Application of MIR129-2 promoter methylation in diagnosis and prognosis of pancreatic cancer

Abstract

The invention discloses application of MIR129-2 promoter methylation in pancreatic cancer diagnosis and prognosis, and belongs to the technical field of gene diagnosis. By detecting the methylation level of the MIR129-2 gene promoter in plasma or tumor tissue of a subject, whether a patient suffers from pancreatic cancer or not can be simply, quickly and accurately diagnosed, and postoperative recurrence and prognosis of the patient can be predicted; therefore, patients suffering from early pancreatic cancer and postoperative high-risk groups can be discriminated in time in clinical practice, valuable reference is provided for treatment strategies, the diagnosis and treatment level of the pancreatic cancer can be improved, and survival prognosis of the pancreatic cancer can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene diagnosis, and specifically relates to an application of MIR129-2 gene promoter methylation as a biomarker in the diagnosis and prognosis of pancreatic cancer. Background Art

[0002] Pancreatic ductal adenocarcinoma (PDAC) is highly malignant and has a very poor prognosis. At present, radical surgery combined with systemic adjuvant chemotherapy is the only way to effectively prolong the life of pancreatic cancer patients. However, due to the insidious onset of pancreatic cancer itself and the lack of typical clinical symptoms in the early stage, about 80% of patients are already in the local advanced stage or have distant metastasis when diagnosed, thus losing the opportunity for surgery; even patients who have undergone radical surgery will mostly experience recurrence and metastasis after surgery, and the 5-year survival rate is only 20-30%. Therefore, early diagnosis and prognosis monitoring of pancreatic cancer are of great value for improving the life expectancy of pancreatic cancer patients. At present, the main diagnosis and prognosis monitoring methods of PDAC include imaging examinations represented by enhanced computed tomography (CT), magnetic resonance imaging (MRI), and endoscopic ultrasound (EUS) and serum tumor marker tests represented by CA19-9. However, traditional methods face problems such as high cost, invasiveness, low sensitivity and specificity. Early diagnosis and prediction of PDAC prognosis are not ideal. There is an urgent need to develop lower-cost and more accurate detection methods to improve the accuracy of PDAC diagnosis and treatment.

[0003] In recent years, liquid biopsy technology represented by circulating tumor DNA (ctDNA) has received widespread clinical attention. ctDNA can carry tumor genetic information such as gene expression levels, gene mutations, and methylation levels, but its heterogeneity is much smaller than that of tumor tissues. Therefore, it is expected to become a biomarker for accurately evaluating minimal residual disease (MRD) in solid tumors. In addition, ctDNA has a short half-life and can be used as a real-time biomarker to reflect the tumor status at a specific moment. It is more suitable for evaluating treatment effects and prognosis monitoring than traditional tumor markers. However, the current popular ctDNA MRD detection focuses on detecting ctDNA mutations. The detection process is cumbersome and costly, and it encounters challenges in clinical application. On the other hand, more and more evidence shows that abnormal DNA methylation significantly promotes the occurrence and development of tumors. DNA methylation has multiple advantages as a marker for early cancer screening. Studies have shown that in early cancer screening, circulating free DNA methylation is significantly superior to circulating free DNA mutations and copy number variations in terms of signal abundance and tissue traceability, and tumor specificity is better than mutation detection. These findings have prompted the development of ctDNA methylation markers to indicate the presence of MRD and identify patients at high risk of recurrence. However, the exploration of methylation biomarkers in the plasma of pancreatic cancer patients is still in its infancy and requires further in-depth research. Therefore, there is an urgent need in this technical field to obtain an effective ctDNA methylation marker to diagnose and predict the prognosis of pancreatic cancer patients. Summary of the invention

[0004] Based on the previous clinical problems of difficulty in early diagnosis of pancreatic cancer and patient prognosis monitoring, the present invention provides a ctDNA-based MIR129-2 promoter methylation detection method to achieve accurate diagnosis of pancreatic cancer, so as to improve the early diagnosis rate of pancreatic cancer patients. In addition, the ctDNA-based MIR129-2 promoter methylation detection method provided by the present invention can also predict postoperative recurrence of pancreatic cancer patients, thereby improving the accuracy of prediction of postoperative recurrence and poor prognosis of pancreatic cancer patients.

