had an increased risk of colorectal cancer by 4% compared with normal individuals in European population samples (OR=1.04, 95% CI: 1.01-1.08, P=3.68 ×10 ^-5 ).

Table 1. Basic information of colorectal cancer patients and normal controls in the European and Chinese populations used in the study

Table 2. Results of risk association analysis of rs4810856 colorectal cancer in European population samples

At the same time, a total of 6,024 colorectal cancer patients with complete medical records and 10,022 normal controls without tumor history were recruited from Beijing and Wuhan, China, and genotyped these individuals. The patients were diagnosed by histopathology, and there was no age limit; the normal controls had no history of tumor, and no signs of tumor after physical examination. At the same time, information such as gender, age, smoking and drinking of the research subjects was collected. Each research subject gave informed consent to participate in this study and donated 2ml of peripheral venous blood for the separation and preparation of lymphocyte genomic DNA. The basic demographic characteristics are shown in Table 1.

We used the population samples recruited from the two places for the two-stage population verification, and calculated the association between the SNP site rs4810856 and the susceptibility to colorectal cancer by using the unconditional logistic regression model for the two-stage population samples, and adjusted the gender, age, Smoking and alcohol use. The results of the two-stage case-control analysis of the Chinese population showed that individuals carrying the rs4810856[C] risk genotype had an increased risk of colorectal cancer by 30% and 14%, respectively (OR=1.30, 95%CI: 1.12-1.49, P= 2.72×10 ^-4 ; OR=1.14, 95% CI: 1.05-1.22, P=3.76×10 ^-4 ). In order to strengthen the testing power of the case-control study, the two-stage samples were combined to test the association between rs4810856 and colorectal cancer susceptibility. It was found that the results of the case-control study in the Chinese population were consistent with the results of colorectal cancer samples in the European population. Individuals carrying the rs4810856[C] risk genotype had an increased risk of colorectal cancer by 16% (OR=1.16, 95% CI: 1.10-1.23, P=9.74×10 ^-7 ), see Table 3 for detailed results.

Table 3. Results of risk association analysis of rs4810856 colorectal cancer in Chinese population samples

*Calculated using Logistic regression model, adjusted for gender, age, smoking and alcohol consumption.

The previous large-sample population association analysis showed that this locus is a risk locus for colorectal cancer, so we used this risk SNP locus to establish a risk prediction model for colorectal cancer. We constructed a formula that considered the three genotypes of SNPs and sex, age, smoking and drinking. Among them, for SNP genotyping, wild homozygous = "1", heterozygous = "2", mutant homozygous = "3"; for gender, male is "1", female is "0"; for Age, greater than or equal to 60 years old is "1", less than 60 years old is "0". In the analysis, the multivariate logistic regression coefficient β was used as the weight, and the formula of risk score based on rs4810856 classification was obtained as follows:

Chinese population risk score=(0.1064×score of gender)+(-0.0121×score of age)+(0.2015×score of smoking)+(-0.1129×score of drinking)+(-0.0648×score of rs8100241 type).

European population risk score = (-0.1889×score of sex)+(-0.0296×score of age)+(-0.0437×score of rs8100241 type)

By drawing the AUC curve, the area under the curve of the model can be obtained as 0.591 and 0.561 for the European population and the Chinese population, respectively, and the results are shown in Figure 3.

In order to functionally analyze the colorectal cancer risk association of rs4810856 marker, the mechanism of action of this SNP site on the occurrence and development of colorectal cancer was explored.

First, we verified the enhancer function regulation function of the region where rs4810856 is located by reporter gene assay. The DNA fragments containing different alleles of rs4810856 were constructed in the reporter gene plasmid vector forward or reverse, and were co-transfected with the internal control plasmid pRL-SV40 into two human colorectal cancer cell lines, SW480 and HCT116, and then the double fluorescence was detected The expression of luciferase gene was compared, and the relative luciferase activity difference among different groups was compared. We found that regardless of the insertion direction of the DNA fragment, the reporter gene activity of the recombinant plasmid containing the rs4810856[C] allele was significantly higher than that of the pGL3-Promoter empty group, indicating that the DNA fragment at the target site has enhancer activity. Compared with the rs4810856[C] genotype group, the reporter gene activity was significantly decreased in the rs4810856[T] genotype group. The above experimental results show that the region where rs4810856 is located has enhancer activity, and the enhancer of rs4810856[C] has a stronger transcriptional activation effect than rs4810856[T]. The results are shown in Figure 4. The results of eQTL analysis showed that the rs4810856 locus with enhancer activity was closely related to the expression levels of target genes PREX1, CSE1L and STAU1 in CRC tissues of population samples, the results are shown in Figure 5.

