ERNA molecular marker for auxiliary diagnosis of intestinal cancer and application of eRNA molecular marker

Abstract

The invention discloses an eRNA molecular marker for auxiliary diagnosis of intestinal cancer and application of the eRNA molecular marker, and is characterized in that the eRNA molecular marker is eRNA42767, and the nucleotide sequence of the eRNA molecular marker is shown as SEQ ID NO.1. The invention also provides application of the eRNA molecular marker in preparation of a kit for auxiliary diagnosis of intestinal cancer, and the kit comprises an eRNA42767 fluorescent quantitative PCR specific amplification primer, the nucleotide sequence of the upstream amplification primer is 5 '-CCTCTCTCTGGCACATGCCTA-3', and the nucleotide sequence of the downstream amplification primer is 5 '-CACATGCGTAGATTCTTGCC-3'. The kit has the advantages that early diagnosis of intestinal cancer patients on the molecular level is conveniently, quickly and efficiently realized, the pertinence, the specificity and the sensitivity are high, and early discovery and timely treatment of intestinal cancer are facilitated.

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 its application. Background Art

[0002] Colorectal cancer (CC) is one of the most common digestive system malignancies with high morbidity and mortality worldwide. Its pathogenesis is complex, often driven by multifactorial, multi-step molecular events. Early symptoms of CC are often subtle, and most patients are diagnosed in the advanced stages, missing the optimal window for treatment. Currently, widely used diagnostic methods include colonoscopy, tissue biopsy, imaging assessment, and serum tumor marker testing (such as the traditional tumor marker carbohydrate antigen CA19-9). However, these methods have limitations in practical application. For example, colonoscopy is an invasive procedure, leading to poor patient compliance and the risk of complications such as bleeding or perforation. Traditional serum markers have limited sensitivity and specificity, making them inadequate for the clinical needs of early, accurate screening and dynamic monitoring of CC.

[0003] In recent years, with the development of high-throughput sequencing and epigenetic technologies, researchers have gradually recognized the important regulatory role of non-coding RNA (ncRNA) in the development and progression of cancer. Among them, enhancer RNA (eRNA), a class of non-coding transcripts originating from active enhancer regions, has been found to regulate the expression of key oncogenes in various tumor types. Not only do eRNAs exhibit tissue- and cell-type-specific expression patterns, but they also reflect enhancer activity and transcriptional regulatory status, playing a crucial role in tumor development, progression, and metastasis. Previous studies have demonstrated that eRNAs exhibit specific expression signatures in malignant tumors such as breast, liver, and lung cancers, and may serve as potential biomarkers for early disease screening, classification, and therapeutic efficacy prediction.

[0004] Currently, molecular diagnostics for colorectal cancer primarily focus on mutations in protein-coding genes (such as KRAS and BRAF) or copy number variations. Research on the expression characteristics of eRNAs in colorectal cancer and their clinical application is still in its infancy. Due to their close association with enhancer activity, eRNAs have the potential to reflect changes in gene regulatory networks specific to colorectal cancer. They are particularly detectable in peripheral fluids such as blood, making them clinically valuable for development as noninvasive molecular biomarkers. Therefore, identifying and screening eRNA molecular markers specifically expressed in colorectal cancer is crucial for achieving early, noninvasive, and accurate diagnosis of 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, wherein the eRNA molecular marker is eRNA42767, and its nucleotide sequence is shown in SEQ ID NO. 1. The location is on human chromosome 2: 173644313-173653943.

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

[0008] The present invention also provides the use of the above-mentioned eRNA molecular marker in the preparation of an auxiliary diagnosis kit for colorectal cancer, wherein the kit includes upstream and downstream amplification primers for eRNA42767 fluorescent quantitative PCR, wherein the nucleotide sequence of the upstream amplification primer for eRNA42767 fluorescent quantitative PCR is shown in SEQ ID NO.2: 5'-CCTCTCTCTGGGACATGCCTA-3'; the nucleotide sequence of the downstream amplification primer for eRNA42767 fluorescent quantitative PCR is shown in SEQ ID NO.3: 5'-CACATGCGTAGAGATTCTTGCC-3'.

