CN116004818A - Early diagnosis markers and kits for colorectal cancer - Google Patents

Abstract

The invention relates to the field of molecular biology and medical diagnosis, in particular to a colorectal cancer early diagnosis marker, in particular to a miRNA marker from plasma exosomes, and application of the marker in preparation of a kit for detecting early colorectal cancer. The colorectal cancer early diagnosis marker is one nucleic acid molecule miRNA or a combination thereof selected from the following: the nucleic acid molecule comprises a nucleic acid molecule encoding plasma exosomes miR-99b-5p and miR-409-3 p; the nucleic acid molecules are up-regulated in target plasma exosomes compared to control samples. The diagnosis kit provided by the invention is beneficial to noninvasively, rapidly and accurately performing early diagnosis of colorectal cancer.

Description

Early diagnosis markers and kits for colorectal cancer

technical field

The present invention relates to the fields of molecular biology and medical diagnosis, in particular to a marker for early diagnosis of colorectal cancer, especially a miRNA marker from plasma exosomes, and the use of the marker in the preparation and detection of early colorectal cancer kits application.

Background technique

Colorectal cancer (CRC) is an epithelial malignant tumor of the colon or rectum, and one of the most common malignant tumors of the gastrointestinal tract. From a global perspective, CRC ranks third overall in cancer incidence, among which women rank second, men rank third, and overall CRC mortality ranks second. In my country, there are 398,000 cases of CRC and 188,000 deaths each year. The disease has caused a great burden on the national economy. The onset of colorectal cancer is hidden, and the clinical symptoms are not obvious in the early stage. Changes in bowel habits and stool properties are often the earliest symptoms in patients, so it is difficult for patients to detect early, resulting in many patients being diagnosed at an advanced stage of the disease. According to reports, compared with patients who were already in the advanced stage when they were discovered, the 5-year survival rate of early CRC can be greatly improved, up to 90%. CRC cells can metastasize to adjacent and distant organs through direct infiltration, lymphatic metastasis, implantation metastasis, and hematogenous metastasis, which can lead to malignant ascites and cancer pain in advanced patients, and seriously reduce the quality of life of patients. Therefore, the prevention and treatment of colorectal cancer focus on early diagnosis and early treatment, timely detection and treatment in the early stage of the disease.

The occurrence of most CRC needs to go through the process of "adenoma-carcinoma", which may last from several years to more than ten years, which provides a great possibility for early clinical intervention on tumors. Advances in early screening and diagnostic techniques have greatly improved the survival rate of CRC patients. From the mid-1970s to 2008–2014, the combined 5-year relative survival rates for all stages increased from 50.6% to 65.1% for CRC and from 48.1% to 68.8% for rectal cancer. Currently, CRC screening in my country mainly includes colonoscopy, fecal immunochemical testing (FIT), multi-target fecal FIT-DNA fecal occult blood detection, and colonic CT imaging. Colonoscopy is a clinical endoscopist who inspects the entire colorectum through a visual lens, and then takes a biopsy of the observed suspicious lesions, and diagnoses the disease through pathological diagnosis. Colonoscopy is currently the gold standard for CRC screening. However, the preoperative preparation of colonoscopy, high risk of complications and medical costs make patient compliance low, hindering the promotion and application of this method as a basic screening project. Moreover, studies have shown that colonoscopy is a very invasive method, which has certain harm to the patient itself. FIT is the detection of human hemoglobin in feces by specific antibodies, suggesting intestinal lesions, so FIT-positive patients need further colonoscopy. Fecal occult blood test (FOBT) is a guaiac-based test that can detect occult blood in feces. However, this method is less sensitive than FIT and is easily affected by diet and drugs. Currently, it is mostly replaced by FIT. A prospective confirmatory study involving 9989 cases reported a diagnostic sensitivity of 92.3% for the differential diagnosis of colorectal cancer by fecal FIT-DNA, and a lower diagnostic sensitivity of 42.4% for advanced adenoma. At present, the detection cost of FIT-DNA is still high, and the diagnostic applicability of the Chinese population still needs further multi-center and large-sample verification. Colon CT imaging requires the subject to prepare the bowel, then fill the colon with gas, and perform a CT scan of the whole colon to observe the entire colon. The diagnosis also needs to be combined with colonoscopy. The application of this examination in population screening requires strict bowel preparation, and due to its certain radiation risk, patient compliance is not high. Regarding serum tumor markers, CEA and CA19-9 are relatively specific molecular markers for colorectal cancer. Although they are helpful for the prognosis of the disease, they are not specific for the diagnosis of colorectal cancer, and they change in the early stage of tumors. It is not obvious and cannot be used for early diagnosis. Therefore, there is an urgent clinical need for a diagnostic marker with high specificity, strong sensitivity, and less trauma for early diagnosis of CRC.

