Molecular marker for early pancreatic tumor detection as well as detection method and application thereof

Abstract

The invention discloses a molecular marker for early pancreatic tumor detection and application thereof. The molecular marker disclosed by the invention is any group of miRNA combinations as follows: a combination 1 is composed of miR-21, miR-30c, miR-141, miR-205 and miR-224; the combination 2 is composed of miR-24, miR-141, miR-155, miR-210 and miR-224, and the combination 2 is composed of miR-24, miR-141, the combination 3 is composed of miR-24, miR-132, miR-146b, miR-210 and miR-221, and the combination 3 is composed of miR-24, miR-132, the combination 4 is composed of the miR-24, the miR-30c, the miR-146b, the miR-197 and the miR-501; and the combination 5 is composed of the miR-23a, the miR-197, the miR-205, the miR-222 and the miR-301.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology and clinical molecular diagnostic marker development, and particularly relates to a molecular marker for early pancreatic tumor detection and application thereof. BACKGROUND

[0002] The pancreas is the second largest digestive gland in the human body, with endocrine and digestive exocrine functions, and is an important digestive organ of the human body. Pancreatic cancer is known as the "king of cancer" due to its strong concealment, rapid disease progression, and high malignancy. Pancreatic cancer generally undergoes a process of transforming from inflammation to cancer, and it takes at least 20 years from mutation to cancerous cell transformation with metastatic ability. Early pancreatic cancer refers to a tumor with a diameter of ≤2 cm, which is limited within the pancreas and has no extrapancreatic invasion or lymph node metastasis. The surgical resection rate and 5-year survival rate of early pancreatic cancer are much higher than those of advanced pancreatic cancer, and early diagnosis is of great significance for the prevention and treatment of pancreatic cancer.

[0003] The most widely used pancreatic cancer diagnostic method in the Pancreatic Cancer Diagnosis and Treatment Guidelines (2022 Edition) is tumor marker detection, imaging examination, and histological or pathological examination. Traditional tumor markers such as CA19-9 have low accuracy, about 30%-60%, and are rarely proven to effectively detect early pancreatic cancer; imaging examinations mainly include abdominal B-ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), endoscopic ultrasonography (EUS), and positron emission tomography (PET). Since the pancreatic cancer tumor is located in the deep part of the human viscera, there are no obvious clinical manifestations in the early stage, and it is difficult to diagnose using radiological imaging during the micro-cancer period, resulting in most pancreatic cancer patients being in the advanced stage at the time of diagnosis, and losing the opportunity for surgical treatment. Histological or pathological examination has the advantage of high accuracy, but it is not suitable for routine detection of asymptomatic patients due to its tissue trauma, resulting in most patients being in the advanced stage. At present, there is a lack of an accurate, timely, and non-invasive early pancreatic cancer diagnostic method.

[0004] Micro ribonucleic acid (miRNA) is a class of non-coding single-stranded small ribonucleic acid molecules about 8 to 24 nucleotides long. It regulates mRNA or polypeptides to achieve multiple gene expression or inhibition, thereby regulating various tumor processes, including cell proliferation, migration, invasion, survival, and metastasis. More and more reports indicate that miRNA is involved in the entire process of transforming pancreatic normal cells from inflammation to cancer, and is closely related to the occurrence and development of pancreatic cancer. Moreover, miRNA is a non-coding short fragment RNA that is not easily degraded in peripheral blood and can stably exist, making it an ideal marker for early screening and early diagnosis of pancreatic cancer.

[0005] Although Chinese patent application CN109423519A and patent CN101942502B disclose the use of micro ribonucleic acid as a diagnostic marker for detecting early clinical pancreatic cancer, the prior art uses a single detection index for judgment, and the precision of the single miRNA invented is not enough, which is not the best marker reflecting the actual situation of the patient in the clinic, and lacks comprehensive consideration of the individualized clinical representation of each miRNA, which may lead to the fact that the detection result of the product cannot truly reflect the actual situation of the patient, resulting in missed diagnosis and misdiagnosis. The inventors disclosed a pancreatic cancer marker combination of 4 miRNAs in Chinese patent CN111575374B, but in actual clinical application, it was found that its application performance did not reach the expected degree, and the clinical application showed that these marker combinations were not the best combination, and they were not compared with the results of traditional tumor marker CA19-9, which may lead to the fact that the detection result of the product cannot truly reflect the actual situation of the early pancreatic cancer patient, and the upper limit of the number of target markers in the combination is 4, which cannot best reflect the superiority of the combination.

