An eRNA molecular marker for assisting in diagnosing intestinal cancer and application thereof

Abstract

The application discloses an eRNA molecular marker for assisting in diagnosing intestinal cancer and application thereof, characterized by that the eRNA molecular marker is eRNA42767, the nucleotide sequence of which is shown in SEQ ID NO. 1, and further provides application of the eRNA molecular marker in preparation of an intestinal cancer auxiliary diagnosis kit, wherein the kit comprises eRNA42767 fluorescent quantitative PCR specific amplification primers, the nucleotide sequence of an upstream amplification primer is 5'-CCTCTCTCTGGGACATGCCTA-3', and the nucleotide sequence of a downstream amplification primer is 5'-CACATGCGTAGAGATTCTTGCC-3', and the advantages are that early diagnosis of intestinal cancer patients at a molecular level can be realized conveniently, quickly and efficiently, the method has strong pertinence, high specificity and high sensitivity, and is favorable for early discovery and timely treatment of intestinal cancer.

Description

TECHNICAL FIELD

[0001] The present application 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

[0002] Intestinal cancer (Colorectal cancer, CC for short) is one of the malignant tumors of the digestive system with high morbidity and mortality worldwide. Its pathogenesis is complex and is often driven by multiple factors and multiple steps of molecular events. The early symptoms of intestinal cancer are not obvious, and most patients are in the middle and advanced stages when diagnosed, missing the best treatment opportunity. The current widely used diagnosis methods in clinical practice include colonoscopy, tissue biopsy, imaging evaluation, and serological tumor marker detection (such as traditional tumor marker carbohydrate antigen CA19-9, etc.). However, these methods have certain limitations in practical application, for example, colonoscopy is an invasive operation with poor patient compliance and risks of complications such as bleeding or perforation; traditional serum markers have limited sensitivity and specificity, making it difficult to meet the clinical needs of early precise screening and dynamic monitoring of intestinal cancer.

[0003] In recent years, with the development of high-throughput sequencing and epigenetic technology, researchers have gradually realized the important regulatory role of non-coding RNA in the occurrence and development of cancer. Among them, enhancer RNA (Enhancer RNA, eRNA for short) as a kind of non-coding transcript derived from active enhancer regions has been found to participate in the regulation of the expression of key cancer genes in various tumor types. eRNA not only has a tissue and cell type-specific expression pattern, but also can reflect the enhancer activity and transcriptional regulation state, and plays an important role in the occurrence, development and metastasis of tumors. Studies have shown that eRNA shows specific expression characteristics in breast cancer, liver cancer, lung cancer and other malignant tumors, and can be used as a potential biomarker for early screening, typing and efficacy prediction of diseases.

[0004] Currently, molecular diagnosis of intestinal cancer still mainly focuses on protein-coding gene mutations (such as KRAS, BRAF) or copy number variations, while the research on the expression characteristics of eRNA in intestinal cancer and its clinical application is still in its infancy. eRNA is closely related to enhancer activity and has the potential to reflect changes in the specific gene regulation network of intestinal cancer, especially in peripheral body fluids such as blood, which has the clinical value of developing into a non-invasive molecular marker. Therefore, exploring and screening eRNA molecular markers with specific expression in intestinal cancer is of great significance for achieving early, non-invasive and precise diagnosis of intestinal cancer. SUMMARY

[0005] The present application discloses an eRNA molecular marker for assisting in diagnosing intestinal cancer and application thereof.

[0006] The present application discloses an eRNA molecular marker for assisting in diagnosing intestinal cancer and application thereof.

[0007] The present application discloses an eRNA molecular marker for assisting in diagnosing intestinal cancer and application thereof.

[0008] The present application discloses an eRNA molecular marker for assisting in diagnosing intestinal cancer and application thereof, wherein the kit comprises an upstream amplification primer and a downstream amplification primer for eRNA42767 fluorescent quantitative PCR, the nucleotide sequence of the upstream amplification primer is shown as SEQ ID NO. 2: 5'-CCTCTCTCTGGGACATGCCTA-3', and the nucleotide sequence of the downstream amplification primer is shown as SEQ ID NO. 3: 5'-CACATGCGTAGAGATTCTTGCC-3'.