MicroRNA (microRNA, miRNA) is a kind of single-stranded small RNA composed of about 18-22 nucleotides without coding function. Studies have confirmed that miRNAs can cause out-of-control changes in biogenesis pathway proteins such as AGO2, Drosha, and Dicer. miRNA plays an important role in the occurrence, development, invasion and metastasis of colorectal cancer. Circulating miRNAs can exist stably in plasma. Many previous studies have considered circulating miRNAs to be a very promising cancer biomarker, because miRNA expression is tissue-specific and miRNA levels may provide a more direct insight into the biological behavior of the disease.

Extracellular vesicles (EVs) refer to secreted vesicles with a membrane structure naturally released from cells, including a heterogeneous population of membrane vesicles from various sources, with diameters ranging from 30-1000 nm. Almost all cells can secrete EVs. More and more studies have found that EVs can be used as messengers for intercellular communication. EVs can carry biological information molecules characteristic of maternal cells, such as DNA, protein, lipid, mRNA, miRNA, and ncRNA, which are widely present in body fluids such as blood, cerebrospinal fluid, urine, amniotic fluid, milk, and exudate. In cancer, the pathophysiological changes of cells such as imbalanced growth of tumor cells, pH changes caused by changes in the microenvironment, cellular hypoxia, and oxidative stress can stimulate tumor cells to produce more EVs than normal cells. CRC-associated EVs can induce cell carcinogenesis by delivering biomolecules to cells. Moreover, tumor-derived EVs can induce angiogenesis and promote cancer cell metastasis. The above-mentioned characteristics of EV show certain advantages in early screening, diagnosis, disease monitoring, and treatment of malignant tumors, and it is expected to become a new clinical tool.

Exosome-derived miRNA (EV-miRNA) has shown high potential as a cancer biomarker in recent years. Tumor cells can produce more exosomes than normal cells, and the exosomes have a relatively high content and high stability, which is very meaningful for finding biomarkers and therapeutic targets. Moreover, tumor cells release a large number of EVs carrying tumor information. Therefore, compared with circulating total miRNAs, EV-miRNA expression profiles may be more tumor-specific and better reflect disease characteristics. At present, many studies have found that EV-miRNA is related to cancer invasion and metastasis. For example, researchers found that exosomal miR-155 secreted by melanoma cells can induce cancer-associated fibroblasts (CAFs) to promote angiogenesis; high levels of exosomal miR -222 is associated with high aggressiveness in breast cancer; miR-21 is dysregulated in different tumor types.

At present, in the research reports on miRNAs derived from blood samples such as serum or plasma exosomes as diagnostic markers for colorectal cancer, many studies have not performed sample verification on differentially expressed exosomal miRNAs; Exosomal miRNAs have been described as diagnostic markers for colorectal cancer, and the detection of a single exosomal miRNA is often insufficient in specificity, sensitivity, and diagnostic value. Therefore, it is necessary to comprehensively evaluate the combination of several plasma exosomal miRNAs and serum tumor markers that have diagnostic value for colorectal cancer, and build a complete evaluation system, so that the combination of exosomal miRNAs can assist the early detection of colorectal cancer. Diagnosis yields tangible application value.

