Early diagnosis markers and kits for colorectal cancer

Abstract

The present invention relates to the fields of molecular biology and medical diagnosis, and more particularly to a marker for the early diagnosis of colorectal cancer, particularly a miRNA marker derived from plasma exosomes, and the use of the marker in the preparation of a kit for detecting early colorectal cancer. The marker for the early diagnosis of colorectal cancer described in the present invention is a nucleic acid molecule selected from the following miRNAs, or a combination thereof: the nucleic acid molecule comprises a nucleic acid molecule encoding plasma exosome miR-99b-5p and miR-409-3p; the nucleic acid molecule is upregulated in target plasma exosomes compared to a control sample. The diagnostic kit provided by the present invention facilitates non-invasive, rapid, and accurate early diagnosis of colorectal cancer.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and medical diagnosis, and specifically to an early diagnostic marker for colorectal cancer, in particular a miRNA marker derived from plasma exosomes, and the use of the marker in preparing a kit for detecting early colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is an epithelial malignancy of the colon or rectum and one of the most common gastrointestinal malignancies. Globally, CRC ranks third in overall cancer incidence, second in women and third in men, and second in overall CRC mortality. In my country, 398,000 cases of CRC and 188,000 deaths occur annually, placing a significant economic burden on the nation. CRC can be insidious, with clinical symptoms often absent in the early stages. Changes in bowel habits and stool characteristics are often the first symptoms, making early detection difficult and leading many patients to be diagnosed at an advanced stage. Early-stage CRC patients who receive timely, standardized treatment have reportedly significantly improved five-year survival rates, reaching up to 90%, compared to those diagnosed at an advanced stage. CRC cells can metastasize to adjacent and distant organs through direct invasion, lymphatic metastasis, implantation, and hematogenous metastasis. This can lead to malignant ascites and cancer-related pain in advanced patients, significantly reducing their quality of life. Therefore, the prevention and treatment of colorectal cancer focuses on early diagnosis and treatment.

[0003] Most CRCs develop through a progression from adenoma to carcinoma, a process that can take several years to over a decade. This offers significant potential for early clinical intervention. Advances in early screening and diagnostic technologies have significantly improved the survival of CRC patients. From the mid-1970s to 2008–2014, the 5-year relative survival rate for all stages of CRC increased from 50.6% to 65.1%, and for rectal cancer from 48.1% to 68.8%. Currently, CRC screening in my country primarily includes colonoscopy, fecal immunochemical test (FIT), multi-target fecal FIT-DNA fecal occult blood test, and colon CT imaging. Colonoscopy involves a clinical endoscopist examining the entire colorectum with a visual camera. Any suspicious lesions observed are then biopsied and confirmed by pathological diagnosis. Colonoscopy is currently the gold standard for CRC screening. However, the required preoperative preparation, high risk of complications, and medical costs have led to low patient compliance, hindering its widespread adoption as a basic screening program. Furthermore, studies have shown that colonoscopy is a highly invasive procedure and carries some risk for the patient. FIT (Fetish Infectious Disease Testing) uses specific antibodies to detect human hemoglobin in stool, indicating intestinal lesions. Therefore, patients with a positive FIT test require further colonoscopy. The fecal occult blood test (FOBT) is a guaiac-based test that detects occult blood in stool. However, this method is less sensitive than the FIT and is susceptible to dietary and medication influences, and is currently being largely replaced by the FIT. A prospective validation study involving 9,989 patients reported that fecal FIT-DNA had a sensitivity of 92.3% for the differential diagnosis of colorectal cancer, while its sensitivity for advanced adenomas was lower, at 42.4%. Currently, the cost of FIT-DNA testing remains high, and its diagnostic applicability in the Chinese population requires further multicenter, large-scale validation. Colonoscopy requires bowel preparation, followed by gas-filled colon for full-colon CT scanning, allowing visualization of the entire colon. Confirmation of the diagnosis also requires a colonoscopy. This test, when used for population screening, requires rigorous bowel preparation and carries a certain radiation risk, resulting in low patient compliance. Regarding serum tumor markers, CEA and CA19-9 are relatively specific molecular markers for colorectal cancer. While helpful in assessing prognosis, they are not specific for colorectal cancer diagnosis and lack obvious changes in the early stages of the tumor, making them unsuitable for early diagnosis. Therefore, there is an urgent clinical need for a diagnostic marker with high specificity, strong sensitivity, and minimal trauma for the early diagnosis of CRC.