[0005] The technical solution provided by the present invention can solve the following technical problems: (1) how to diagnose patients with pancreatic cancer at an early stage, so as to start treatment as early as possible and improve the prognosis of pancreatic cancer; and (2) how to accurately predict the risk of recurrence of patients after pancreatic cancer surgery, so as to improve the treatment effect and promote individualized precision treatment.

[0006] The present invention includes the following technical solutions:

[0007] In a first aspect, the present invention provides a method for detecting the methylation level of the MIR129-2 gene promoter in at least one of the following aspects:

[0008] a1) Application in the preparation and / or screening of products for diagnosis or auxiliary diagnosis of pancreatic cancer;

[0009] a2) Application in the preparation and / or screening of products for evaluating or assisting in evaluating the therapeutic efficacy of pancreatic cancer;

[0010] a3) Use in the preparation and / or screening of products for identifying patients with a high risk of recurrence after pancreatic cancer surgery;

[0011] a4) Application in the preparation and / or screening of products for assessing the prognosis and survival of pancreatic cancer patients.

[0012] In the present invention, the pancreatic cancer is selected from pancreatic ductal adenocarcinoma (PDAC).

[0013] The products include but are not limited to reagents, kits, chips, test strips, membrane strips, systems or detection platforms.

[0014] The material for detecting the methylation level of the MIR129-2 gene promoter includes any reagent required for detecting the methylation level of the MIR129-2 gene promoter by qMSP method (quantitative methylation-specific PCR), RT-PCR method, RT-qPCR method, biochip detection method, and Southern blotting method.

[0015] In a specific embodiment of the present invention, the substance for detecting the methylation level of the MIR129-2 gene promoter is a reagent for detecting the methylation level of the MIR129-2 gene promoter by the qMSP method.

[0016] Furthermore, the reagents include pre-amplification primers and probes targeting the methylation of the MIR129-2 gene promoter.

[0017] In a specific embodiment of the present invention, the primers include a forward pre-amplification primer as shown in SEQ ID NO.1 and a reverse pre-amplification primer as shown in SEQ ID NO.2; the nucleotide sequence of the probe is shown in SEQ ID NO.3.

[0018] In a second aspect, the present invention provides a primer and probe combination for detecting the methylation level of the MIR129-2 gene promoter, characterized in that the primers include a forward pre-amplification primer shown in SEQ ID NO.1 and a reverse pre-amplification primer shown in SEQ ID NO.2; and the probe is selected from the sequence shown in SEQ ID NO.3.

[0019] Preferably, the 5' end of the probe sequence is labeled with a fluorescent group, and the 3' end is labeled with a quencher group; the fluorescent group is selected from one of VIC, ROX, FAM, Cy5 or HEX, and the quencher group is selected from BHQ or MGB.

[0020] Specifically, the nucleotide information of the primers and probes for MIR129-2 gene promoter methylation involved in the present invention is as follows:

[0021]

[0022] In the table, "F" indicates a forward preamplification primer; "R" indicates a reverse preamplification primer; and "P" indicates a probe.

[0023] In a third aspect, the present invention provides a kit for detecting the methylation level of the MIR129-2 gene promoter, characterized in that the kit comprises the primer and probe combination described in the second aspect of the present invention.

[0024] Preferably, the kit further comprises specific primers and probes for an internal reference gene, and the internal reference gene is selected from ACTB or GAPDH.

[0025] In a specific embodiment of the present invention, the internal reference gene is selected from ACTB.

[0026] The primers for the internal reference gene ACTB include the forward amplification primer shown in SEQ ID NO.4 and the reverse amplification primer shown in SEQ ID NO.5; the nucleotide sequence of the probe is shown in SEQ ID NO.6.