[0006] Therefore, it is of great significance to develop more efficient and sensitive miRNA combinations and detection methods for the early diagnosis, treatment and prognosis of pancreatic cancer. SUMMARY

[0007] In view of the problems of difficulty in early diagnosis of pancreatic cancer and low sensitivity and specificity of existing biomarkers, the purpose of the present application is to provide a molecular marker for early pancreatic tumor detection and its application.

[0008] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0009] The first aspect of the present application provides a molecular marker for early pancreatic cancer detection, and the molecular marker is any one of the following miRNA combinations:

[0010] Combination 1: composed of miR-21, miR-30c, miR-141, miR-205 and miR-224;

[0011] Combination 2: composed of miR-24, miR-141, miR-155, miR-210 and miR-224;

[0012] Combination 3: composed of miR-24, miR-132, miR-146b, miR-210 and miR-221;

[0013] Combination 4: composed of miR-24, miR-30c, miR-146b, miR-197 and miR-501;

[0014] Combination 5: consisting of miR-23a, miR-197, miR-205, miR-222 and miR-301.

[0015] According to a preferred embodiment, the molecular marker is miRNA in serum or plasma.

[0016] The second aspect of the present application provides use of a reagent for detecting the above-mentioned molecular marker in the preparation of a product for the diagnosis or auxiliary diagnosis of early pancreatic cancer.

[0017] According to a preferred embodiment, the product comprises a reagent, a chip, a test paper or a kit.

[0018] According to a preferred embodiment, the product comprises a portable test paper, a digital test strip, a digital test card and a detector.

[0019] The third aspect of the present application provides a kit for the diagnosis or auxiliary diagnosis of early pancreatic cancer, the kit comprising a reagent for detecting the expression amount of the above-mentioned molecular marker.

[0020] According to a preferred embodiment, the reagent in the kit comprises a miRNA probe.

[0021] According to a preferred embodiment, the probe sequence corresponding to the above-mentioned molecular marker in the kit is as follows:

[0022] The probe sequence corresponding to miR-21 is shown as SEQ ID NO. 1,

[0023] The probe sequence corresponding to miR-30c is shown as SEQ ID NO. 13,

[0024] The probe sequence corresponding to miR-141 is shown as SEQ ID NO. 21,

[0025] The probe sequence corresponding to miR-205 is shown as SEQ ID NO. 37,

[0026] The probe sequence corresponding to miR-224 is shown as SEQ ID NO. 53,

[0027] The probe sequence corresponding to miR-24 is shown as SEQ ID NO. 9,

[0028] The probe sequence corresponding to miR-155 is shown as SEQ ID NO. 29,

[0029] The probe sequence corresponding to miR-210 is shown as SEQ ID NO. 41,

[0030] The probe sequence corresponding to miR-132 is shown as SEQ ID NO. 17,

[0031] The probe sequence corresponding to miR-146b is shown as SEQ ID NO. 25,

[0032] The probe sequence corresponding to miR-221 is shown as SEQ ID NO. 45,

[0033] The probe sequence corresponding to miR-197 is shown as SEQ ID NO. 33,

[0034] The probe sequence corresponding to miR-501 is shown as SEQ ID NO. 57,

[0035] The probe sequence corresponding to miR-23a is shown as SEQ ID NO. 5,

[0036] The probe sequence corresponding to miR-222 is shown as SEQ ID NO. 49,

[0037] The probe sequence corresponding to miR-301 is shown as SEQ ID NO. 61.

[0038] According to a preferred embodiment, the reagent in the kit further comprises miRNA primers, the miRNA primers comprising reverse transcription primers, quantitative PCR forward primers and reverse primers.

[0039] According to a preferred embodiment, the miRNA primer sequences corresponding to the above-mentioned molecular markers are as follows:

[0040] The sequences of the quantitative PCR forward primers and reverse primers, and reverse transcription primers corresponding to miR-21 are shown as SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4, respectively;

[0041] The sequences of the quantitative PCR forward primers and reverse primers, and reverse transcription primers corresponding to miR-30c are shown as SEQ ID NO. 14, SEQ ID NO. 15, and SEQ ID NO. 16, respectively;

[0042] The sequences of the quantitative PCR forward primers and reverse primers, and reverse transcription primers corresponding to miR-141 are shown as SEQ ID NO. 22, SEQ ID NO. 23, and SEQ ID NO. 24, respectively;

[0043] The sequences of the quantitative PCR forward primers and reverse primers, and reverse transcription primers corresponding to miR-205 are shown as SEQ ID NO. 38, SEQ ID NO. 39, and SEQ ID NO. 40, respectively;

[0044] The sequences of the forward primer and the reverse primer, and the reverse transcription primer corresponding to miR-224 for quantitative PCR are shown in SEQ ID NO. 54, SEQ ID NO. 55, and SEQ ID NO. 56, respectively;