It is disclosed in the Chinese invention patent application (publication number: CN112779336A, publication date: May 11, 2021) titled "Diagnostic Kit for Early Metastasis of Colorectal Cancer Based on Exosomal LncCLDN23 Expression Level" and application number CN202110138441.8 A diagnostic kit for early metastasis of colorectal cancer based on the expression level of exosomal LncCLDN23 was developed, which included primers for real-time quantitative PCR amplification of exosomal LncCLDN23. Detection of exosomal LncCLDN23 in plasma by real-time quantitative PCR 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, so as to provide an important basis for early diagnosis and screening of colorectal cancer. A Chinese invention patent application titled "Application of an exosomal 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 2021 The application of an exosomal membrane protein as a diagnostic marker for colon cancer and a kit for the early diagnosis of colon cancer were disclosed. The patent has determined through experiments that the CD33 and CD147 proteins of 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 exosomal miRNA as an early diagnostic marker for colorectal cancer. The Chinese invention patent application (publication number: CN109439749A, publication date: March 8, 2019) entitled "Exosomal miRNA markers and diagnostic kits for the diagnosis of colorectal cancer" and application number CN201811126210.X Exosomal miRNA markers for the diagnosis of colorectal cancer were selected from let-7b-3p, let-7b-5p, miR-150-3p, miR-145-3p, miR-139-3p , at least one of miR-139-5p, miR-15b-3p, miR-125a-5p, miR-140-5p, miR-342-3p, miR-132-5p and miR-92b-5p. The patent is based on the non-invasive detection technology for the diagnosis and recurrence monitoring of colorectal cancer. It has high sensitivity and high specificity in early colorectal cancer. The combined AUC of multiple miRNA markers can reach up to 0.99, which has extremely superior diagnostic performance . However, this patent does not screen highly differentially expressed plasma exosomal miRNAs and combine them with serum tumor markers to improve the sensitivity and specificity of early diagnosis of colorectal cancer.

So far, it has been reported that miR-99b-5p and miR-409-3p play an important role in the occurrence and development of gastric cancer, prostate cancer, lung cancer and other tumors. However, regarding miR-99b-5p and miR-409 The expression and early diagnosis of -3p in plasma exosomes of patients with colorectal cancer have not been specifically reported.

Contents of the invention

technical problem

The purpose of the present invention is to provide a colorectal cancer early diagnosis marker and application method to form a combined result evaluation system, which is beneficial to non-invasive, Fast and accurate early diagnosis of colorectal cancer.

Technical solutions

The first aspect of the present invention provides a marker for early diagnosis of colorectal cancer, which is a nucleic acid molecule miRNA selected from the following or a combination thereof: the nucleic acid molecule includes encoding plasma exosomal miR-99b-5p and miR- A nucleic acid molecule of 409-3p; the expression of the nucleic acid molecule in target plasma exosomes is up-regulated compared with the control sample.

The second aspect of the present invention provides a diagnostic kit for identifying early colorectal cancer, said diagnostic kit including the early diagnostic markers for colorectal cancer described in the present invention.

At least one of the nucleic acid molecules of the present invention is differentially expressed in exosomes between target plasma and healthy control plasma. Taking the plasma samples of healthy volunteers as the control, miR-99b-5p was highly expressed in the plasma exosomes of colorectal cancer patients, and the early stage was higher than the late stage; taking the plasma samples of healthy volunteers as the control, miR-409-3p was highly expressed in the colorectal cancer patients’ plasma exosomes. Highly expressed in plasma exosomes of cancer patients.

Therefore, the characteristic of differential expression of at least one nucleic acid molecule in the two exosomal miRNAs is an indication of the presence of colorectal cancer.