[0004] MicroRNA (miRNA) is a type of single-stranded small RNA composed of approximately 18-22 nucleotides that does not have a coding function. Studies have confirmed that miRNA can cause uncontrolled changes in proteins in the biogenesis pathway, such as AGO2, Drosha, and Dicer. MiRNA plays an important role in the occurrence, development, invasion, and metastasis of colorectal cancer. Circulating miRNA can exist stably in plasma. Many previous studies have considered circulating miRNA to be a very promising cancer biomarker because miRNA expression is tissue-specific and miRNA levels may provide a more direct understanding of the biological behavior of the disease.

[0005] Extracellular vesicles (EVs) are secreted membrane-bound vesicles naturally released from cells. They comprise a heterogeneous population of membrane vesicles from various sources, with diameters ranging from 30 to 1000 nm. Almost all cells can secrete EVs. A growing number of studies have shown that EVs can function as messengers for intercellular communication. EVs can carry bioinformatics molecules characteristic of maternal cells, such as DNA, proteins, lipids, mRNA, miRNA, and ncRNA. They are widely present in body fluids such as blood, cerebrospinal fluid, urine, amniotic fluid, breast milk, and exudates. In cancer, pathophysiological changes such as imbalanced tumor cell growth, pH shifts caused by altered microenvironment, cellular hypoxia, and oxidative stress can stimulate tumor cells to produce more EVs than normal cells. CRC-associated EVs can deliver biomolecules to cells, triggering cell carcinogenesis. Furthermore, tumor-derived EVs can induce angiogenesis and promote cancer cell metastasis. These properties of EVs offer advantages in the early screening, diagnosis, monitoring, and treatment of malignant tumors, making them a promising new clinical tool.

[0006] Exosome-derived miRNA (EV-miRNA) has shown great potential as a cancer biomarker in recent years. Tumor cells produce more exosomes than normal cells, and exosomes are relatively abundant and stable, making them highly valuable for the search for biomarkers and therapeutic targets. Furthermore, tumor cells release large numbers of EVs carrying tumor information. Therefore, compared to circulating total miRNA, EV-miRNA expression profiles may be more tumor-specific and better reflect disease characteristics. Numerous studies have linked EV-miRNAs to cancer invasion and metastasis. For example, researchers have found that exosomal miR-155 secreted by melanoma cells can induce angiogenesis in cancer-associated fibroblasts (CAFs); high levels of exosomal miR-222 are associated with increased invasiveness in breast cancer; and miR-21 is dysregulated in different tumor types.

[0007] Currently, many studies investigating the use of exosome-derived miRNAs from blood samples, such as serum or plasma, as diagnostic markers for colorectal cancer have not validated differentially expressed exosomal miRNAs. Furthermore, most studies have only described single exosomal miRNAs as diagnostic markers for colorectal cancer. The detection of single exosomal miRNAs often lacks specificity, sensitivity, and diagnostic value. Therefore, it is necessary to comprehensively evaluate combinations of several plasma exosomal miRNAs with diagnostic value for colorectal cancer, along with serum tumor markers, and to establish a comprehensive evaluation system to ensure the practical application of exosomal miRNA combinations in assisting the early diagnosis of colorectal cancer.

[0008] The Chinese invention patent application entitled "Diagnostic kit for early metastasis of colorectal cancer based on the expression level of exosomal LncCLDN23" and application number CN202110138441.8 (publication number: CN112779336A, publication date: May 11, 2021) discloses a diagnostic kit for early metastasis of colorectal cancer based on the expression level of exosomal LncCLDN23. The kit includes real-time quantitative PCR amplification primers for exosomal LncCLDN23. The content of exosomal LncCLDN23 in plasma is detected by real-time quantitative PCR, which provides an important reference for predicting early metastasis of colorectal cancer. This patent does not select exosomal miRNA as a marker to detect its expression level, thereby providing an important basis for early diagnosis and screening of colorectal cancer. The Chinese invention patent application entitled "Application of an exosome membrane protein as a diagnostic marker for colon cancer and a kit for early diagnosis of colon cancer" with application number CN202110039502.5 (publication number: CN112379096A, publication date: February 19, 2021) discloses the application of an exosome membrane protein as a diagnostic marker for colon cancer and a kit for early diagnosis of colon cancer. The patent experimentally determined that the CD33 and CD147 proteins in exosomes can be used as diagnostic markers for colon cancer, and their detection can achieve early and accurate diagnosis of colon cancer. The patent also did not select exosome miRNA as an early diagnostic marker for colorectal cancer. The Chinese invention patent application entitled “Exosomal miRNA Markers and Diagnostic Kits for Diagnosis of Colorectal Cancer” and application number CN201811126210.X (publication number: CN109439749A, publication date: March 8, 2019) provides exosomal miRNA markers for diagnosis of colorectal cancer, wherein the markers are selected from at least one of let-7b-3p, let-7b-5p, miR-150-3p, miR-145-3p, miR-139-3p, miR-139-5p, miR-15b-3p, miR-125a-5p, miR-140-5p, miR-342-3p, miR-132-5p and miR-92b-5p. This patent, based on non-invasive detection technology, develops a method for diagnosing and monitoring colorectal cancer recurrence. It demonstrates high sensitivity and specificity in early-stage colorectal cancer, with a combined AUC of up to 0.99 for multiple miRNA markers, demonstrating exceptional diagnostic performance. However, this patent does not screen for highly differentially expressed plasma exosomal miRNAs and combine them with serum tumor markers to enhance the sensitivity and specificity of early colorectal cancer diagnosis.