[0045] The sequences of the forward primer and the reverse primer, and the reverse transcription primer corresponding to miR-24 for quantitative PCR are shown in SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12, respectively;

[0046] The sequences of the forward primer and the reverse primer, and the reverse transcription primer corresponding to miR-155 for quantitative PCR are shown in SEQ ID NO. 30, SEQ ID NO. 31, and SEQ ID NO. 32, respectively;

[0047] The sequences of the forward primer and the reverse primer, and the reverse transcription primer corresponding to miR-210 for quantitative PCR are shown in SEQ ID NO. 42, SEQ ID NO. 43, and SEQ ID NO. 44, respectively;

[0048] The sequences of the forward primer and the reverse primer, and the reverse transcription primer corresponding to miR-132 for quantitative PCR are shown in SEQ ID NO. 18, SEQ ID NO. 19, and SEQ ID NO. 20, respectively;

[0049] The sequences of the forward primer and the reverse primer, and the reverse transcription primer corresponding to miR-146b for quantitative PCR are shown in SEQ ID NO. 26, SEQ ID NO. 27, and SEQ ID NO. 28, respectively;

[0050] The sequences of the forward primer and the reverse primer, and the reverse transcription primer corresponding to miR-221 for quantitative PCR are shown in SEQ ID NO. 46, SEQ ID NO. 47, and SEQ ID NO. 48, respectively;

[0051] The sequences of the forward primer and the reverse primer, and the reverse transcription primer corresponding to miR-197 for quantitative PCR are shown in SEQ ID NO. 34, SEQ ID NO. 35, and SEQ ID NO. 36, respectively;

[0052] The sequences of the forward primer and the reverse primer, and the reverse transcription primer corresponding to miR-501 for quantitative PCR are shown in SEQ ID NO. 58, SEQ ID NO. 59, and SEQ ID NO. 60, respectively;

[0053] The sequences of the forward primer and the reverse primer, and the reverse transcription primer corresponding to miR-23a for quantitative PCR are shown in SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, respectively;

[0054] The sequences of the forward primer and reverse primer of quantitative PCR corresponding to miR-222 and reverse transcription primer are shown in SEQ ID NO. 50, SEQ ID NO. 51 and SEQ ID NO. 52, respectively.

[0055] The sequences of the forward primer and reverse primer of quantitative PCR corresponding to miR-301 and reverse transcription primer are shown in SEQ ID NO. 62, SEQ ID NO. 63 and SEQ ID NO. 64, respectively.

[0056] The beneficial effects of the present application are:

[0057] (1) The miRNAs selected in the present application are the most effective biomarker combinations found through systematic research (all closely related to the occurrence, development and diffusion of pancreatic cancer), and repeatedly verified by different samples, multiple research centers and clinical centers. Five groups of miRNA combinations (5 miRNAs in each group) are proposed as diagnostic biomarkers for pancreatic cancer, providing theoretical support for early pancreatic tumor molecular diagnosis and precise medication.

[0058] (2) The present application establishes an early pancreatic cancer detection marker composed of 5 miRNAs. The detection accuracy is comprehensively judged by combining the combined detection results of the 5 miRNA combinations with the conventional pathological results, and the detection accuracy is high (the AUC of the 5 miRNAs is all > 95%). The detection method is the best molecular marker for clinical diagnosis determined on the basis of more than 1000 cases of verification.

[0059] (3) The molecular marker for early pancreatic cancer detection of the present application is the optimal biomarker combination obtained after comparative analysis of the results of the traditional tumor markers CEA and CA19-9. The diagnostic sensitivity and specificity are superior to the early pancreatic cancer detection markers reported in the prior art.

[0060] The molecular marker and kit for early pancreatic cancer detection / diagnosis provided by the present application can be used for early pancreatic cancer screening and differential diagnosis, disease complication occurrence and recurrence monitoring, efficacy, drug efficacy and precise medication guidance evaluation, and has the advantages of wide detection spectrum, high sensitivity, good specificity, low detection cost, convenient sampling, easy sample storage, etc. It can be widely used for early pancreatic cancer screening and prognosis related work, and improves the individual differences, low specificity and low sensitivity caused by the instability of single marker or the currently widely used biomarker itself, significantly improves the clinical detection rate of early pancreatic cancer, and reduces the misdiagnosis rate and missed diagnosis rate of pancreatic cancer. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1ROC curves of 5 different miRNAs combinations, A: ROC curve of combination 1; B: ROC curve of combination 2; C: ROC curve of combination 3; D: ROC curve of combination 4; E: ROC curve of combination 5; F: ROC curve of combination 6.