In the third aspect of the present invention, the diagnostic kit for identifying early colorectal cancer according to the present invention further includes the serum tumor marker CEA.

technical effect

The present invention screens exosomal miRNAs differentially expressed in colorectal cancer patients in plasma exosomes to determine those exosomal miRNAs that have the potential to be diagnostic biomarkers for colorectal cancer, and then identify candidate exosomal miRNAs Validation of plasma samples was performed. The results showed that miR-99b-5p and miR-409-3p were highly expressed in colorectal cancer. Moreover, the inventors found that the expression of miR-99b-5p was higher in the early stage than in the late stage, and higher in the colon than in the rectum. It is suggested that miR-99b-5p plays an important role in early colorectal cancer. At the same time, the inventors also found that the exosomal miRNA combination can be used for the differential diagnosis of early colorectal cancer, and the area under the ROC curve (Area under the ROC curve, AUC) of miR-99b-5p and miR-409-3p combination is 0.741 ( 95%CI=0.592-0.891, Sensitivity=77.3%, Specificity=78.3%, P=0.006); miR-99b-5p, miR-409-3p combined with CEA AUC is 0.812 (95%CI=0.692-0.944, Sensitivity =90.9%, Specificity=65.2%, P<0.001). The results show that the differential diagnosis ability of exosomal miRNA combination is higher than that of single exosomal miRNA, which has the potential as a marker for early screening of colorectal cancer. The invention will help to promote plasma exosomal miRNA as a non-invasive diagnostic biomarker for early diagnosis of colorectal cancer.

In the present invention, the expression levels of exosomal miR-99b-5p and miR-409-3p in the plasma of colorectal cancer patients and healthy persons are jointly detected to analyze exosomal miR-99b-5p, miR-409-3p and serum tumor The area under the curve of the specificity, sensitivity, and receiver characteristic curves of the marker CEA alone and in combination for the diagnosis of colorectal cancer, and the exosomal miRNA expression level nodes for the diagnosis of colorectal cancer were obtained.

The advantages of the present invention are:

(1) Exosomal miRNAs are a new type of liquid biopsy biomarkers, which are more sensitive, stable and non-invasive, require less sample volume, and are easily accepted by patients. Their successful development is helpful for the auxiliary diagnosis of colorectal cancer .

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

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

(4) Combined with CEA, a specific serum marker for colorectal cancer, it is more targeted, has better sensitivity and specificity, and is more convincing.

Description of drawings

Figure 1A and Figure 1B are electron microscope (TEM) identification images of exosomes extracted in the present invention. A: scale bar 500 nm; B: scale bar 100 nm.

Fig. 1C is a graph of nanoparticle tracking analysis (NTA) of exosomes extracted in the present invention. Left: particle size distribution of sample EV particles; right: the display of particles in the detection window.

Figure 1D is a Western Blotting identification diagram of the exosomes extracted in the present invention.

Figure 2 shows the average relative expression levels of miRNAs in 16 plasma EV-RNA samples.

Figure 3 shows the expression levels of miR-99b-5p and miR-409-3p in plasma exosomes (EV).

Figure 4A and Figure 4B are ROC curve analysis of the diagnostic performance of miR-99b-5p and miR-409-3p in identifying colorectal cancer.

Figure 4C and Figure 4D are ROC curve analysis of the diagnostic efficacy of miR-99b-5p and miR-409-3p in identifying early colorectal cancer.

Figure 4E is the ROC curve analysis of the diagnostic performance of CEA in identifying early colorectal cancer.

Figure 5A is the ROC curve analysis of the diagnostic efficiency of the combination of miR-99b-5p and miR-409-3p in identifying early colorectal cancer.

Figure 5B is the ROC curve analysis of the diagnostic performance of miR-99b-5p, miR-409-3p and CEA combination in identifying early colorectal cancer.

Detailed ways

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

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 reagent Box (Tiangen Company, China), miRcute Enhanced miRNA Fluorescent Quantitative Detection Kit (Tiangen Company, China) for real-time fluorescent quantitative PCR for miRNA quantification, and Illumina HiSeqTM2500 (Illumina, USA) for miRNA sequencing.

