An eRNA molecular marker for auxiliary diagnosis of colorectal cancer and its application

Abstract

The present invention discloses an eRNA molecular marker for auxiliary diagnosis of colorectal cancer and its application. The eRNA molecular marker is characterized in that the eRNA molecular marker is eRNA58704, and its nucleotide sequence is shown in SEQ ID NO.1. The invention also provides the application of the eRNA molecular marker in the preparation of a kit for auxiliary diagnosis of colorectal cancer. The kit comprises eRNA58704 fluorescent quantitative PCR specific amplification primers, the nucleotide sequence of the upstream primer is 5'-AAGTCAGGTTCATTTTCCGTGC-3', the nucleotide sequence of the downstream primer is 5'-AGCAACCAACGCTCTTGTCA-3', and also comprises carbohydrate antigen CA19-9. The kit has the advantages of convenient, fast and efficient early diagnosis of colorectal cancer patients at the molecular level, strong pertinence, strong specificity and high sensitivity, and is conducive to the early detection and timely treatment of colorectal cancer.

Description

Technical Field

[0001] The present invention relates to an auxiliary diagnosis method for intestinal cancer, and in particular to an eRNA molecular marker for auxiliary diagnosis of intestinal cancer and application thereof. Background Art

[0002] Colorectal cancer is a malignant tumor that is highly heterogeneous at the molecular level and in clinical manifestations. Its high-risk factors include genetics, environment, diet, inflammatory bowel disease, gender, etc. The diagnosis of colorectal cancer relies on endoscopy, pathological biopsy, CT imaging, magnetic resonance imaging, ultrasound examination and other means, and a comprehensive judgment is made in combination with clinical manifestations. The diagnosis rate of early colorectal cancer is low, and many colorectal cancer patients are already in the middle and late stages of the disease when they first visit the doctor, so the prognosis is poor. Studies have shown that early diagnosis and early treatment are the key to improving the survival rate of colorectal cancer patients. If surgical treatment is performed in time in the early stage of colorectal cancer, the patient's five-year survival rate can be significantly improved to more than 90%. Therefore, the current focus of colorectal cancer research is to discover stable and effective early diagnostic markers, as well as to deeply explore the pathogenesis of colorectal cancer in order to reduce the mortality rate of patients and improve their quality of life.

[0003] Early colorectal cancer usually has no obvious symptoms, and its symptoms are often similar to those of other gastrointestinal diseases, which makes screening and diagnosis complicated. Tissue biopsy has always been considered the main method for cancer diagnosis. However, because it can only obtain a small part of the tissue, it is often difficult to fully reflect the heterogeneity of the tumor or monitor the dynamic changes of the tumor in real time. In addition, as an invasive operation, tissue biopsy may increase the risk of cancer cell metastasis, thereby adversely affecting the patient's survival and prognosis. In contrast, liquid biopsy, due to its minimally invasive nature, provides broader prospects for the diagnosis and dynamic monitoring of cancer. The traditional tumor marker carbohydrate antigen CA19-9 currently used for auxiliary diagnosis of colorectal cancer has limited sensitivity and specificity, which is difficult to meet the needs of early diagnosis. Therefore, it is urgent to find an ideal tumor marker that can be used for the early diagnosis of colorectal cancer.

[0004] Enhancer RNA (eRNA) is a newly discovered class of non-coding RNA that has unique biological functions in gene expression regulation and disease processes. Enhancers are DNA fragments in the genome that can significantly increase the expression of target genes, usually located upstream or downstream of the target gene. eRNA is transcribed from the enhancer region and is usually between 6kb and 9kb in length. Its production indicates the activation state of the enhancer. Studies in recent years have shown that eRNA exhibits potential biomarker effects in a variety of diseases such as breast cancer, gastric cancer, and cervical cancer, while there are fewer studies on the association between eRNA and colorectal cancer. eRNA has therapeutic potential in non-invasive detection in body fluids such as blood, saliva, and urine. Therefore, it is of great significance to develop eRNA molecular markers for auxiliary diagnosis of colorectal cancer and to reflect the diagnosis of early molecular changes in colorectal cancer. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an eRNA molecular marker with strong specificity and high sensitivity for auxiliary diagnosis of colorectal cancer and its application.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: an eRNA molecular marker for auxiliary diagnosis of colorectal cancer, the eRNA molecular marker is eRNA58704, and its nucleotide sequence is shown in SEQ ID NO. 1. The location is on human chromosome 4: 74539992-74547511.