[0009] Compared with existing technologies, the present invention offers the following advantages: It is the first to disclose an eRNA molecular marker for the auxiliary diagnosis of colorectal cancer and its application. The eRNA42767 molecular marker is highly expressed in the plasma of colorectal cancer patients. Using peripheral blood samples, plasma detection of eRNA42767 allows for convenient, rapid, and efficient early diagnosis of colorectal cancer at the molecular level. This method is highly targeted, sensitive, and accurate, improving the detection rate of colorectal cancer and facilitating its early detection and timely treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 In the figure, A shows the t-SNE (t-distributed stochastic neighbor embedding) analysis results of tumor epithelial cells of 14 cancer types, and B shows the log2 fold of the average expression of eRNA in the colorectal cancer cell population relative to the average expression of all other cancer cell types. The small blue dotted square on the left represents the iconic eRNA in colorectal cancer, and the large blue dotted square on the right represents the expression pattern of the iconic eRNA in colorectal cancer in different cancers. 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; Figure 3In the figure, A is an agarose gel electrophoresis diagram of the amplified fragment of eRNA42767, including the complete 5' end sequence, the middle sequence and the complete 3' end sequence; B is a schematic diagram of the principle of designing specific upstream and downstream amplification primers for fluorescent quantitative PCR based on the eRNA42767 sequence; Figure 4 is the lg value of the pUC57-eRNA42767 recombinant plasmid copy number and C Linear standard curve of q value; Figure 5 Statistical analysis results of eRNA42767 copy numbers in plasma of healthy subjects and colorectal cancer patients; Figure 6 The ROC curve and diagnostic value of eRNA42767 in the plasma of colorectal cancer patients. DETAILED DESCRIPTION

[0011] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0012] Specific Example 1: Screening of eRNA molecular markers.

[0013] By collecting publicly available full-length single-cell sequencing databases including SMARTer, Smart-seq, Smart-seq2, Quartz-seq, RamDA-seq, SUPeR-seq, MATQ-seq and Tang's method, we collected tumor epithelial cells of 14 cancer types, such as Figure 1The following are shown: breast cancer subtypes (Luminal A, Luminal B), pancreatic adenocarcinoma (PAAD), oral squamous cell carcinoma (OSCC), triple-negative breast cancer (TNBC), myxoid liposarcoma (MLPS), HER2-positive breast cancer (Human epidermal growth factor receptor 2, HER2), glioblastoma multiforme (GBM), hepatocellular carcinoma (HCC), renal cell carcinoma (RCC), neuroblastoma (NB), multiple myeloma (MM), lung cancer (LC), and colorectal cancer (CC).

[0014] Furthermore, based on enhancer databases such as ENCODE, FANTOM, and Roadmap, and eRNA databases such as HeRA, eRic, HACER, and RAEdb, a human eRNA collection was established. The findallmarker function in the Seurat package in the R language was then used to calculate and identify the characteristic genes of specific cell populations. The corresponding specifically expressed marker eRNAs in tumor epithelial cells of 14 cancer types were calculated (the average expression level of the eRNA in the specified cell type relative to the average expression level of all other cell types was log2 times greater than 0.5). Figure 1 As shown in Figure 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.

[0015] like Figure 1 As shown in B, the characteristic eRNAs of the colorectal cancer cell population were calculated by the findallmarker function. The average expression level of eRNA42767 in the colorectal cancer cell population was >0.5 log2 times the average expression level of all other cancer cell types, and eRNA42767 was screened out as one of the marker eRNAs in the colorectal cancer cell population. Figure 1The color gradient of the circles in middle B represents the average expression level of eRNA, and the size gradient of the circles represents the percentage of cells expressing characteristic eRNA in the intestinal cancer cell population among the total number of cells of this tumor type. Figure 2 The differential expression of eRNA42767 in rectal adenocarcinoma and colon adenocarcinoma from The Cancer Genome Atlas (TCGA) is shown. Figure 2 The red, green and grey dots 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 eRNA42767 is significantly highly expressed in rectal adenocarcinoma tissues. Figure 2 As shown in Figure B, a volcano plot of the TCGA-colon adenocarcinoma dataset shows that eRNA42767 is significantly overexpressed in colon adenocarcinoma tissues. eRNA42767 appears as an upregulated signature gene in both cancer types, suggesting its potential diagnostic or prognostic value in colorectal cancer.

[0016] Specific Example 2: Diagnostic verification of eRNA molecular markers.

[0017] 1. Colorectal Cancer Patient Data Collection: This study collected plasma samples from 78 colorectal cancer patients and 65 healthy individuals at the First Affiliated Hospital of Ningbo University. Informed consent was obtained from the patients or their families. Complete patient information was maintained, and a clinical data database was established in accordance with regulations.

[0018] Inclusion criteria: (1) patients diagnosed with colorectal cancer by endoscopic examination, histopathological evidence, etc.; (2) patients in the active stage of the disease (moderate to severe); (3) age range 18 to 75 years.

[0019] Exclusion criteria: (1) patients with other types of inflammatory bowel disease such as ulcerative colitis; (2) patients with serious complications such as intestinal perforation, intra-abdominal abscess, and severe infection; (3) patients currently using immunosuppressants, corticosteroids, and other drugs; (4) patients with severe diseases such as severe liver or kidney damage, cardiovascular disease, etc.; (5) patients who had undergone intestinal surgery within 3 to 6 months before the study.

