Application of hsa_circ_0017469 in the preparation of products for early screening and prognosis assessment of colorectal cancer

Abstract

The present invention provides the use of hsa_circ_0017469 in the preparation of products for early screening and prognosis assessment of colorectal cancer. The present invention uses Arraystar CircRNA chips to screen and identify CircRNAs in CRC cancer and paracancerous tissues, and further expands the amount of tissue samples and clinical plasma samples for verification, proving that hsa_circ_0017469 expression in cancer tissues of CRC patients is significantly increased, and can be used for colorectal cancer screening and postoperative prognosis risk assessment, and develops methods and products for visual detection and real-time quantitative PCR detection of hsa_circ_0017469, providing means for colorectal cancer screening and postoperative prognosis risk assessment.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and medicine, and in particular to the application of hsa_circ_0017469 in the preparation of products for early screening and prognosis evaluation of colorectal cancer. Background Art

[0002] In recent years, the incidence of colorectal cancer (CRC) in my country has shown a clear upward trend. my country's population accounts for only 17% of the world's total population, but CRC patients account for 31% of the world's total, and it is still growing at an average annual rate of 4% to 5%. Colorectal cancer is a cancer that can benefit most from screening. Actively carrying out secondary prevention-colorectal cancer screening plays an important role in the prevention and treatment of colorectal cancer. For patients who have been diagnosed with colorectal cancer, individualized treatment is the key development direction of tertiary prevention in the future. The traditional screening methods for colorectal cancer are colonoscopy and fecal occult blood test. Colonoscopy is the gold standard for clinical diagnosis of colorectal cancer, but it is an invasive examination and patient compliance is poor. Fecal occult blood test is currently a widely used screening method. It is safe and non-invasive, and the subjects have good compliance, but the detection sensitivity is low and the specificity is poor, which is easy to cause misdiagnosis. By screening tumor markers in the blood, common tumor markers include carcinoembryonic antigen, CA125 and CA199, etc. This indicator is affected by factors such as inflammation and stress in the body and has no obvious specificity. On the other hand, the treatment of CRC includes radical surgical resection, chemotherapy, radiotherapy, targeted therapy and immunotherapy, among which radical surgical resection is the most effective method, but local recurrence after surgery (60% to 80% of CRC patients relapse within 2 years after surgery) and distant metastasis are still the current problems. Therefore, it is particularly important to take effective measures after surgery to timely evaluate and predict tumor recurrence and metastasis. The prognosis of CRC patients can be evaluated by combining tumor staging and various clinical pathological characteristics, but there are still significant differences in the prognosis of patients with the same tumor stage and similar clinical pathological characteristics. Therefore, traditional staging and grading cannot accurately judge the prognosis of patients. At present, precision treatment of CRC guided by molecular marker characteristics plays an increasingly important role in clinical practice. Actively exploring and discovering new molecular markers of CRC related to recurrence and metastasis or treatment efficacy evaluation can not only predict recurrence and metastasis and evaluate treatment efficacy, but also carry out molecular targeted intervention on targets, greatly improving the prognosis of patients.

[0003] Domestic and foreign CRC-related consensus and guidelines emphasize that the specific classification of molecular markers is an important factor affecting the treatment decision of metastatic CRC (mCRC), and unanimously recommend RAS, BRAF, MMR / MSI, etc. as routine molecular markers to guide treatment and judge prognosis. However, in the era of precision medicine where CRC can no longer be treated uniformly as a single disease, the detection rate of molecular markers in CRC patients is only 20% to 30%, especially in second- and third-tier cities or areas with underdeveloped medical levels. The detection rate is even lower, which greatly affects the diagnosis of CRC patients and the choice of individualized treatment plans. The reason is that on the one hand, the types of CRC molecular markers that can be used are limited, and on the other hand, there are practical problems such as long molecular marker detection cycle and high price.