[0007] The present invention also provides the use of the eRNA molecular marker in preparing a kit for auxiliary diagnosis of colorectal cancer.

[0008] Furthermore, the kit includes upstream and downstream primers for fluorescent quantitative PCR amplification of eRNA58704. The nucleotide sequence of the upstream primer for fluorescent quantitative PCR amplification of eRNA58704 is shown in SEQ ID NO.2: 5'-AAGTCAGGTTCATTTTCCGTGC-3'; the nucleotide sequence of the downstream primer for fluorescent quantitative PCR amplification of eRNA58704 is shown in SEQ ID NO.3: 5'-AGCAACCAACGCTCTTGTCA-3'.

[0009] Furthermore, the kit includes the eRNA58704 molecular marker and the carbohydrate antigen CA19-9.

[0010] Compared with the prior art, the advantages of the present invention are: the present invention discloses for the first time an eRNA molecular marker for auxiliary diagnosis of colorectal cancer and its application, and the eRNA58704 molecular marker is highly expressed in the plasma of colorectal cancer patients. Using the collected peripheral blood specimens, the detection of eRNA58704 in the plasma alone or in combination with the carbohydrate antigen CA19-9 can conveniently, quickly and efficiently diagnose colorectal cancer patients at the molecular level in an early stage, with strong pertinence, high sensitivity, and high accuracy of the test results, which improves the detection rate of colorectal cancer and is conducive to the early detection and timely treatment of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 In the figure, A is the t-SNE (t-distributed stochastic neighbor embedding) analysis result of tumor epithelial cells of 14 cancers, B is the log2 multiple result of the average expression of eRNA58704 in the colorectal cancer cell group relative to the average expression of all other cancer cell types, the small black dotted box on the left represents the iconic eRNA in colorectal cancer, and the large black dotted box on the right represents the expression pattern of the iconic eRNA in colorectal cancer in different cancers;

[0012] Figure 2 In the figure, A is the volcano map of the TCGA-rectal adenocarcinoma dataset, and B is the volcano map of the TCGA-colon adenocarcinoma dataset;

[0013] Figure 3 In the figure, A is an agarose gel electrophoresis diagram showing the cDNA end amplification band at the 5' end, the middle sequence band and the cDNA end amplification band at the 3' end of eRNA58704, and B is the upstream and downstream amplification primers designed at 3369-3566 bp on the eRNA58704 genome;

[0014] Figure 4 is the lg value of the pUC57-eRNA58704 recombinant plasmid copy number and C Linear standard curve of q value;

[0015] Figure 5 The statistical analysis results of the eRNA58704 copy number in the plasma of healthy people and colorectal cancer patients;

[0016] Figure 6 is the area under the ROC curve and diagnostic value of eRNA58704 in colorectal cancer patients;

[0017] Figure 7 The area under the ROC curve and the combined diagnostic value of eRNA58704 and CA19-9 in colorectal cancer. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below with reference to the accompanying drawings.

[0019] Specific example 1: Screening out eRNA58704 as a molecular marker for colorectal cancer.

[0020] By collecting public full-length single-cell sequencing databases including SMARTer, Smart-seq, Smart-seq2, Quartz-seq, RamDA-seq, SUPeR-seq, MATQ-seq and Tang's method, tumor epithelial cells of 14 cancers were collected, such as Figure 1 The following are shown: colorectal cancer (CC), neuroblastoma (NB), multiple myeloma (MM), lung cancer (LC), breast cancer subtypes including luminal B and luminal A; pancreatic cancer (PAAD), oral squamous cell carcinoma (OSCC), triple-negative breast cancer (TNBC), myxoid liposarcoma (MLPS), HER2-positive breast cancer (Human epidermal growth factor receptor 2), glioblastoma multiforme (GBM), hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC).

[0021] We further established a human eRNA collection based on enhancer databases such as ENCODE, FANTOM, and Roadmap, and eRNA databases such as HeRA, eRic, HACER, and RAEdb. We then used the findallmarker function in the Seurat software package in the R language to calculate and identify the characteristic genes of specific cell populations, and screened out the corresponding specifically expressed marker eRNAs in tumor epithelial cells of 14 types of cancer. The average expression of this eRNA in the specified cell type relative to the log2 multiple of other cell types is >0.5. Figure 1As shown in A, the t-SNE (t-distributed stochastic neighbor embedding) analysis results of tumor epithelial cells of 14 types of cancer are displayed, and different colors represent different cancer types.