[0009] So far, it has been reported that miR-99b-5p and miR-409-3p are of great significance in the occurrence and development of gastric cancer, prostate cancer, lung cancer and other tumors. However, there have been no specific research reports on the expression of miR-99b-5p and miR-409-3p in plasma exosomes and early diagnosis of colorectal cancer patients. Summary of the Invention

[0010] Technical issues

[0011] The purpose of the present invention is to provide a marker for early diagnosis of colorectal cancer and its application method to address the problems of low specificity and low sensitivity of current blood markers for diagnosing colorectal cancer, so as to form a combined result evaluation system, which is conducive to the non-invasive, rapid and accurate early diagnosis of colorectal cancer.

[0012] Technical Solution

[0013] In the first aspect of the present invention, an early diagnostic marker for colorectal cancer is provided, which is a nucleic acid molecule selected from the following miRNA or a combination thereof: the nucleic acid molecule comprises a nucleic acid molecule encoding plasma exosome miR-99b-5p and miR-409-3p; the nucleic acid molecule is upregulated in the target plasma exosomes compared to the control sample.

[0014] In a second aspect, the present invention provides a diagnostic kit for identifying early colorectal cancer, wherein the diagnostic kit comprises the early diagnosis marker for colorectal cancer according to the present invention.

[0015] At least one of the nucleic acid molecules described herein is differentially expressed in exosomes from the target plasma and healthy control plasma. Using plasma samples from healthy volunteers as a control, miR-99b-5p is highly expressed in exosomes from the plasma of patients with colorectal cancer, with higher expression in early-stage exosomes than in late-stage exosomes. Furthermore, miR-409-3p is highly expressed in exosomes from the plasma of patients with colorectal cancer, using plasma samples from healthy volunteers as a control.

[0016] Therefore, the characteristic of differential expression of at least one nucleic acid molecule in the two exosomal miRNAs is indicative of the presence of colorectal cancer.

[0017] In a third aspect of the present invention, the diagnostic kit for identifying early colorectal cancer further comprises the serum tumor marker CEA.

[0018] Technical Effects

[0019] The present invention screened exosomal miRNAs differentially expressed in plasma exosomes from patients with colorectal cancer to identify those with potential as diagnostic biomarkers for colorectal cancer. These candidate exosomal miRNAs were then validated in plasma samples. The results showed that miR-99b-5p and miR-409-3p were highly expressed in colorectal cancer. Furthermore, the inventors found that miR-99b-5p expression was higher in early-stage than late-stage disease, and higher in the colon than in the rectum. This suggests that miR-99b-5p may be of significant significance in early-stage colorectal cancer. At the same time, the inventors also found that exosomal miRNA combinations can be used to differentially diagnose early colorectal cancer. The area under the ROC curve (AUC) for the combination of miR-99b-5p and miR-409-3p was 0.741 (95% CI = 0.592-0.891, Sensitivity = 77.3%, Specificity = 78.3%, P = 0.006); the AUC for the combination of miR-99b-5p, miR-409-3p and CEA was 0.812 (95% CI = 0.692-0.944, Sensitivity = 90.9%, Specificity = 65.2%, P < 0.001). The results show that the differential diagnostic ability of the exosomal miRNA combination is higher than that of a single exosomal miRNA, and it has the potential to serve as a marker for early screening of colorectal cancer. The present invention will help promote the use of plasma exosomal miRNA as a non-invasive diagnostic biomarker for early diagnosis of colorectal cancer.