[0027] Specifically, the specific nucleotide information of the primers and probes for the internal reference gene ACTB involved in the present invention is as follows:

[0028]

[0029] In the table, "F" indicates forward amplification primer; "R" indicates reverse amplification primer; and "P" indicates probe.

[0030] Special base description: I represents hypoxanthine.

[0031] Preferably, the kit further comprises a conversion reagent, which is a reagent that converts unmethylated cytosine in DNA into uracil, leaving 5-MeC unaffected. The conversion reagent is selected from hydrazine salts, bisulfite salts, bisulfite salts, or compounds that can produce hydrazine salts, bisulfite salts, or bisulfite salts under appropriate conditions.

[0032] In a specific embodiment of the present invention, the bisulfite is selected from sodium bisulfite.

[0033] In a specific embodiment of the present invention, the bisulfite is selected from sodium metabisulfite, potassium bisulfite, cesium bisulfite or ammonium bisulfite.

[0034] Preferably, the kit also includes other reagents required for PCR amplification.

[0035] In a fourth aspect, the present invention provides a system, characterized in that the system at least comprises:

[0036] A detection module, the detection module comprising a substance for detecting the methylation level of the MIR129-2 gene promoter in the sample to be tested; and

[0037] Analysis module: The analysis module includes judging the methylation level of the MIR129-2 gene promoter according to the RQ value obtained by the detection module.

[0038] Preferably, the analysis module has an embedded model for determining the methylation level of the MIR129-2 gene promoter, and the model is: Y=((RQ value*ng*1000) / 3.3) / ML;

[0039] Y represents the methylated haploid genome equivalent per milliliter (mhGE / ML); RQ value is the output value of the real-time PCR thermal cycler.

[0040] The judgment rules are:

[0041] When mhGE / ML≥8, the MIR129-2 gene promoter methylation was positive; when mhGE / ML<8, the MIR129-2 promoter methylation was negative.

[0042] The sample to be tested is selected from blood, serum, plasma or pancreatic cancer tissue.

[0043] Furthermore, the present invention determines at least one of the following items of the subject based on the MIR129-2 gene promoter methylation level obtained by the analysis module:

[0044] b1) Diagnosis of pancreatic cancer;

[0045] b2) Evaluate or assist in evaluating the therapeutic efficacy of pancreatic cancer;

[0046] b3) Identify patients with high risk of recurrence after pancreatic cancer surgery;

[0047] b4) To evaluate the prognosis and survival of patients with pancreatic cancer.

[0048] The present invention found that the MIR129-2 gene promoter was highly methylated in pancreatic cancer patients and pancreatic cancer patients with poor prognosis, indicating that the methylation degree of the MIR129-2 gene promoter is closely related to the diagnosis and prognosis of pancreatic cancer patients. Based on this, the present invention provides a ctDNA-based MIR129-2 gene promoter methylation detection method, which can be used for accurate diagnosis of pancreatic cancer and prediction of postoperative recurrence of pancreatic cancer patients, improve clinical diagnosis and treatment efficacy, and promote individualized precision treatment.

[0049] By detecting the methylation level of the MIR129-2 promoter in the plasma of subjects, the present invention can simply, rapidly, and accurately diagnose whether a patient has pancreatic cancer and predict the postoperative recurrence and prognosis of the patient, so as to timely identify patients with early-stage pancreatic cancer and high-risk populations after surgery in clinical practice and provide valuable references for treatment strategies, which helps to improve the diagnosis and treatment level of pancreatic cancer and improve the survival prognosis of pancreatic cancer.

[0050] The technical solution provided by the present invention has the following advantages:

[0051] (1) It can be detected using a blood sample, the sample acquisition is simple and convenient, and the cost is low;

[0052] (2) By only detecting the same index (the methylation level of the MIR129-2 promoter in plasma), predictive information on the diagnosis of pancreatic cancer and postoperative recurrence can be obtained, with a wide application range and convenient operation;

[0053] (3) The accuracy of early diagnosis is high, the sensitivity is 87.34%, the specificity is 96.61%, and the area under the corresponding ROC curve (AUC) is 0.952;

[0054] (4) The prediction of postoperative recurrence and prognosis of pancreatic cancer patients is accurate. Compared with postoperative plasma MIR129-2 promoter methylation negative patients, the disease progression risk of postoperative plasma MIR129-2 promoter methylation positive patients increases by 15.35 times, and the progression-free survival period is significantly shorter.