[0022] like Figure 1 As shown in B, the characteristic eRNAs of the colorectal cancer cell population were screened out by the findallmarker function, and it was found that the average expression level of eRNA58704 in the colorectal cancer cell population was >0.5 of the log2 multiple of other cancer cell types, confirming that it was one of the marker eRNAs of the colorectal cancer cell population. Figure 1 The circle color gradient in B represents the average expression level of eRNA, and the circle size gradient represents the proportion of cells expressing characteristic eRNA in the colorectal cancer cell population to the total number of cells of this tumor type. Figure 2 The differential expression of eRNA58704 in rectal adenocarcinoma and colon adenocarcinoma from The Cancer Genome Atlas (TCGA) is shown. Figure 2 The pink, blue, and black dots in the figure correspond to up-regulated, down-regulated, and no significant difference in gene expression, respectively. Figure 2 As shown in A, the volcano map of the TCGA-rectal adenocarcinoma dataset shows that eRNA58704 is significantly highly expressed in rectal adenocarcinoma tissues. Figure 2 As shown in B, the volcano plot of the TCGA-colon adenocarcinoma dataset shows that eRNA58704 is significantly overexpressed in colon adenocarcinoma tissues. eRNA58704 is an upregulated signature gene in both cancer types, indicating its potential diagnostic or prognostic value in colorectal cancer.

[0023] Specific Example 2: The diagnostic value of eRNA58704 as a molecular marker for colorectal cancer.

[0024] 1. Collection of plasma from healthy people and colorectal cancer patients: This study collected plasma samples from 159 healthy people and 93 colorectal cancer patients from the First Affiliated Hospital of Ningbo University. In the healthy control group, there were 65 males and 94 females with an average age of 40.8±11.5 years; in the colorectal cancer patient group, there were 54 males and 39 females with an average age of 66.4±12.5 years. The relevant information of all participants has been fully recorded and organized into the clinical data database in accordance with regulations. Informed consent has been obtained from the individual or his family, and an information database of the patient's clinical pathological data has been collected and established.

[0025] Inclusion criteria: ① Patients diagnosed with colorectal adenocarcinoma by pathological histological examination. ② Adult patients over 18 years old. ③ Patients voluntarily participated in this study, signed informed consent, and were able to provide plasma samples that met the requirements. Exclusion criteria: ① Patients with other active malignant tumors in the past or currently. ② Patients with severe organ dysfunction such as heart, liver, and kidney, or other serious diseases. ③ Pregnant or lactating women. ④ Patients who have received anti-tumor treatments such as chemotherapy, radiotherapy, and immunotherapy in the past 4 weeks. ⑤ Patients who have undergone major surgery or have a history of severe trauma recently (such as within 6 months). ⑥ Patients with serious diseases that affect the blood circulation system (such as severe anemia, leukemia).

[0026] 2. Separation of plasma: Peripheral blood samples were collected from healthy people and colorectal cancer patients, approximately 5 mL for each sample. The blood samples were placed in a vacuum blood collection tube containing an anticoagulant and immediately placed in a 4°C refrigerator. The samples were allowed to stand for 30 minutes and centrifuged at 3000 rpm for 10 minutes at 4°C to remove residual blood cell components in the plasma. The upper plasma was aspirated and transferred to enzyme-free centrifuge tubes. The obtained plasma was packaged and stored in a -80°C refrigerator.