Example 1: Extraction of subjects and samples

The samples were provided by the Third Affiliated Hospital of Guangzhou Medical University. Among them, there were 12 cases of colorectal cancer patients (CRC) and 4 cases of healthy subjects (Healthy control, HC).

Example 2: Extraction of exosomes

Use EDTA anticoagulant tubes to collect 4ml of fasting peripheral blood from CRC patients and healthy subjects. Immediately after drawing the blood, mix it upside down 5-8 times, place it at room temperature, and centrifuge it at 3500rpm for 10min within 2 hours. Store the tube (do not absorb the blood cells in the lower layer of the plasma), and store it in a negative 80°C refrigerator until use.

Plasma exosomes were extracted using exoRNeasy Midi Kit (Qiagen, Germany) and Buffer XE (Qiagen, Germany). Follow the instructions of the kit, and the specific steps are as follows:

(1) Dissolve the plasma frozen at -80°C at room temperature, and then perform 2 steps of plasma pretreatment:

Centrifuge: 3000g, 4°C, 15min, take the supernatant; 16000g, 4°C, 20min, take 1ml of the supernatant.

(2) Add the plasma sample and XBP to Buffer XBP according to the volume ratio of 1:1, gently invert the test tube 5 times, mix thoroughly, and warm the mixture to room temperature.

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

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

(5) Add Buffer XE 400ul, incubate for 1min, centrifuge at 500g, 5min, room temperature.

(6) Add the eluent to the membrane again, incubate for 1 min, centrifuge at 3000 g, 5 min, at room temperature.

(7) The obtained eluate, that is, the plasma EV extraction sample, was subpackaged and frozen at -80°C.

Referring to Figure 1A-D, the identification results of the extracted exosomes are as follows:

1) Electron microscopy shows: as shown in Figure 1A-B, the exosomes have complete morphology and structure, with double-sided membrane structure and uniform distribution;

2) Nanoparticle tracking analysis (NTA) results: as shown in Figure 1C, the average particle size (diameter) of exosomes is around 168nm;

3) Western blot analysis: As shown in Figure 1D, exosomes expressed EV characteristic proteins CD63, CD81, TSG101 and Alix protein.

Example 3: Extraction of exosome miRNA

ExoRNeasy Midi Kit was used to extract exosomal miRNA from plasma. Follow the instructions of the kit for operation.

(1) Plasma is thawed at room temperature, plasma pretreatment steps: 2 times of centrifugation: 1ml plasma.

3000g, 4°C, 15min, take the supernatant → 900ul;

16000g, 4°C, 20min, take the supernatant → take 800ul of the supernatant.

(2) Sample: XBP=1:1 add Buffer XBP. Mix well.

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

Centrifugation: 500g, 1min, room temperature;

Centrifuge again: 3000g, 1min, room temperature;

Discard the flow-through and return the column to the same collection tube. Make sure all liquid has passed through the membrane.

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

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

(6) Add 700 μl of QIAzol Lysis Solution to the membrane. 3000g, 5min, room temperature; to collect the lysate, and then completely transferred to the EP tube. Add artificially synthesized 5' phosphorylated RNA, that is, 10ul (2×10 ^-7 uM) of the external reference cel-miR-39-3p.

(7) Briefly vortex the test tube containing the lysate and incubate at room temperature for 5 min.

(8) Add 90 μl of chloroform to the test tube containing the lysate, and close the cap tightly. Shake vigorously for 15 seconds, then incubate at room temperature for 3 min.

(9) Centrifuge at 12000g for 15min at 4°C. After centrifugation, the sample was separated into three phases: an upper colorless aqueous phase containing RNA; a thin white phase; and a lower red organic phase. RNA is present in the upper aqueous phase.

(10) Transfer the upper aqueous phase to a new EP tube. 250ul/sample, avoid transferring any interphase material. Add 2 volumes of absolute ethanol, namely 500ul, and mix well.