[0055] (5) The prediction efficacy of postoperative recurrence and prognosis is superior to that of traditional serological tumor markers. Even in patients with negative CA19-9, the disease progression risk of plasma MIR129-2 promoter methylation positive patients is still 10.45 times higher than that of negative patients, and MIR129-2 promoter methylation is still an important prognostic factor.

[0056] Therefore, using MIR129-2 promoter methylation as a biomarker for the diagnosis and prognosis of pancreatic cancer provided by the present invention has higher diagnostic and prognostic prediction efficacy than traditional serological tumor markers, with low cost, simple operation, can significantly reduce medical expenses, and benefit the majority of patients. Description of the Drawings

[0057] Figure 1 It is a differential analysis diagram using pancreatic ductal adenocarcinoma DNA methylation data in the TCGA database, where MIR129-2 is the only miRNA.

[0058] Figure 2 It is a survival analysis diagram using TCGA data. Patients with a high MIR129-2 promoter methylation level have a worse prognosis than those with a low methylation level.

[0059] Figure 3 This is a graph of the methylation level of the MIR129-2 promoter in pancreatic ductal adenocarcinoma, pancreatic benign tumors, and adjacent normal pancreatic tissues. In 179 PDAC tissues, the methylation level of MIR129-2 was significantly higher than that in other pancreatic lesions and normal pancreatic tissues.

[0060] Figure 4 The receiver operating characteristic (ROC) curve analysis of MIR129-2 methylation level shows that tissue MIR129-2 methylation level can effectively distinguish PDAC from normal tissue samples. The area under the curve (AUC) is 0.994 (95% CI: 0.988-1), corresponding to a sensitivity of 97.77% and a specificity of 98.71%.

[0061] Figure 5 The figure shows the receiver operating characteristic (ROC) curve analysis of plasma MIR129-2 promoter methylation levels in patients with pancreatic ductal adenocarcinoma and healthy volunteers. The plasma MIR129-2 methylation level can effectively distinguish PDAC patients from healthy volunteers. The area under the curve (AUC) is 0.952 (95% CI: 0.928-0.979), corresponding to a sensitivity of 87.34% and a specificity of 96.61%.

[0062] Figure 6 A multivariate COX proportional hazard model was used to evaluate the relationship between postoperative plasma MIR129-2 promoter methylation level and disease progression. Compared with patients with negative postoperative plasma MIR129-2 methylation, patients with positive postoperative plasma MIR129-2 methylation had a 15.35-fold increased risk of disease progression (HR=15.35, 95% CI: 4.64-50.79, P<0.001).

[0063] Figure 7 This is an analysis chart of progression-free survival (PFS) in pancreatic cancer patients. The progression-free survival (PFS) of subjects with negative plasma MIR129-2 methylation after surgery was significantly longer than that of positive subjects (median survival of negative patients: NA, 95% CI: NA-NA vs median survival of positive patients: 173.00, 95% CI: 147.00-NA).

[0064] Figure 8The figure shows the survival analysis of patients with positive and negative plasma MIR129-2 methylation in CA19-9 negative patients. Compared with the methylation positive group, the survival rate of the methylation negative group remained high throughout the follow-up period. Moreover, the gap between the survival rates of the two groups gradually widened (HR = 10.45, 95% CI: 2.27-48.08, p < 0.001. This shows that even in the case of negative CA19-9, MIR129-2 methylation can still affect the prognosis of PDAC. DETAILED DESCRIPTION

[0065] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0066] Example 1 Primers and probes for amplification of MIR129-2 gene promoter methylation

[0067] The selection of the MIR129-2 gene methylation detection region will affect the diagnosis and prognosis of pancreatic cancer. The detection region can be selected from the MIR129-2 gene region or the MIR129-2 gene promoter region. The present invention preferably uses the MIR129-2 gene promoter methylation region as the detection target. Further, the present invention obtains amplification primers according to the detection target design. The primers designed in different regions of the detection target have obvious differences in the detection results. After optimization and screening, the present invention provides amplification primers and probes for MIR129-2 gene promoter methylation as shown in the following table.