[0020] 2. Separate plasma: Draw 5 mL of peripheral blood into an EDTA-anticoagulant blood collection tube and immediately place it in a 4°C refrigerator. Let it stand for 30 minutes. Centrifuge it at 3000 rpm for 10 minutes at 4°C to remove the residual blood cell components in the plasma. Aspirate the upper layer of plasma and transfer it to an enzyme-free centrifuge tube. Aliquot the obtained plasma samples and freeze them in a -80°C refrigerator.

[0021] 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, and draw 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 minutes 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.

[0022] 4. Synthesize cDNA: Follow the instructions of the Polestar 1st cDNA Synthesis Kit (gDNA removal) produced by Beijing Baoying Tonghui Biotechnology Co., Ltd. and prepare the reverse transcription reaction solution according to the following composition formula: Reverse transcription reaction system: 7 µL enzyme-free water, 4 µL 5× Polestar RT MasterMix (with dsDNase), 20 µM concentration of OligodT (18) 1 µL of culture medium, 8 µL of total RNA, for a total volume of 20 µL.

[0023] cDNA synthesis reaction procedure: 25℃ for 10 min, 55℃ for 60 min, and 85℃ for 5 min. The reverse transcribed cDNA was stored at -20℃. If the next step of the fluorescent quantitative PCR experiment is directly continued, the sample was added according to the PCR system.

[0024] 5. Rapid amplification of cDNA ends (RACE) was used to determine the true full length of eRNA42767, whose nucleotide sequence is shown in SEQ ID NO. 1. The specific process is as follows: RNA was extracted using a Trizol extraction kit (purchased from Shanghai Sangon Biotechnology, Catalog No. B511321). 1.5% agarose gel and 1× Tris-acetate electrophoresis buffer were used for observation and photography under UV light. The 5'-RACE kit was purchased from Shanghai Sangon Biotechnology (Catalog No. B605102). The nucleotide sequence of the upstream primer for 5'-RACE specific amplification is shown in SEQ ID NO. 4: 5'-CTCATTAGATATACAGAATCCTTCACAG-3', and the nucleotide sequence of the downstream primer for 5'-RACE specific amplification is shown in SEQ ID NO. 5: 5'- CCTTGTCCATCATGATCACTCAAC-3'.

[0025] 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'-GCATACTATTCTGTGGCATATATGTAC-3', and the nucleotide sequence of the downstream primer for 3'-RACE specific amplification is shown in SEQ ID NO. 7: 5'-TACACCAAGGTGCTTCATCATTC-3'.

[0026] The nucleotide sequence of the upstream primer of the eRNA42767 intermediate sequence fragment 1 (PCR amplification product length 5192 bp) is shown in SEQ ID NO. 8: 5'-CATATCTACTCTTACCAAGGTCTGAAA-3', and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 9: 5'-CCTTCATTTAAGCCTCCATCTCTA-3'.

[0027] The nucleotide sequence of the upstream primer of the eRNA42767 intermediate sequence fragment 2 (PCR amplification product length 3096 bp) is shown in SEQ ID NO.10: 5'-GGGAGGAGAGAGAAAAAGAAAATAGA-3', and the nucleotide sequence of its downstream primer is shown in SEQ ID NO.11: 5'-GCAACCCATACATGGTAATTCTCA-3'. These two intermediate sequences have overlapping sequences and can cover the sequence of the entire intermediate region.

[0028] The results are as follows Figure 3 As shown in A, agarose gel electrophoresis clearly shows the amplified fragment, including the complete 5' terminal sequence, the middle sequence and the complete 3' terminal sequence. The rapid amplification of cDNA ends technique proves that the complete 5' terminal sequence of eRNA42767 is 444 bp and the complete 3' terminal sequence is 1793 bp.

[0029] Further design upstream and downstream amplification primers for eRNA42767 fluorescence quantitative PCR, such as Figure 3 As shown in Figure B, upstream and downstream primers for quantitative PCR were designed at positions 8384-8514 bp of the eRNA42767 sequence. The nucleotide sequence of the upstream primer for quantitative PCR of eRNA42767 is shown in SEQ ID NO. 2: 5'-CCTCTCTCTGGGACATGCCTA-3'; the nucleotide sequence of the downstream primer for quantitative PCR of eRNA42767 is shown in SEQ ID NO. 3: 5'-CACATGCGTAGAGATTCTTGCC-3'. TA cloning sequencing revealed a 131 bp PCR product that matched the sequence at position 8384-8514 of eRNA42767, demonstrating the specificity of the designed upstream and downstream primers.