In order to further explore the effects of rs4810856 single-base gene editing and target genes on the proliferation of colorectal cancer cells, we conducted cell proliferation experiments, colony formation experiments, and subcutaneous tumor formation experiments in nude mice. In the cell proliferation experiment, we used CRISPR/Cas9 to construct rs4810856 single nucleotide mutant cell lines in SW480 and HCT116 cells as an experimental model to study the effect of rs4810856 on the malignant phenotype of colorectal cancer cells. The construction process is shown in Figure 6. Show. Compared with the rs4810856[T] cell line, the proliferation ability of the cells carrying the rs4810856[C] cell line was significantly stronger, and the results are shown in Figure 7. Then, we used the stably transfected cell lines with overexpression of PREX1, CSE1L and STAU1 respectively and combined overexpression as experimental models to detect the effect of target genes on the proliferation ability of colorectal cancer cells. The experiment found that compared with the cells of the blank control group, the clone formation ability of the cell lines overexpressing the target genes was significantly enhanced, and the clone formation ability of the cell lines overexpressing the target genes was the strongest. The results are shown in Figure 8. In the subcutaneous tumor formation experiment in nude mice in vivo, we injected the stable cell line into the subcutaneous of nude mice, regularly measured the volume of subcutaneous tumors in different groups of nude mice, and drew the growth curve of subcutaneous tumor formation. We found that compared with the nude mice injected with blank control cell lines, the nude mice injected with PREX1, CSE1L and STAU1 overexpressed cell lines respectively had significantly larger subcutaneous tumor volume, while the nude mice injected with target gene combined with overexpressed cell lines , the subcutaneous tumor had the largest volume, and the results are shown in Figure 9. The results of cell phenotype and subcutaneous tumor formation in nude mice showed that PREX1, CSE1L and STAU1 could exert a certain synergistic effect to promote the proliferation of colorectal cancer cells.

Combining the above-mentioned large-scale population analysis, bioinformatics analysis and rigorous experimental design, the results show that the rs4810856 risk locus located in the chromatin 20q13. Individuals with genetic variants significantly increase the risk of colorectal cancer. And its mechanism of action is to inhibit the chromatin interaction between its region and the promoter of the target gene by affecting the binding ability of rs4810856 and the transcription factor ZEB1, thereby reducing the expression levels of the target genes PREX1, CSE1L and STAU1, and inhibiting the AKT signaling pathway Activated to reduce the risk of colorectal cancer. Therefore, the rs4810856 risk locus located in the non-coding region is closely related to the risk of colorectal cancer, and it is expected to be clinically applied to assist in the early diagnosis of colorectal cancer and early detection of high-risk groups of colorectal cancer.

experimental method:

1. DNA extraction from peripheral blood:

We used conventional phenol-chloroform method to extract DNA, the specific steps are as follows:

1) Take about 3ml of anticoagulated blood, centrifuge at 5,000×g for 15min at room temperature, discard the upper layer, and keep about 0.3ml of blood cells. Add 0.5ml freshly prepared extraction buffer with a final concentration of 20μg/ml RNase, mix well and incubate at 37°C for 1h.

2) Add proteinase K at a final concentration of 100 μg/ml, mix well and incubate overnight at 37°C.

3) Add 0.7 ml of phenol (pH=7.0) equilibrated with Tris buffer solution to each tube, mix well, and centrifuge at 8,000×g for 15 min at room temperature.

4) Transfer the upper layer liquid to another 1.5ml centrifuge tube, add an equal volume of phenol-chloroform (1:1) 0.7ml, mix well for 15min; centrifuge at 8,000×g for 15min at room temperature.

5) Transfer the upper layer liquid to another clean 1.5ml centrifuge tube, add 10% volume of 10M ammonium acetate solution, add 2 times the volume of pre-cooled absolute ethanol, and stand at -20°C for 2h to precipitate DNA.

6) The precipitated DNA was washed with 75% ethanol, centrifuged at 12,000×g for 15 minutes, and the upper layer was discarded; then washed with 75% ethanol, centrifuged at 12,000×g for 15 minutes, and the upper layer was discarded.

7) Place the tubes upside down on absorbent paper. After the ethanol has evaporated, add appropriate TE buffer solution to each tube, place at 4°C for a week, and store at -20°C for later use.

2. Genotyping

The genotyping platform used was TaqMan genotyping technology (ABI 7900HT Real Time PCR system, Applied Biosystems), and the 5 μl PCR reaction system is shown in Table 4:

Table 4. TaqMan Genotyping System Preparation

The reaction conditions are: pre-denaturation at 95°C for 10 min, followed by 45 cycles of 95°C for 15 sec and 60°C for 1 min, and cooling to 4°C.