[0068] Table 1 Nucleotide information of primers and probes for MIR129-2 gene promoter methylation

[0069]

[0070] Example 2 Clinical study plan and conclusion

[0071] This study was approved by the Human Subjects Ethics Committee of Zhongshan Hospital of Fudan University, the study protocol followed the guidelines established by the Declaration of Helsinki, and written informed consent was obtained from all subjects.

[0072] 1. Research subjects

[0073] The research objects of this embodiment are as follows:

[0074] Cohort 1: paraffin samples of pancreatic tumor tissues, including 179 pancreatic ductal adenocarcinoma (PDAC) samples, 46 pancreatic cystadenoma (MCN and SCN) samples, 19 pancreatic solid pseudopapillary tumor (SPT) samples, 36 pancreatic neuroendocrine tumor (NET) samples, 34 pancreatic intraductal papillary neoplasia (IPMN) samples and 306 adjacent normal tissue samples.

[0075] Cohort 2: plasma samples, including 157 patients with pancreatic ductal adenocarcinoma and 59 healthy volunteers.

[0076] Cohort 3: plasma samples, including 113 samples collected short-term (2-4 weeks) after surgery and 167 samples collected long-term (every three months) after surgery from 114 patients with pancreatic ductal adenocarcinoma.

[0077] 2. Research Methods

[0078] (1) Tissue paraffin samples

[0079] Genomic DNA was extracted using the HiPure FFPE DNA Kit (Magen #D3226) and DNA concentration was quantified using the Qubit 3.0 Fluorometer (Thermo Fisher Scientific, USA). TM The kit (ZYMO RESEARCH, catalog number D5006) was used to perform bisulfite conversion on 10 to 50 ng of extracted DNA according to the manufacturer's protocol. The converted DNA was then treated with a quantitative methylation-specific PCR (qMSP) assay, which included a preamplification step and subsequent quantitative PCR on an ABI 7500 real-time PCR thermal cycler. The reference gene ACTB was used as a control to ensure the quality of the experiment. The test should be repeated for each sample to reduce errors. The RQ value output by the final ABI 7500 real-time PCR thermal cycler is the methylation rate.

[0080] (2) Plasma samples

[0081] 8-10 ml of whole blood was collected from the subjects, and about 3-4 ml of plasma was separated by centrifugation. ctDNA was extracted from the plasma using the QIAamp circulating nucleic acid extraction kit (Qiagen, Hilden, Germany) and the DNA concentration was quantitatively analyzed using the Qubit 3.0 fluorometer (ThermoFisher Scientific, USA). Subsequently, the EZ DNA Methylation-Gold TMThe kit (ZYMO RESEARCH, catalog number D5006) was used to perform bisulfite conversion on 10 to 50 ng of extracted DNA according to the manufacturer's protocol. The converted DNA was then processed using a quantitative methylation-specific PCR (qMSP) assay, which included a preamplification step and subsequent quantitative PCR on an ABI 7500 real-time PCR thermal cycler. The reference gene ACTB was used as a control to ensure the quality of the experiment. The test should be repeated for each sample to reduce errors.

[0082] The methylation rate of plasma samples was represented by the haploid genome equivalent per milliliter of methylation (mhGE / ML). The calculation method of mhGE / ML is as follows: ((RQ value*ng*1000) / 3.3) / ML. The RQ value is directly output by the ABI 7500 real-time PCR thermal cycler. If mhGE / ML≥8, the MIR129-2 promoter is positive for methylation, and if mhGE / ML<8, the MIR129-2 promoter is negative for methylation.