[0030] 6. Preparation of plasmid standards: The PCR product of eRNA42767 was sequenced by TA cloning. The resulting 131 bp base sequence was cloned into the pUC57 plasmid to obtain the pUC57-eRNA42767 recombinant plasmid (insert base 131 bp, total plasmid length 2835 bp), which was prepared by General Biotechnology (Anhui) Co., Ltd.

[0031] 7. UV spectrophotometer measurement of recombinant plasmid: Dissolve the pUC57-eRNA42767 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 219.31 ng / μL, and the OD 260 The value is 4.386.

[0032] 8. Absolute quantitative PCR: Perform gradient dilution of the high-concentration standard and calculate the copy number in 1 μL of plasmid standard according to the formula: , where the DNA length is the full length of the plasmid, including the backbone vector. The copy numbers of the eight dilution gradients of the pUC57-eRNA42767 recombinant plasmid were calculated, and the concentrations were 7.06×10 9 copies / μL, 7.06×10 8 copies / μL, 7.06×10 7 copies / μL, 7.06×10 6 copies / μL, 7.06×10 5 copies / μL, 7.06×10 4 copies / μL, 7.06×10 3copies / μL and 7.06×10 2 Each gradient of plasmid standard was repeated 3 times and loaded into a 96-well PCR reaction plate. Amplification was performed according to the reaction system and reaction procedure of real-time quantitative PCR amplification to obtain 8 plasmids with different copy numbers. C q value.

[0033] The real-time quantitative PCR reaction system was formulated as follows: 10.9 µL enzyme-free water, 1 µL cDNA, 0.3 µL 10 µM upstream primer for quantitative PCR, 0.3 µL 10 µM downstream primer for quantitative PCR, and 12.5 µL GoTaq qPCR Master Mix 2×, for a total volume of 25 µL. The real-time quantitative PCR protocol was as follows: 1 cycle of initial denaturation at 95°C for 5 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing at 62°C for 30 seconds, and extension at 72°C for 30 seconds. A standard curve for the pUC57-eRNA42767 recombinant plasmid was automatically generated using the fluorescence measurement system based on the fluorescence changes.

[0034] The results are as follows Figure 4 As shown, the linear equation of the pUC57-eRNA42767 recombinant plasmid is Y = -3.804X + 46.06, where Y is the value obtained by real-time quantitative PCR detection. C q value, 10 X The corresponding copy number in the test sample. 2 is 0.9957, indicating that within the range of plasmid dilution concentration, the lg value of the pUC57-eRNA42767 recombinant plasmid copy number is C The q values showed a good linear relationship. The amplification efficiency E was 83.18%, indicating that the standard curves established with each recombinant plasmid could accurately reflect the amplification of the target product.

[0035] The absolute quantitative PCR was used to obtain the C The q value, that is, the Y value in the formula, is calculated according to the formula Y = -3.804X +46.06, and the copy number of eRNA42767 in 1μL cDNA is 10 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 eRNA42767 in 1 mL plasma was 80×10 X Calculation. The copy number of eRNA42767 in 1 mL of plasma from healthy subjects and colorectal cancer patients was statistically analyzed. The results are as follows Figure 5As shown, eRNA42767 was significantly overexpressed in the plasma of colorectal cancer patients, and the cutoff value for distinguishing healthy people from colorectal cancer patients was 284,500 copies / mL.

[0036] 9. Use ROC curve to analyze the diagnostic ability of the eRNA42767 molecular marker: Based on the levels of eRNA42767 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 eRNA42767 levels in distinguishing healthy subjects from colorectal cancer patients, and evaluate the diagnostic value of eRNA42767 in judging colorectal cancer patients.

[0037] The results are as follows Figure 6 As shown in the data, the diagnostic value of eRNA42767 in colorectal cancer plasma is: the area under the ROC curve is 0.868, the sensitivity is 0.833, the specificity is 0.723, the positive predictive value is 0.783, and the negative predictive value is 0.783, which can be effectively used for the early diagnosis of colorectal cancer.

[0038] 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 persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. An eRNA molecular marker for assisting the diagnosis of colorectal cancer, characterized by: The eRNA molecular marker is eRNA42767, and the nucleotide sequence of eRNA42767 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. 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 amplification primers for eRNA42767 fluorescent quantitative PCR. The nucleotide sequence of the upstream amplification primer for eRNA42767 fluorescent quantitative PCR is shown in SEQ ID NO.2: 5'-CCTCTCTCTGGGACATGCCTA-3'; the nucleotide sequence of the downstream amplification primer for eRNA42767 fluorescent quantitative PCR is shown in SEQ ID NO.3: 5'-CACATGCGTAGAGATTCTTGCC-3'.

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