[0009] Compared with the prior art, the present application has the advantages that the present application discloses an eRNA molecular marker for assisting in diagnosing intestinal cancer and application thereof for the first time, and the eRNA42767 molecular marker is highly expressed in the plasma of intestinal cancer patients. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 In the figure, A is the t-SNE (t-distributed Stochastic Neighbour Embedding) analysis result of the tumor epithelial cells of 14 kinds of cancers, B is the log2 fold result of the average expression amount of eRNA in the intestinal cancer cell group relative to the average expression amount of all other cancer cell types, the left blue dotted small box represents the eRNA marker in colorectal cancer, and the right blue dotted large box represents the expression mode of the eRNA marker in colorectal cancer in different cancers.

[0011] Figure 2 In the figure, A is the volcano plot of the TCGA-rectal adenocarcinoma data set, and B is the volcano plot of the TCGA-colon adenocarcinoma data set.

[0012] Figure 3 A is the agarose gel electrophoresis map of eRNA42767 amplified fragment, including 5' end complete sequence, middle sequence and 3' end complete sequence, B is the principle diagram of specific upstream and downstream amplification primers for designing fluorescence quantitative PCR according to eRNA42767 sequence;

[0013] Figure 4 is the linear standard curve of lg value of pUC57-eRNA42767 recombinant plasmid copy number and q value; C

[0014] Figure 5 is the statistical analysis result of eRNA42767 copy number in plasma of healthy people and intestinal cancer patients;

[0015] Figure 6 is the ROC curve and diagnostic value of eRNA42767 in plasma of intestinal cancer patients. DETAILED DESCRIPTION

[0016] The application will be further described in detail in the following embodiment combined with the drawings.

[0017] Specific embodiment one: screening of eRNA molecular marker.

[0018] By collecting published full-length single cell sequencing databases including SMARTer, Smart-seq, Smart-seq2, Quartz-seq, RamDA-seq, SUPeR-seq, MATQ-seq and Tang's method, 14 kinds of tumor epithelial cells of cancers such as lung cancer, breast cancer, liver cancer, stomach cancer, colon cancer, prostate cancer, ovarian cancer, pancreatic cancer, brain cancer, cervical cancer, bladder cancer, head and neck cancer, esophageal cancer and skin cancer were collected. Figure 1 ​The 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).

[0019] 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.

[0020] 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.

[0021] Specific Example 2: Diagnostic verification of eRNA molecular markers.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 3. Extraction of plasma RNA: Using Trizol LS reagent from Invitrogen, 250 μL of plasma sample was taken into a nuclease-free 1.5 mL centrifuge tube, 750 μL of Trizol LS reagent was added, the centrifuge tube cap was tightly closed, vortexed for 15 s, and placed in a 4°C refrigerator for 10 min; after taking it out, it was centrifuged for 10 s, 200 μL of chloroform was added, the centrifuge tube cap was tightly closed, and it was shaken by hand for 6 times, placed in a 4°C centrifuge for 3 min, then centrifuged at 12000 rpm for 15 min at 4°C, so that the liquid in the tube was clearly divided into 3 layers; a nuclease-free 1.5 mL centrifuge tube was prepared, 500 μL of isopropanol was added, the centrifuged sample was taken out, 500 μL of the upper transparent liquid was taken into the centrifuge tube with isopropanol prepared in advance, the centrifuge tube cap was tightly closed, and it was mixed by inverting, and placed in a 4°C refrigerator for 10 min, then centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was carefully aspirated; 1 mL of pre-cooled 75% ethanol was added to the centrifuge tube, the centrifuge tube cap was tightly closed, and the centrifuge tube was inverted to wash the precipitate; after centrifugation at 12000 rpm for 3 min at 4°C, the supernatant was aspirated, and the centrifuge tube was placed in the centrifuge again, centrifuged at 12000 rpm for 1 min at 4°C, and the supernatant was aspirated; dried for 3 min, added 8 μL of enzyme-free water, and mixed by blowing; 8 μL of total RNA was taken for reverse transcription.