[0083] (3) Collect clinical pathological information and postoperative follow-up data of pancreatic cancer patients

[0084] Routine clinical information includes age, gender, and carbohydrate antigen 19-9 (CA19-9) levels. Postoperative pathology reports include tumor size, location, differentiation, nerve invasion, vascular invasion, and lymph node metastasis. Postoperative adjuvant therapy includes chemotherapy regimens and radiotherapy.

[0085] Follow-up was performed every 3 months in the first year, every 3-6 months in the second year, and then annually. Follow-up included blood routine, liver function tests, serum tumor markers, and abdominal enhanced CT or magnetic resonance imaging. Recurrence-free survival (RFS) was defined as the time interval from the date of surgery to the date of recurrence (or the last follow-up).

[0086] (4) Screening of potential ctDNA methylation indicators

[0087] DNA methylation data of pancreatic ductal adenocarcinoma patients were obtained from the TCGA database. The coefficient of variation (CV) of each CpG checkpoint was calculated based on the methylation β value in the methylation data matrix, and highly variable CpG checkpoints with a CV greater than 5 were screened for differential analysis. Finally, the only miRNA marker MIR129-2 promoter methylation ( Figure 1)。Relevant clinical information was collected from the TCGA-PDAC dataset, and patient samples were grouped according to the median methylation level of the MIR129-2 gene, thus dividing them into a hypermethylation group and a hypomethylation group. The Cox proportional hazards regression model and Kaplan-Meier survival curves were used to evaluate the relationship between methylation grouping and patient survival ( Figure 2 )。

[0088] (5) Analyze the correlation between MIR129-2 promoter methylation and pathological diagnosis in tissue samples

[0089] Process the paraffin samples of the tissues in cohort 1 according to the method described in (1) and perform quantitative methylation-specific PCR (qMSP) detection to obtain the methylation rate (RQ value) of the tissue samples. Compare the differences in methylation rate levels among samples with different pathological diagnoses, and draw the receiver operating characteristic curve (ROC) for the correlation between MIR129-2 methylation and clinical diagnosis. The results show that tissue MIR129-2 methylation is a specific marker for pancreatic ductal adenocarcinoma ( Figure 3 ), AUC = 0.994 (CI: 0.988 - 1), sensitivity is 97.77%, and specificity is 98.71% ( Figure 4 )。

[0090] (6) Analyze the correlation between MIR129-2 promoter methylation and pathological diagnosis in plasma samples

[0091] Process the plasma samples in cohort 2 according to the method described in (2) and perform quantitative methylation-specific PCR (qMSP) detection to obtain the methylation rate (RQ value) of the tissue samples. Compare the differences in methylation rate levels between pancreatic ductal adenocarcinoma patients and healthy volunteers, and draw the receiver operating characteristic curve (ROC) for the correlation between MIR129-2 methylation and clinical diagnosis. The results show that tissue MIR129-2 methylation is a specific marker for pancreatic ductal adenocarcinoma, AUC = 0.952 (CI: 0.928 - 0.979), sensitivity is 87.34%, and specificity is 96.61% ( Figure 5 )。

[0092] (7) Analyze the correlation between MIR129-2 promoter methylation and postoperative recurrence of pancreatic cancer in plasma samples

[0093] Plasma samples from cohort 3 were processed as described in (2) and quantitative methylation-specific PCR (qMSP) was performed to obtain tissue sample methylation rates (mhGE / ML). A multivariate COX proportional hazard model was constructed to evaluate the relationship between postoperative plasma MIR129-2 promoter methylation level and disease progression. Compared with patients with negative postoperative plasma MIR129-2 methylation, patients with positive postoperative plasma MIR129-2 methylation had a 15.35-fold increased risk of disease progression (HR = 15.35, 95% CI: 4.64-50.79, P < 0.001) ( Figure 6 ). The progression-free survival of pancreatic cancer patients with different methylation levels was also analyzed. The results showed that the progression-free survival (PFS) of subjects with negative plasma MIR129-2 methylation after surgery was significantly longer than that of subjects with positive plasma MIR129-2 methylation (median survival of negative patients: NA, 95% CI: NA-NA vs median survival of positive patients: 173.00, 95% CI: 147.00-NA) ( Figure 7 ).