[0020] The present invention jointly detects the expression levels of exosomal miR-99b-5p and miR-409-3p in the plasma of colorectal cancer patients and healthy subjects, analyzes the specificity, sensitivity and area under the curve of the receiver operating characteristic curve of exosomal miR-99b-5p, miR-409-3p and serum tumor marker CEA for diagnosing colorectal cancer alone and in combination, and obtains the exosomal miRNA expression level node for diagnosing colorectal cancer.

[0021] The advantages of the present invention are:

[0022] (1) Exosomal miRNAs are a new type of liquid biopsy biomarker that is more sensitive, stable, and non-invasive, requires less sample volume, and is easily accepted by patients. Its successful development will help in the auxiliary diagnosis of colorectal cancer.

[0023] (2) For the first time, we identified exosomal miR-99b-5P and miR-409-3p as markers for the early diagnosis of colorectal cancer, and established a new early diagnosis and detection index for colorectal cancer.

[0024] (3) Products designed based on multiple miRNAs have better sensitivity and specificity than products designed based on single miRNAs, and can reduce the errors caused by individual expression differences of a single indicator, making the results more accurate.

[0025] (4) Combined with CEA, a specific serum marker for colorectal cancer, it is more targeted, has better sensitivity and specificity, and is more convincing. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A and Figure 1B Electron microscopy (TEM) images of the exosomes extracted in this invention. A: Scale bar 500 nm; B: Scale bar 100 nm.

[0027] Figure 1C Nanoparticle tracking analysis (NTA) images of exosomes extracted in this study. Left: Particle size distribution of sample EVs; Right: Particles displayed in the detection window.

[0028] Figure 1D This is a Western Blotting identification image of the exosomes extracted in the present invention.

[0029] Figure 2 is the average relative expression level of miRNA in 16 plasma EV-RNA samples.

[0030] Figure 3 is the expression level of miR-99b-5p and miR-409-3p in plasma exosomes (EVs).

[0031] Figure 4A and Figure 4B ROC curve analysis was performed to analyze the diagnostic efficacy of miR-99b-5p and miR-409-3p in identifying colorectal cancer.

[0032] Figure 4C and Figure 4D ROC curve analysis was performed to analyze the diagnostic efficacy of miR-99b-5p and miR-409-3p in identifying early colorectal cancer.

[0033] Figure 4E The ROC curve was used to analyze the diagnostic performance of CEA in identifying early colorectal cancer.

[0034] Figure 5A ROC curve analysis was performed to analyze the diagnostic efficacy of the combination of miR-99b-5p and miR-409-3p in identifying early colorectal cancer.

[0035] Figure 5B ROC curve analysis was performed to analyze the diagnostic efficacy of the combination of miR-99b-5p, miR-409-3p and CEA in identifying early colorectal cancer. DETAILED DESCRIPTION

[0036] The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0037] Exosome extraction kit exoRNeasy Midi Kit (Qiagen, Germany), Buffer XE (Qiagen, Germany), protein concentration detection kit Pierce TM BCA Protein Assay Kit (Thermo, USA), miRcute enhanced miRNA cDNA first-strand synthesis kit (Tiangen, China), and miRcute enhanced miRNA fluorescence quantitative detection kit (Tiangen, China) were used for miRNA quantification by real-time fluorescence quantitative PCR, and Illumina HiSeqTM2500 (Illumina, USA) was used for miRNA sequencing.

[0038] Example 1: Subjects and sample extraction

[0039] The samples were provided by the Third Affiliated Hospital of Guangzhou Medical University, including 12 patients with colorectal cancer (CRC) and 4 healthy controls (HC).

[0040] Example 2: Extraction of exosomes

[0041] 4 ml of fasting peripheral blood was collected from CRC patients and healthy subjects using EDTA anticoagulant tubes. Immediately after blood collection, the tubes were mixed by inversion 5-8 times, placed at room temperature, and centrifuged at 3500 rpm for 10 min within 2 hours. The supernatant, i.e., plasma, was carefully collected into cryopreservation tubes (do not aspirate the blood cells below the plasma) and stored in a -80°C refrigerator until use.