(11) Pipette the sample (including any precipitate that may have formed) into the RNeasyMinElute spin column in a 2ml collection tube. Gently close the lid, centrifuge: 12000g, 30s, room temperature. Discard the flow-through. Reuse the collection tube in the next step. Repeat this step for the remaining samples.

(12) Add 700 μl buffer RWT to the RNeasy MinElute spin column. Gently close the lid and centrifuge at 12000g for 30s at room temperature. Discard flow through. Reuse the collection tube in the next step.

Example 4: NGS sequencing analysis of miRNA

The extracted plasma exosomal miRNAs were sequenced for library construction.

(1) Sample RNA First, the RNA concentration was detected with Qubit (Bio-Rad, USA) and Agilent2200 (Agilent, USA).

(2) Library quality inspection was performed by Agilent 2200TapeStation (Agilent, USA).

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

(4) Calculating the expression levels of the identified miRNAs. The miRNA expression correction method RPM (the number of reads per million of miRNA) = calculate the number of reads per million Clean Reads compared to a specific miRNA (the number of reads per million clean tag).

(5) Use the edgeR R language software package to analyze the difference in miRNA expression. The average RPM expression of abnormally expressed miRNAs in samples between groups is converted to log2 (RPM), and the average relative expression of miRNAs in plasma EV-RNA samples is plotted. The expression level bar graph is shown in Figure 2. The differentially expressed miRNAs among the comparison groups were obtained through the two levels of difference fold (|log2(Fold Change)|>1) and significance level (P value<0.05): miR-99b-5p and miR-409-3p.

Example 5: Real-time fluorescent quantitative PCR verification of differentially expressed miRNAs

The expression levels of miR-99b-5p and miR-409-3p were quantitatively detected by real-time fluorescence quantitative PCR (RT-qPCR). For the Cq values obtained by RT-qPCR, the relative quantification of miRNA expression was performed using the 2-ΔCq method. The formula of 2-ΔCq is: ΔCq=Cq(candidate miRNA)-Cq(reference miRNA), so as to obtain the fold change of miRNA expression level in the CRC group relative to the healthy control group, as shown in Figure 3.

Example 6: Verification of the early diagnostic efficacy of miRNA combinations for colorectal cancer

Exosomal miR-99b-5p, miR-409-3p, tumor marker CEA and their combination were used as detection objects, and the ROC curve was drawn through the "ROC curve graph" function of SPSS software, see Figure 4A-E and Figure 5A- b. Obtain 95% confidence interval (95% Confidence interval, 95% CI) and area under ROC curve (Area under ROC curve, AUC). Sensitivity and specificity for ROC curve cutoffs were determined using the maximum value of Youden's index. For early colorectal cancer, the analysis showed:

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

(2) Combination of miR-99b-5p and miR-409-3p: AUC=0.741, 95%CI=0.592-0.891, Sensitivity (sensitivity)=77.3%, Specificity (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 (sensitivity)=90.9%, Specificity (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 90.9% for early colorectal cancer, and has extremely high diagnostic efficiency.

Based on the above data and conclusions, combined with the prior art in this field, a single miR-99b-5p or miR-409-3p, a combination of miR-99b-5p or miR-409-3p, or miR-99b-5p The combination of , miR-409-3p and CEA can all be used to construct a diagnostic kit for distinguishing a colorectal cancer patient from a healthy individual for detecting early colorectal cancer.

Claims (3)

1. A marker for early diagnosis of colorectal cancer, characterized in that, the marker is a nucleic acid molecule miRNA selected from the following or a combination thereof: the nucleic acid molecule comprises encoding plasma exosome miR-99b-5p and miR-409-3p nucleic acid molecules; the expression of the nucleic acid molecules in the target plasma exosomes is up-regulated compared with the control sample. 2. A diagnostic kit for identifying early colorectal cancer, characterized in that: said diagnostic kit comprises the early diagnostic markers for colorectal cancer as claimed in claim 1. 3. The diagnostic kit for identifying early colorectal cancer according to claim 2, characterized in that: it also includes the serum tumor marker CEA.

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