[0004] Circular RNA (circRNA) is a special and widely existing stable RNA molecule that can participate in the regulation of mammalian gene expression and is associated with a variety of diseases including cancer. Unlike traditional linear RNA (linear RNA, containing 5' and 3' ends), circRNA molecules have a closed circular structure, are not affected by RNA exonucleases, are more stable in expression, and are not easily degraded. In recent years, a large number of studies have shown that circRNA is closely related to the growth and development of organisms, stress response, disease occurrence and development, and predicts that it has application prospects in disease diagnostic markers. Circular RNA is extremely stable in biological cells and is a popular target for the treatment of human diseases. Its application potential is very large, and it has opened up new ideas in the field of biomedicine. Circular RNA with clear functions can be used as biomarkers for the diagnosis of certain diseases, as well as therapeutic targets for certain important diseases, especially immune diseases and cancer. It is widely used in the treatment of infectious diseases, rare diseases, blood diseases, and tumors, and has both research and application value. Many circRNAs have been identified as oncogenes or tumor suppressors that mediate the occurrence, metastasis, and chemotherapy resistance of CRC. CircRNA in tissues or exosomes can be used as a biomarker for diagnosis and prognosis and a potential therapeutic target in the clinical practice of CRC. The present invention provides a circRNA related to CRC, which can be used for early screening and prognosis evaluation of CRC. Summary of the invention

[0005] In order to overcome the defects in the prior art, the present invention provides the use of hsa_circ_0017469 in the preparation of products for early screening and prognosis evaluation of colorectal cancer, and provides a kit and method for detecting hsa_circ_0017469 and products for early screening and prognosis evaluation of colorectal cancer.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention is to provide the use of a reagent for detecting hsa_circ_0017469 in the preparation of a product for early screening or prognosis evaluation of colorectal cancer, wherein the product comprises a reagent for detecting hsa_circ_0017469.

[0008] Furthermore, the above-mentioned reagents for detecting hsa_circ_0017469 include one or more of qRT-PCR detection reagents, Northern blot detection reagents, droplet digital PCR detection reagents, CRISPR / Cas13a-RPA detection reagents, high-throughput sequencing technology detection reagents, nucleic acid hybridization technology detection reagents, and gene chip technology detection reagents.

[0009] Furthermore, the above-mentioned qRT-PCR detection reagent includes primers with sequences of SEQ ID NOs.1-2.

[0010] Furthermore, the above-mentioned CRISPR / Cas13a-RPA detection reagent includes primers with sequences of SEQ ID NO.5 to 6 and CrRNA with a sequence of SEQ ID NO.7.

[0011] Furthermore, the above product also includes reagents for detecting CEA.

[0012] The second aspect of the present invention is to provide a method for visually detecting hsa_circ_0017469 based on CRISPR / Cas13a-RPA technology, wherein the sequences of the primers used are SEQ ID NO.5-6, and the CrRNA used is SEQ ID NO.7. After the reaction is completed, it is placed under blue light for observation.

[0013] Further, the reaction system used includes NTP, crRNA, Cas 13a, RNase inhibitor, T7 RNA polymerase, primer, ssRNA reporter, DNA template, MgOAc, T4 uvsX recombinase, SSB and strand displacement DNA polymerase; preferably including the following components: 2 μL T4 uvsX (1 mM), 3 μL SSB (0.5 mM), 2 μL Bsu (0.5 mM), 3 μL NTP (1 mM), 2 μL crRNA (40 nM), 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 2 μL ssRNA reporter (80 nM), 1 μL template, 4 μL Cas13a (80 nM), 1 μL RNase inhibitor (40 U), 1 μL T7 RNA polymerase (30 U) and 2 μL MgOAc (0.1 mM).

[0014] Furthermore, the reaction procedure adopted was 37°C for 15 min.

[0015] The third aspect of the present invention is to provide a system for early screening or prognosis assessment of colorectal cancer, comprising:

[0016] Module for detecting the expression of hsa_circ_0017469;

[0017] A data processing module, receiving the relative expression value of hsa_circ_0017469 of the patient sent by the above-mentioned module for detecting the expression of hsa_circ_0017469, and comparing it with the judgment threshold; and a conclusion output module, configured to receive the comparison result sent by the above-mentioned data processing module, and judge the comparison result according to a predetermined judgment condition; determine whether the patient is a colorectal cancer patient or evaluate the prognosis of the colorectal cancer patient.

[0018] Furthermore, the module for detecting the expression level of hsa_circ_0017469 is constructed based on one or more of qRT-PCR, Northern blot, droplet digital PCR, CRISPR / Cas13a-RPA, high-throughput sequencing technology, nucleic acid hybridization technology, and gene chip technology.