[0027] 4. Synthesis of cDNA: According to the instructions of Polestar 1st cDNA Synthesis Kit (gDNA removal) produced by Beijing Baoyin Tonghui Biotechnology Co., Ltd., the reverse transcription reaction solution was prepared according to the following component formula:

[0028] Reverse transcription reaction system: enzyme-free water 7 μL, 5×Polestar RT MasterMix (with dsDNase) 4 μL, OligodT 1 μL at a concentration of 20 μM, total RNA 8 μL, total volume 20 μL. (18)

[0029] cDNA synthesis reaction program: 25°C for 10 min, 55°C for 60 min, 85°C for 5 min, the cDNA after reverse transcription was stored at -20°C, and if the next step was to continue the fluorescence quantitative PCR experiment, the sample was added according to the PCR system.

[0030] 5. The real full length of eRNA42767 was determined by using cDNA end rapid amplification technology (Rapid amplification of cDNA ends, abbreviated as RACE), and the nucleotide sequence is shown in SEQ ID NO. 1. The specific process is as follows:

[0031] ​RNA was extracted using Trizol extraction kit (purchased from Shanghai Biotechnology, item number B511321), 1.5% agarose, 1x Tris-acetic acid electrophoresis buffer, observed and photographed under ultraviolet transmission light. The 5'-RACE kit was purchased from Shanghai Biotechnology (item number B605102), the nucleotide sequence of the 5'-RACE specific upstream primer was shown in SEQ ID NO. 4: 5'-CTCATTAGATATACAGAATCCTTCACAG-3', and the nucleotide sequence of the 5'-RACE specific downstream primer was shown in SEQ ID NO. 5: 5'-CCTTGTCCATCATGATCACTCAAC-3'.

[0032] The 3'-RACE kit was purchased from Shanghai Biotechnology (item number B605101), the nucleotide sequence of the 3'-RACE specific upstream primer was shown in SEQ ID NO. 6: 5'-GCATACTATTCTGTGGCATATATGTAC-3', and the nucleotide sequence of the 3'-RACE specific downstream primer was shown in SEQ ID NO. 7: 5'-TACACCAAGGTGCTTCATCATTC-3'.

[0033] The nucleotide sequence of the upstream primer of the eRNA42767 intermediate sequence fragment 1 (PCR amplification product length of 5192bp) was shown in SEQ ID NO. 8: 5'-CATATCTACTCTTACCAAGGTCTGAAA-3', and the nucleotide sequence of the downstream primer was shown in SEQ ID NO. 9: 5'-CCTTCATTTAAGCCTCCATCTCTA-3'.

[0034] The nucleotide sequence of the upstream primer of the eRNA42767 intermediate sequence fragment 2 (PCR amplification product length of 3096bp) was shown in SEQ ID NO. 10: 5'-GGGAGGAGAGAGAAAAAGAAAATAGA-3', and the nucleotide sequence of the downstream primer was shown in SEQ ID NO. 11: 5'-GCAACCCATACATGGTAATTCTCA-3', and the two intermediate sequence fragments had overlapping sequences and could cover the entire intermediate sequence.

[0035] The results are shown in Figure 2A. Figure 3 As shown in Figure 2A, agarose gel electrophoresis clearly showed the amplified fragments, including the 5' end complete sequence, the intermediate sequence and the 3' end complete sequence, and the cDNA end rapid amplification technology proved that the 5' end complete sequence of eRNA42767 was 444bp, and the 3' end complete sequence was 1793bp.

[0036] The upstream and downstream amplification primers for the eRNA42767 fluorescent quantitative PCR were further designed, as shown in Table 1. Figure 3 The upstream and downstream amplification primers for the eRNA42767 fluorescent quantitative PCR were further designed, as shown in Table 1.

[0037] 6. Preparation of plasmid standard: After T-A cloning and sequencing of the eRNA42767 PCR product, the 131 bp base sequence obtained by sequencing was cloned into pUC57 plasmid to obtain a pUC57-eRNA42767 recombinant plasmid (inserted base 131 bp, plasmid full length 2835 bp), which was prepared by General Biotech (Anhui) Co., Ltd.