[0042] Plasma exosomes were extracted using the exoRNeasy Midi Kit (Qiagen, Germany) and Buffer XE (Qiagen, Germany). The specific steps were as follows:

[0043] (1) Plasma frozen at -80°C was thawed at room temperature, and then two plasma pretreatment steps were performed:

[0044] Centrifugation: 3000g, 4℃, 15min, take the supernatant; 16000g, 4℃, 20min, take 1ml of supernatant.

[0045] (2) Add the plasma sample and XBP to Buffer XBP at a volume ratio of 1:1. Gently invert the tube 5 times to mix thoroughly and allow the mixture to warm to room temperature.

[0046] (3) Add the sample / XBP mixture to the spin column and centrifuge at 500 g for 1 min at room temperature. Discard the flow-through and return the column to the same collection tube. Ensure that all liquid has passed through the membrane. Note: If any liquid remains on the membrane, spin again at 3000 g for 1 min to ensure that all liquid has passed through the membrane.

[0047] (4) Add 3.5 ml of XWP buffer to the spin column and centrifuge at 3000 g for 5 min to wash the spin column and remove any residual buffer. Discard the flow-through and collection tube and transfer the spin column to a new collection tube.

[0048] (5) Add 400 μl of Buffer XE, incubate for 1 min, and centrifuge at 500 g for 5 min at room temperature.

[0049] (6) Add the eluate to the membrane again, incubate for 1 min, and centrifuge at 3000g for 5 min at room temperature.

[0050] (7) The obtained eluate, i.e., the plasma EV extraction sample, was aliquoted and frozen at -80°C.

[0051] See also Figure 1A -D, the identification results of the extracted exosomes are as follows:

[0052] 1) Electron microscopy shows: Figure 1A -B shows that the exosomes have complete morphological structure, bilateral membrane structure, and are evenly distributed;

[0053] 2) Nanoparticle tracking analysis (NTA) results: Figure 1C As shown, the average particle size (diameter) of exosomes is about 168 nm;

[0054] 3) Western blot analysis: Figure 1D As shown, exosomes express EV characteristic proteins CD63, CD81TSG101 and Alix protein.

[0055] Example 3: Extraction of exosomal miRNA

[0056] Exosome miRNA was extracted from plasma using the exoRNeasy Midi Kit. Follow the kit instructions.

[0057] (1) Plasma was thawed at room temperature. Plasma pretreatment steps: 2 centrifugations: 1 ml plasma.

[0058] 3000g, 4℃, 15min, take the supernatant → 900ul;

[0059] 16000g, 4℃, 20min, take the supernatant → take 800ul of supernatant.

[0060] (2) Add Buffer XBP at a ratio of 1:1. Mix thoroughly.

[0061] (3) Add the sample / XBP mixture to the spin column,

[0062] Centrifugation: 500 g, 1 min, room temperature;

[0063] Centrifuge again: 3000 g, 1 min, room temperature;

[0064] Discard the flow-through and return the column to the same collection tube. Ensure that all the liquid has passed through the membrane.

[0065] (4) Add 3.5 ml of XWP buffer to the spin column to wash the column and remove residual buffer. Centrifuge: 3000 g, 1 min, room temperature; discard the flow-through and collection tube.

[0066] (5) Transfer the spin column to a fresh collection tube.

[0067] (6) Add 700 μl of QIAzol lysis buffer to the membrane. Centrifuge at 3000 g for 5 min at room temperature to collect the lysate, and then transfer it completely to an EP tube. Add 10 μl of artificially synthesized 5' phosphorylated RNA, i.e., the external reference cel-miR-39-3p (2×10 -7 uM).

[0068] (7) Briefly vortex the tube containing the lysate and incubate at room temperature for 5 minutes.

[0069] (8) Add 90 μl of chloroform to the tube containing the lysate and securely cap the tube. Shake vigorously for 15 seconds and then incubate at room temperature for 3 minutes.

[0070] (9) Centrifuge at 12,000 g for 15 min at 4°C. After centrifugation, the sample separates into three phases: an upper colorless aqueous phase containing RNA, a thin white interphase; and a lower red organic phase. RNA is present in the upper aqueous phase.

[0071] (10) Transfer the upper aqueous phase to a new EP tube. 250 μl / sample, avoiding transfer of any interphase material. Add 2 volumes of anhydrous ethanol, i.e., 500 μl, and mix thoroughly.

[0072] (11) Pipette the sample (including any precipitate that may have formed) into an RNeasy MinElute spin column in a 2 ml collection tube. Gently close the lid and centrifuge at 12,000 g for 30 seconds at room temperature. Discard the flow-through. Reuse the collection tube in the next step. Repeat this step for the remaining samples.