[0019] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0020] The present invention uses Arraystar CircRNA chip to screen and identify CircRNA in CRC cancer and adjacent tissues, and further expands the amount of tissue samples and clinical plasma samples for verification, proving that the expression of hsa_circ_0017469 is significantly increased in the cancer tissues of CRC patients, and can be used for colorectal cancer screening and postoperative prognosis risk assessment. A method and product for visual detection and real-time quantitative PCR detection of hsa_circ_0017469 are developed, providing a means for colorectal cancer screening and postoperative prognosis risk assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It shows that hsa_circ_0017469 has significant differences in the circRNA expression profiles of CRC patients' cancer tissues and adjacent tissues; Figure A shows the circRNA expression profiles of CRC patients' cancer tissues and adjacent tissues, and Figure B shows the volcano map of the differential circRNAs of CRC patients' cancer tissues and adjacent tissues;

[0022] Figure 2 The experimental results of Sanger sequencing verification and RNase R enzyme digestion of hsa_circ_0017469 are shown. The sequencing results verified the sequences at the cyclization site and the trans-cleavage site, and determined that hsa_circ_0017469 ( Figure 2 A); hsa_circ_0017469 treated with RNase R is less digestible than linear RNA and has molecular stability ( Figure 2 B);

[0023] Figure 3 The expression levels of hsa_circ_0017469 in different groups are shown; Figure A shows the RT-PCR results of hsa_circ_0017469 in cancer tissues of 120 patients with colorectal cancer after surgery; Figure B shows the comparison results of hsa_circ_0017469 levels in plasma samples of 120 patients with colorectal cancer before surgery, healthy control group, benign lesion group, and other tumor groups;

[0024] Figure 4 The ROC curves of the marker screening colorectal cancer model are shown; Figure A is the ROC curve of the hsa_circ_001746 colorectal cancer screening model, Figure B is the ROC curve of the CEA colorectal cancer screening model, and Figure C is the ROC curve of the hsa_circ_0017469 and CEA combined colorectal cancer screening model;

[0025] Figure 5 The prognostic risk model (Figure A), survival curve analysis results (Figure B), and tumor metastasis analysis results (Figure C) are shown;

[0026] Figure 6 The results of visual identification of hsa_circ_0017469 are shown; Circ_0017469 represents the test results of plasma samples from patients with colorectal cancer, and Positive (positive control) represents the hsa_circ_0017469 standard (1×10 3 copies / μL) test results, and Negative (negative control) is the test result of samples without hsa_circ_0017469. DETAILED DESCRIPTION

[0027] The present invention is described in detail and specifically below through specific embodiments and drawings to provide a better understanding of the present invention, but the following embodiments do not limit the scope of the present invention.

[0028] The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.

[0029] Example 1

[0030] In this example, the target circular RNA was screened and determined by using the Arraystar circular RNA chip. The specific experimental steps and results are as follows:

[0031] 1. Arraystar circRNA chip was used to detect the expression changes of circRNA in 3 pairs of CRC and adjacent tissues, and it was found that the circRNA expression profiles of CRC patient cancer tissues and adjacent tissues were significantly different. 90 differentially expressed circRNAs were screened out, of which 58 were upregulated and 32 were downregulated (screening conditions: fold_change ≥ 1.5, P-value < 0.05). qPCR was used to further detect 4 significantly upregulated circRNAs, and finally hsa_circ_0017469 was determined as the target of subsequent research (such as Figure 1 ).

[0032] 2. Sanger sequencing was used to verify the sequence at the cyclization site and trans-splicing site of hsa_circ_0017469. hsa_circ_0017469 is located at chr10 (p15.3), with the gene symbol WDR37, and is cyclized by exon 11, exon 12, and exon 13. The sequencing results showed that the splicing site was AGCA and the sequencing sequence was consistent with expectations, and was determined to be hsa_circ_0017469 ( Figure 2 A). hsa_circ_0017469 has molecular stability after RNase R treatment ( Figure 2 B). The specific steps are as follows: dilute the RNase R stock solution to 2U / μl, and prepare the following system: 2μl buffer, 5μl RNase R, 13μl RNA. After mixing, incubate the entire reaction system at 37℃ for 30 minutes, and finally incubate at 70℃ for 10 minutes to inactivate RNaseR.