[0027] 3. Extraction of plasma RNA: Use Trizol LS reagent from Invitrogen, USA, pipette 250 μL of plasma sample into a 1.5 mL nuclease-free centrifuge tube, add 750 μL of Trizol LS reagent, fasten the cap of the centrifuge tube, vortex for 15 seconds, and place in a 4°C refrigerator for 10 minutes; take it out and centrifuge it for 10 seconds, add 200 μL of chloroform, fasten the cap of the centrifuge tube, shake it by hand 6 times, place it in a 4°C centrifuge for 3 minutes, and centrifuge it at 4°C, 12000 rpm for 15 minutes to separate the liquid in the tube into 3 clear layers; prepare a 1.5 mL nuclease-free centrifuge tube, add 500 μL of isopropanol, take out the centrifuged specimen, pipette 500 μL of the upper transparent liquid into the prepared centrifuge tube with isopropanol, fasten the cap of the centrifuge tube, invert and mix, let it stand at 4°C for 10 minutes, and then centrifuge it at 4°C, 12000 rpm for 10 min, carefully discard the supernatant; add 1 mL of pre-cooled 75% ethanol to the centrifuge tube, fasten the centrifuge tube cap, and turn the centrifuge tube upside down to wash the precipitate; centrifuge at 4°C, 12000 rpm for 3 min, discard the supernatant, and place the centrifuge tube in the centrifuge again, centrifuge at 4°C, 12000 rpm for 1 min and discard the supernatant; dry for 3 min, add 8 μL of enzyme-free water, and mix by pipetting; take 8 μL of total RNA for reverse transcription.

[0028] 4. Reverse transcription and cDNA synthesis: According to the instructions of Polestar 1st cDNA Synthesis Kit (gDNA removal) produced by Beijing Baoying Tonghui Biotechnology Co., Ltd., the reverse transcription reaction solution was prepared according to the following component formula.

[0029] Reverse transcription reaction system: 7 µL enzyme-free water, 4 µL 5× Polestar RT MasterMix (containing dsDNase), 20 µM concentration of Oligo dT (18) Primer 1 µL and total RNA 8 µL, total volume 20 µL. cDNA synthesis reaction procedure: 25℃10 min, 55℃60 min, 85℃5min, reverse transcribed cDNA stored at -20℃, if directly proceed to the next step of fluorescent quantitative PCR experiment, add samples according to the PCR system.

[0030] 5. Rapid amplification of cDNA ends (RACE) was used to determine the full-length sequence of eRNA58704, whose nucleotide sequence is shown in SEQ ID NO.1. The specific process is as follows:

[0031] First, RNA was extracted using Trizol extraction kit (purchased from Shanghai Sangon Biotechnology, catalog number B511321), 1.5% agarose, 1× Tris-acetate electrophoresis buffer, and observed and photographed under ultraviolet transmission light. The 5'-RACE kit was purchased from Shanghai Sangon Biotechnology (catalog number B605102), and the nucleotide sequence of the upstream primer for 5'-RACE specific amplification was shown in SEQ ID NO.4: 5'-TACTTCAAATAGGCCAGCAGCT-3', and the nucleotide sequence of the downstream primer for 5'-RACE specific amplification was shown in SEQ ID NO.5: 5'-AGTTTTTCCTATTGCCAGCTACTTC-3'.

[0032] The 3'-RACE kit was purchased from Shanghai Sangon Biotechnology (Cat. No. B605101). The nucleotide sequence of the upstream primer for 3'-RACE specific amplification is shown in SEQ ID NO.6: 5'-GTGCCTTCTTAAAAAAAATCTGTGA-3', and the nucleotide sequence of the downstream primer for 3'-RACE specific amplification is shown in SEQ ID NO.7: 5'-CTCCTCTGGCTGACCTGATTCTT-3'.

[0033] The nucleotide sequence of the upstream primer of the eRNA58704 intermediate sequence fragment 1 (PCR amplification product length 884 bp) is shown in SEQ ID NO.8: 5'-CCTCCATTTGCCAAAACCTCA-3', and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.9: 5'-GTAGTACCAACCTCATAGAGTTGCAG-3'.

[0034] The nucleotide sequence of the upstream primer of the eRNA58704 intermediate sequence fragment 2 (PCR amplification product length 910 bp) is shown in SEQ ID NO.10: 5'-TAATGTCCATATTTGTGAGGCAGTT-3', and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.11: 5'-AAAACAGCTATAACGCCCTTCAC-3'.

[0035] The nucleotide sequence of the upstream primer of the middle sequence fragment 3 of eRNA58704 (the PCR amplification product length is 963 bp) is shown in SEQ ID NO.12: 5'-TCATTCATTTATCAACAAGCATTTATC-3', and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.13: 5'-GAATGTCTCAATTGCTCAGTTTATG-3'.