[0073] (12) Add 700 μl of Buffer RWT to the RNeasy MinElute spin column. Gently close the cap and centrifuge at 12,000 g for 30 seconds at room temperature. Discard the flow-through. Reuse the collection tube in the next step.

[0074] Example 4: NGS sequencing analysis of miRNA

[0075] The extracted plasma exosome miRNA was sequenced.

[0076] (1) Sample RNA was first analyzed for RNA concentration using Qubit (Bio-Rad, USA) and Agilent 2200 (Agilent, USA).

[0077] (2) The library quality control was performed using Agilent 2200 TapeStation (Agilent, USA).

[0078] (3) Illumina HiSeqTM2500 (Illumina, USA) was used for sequencing identification.

[0079] (4) Calculate the expression level of the identified miRNA. The miRNA expression level calibration method RPM (the number of reads per million of miRNA) = calculate the number of reads per million clean reads that map to a specific miRNA (the number of reads per million clean tag).

[0080] (5) The edgeR R language software package was used to perform differential analysis of miRNA expression. The average RPM expression of abnormally expressed miRNAs between groups was converted to log2 (RPM), and a bar graph of the average relative expression of miRNAs in plasma EV-RNA samples was drawn. Figure 2 The differentially expressed miRNAs between the comparison groups were obtained by two levels: |log2(Fold Change)|>1) and significance level (P value<0.05): miR-99b-5p and miR-409-3p.

[0081] Example 5: Real-time fluorescence quantitative PCR verification of differentially expressed miRNAs

[0082] The expression levels of miR-99b-5p and miR-409-3p were quantitatively detected by real-time fluorescence quantitative PCR (RT-qPCR). The Cq values ​​obtained by RT-qPCR were used to perform relative quantification of miRNA expression using the 2-ΔCq method. The 2-ΔCq formula is: ΔCq = Cq (candidate miRNA) - Cq (reference miRNA), thereby obtaining the fold change in miRNA expression levels in the CRC group relative to the healthy control group. Figure 3 .

[0083] Example 6: Validation of miRNA combination for early diagnosis of colorectal cancer

[0084] Exosomal miR-99b-5p, miR-409-3p, tumor marker CEA and their combination were used as detection objects, and the ROC curve was drawn using the "ROC curve graph" function of SPSS software. Figure 4A -E and Figure 5A -B. 95% confidence intervals (95% CI) and areas under the ROC curves (AUCs) were obtained. The sensitivity and specificity corresponding to the cutoff values ​​of the ROC curves were determined using the maximum Youden index. For early colorectal cancer, the analysis showed:

[0085] (1) miR-99b-5p: AUC = 0.735, 95% CI = 0.587-0.884, Sensitivity = 72.7%, Specificity = 78.3%, P = 0.007; miR-409-3p: AUC = 0.611, 95% CI = 0.443-0.778, Sensitivity = 63.6%, Specificity = 60.9%, P = 0.206; CEA: AUC = 0.820, 95% CI = 0.695-0.945, Sensitivity = 72.7%, Specificity = 87%, P < 0.001, see Figure 4C .

[0086] (2) Combination of miR-99b-5p and miR-409-3p: AUC = 0.741, 95% CI = 0.592-0.891, Sensitivity = 77.3%, Specificity = 78.3%, P = 0.006, see Figure 5A ; miR-99b-5p and miR-409-3p combined with CEA: AUC = 0.812, 95% CI = 0.679-0.945, Sensitivity = 90.9%, Specificity = 65.2%, P < 0.001, see Figure 5B The data results showed that the combination of miR-99b-5p, miR-409-3p and CEA has a sensitivity of up to 90.9% for early colorectal cancer, with extremely high diagnostic efficacy.

[0087] Based on the above data and conclusions, and in combination with the existing technology in this field, miR-99b-5p or miR-409-3p alone, a combination of miR-99b-5p or miR-409-3p, or a combination of miR-99b-5p, miR-409-3p and CEA can be used to construct a diagnostic kit for distinguishing a colorectal cancer patient from a healthy individual, for detecting early colorectal cancer.

Claims

1. The use of a reagent for detecting the expression level of early diagnosis markers for colorectal cancer for the preparation of a diagnostic kit for identifying early colorectal cancer, wherein the early diagnosis markers for colorectal cancer are nucleic acid molecules encoding plasma exosomal miR-99b-5p and miR-409-3p and the serum tumor marker CEA.

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