[0033] The above research results suggest that hsa_circ_0017469 is upregulated in CRC cancer tissues, is formed by exon cyclization, is not easily degraded by nuclease exonuclease RNaseR, and is highly stable.

[0034] Example 2

[0035] This example verifies that hsa_circ_0017469 can be used for early screening and prognosis assessment of colorectal cancer. The specific experimental steps and results are as follows:

[0036] 1. Collection of Clinical Samples

[0037] Preoperative plasma, postoperative cancer and adjacent tissue samples from 120 patients with colorectal cancer, 60 plasma specimens from benign lesions (including colon polyps, colorectal adenomas, ulcerative colitis, viral colitis, prostatic hyperplasia, and gastritis), and 60 plasma specimens from other tumors (gastric cancer, liver cancer, esophageal cancer, pancreatic cancer, prostate cancer, and peritoneal tumors) were collected.

[0038] 2. RNA extraction from colorectal cancer tissue and adjacent tissues

[0039] 10-30 mg of colorectal cancer tissue was cut and transferred to lysis buffer. The tissue was ground at 4.0 m / s for 30 s using a FastPrep instrument. RNA from cancer tissue and adjacent tissue was extracted using a commercial kit.

[0040] 3. Extraction of Total RNA from Plasma

[0041] First, add 250 μL of plasma, 750 μL of Trizol reagent, and 200 μL of chloroform to a 1.5 mL test tube that has been free of RNase. After vigorous shaking for 15 seconds, let stand at room temperature for 3 minutes, and centrifuge at 120,000 rpm at 4°C for 15 minutes. Pipette the upper aqueous layer into a new centrifuge tube, add 500 μL of isopropanol, mix by inversion, let stand at room temperature for 10 minutes, and centrifuge at 120,000 rpm at 4°C for 10 minutes. Discard the supernatant and add 1 mL of 75% ethanol, wash thoroughly to suspend the precipitate, centrifuge at 120,000 rpm at 4°C for 5 minutes, and discard the supernatant. Let dry at room temperature for 10 minutes, and add 50 μL of RNase-free water to dissolve the RNA. Use NanoDrop2000 UV spectrophotometer to measure the optical density of total RNA at 260 nm and 280 nm.

[0042] 4. Reverse Transcription

[0043] Two-step reverse transcription was performed to obtain cDNA. First, the system was prepared: 2 μL of 5×gDNAEraser Buffer, 1 μL of gDNAEraser, and 7 μL of RNA. Heat at 42°C for 2 min.

[0044] Then add the following to the above reaction system: PrimeScirpt RT Enzyme MixⅠ1μL, RT Primer Mix4μL, 5× PrimeScirpt Buffer 24μL, RNase Free dH2O 1μl. The reaction program is 42℃15min, 85℃5s.

[0045] 5. PCR detection

[0046] The 20μL PCR reaction detection system is: 2×TB Green Taq Mix 10μL, upstream primer 0.4μL at a concentration of 10μM, downstream primer 0.4μL at a concentration of 10μM, ROX reference dye 0.4μL, cDNA 2μL, sterile water 6.8μL. The instrument is Applied Biosystems 7500, and the reaction program is: 95℃30s; 95℃5s, 62℃34s, 40 cycles, and fluorescence detection at 62℃. GAPDH is used as an internal reference, and the primer information is shown in Table 1.

[0047] Table 1 hsa_circ_0017469 and internal reference PCR primer information

[0048]

[0049] For all RT-qPCR results, use First, calculate △CT = target gene CT value - reference gene CT value. Then calculate the , and their average value is defined as The final calculation All statistical results are based on Conduct analysis

[0050] according to Figure 3 It can be seen that the level of hsa_circ_0017469 in cancer tissues of 120 patients with colorectal cancer after surgery was significantly higher than that in adjacent tissues (P<0.001) ( Figure 3 A), the hsa_circ_0017469 in the plasma samples of the above colorectal cancer patients before surgery was significantly higher than that in the healthy control group (P<0.01), the benign lesion group (P<0.001) and the other tumor group (P<0.01) ( Figure 3 B).