[0036] The nucleotide sequence of the upstream primer of the eRNA58704 intermediate sequence fragment 4 (PCR amplification product length 967 bp) is shown in SEQ ID NO.14: 5'-AATGGTTAATGTGTGCTGTATGGAT-3', and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.15: 5'-CTGTGTACAATGCCTAAAGGAAGTC-3'.

[0037] The nucleotide sequence of the upstream primer of the middle sequence fragment 5 of eRNA58704 (the PCR amplification product length is 993 bp) is shown in SEQ ID NO.16: 5'-GAGACTGGGAAGACTCACACTTCTG-3', and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.17: 5'-CTCCAAAGAATGTATACAAGAACACTC-3'.

[0038] The nucleotide sequence of the upstream primer of the eRNA58704 intermediate sequence fragment 6 (PCR amplification product length 970 bp) is shown in SEQ ID NO.18: 5'-CTGTAAGAGGTCAAAGGAGGGTG-3', and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.19: 5'-TAGGGAGGATCTGATGGGGTTAG-3'.

[0039] The nucleotide sequence of the upstream primer of the eRNA58704 intermediate sequence fragment 7 (PCR amplification product length 927 bp) is shown in SEQ ID NO. 20: 5'-AGATGTTCTCTGTTGCTCAGGATAG-3', and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 21: 5'-TAGGTGTCACTGGACAAATGACTG-3'. These 7 amplified fragments have overlapping regions, covering the entire intermediate sequence.

[0040] like Figure 3 As shown in A, the agarose gel electrophoresis results show the amplified fragments at the 5' end, the middle sequence and the 3' end. The rapid amplification of cDNA ends technique determined that the 5' end length of eRNA58704 was 416 bp and the 3' end length was 391 bp.

[0041] The specific primers for quantitative PCR amplification of eRNA58704 were further designed, such as Figure 3 As shown in B, upstream and downstream primers for fluorescence quantitative PCR amplification of eRNA58704 were designed at the 3369-3566bp position of the eRNA58704 sequence. The nucleotide sequence of the upstream primer for fluorescence quantitative PCR amplification of eRNA58704 is shown in SEQ ID NO.2: 5'-AAGTCAGGTTCATTTTCCGTGC-3', and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3: 5'-AGCAACCAACGCTCTTGTCA-3'. The TA cloning sequencing results showed that the PCR product was 198bp in length, which matched the sequence at 3369-3566bp of eRNA58704, proving that the designed primers were highly specific.

[0042] 6. Preparation of plasmid standards: After the PCR product of eRNA58704 was sequenced by TA cloning, the base sequence 198bp obtained by sequencing was cloned into the pUC57 plasmid to obtain pUC57-eRNA58704 (inserted base 198bp, full length of plasmid 2902bp), which was prepared by General Biotechnology (Anhui) Co., Ltd.

[0043] 7. UV spectrophotometer measurement of recombinant plasmid: Dissolve pUC57-eRNA58704 recombinant plasmid powder in 50 μl of enzyme-free water to obtain the first high-concentration standard, and then measure its concentration and OD 260 The concentration of the recombinant plasmid pUC57-eRNA42767 was 216.88 ng / μL, and the OD 260 The value is 4.338.

[0044] 8. Absolute quantitative PCR: Perform gradient dilution of high-concentration standard products and calculate the copy number in 1 μL of plasmid standard products according to the formula , where the DNA length is the full length of the plasmid, including the backbone vector, and the copy numbers of the 7 dilution gradients of the pUC57-eRNA58704 recombinant plasmid were calculated, with concentrations of 6.82×10 9 copies / μL, 6.82×10 8 copies / μL, 6.82×10 7 copies / μL, 6.82×10 6 copies / μL, 6.82×10 5 copies / μL, 6.82×10 4 copies / μL and 6.82×10 3 Each gradient of plasmid standard was set up with 3 replicates, and the samples were added to the 96-well PCR reaction plate. The amplification was performed according to the reaction system and reaction procedure of real-time quantitative PCR amplification to obtain 7 different copy numbers. C q value.