The primers and probes used in the reaction are as follows:

rs4810856 primer:

Forward primer: caatacctctcattagttatgcctaagc (SEQ ID NO: 1)

Reverse primer: cagggaggagtctggtattttttaata (SEQ ID NO: 2)

rs4810856 probe:

Forward probe: fam-acctaccatctttc-mgb (SEQ ID NO: 3)

Reverse probe: vic-acctaccacctttc-mgb (SEQ ID NO: 4)

3. Cell Proliferation Experiment

1) Cell count

Before conducting CCK-8 proliferation experiments and colony formation experiments, cell counting is required to ensure that an appropriate number of cells are inoculated in different groups of well plates. In this experiment, a hemocytometer is used for cell counting. The counting plate is composed of two counting pools of the same size, and each counting pool is composed of nine 1mm×1mm squares, and each square can hold a liquid volume of 0.1mm3. The specific counting steps are as follows:

① Digest the cell plate that has been cultured for a period of time to obtain a cell suspension, mix as much as possible to disperse the cells.

② After sterilizing the counting plate, cover it with a coverslip.

③Pipe 10mL of cell suspension and slowly drop it into the counting plate along the edge of the slide to ensure that there are no air bubbles between the cover glass and the counting plate).

④Stay for 1 min, observe the counting plate under a microscope, and count the squares at the four corners according to the following principles: a. For the cells that press the edge of the square, only count the cells on the upper and left sides; b. If the cells are clustered block, it is counted as one cell.

⑤Concentration of cell suspension = total number of cells in the four-corner square/4×104 cells/mL.

2) CCK-8 cell proliferation assay

In this study, CCK-8 kit (Dojindo, Japan) was used to detect the proliferation ability of colorectal cancer cells.

① Aspirate 100mL of cell suspension and dilute it according to the counting concentration to about 2,000 cells, and inoculate it in a 96-well plate.

② Cell viability was detected at four time points of 24h, 48h, 72h and 96h. Add 10mL of CCK-8 reagent to each well, shake it up and down, left and right to mix gently, and put it back into the cell culture incubator to continue culturing for 1.5h.

③ Use a microplate reader to measure the absorbance, the wavelength is set to 450nm, draw the cell proliferation curves of different experimental groups according to the absorbance values at four time points to compare the differences.

3) Colony formation experiment

① After 36 hours of transfection, cells were counted and diluted to an appropriate concentration. Add 2 mL of cell suspension to each well of the 6-well plate to make the cell volume about 1,000, and continue to culture for about 2 weeks.

② During the period, observe the cell morphology and replace the fresh medium every 2-3 days. If cell clones are observed under the microscope, fixation staining can be performed.

③Take out the 6-well plate from the incubator, suck off the waste liquid and slowly add PBS to wash it twice. After aspirating the PBS in each well, add 2mL of methanol solution and let it stand at room temperature for 30min.

④ After absorbing the methanol, add 0.25% crystal violet solution, and place it in the dark for 30 minutes for staining.

⑤ After the staining is completed, suck off the waste liquid in the well, and wash the orifice plate with double distilled water until the field of view is clear, and take pictures for preservation.

In summary, we systematically integrated multi-omics data including ATAC-seq, CHIP-seq, RNA-seq, and Hi-C by using the ABC model, thereby constructing a colorectal cancer enhancer-target gene mapping map. Further, combined with large-scale case-control studies of European populations and Chinese populations, biological experiments and other technical methods. It was found that rs4810856 located in the 20q13.13 region had the strongest gene expression regulation effect, and individuals carrying the [C] allele had a significantly increased risk of colorectal cancer compared with rs4810856[T] individuals. Mechanistically, we found that rs4810856 located in the chromatin 20q13.13 region can simultaneously regulate three target genes: PREX1, CSE1L and STAU1. Moreover, the binding ability of rs4810856[C] to the transcription factor ZEB1 is stronger than that of rs4810856[T] carriers, which affects the chromatin interaction between its region and the promoter of the target gene, thereby promoting the expression levels of the target genes PREX1, CSE1L and STAU1 , and affect the activation of the AKT signaling pathway, thereby promoting the risk of colorectal cancer. Therefore, the rs4810856 mutation located in the non-coding region is closely related to the risk of colorectal cancer, and it is expected to be clinically applied to assist in the early diagnosis of colorectal cancer and early detection of colorectal cancer patients.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

Claims (1)

1. The application of the detection reagent of enhancer functional site rs4810856 in the preparation of colorectal cancer auxiliary diagnosis kit.

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