[0051] like Figure 4 The RT-qPCR results showed that hsa_circ_0017469 was significantly upregulated in the plasma of colorectal cancer patients compared with the healthy physical examination group, benign lesion group and other tumor groups. The area under the ROC curve (AUC) of hsa_circ_0017469 was 0.754 ( Figure 4 A), the area under the CEA curve is 0.708 ( Figure 4 B), indicating that hsa_circ_0017469 has better potential for diagnosing colorectal cancer than CEA, and the area under the ROC curve of hsa_circ_0017469 combined with CEA is 0.844 ( Figure 4C). Therefore, hsa_circ_0017469 combined with CEA can be used as a marker combination to assist colorectal cancer screening and for early screening of colorectal cancer.

[0052] 6. Constructing prognostic risk model, survival curve analysis and tumor distant metastasis analysis

[0053] By detecting the changes in the expression level of hsa_circ_0017469 in the patient's tumor plasma, it is used to predict the prognosis and mortality risk of patients after colorectal cancer surgery, thereby improving the accuracy of predicting prognosis through plasma specimens of colorectal cancer patients, which is of great significance for the patient's further diagnosis and treatment plan and prolonging the patient's survival. Multivariate analysis showed that hsa_circ_0017469 was an independent risk factor for survival and was significantly associated with the risk of death ( Figure 5 A). Survival curve analysis showed that hsa_circ_0017469 can be used as a new marker for the prognosis assessment of CRC patients after surgery ( Figure 5 B). The plasma hsa_circ_0017469 level in patients with distant metastasis of colorectal cancer at stage M1 was detected and compared with that in patients with non-metastatic colorectal cancer at stage M0. There was a significant difference in the levels of hsa_circ_0017469 between the two groups (P<0.01). The results showed that plasma hsa_circ_0017469 can be used to monitor postoperative recurrence and metastasis of colorectal cancer patients ( Figure 5 C).

[0054] Example 3

[0055] This example provides a method for visual identification of circular RNA hsa_circ_0017469, and the specific steps are as follows:

[0056] (1) Design of hsa_circ_0017469RPA primers and crRNA of Cas13a of the amplified product. The sequence information is shown in Table 2.

[0057] Table 2 hsa_circ_0017469RPA primers and CrRNA sequence information

[0058]

[0059] (2) CRISPR / Cas13a-RPA Visualization Detection

[0060] The total volume of the reaction system was 25 μL, including: 3 μL NTP (1 mM), 2 μL crRNA (40 nM), 4 μL Cas 13a (80 nM), 1 μL RNase (40 U) inhibitor, 1 μL T7 RNA polymerase (30 U), 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 2 μL ssRNA reporter (80 nM), 1 μL DNA template (80 nM), 2 μL MgOAc (0.1 mM), 2 μL recombinase (T4uvsX), 3 μL single-stranded DNA binding protein (SSB) (0.5 mM) and 2 μL strand displacement DNA polymerase (Bsu) (0.5 mM); the reaction procedure was: 37°C for 15 min, detected on a blue light (gel cutting instrument).

[0061] The results are as follows Figure 6 As shown, under blue light (470 nm), the circular RNA hsa_circ_0017469 detection tube appears green.

[0062] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modification and substitution of the present invention are also within the scope of the present invention. Therefore, the equalization and modification made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. Use of a reagent for detecting hsa_circ_0017469 in the preparation of a product for early screening or prognosis evaluation of colorectal cancer, characterized in that: The product includes reagents for detecting hsa_circ_0017469; the reagents for detecting hsa_circ_0017469 include reagents for qRT-PCR detection and reagents for CRISPR / Cas13a-RPA detection.

2. The use according to claim 1, characterized in that: The qRT-PCR detection reagents include primers with sequences of SEQ ID NO.1-2.

3. The use according to claim 1, characterized in that: The CRISPR / Cas13a-RPA detection reagent includes primers with sequences of SEQ ID NO.5 to 6 and CrRNA with a sequence of SEQ ID NO.

7.

4. A system for early screening or prognosis assessment of colorectal cancer, characterized in that: include: A module for detecting the expression level of hsa_circ_0017469; the module for detecting the expression level of hsa_circ_0017469 is constructed based on qRT-PCR technology; A data processing module receives the relative expression value of hsa_circ_0017469 of the patient sent by the module for detecting the expression of hsa_circ_0017469, and compares it with the judgment threshold; and The conclusion output module is configured to receive the comparison result sent by the data processing module and judge the comparison result according to a predetermined judgment condition; determine whether the patient is a colorectal cancer patient or evaluate the prognosis of the colorectal cancer patient.

Images