[0045] The reaction system formula of real-time quantitative PCR amplification is as follows: 10.9 µL enzyme-free water, 1 µL cDNA, 0.3 µL upstream primer of fluorescence quantitative PCR amplification with a concentration of 10 µM, 0.3 µL downstream primer of fluorescence quantitative PCR amplification with a concentration of 10 µM, 12.5 µL GoTaq®qPCR Master Mix 2×, total volume is 25 µL. The reaction program of real-time quantitative PCR amplification is as follows: 95°C pre-denaturation for 5 minutes, 1 cycle; 95°C denaturation for 15 seconds, 62°C annealing for 30 seconds, 72°C extension for 30 seconds, 40 cycles. The fluorescence quantitative PCR determination system automatically generates the standard curve of the pUC57-eRNA58704 recombinant plasmid according to the change law of fluorescence value.

[0046] The results are as follows Figure 4 As shown, the linear equation of the pUC57-eRNA58704 recombinant plasmid is Y = -4.042X +46.85, where Y is the value obtained by real-time quantitative PCR detection. C q value, 10X The corresponding copy number in the test sample. The determination coefficient R 2 is 0.9963, indicating that within the range of plasmid dilution concentration, the lg value of the pUC57-eRNA58704 recombinant plasmid copy number is C The q value has a good linear relationship. The amplification efficiency E is 76.77%, indicating that the standard curve established based on the recombinant plasmid standard can accurately reflect the amplification of the target product.

[0047] The absolute quantitative PCR was used to obtain the C The q value, i.e. the Y value in the formula, is calculated as 10 copies of eRNA58704 in 1 μL cDNA according to the formula Y = -4.042X +46.85 X Since 8 μL RNA was extracted from 250 μL plasma, and then all 8 μL RNA was used for reverse transcription to obtain 20 μL cDNA, and only 1 μL cDNA was used for real-time quantitative PCR amplification, it was inferred that the copy number of eRNA58704 in 1 mL plasma was 80×10 X According to this method, the copy number of eRNA58704 in 1 mL of plasma of healthy people and colorectal cancer patients was statistically analyzed. The results are as follows Figure 5 As shown, eRNA58704 was significantly overexpressed in the plasma of colorectal cancer patients, and the cutoff value for distinguishing healthy people from colorectal cancer patients was 173,044 copies / mL.

[0048] 9. Use ROC curve to analyze the diagnostic value of eRNA58704 as a molecular marker for colorectal cancer: According to the levels of eRNA58704 in the plasma of healthy subjects and colorectal cancer patients, analyze the area under the ROC curve, sensitivity, specificity, positive predictive value and negative predictive value of eRNA58704 level in distinguishing healthy subjects from colorectal cancer patients, and evaluate the diagnostic value of eRNA58704 in judging colorectal cancer patients.

[0049] The results are as follows Figure 6 As shown in the figure, the diagnostic value of eRNA58704 in the plasma of colorectal cancer patients is: the area under the ROC curve is 0.757, the sensitivity is 0.806, the specificity is 0.629, the positive predictive value is 0.560, and the negative predictive value is 0.847, which can be effectively used for the early screening and diagnosis of colorectal cancer. Figure 7 As shown, the combined use of eRNA58704 and CA19-9 can significantly improve the diagnostic accuracy of colorectal cancer, and the area under the ROC curve of combined diagnosis is 0.818.

[0050] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. An eRNA molecular marker for assisting the diagnosis of colorectal cancer, characterized in that: The eRNA molecular marker is eRNA58704, and the nucleotide sequence of the eRNA58704 is shown in SEQ ID NO.

1.

2. Use of the eRNA molecular marker according to claim 1 in preparing a kit for auxiliary diagnosis of colorectal cancer.

3. The use of the eRNA molecular marker according to claim 2 in preparing a kit for auxiliary diagnosis of colorectal cancer, characterized in that: The kit includes upstream and downstream primers for eRNA58704 fluorescence quantitative PCR amplification. The nucleotide sequence of the upstream primer for eRNA58704 fluorescence quantitative PCR amplification is shown in SEQ ID NO.2: 5'-AAGTCAGGTTCATTTTCCGTGC-3'; the nucleotide sequence of the downstream primer for eRNA58704 fluorescence quantitative PCR amplification is shown in SEQ ID NO.3: 5'-AGCAACCAACGCTCTTGTCA-3'.

4. The use of the eRNA molecular marker according to claim 2 in preparing a kit for auxiliary diagnosis of colorectal cancer, characterized in that: The kit comprises the eRNA58704 molecular marker and the carbohydrate antigen CA19-9.

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