[0094] (8) Analysis of the correlation between MIR129-2 promoter methylation and postoperative recurrence of pancreatic cancer in plasma samples of patients with postoperative CA19-9 negative

[0095] Plasma samples from cohort 3 were processed according to the method described in (2) and quantitative methylation-specific PCR (qMSP) was performed to obtain the methylation rate (mhGE / ML) of tissue samples. The survival difference between patients with positive and negative plasma MIR129-2 methylation was analyzed in CA19-9-negative patients. Compared with the methylation-positive group, the survival rate of the methylation-negative group remained high throughout the follow-up period. Moreover, the gap between the survival rates of the two groups gradually widened (HR=10.45, 95% CI: 2.27-48.08, p<0.001. This shows that even in the case of negative CA19-9, MIR129-2 methylation can still affect the prognosis of PDAC ( Figure 8 ).

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a substance for detecting the methylation level of the MIR129-2 gene promoter in at least one of the following: a1) Application in the preparation and / or screening of products for diagnosis or auxiliary diagnosis of pancreatic cancer; a2) Application in the preparation and / or screening of products for evaluating or assisting in evaluating the therapeutic efficacy of pancreatic cancer; a3) Use in the preparation and / or screening of products for identifying patients with a high risk of recurrence after pancreatic cancer surgery; a4) Application in the preparation and / or screening of products for assessing the prognosis and survival of pancreatic cancer patients.

2. The use according to claim 1, characterized in that: The material for detecting the methylation level of the MIR129-2 gene promoter includes any reagent required for detecting the methylation level of the MIR129-2 gene promoter by qMSP method, RT-PCR method, RT-qPCR method, biochip detection method, and Southern blotting method.

3. The use according to claim 2, characterized in that: The substance for detecting the methylation level of the MIR129-2 gene promoter is a reagent for detecting the methylation level of the MIR129-2 gene promoter by the qMSP method.

4. A primer and probe combination for detecting the methylation level of the MIR129-2 gene promoter, characterized in that: The primers include the forward pre-amplification primer shown in SEQ ID NO.1 and the reverse pre-amplification primer shown in SEQ ID NO.2; the probe is selected from the sequence shown in SEQ ID NO.

3.

5. The primer and probe combination according to claim 4, characterized in that: The 5' end of the probe sequence is labeled with a fluorescent group, and the 3' end is labeled with a quenching group; the fluorescent group is selected from one of VIC, ROX, FAM, Cy5 or HEX, and the quenching group is selected from BHQ or MGB.

6. A kit for detecting the methylation level of the MIR129-2 gene promoter, characterized in that: The kit comprises the primer and probe combination according to any one of claims 3-4.

7. The kit according to claim 6, characterized in that The kit also includes specific primers and probes for internal reference genes, and the internal reference gene is selected from ACTB or GAPDH.

8. The kit according to claim 6, characterized in that The kit further comprises a conversion reagent, which is selected from hydrazine salts, bisulfite salts, bisulfite salts, or compounds that can generate hydrazine salts, bisulfite salts, or bisulfite salts under appropriate conditions.

9. A system, characterized in that The system comprises at least: A detection module, the detection module comprising the substance for detecting the methylation level of the MIR129-2 gene promoter in the sample to be tested as described in claim 1; and Analysis module: The analysis module includes judging the methylation level of the MIR129-2 gene promoter according to the RQ value obtained by the detection module.

10. The system according to claim 9, characterized in that The analysis module has a built-in model for determining the methylation level of the MIR129-2 gene promoter, and the model is: Y=((RQ value*ng*1000) / 3.3) / ML; Y represents the methylated haploid genome equivalent per milliliter (mhGE / ML); RQ value is the output value of the real-time PCR thermal cycler; The judgment rules are: When mhGE / ML≥8, the MIR129-2 gene promoter methylation was positive; when mhGE / ML<8, the MIR129-2 promoter methylation was negative.

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