[0038] 7. Measurement of recombinant plasmid by ultraviolet spectrophotometer: 50 microliters of enzyme-free water was used to dissolve the pUC57-eRNA42767 recombinant plasmid dry powder to obtain the first high concentration standard, and then the concentration and OD 260 value were measured. The concentration of the recombinant plasmid pUC57-eRNA42767 was 219.31 ng / μL, and the OD 260 value was 4.386.

[0039] 8. Absolute quantitative PCR: The high concentration standard was gradiently diluted, and the copy number in 1 μL of plasmid standard was calculated according to the formula: wherein the DNA length is the full length of the plasmid, including the backbone vector, and the copy numbers of the 8 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 x 10 2 copies / μL, 3 replicates were set for each gradient, and the plasmid standard was added to the PCR reaction plate of the 96-well plate, and amplification was performed according to the reaction system and reaction procedure of real-time quantitative PCR amplification, and 8 different copy numbers corresponding to C q values were obtained.

[0040] The reaction system formula of real-time quantitative PCR amplification is as follows: 10.9 μL of enzyme-free water, 1 μL of cDNA, 0.3 μL of 10 μM fluorescent quantitative PCR upstream amplification primer, 0.3 μL of 10 μM fluorescent quantitative PCR downstream amplification primer, 12.5 μL of GoTaq qPCR Master Mix 2x, and the total volume is 25 μL. The reaction procedure 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 pUC57-eRNA42767 recombinant plasmid according to the change rule of fluorescence value.

[0041] The results are shown in Figure 4 The linear equation of pUC57-eRNA42767 recombinant plasmid is Y = -3.804X + 46.06, wherein Y is the C q value obtained by real-time quantitative PCR detection, X 10 is the corresponding copy number in the sample. The determination coefficient R 2 is 0.9957, indicating that the lg value of pUC57-eRNA42767 recombinant plasmid has a good linear relationship with the C q value in the range of plasmid dilution concentration. The amplification efficiency E is 83.18%, which shows that the standard curve established by each recombinant plasmid can accurately reflect the amplification of the target product.

[0042] The C q value of each sample is obtained by absolute quantitative PCR, that is, the Y value in the formula, and the copy number of eRNA42767 in 1 μL of cDNA is calculated according to the formula Y = -3.804X + 46.06 X . Since 8 μL of RNA is extracted from 250 μL of plasma, and then 8 μL of RNA is used for reverse transcription to obtain 20 μL of cDNA, only 1 μL of cDNA is used for real-time quantitative PCR amplification, so it is inferred that the copy number of eRNA42767 in 1 mL of plasma is calculated according to 80 x 10 X . The copy number of eRNA42767 in 1 mL of plasma of healthy people and intestinal cancer patients was statistically analyzed, and the results are shown in Figure 5As shown in Table 2, eRNA42767 was significantly higher expressed in plasma of colorectal cancer patients, and the cutoff value for distinguishing healthy people from colorectal cancer patients was 284,500 copies / mL.

[0043] 9. Analysis of the diagnostic ability of eRNA42767 molecular marker by ROC curve: According to the level of eRNA42767 in the plasma of healthy people and colorectal cancer patients, the area under the ROC curve, sensitivity, specificity, positive predictive value and negative predictive value of eRNA42767 level for distinguishing healthy people from colorectal cancer patients were analyzed to evaluate the diagnostic value of eRNA42767 in judging colorectal cancer patients.

[0044] The results are shown in Table 3. Figure 6 As shown in Table 3, the diagnostic value of eRNA42767 in plasma of colorectal cancer was: the area under the ROC curve was 0.868, the sensitivity was 0.833, the specificity was 0.723, the positive predictive value was 0.783, and the negative predictive value was 0.783, which could be effectively used for early diagnosis of colorectal cancer.

[0045] The above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present application should also be within the scope of the present application.

Claims

1. An eRNA molecular marker for assisting in 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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