[0062] Figure 2 is the stratification statistical result of each case of pancreatic cancer of combination 1 of the application.

[0063] Figure 3 is the stratification statistical result of each case of pancreatic cancer of combination 2 of the application.

[0064] Figure 4 is the stratification statistical result of each case of pancreatic cancer of combination 3 of the application.

[0065] Figure 5 is the stratification statistical result of each case of pancreatic cancer of combination 4 of the application.

[0066] Figure 6 is the stratification statistical result of each case of pancreatic cancer of combination 5 of the application.

[0067] Figure 7 is the gene amplification fold result of 5 marker combinations of the application.

[0068] Figure 8 is the ROC curve of different target combinations.

[0069] Figure 9 Comparison of ROC curves of marker combinations of the application and pancreatic cancer marker detection in the prior art, A: comparison of ROC curves of 5 combinations and pancreatic cancer marker detection in the prior art; B: comparison of ROC curves of combination 1 and pancreatic cancer marker detection in the prior art; C: comparison of ROC curves of combination 2 and pancreatic cancer marker detection in the prior art; D: comparison of ROC curves of combination 3 and pancreatic cancer marker detection in the prior art; E: comparison of ROC curves of combination 4 and pancreatic cancer marker detection in the prior art; F: comparison of ROC curves of combination 5 and pancreatic cancer marker detection in the prior art. DETAILED DESCRIPTION

[0070] In order to make the objectives, technical solutions and advantages of the present application clearer, the following further describes the present application with reference to examples. It should be understood that the specific examples described herein are only used to explain the present application and should not be used to limit the present application.

[0071] The methods involved in the following examples are all conventional methods unless otherwise specified; the reagents and materials used are all conventional reagents and materials in the art unless otherwise specified, and can be obtained by commercial purchase.

[0072] Example 1

[0073] The inventive concept of the present application is that miRNA is involved in the whole process of normal cell transformation from inflammation to cancer in pancreas, is closely related to the occurrence and development of pancreatic cancer, and miRNA is a non-coding short fragment RNA which is not easy to be degraded in peripheral blood and is an ideal marker for early screening and early diagnosis of pancreatic cancer. Since the abnormal expression of miRNA is reflected in each stage of the pathogenesis of pancreatic cancer, the differential expression of miRNA according to different pathological types is used to detect the characteristic diseases of each stage, which is a technical means with excellent sensitivity and specificity to distinguish whether the patient has pancreatic benign disease or pancreatic cancer. Therefore, based on the research reports of a variety of miRNAs closely related to the occurrence, development and spread of pancreatic cancer, and according to the expression intensity difference of a variety of miRNAs in early pancreatic cancer cells shown by the existing research, the inventors determined that the pancreatic cancer marker miRNAs are selected from: miR-21, miR-23a, miR-24, miR-30c, miR-132, miR-141, miR-146b, miR-155, miR-197, miR-205, miR-210, miR-221, miR-222, miR-224 and miR-501. At the same time, the above mRNA is expressed in whole blood, urine, saliva and the like in addition to serum and plasma. The above content can be obtained according to the following reference:

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[0132] Further, the inventors, through clinical multicenter verification, comprehensively analyze the medical background, BMI (obesity index), living habits (drinking and smoking), clinical metabolic indicators, and their variability in individual and follow-up individuals, and use big data comprehensive analysis methods (such as comparing para-cancer tissues, using machine learning algorithms to find miRNAs that are highly differentially expressed in early pancreatic cancer tissues, and using artificial intelligence deep learning and other optimization algorithms to find specific miRNAs expressed in different pathological periods of pancreatic cancer) to calculate the contribution of each miRNA to the carcinogenesis of early pancreatic cells, and to optimize and verify the grouping of miRNA combinations, and finally divide the above candidate miRNAs into 5 combinations:

[0133] Combination 1: miR-21 / miR-30c / miR-141 / miR-205 / miR-224;

[0134] Combination 2: miR-24 / miR-141 / miR-155 / miR-210 / miR-224;

[0135] Combination 3: miR-24 / miR-132 / miR-146b / miR-210 / miR-221;

[0136] Combination 4: miR-24 / miR-30c / miR-146b / miR-197 / miR-501;

[0137] Combination 5: miR-23a / miR-197 / miR-205 / miR-222 / miR-301.

[0138] I. Experimental samples and main reagents

[0139] 1. Collection of experimental samples

[0140] The serum samples were collected from patients at Shanghai Changhai Hospital, the First Affiliated Hospital of Dalian Medical University, Peking Union Medical College Hospital of Chinese Academy of Medical Sciences, and Second Xiangya Hospital of Central South University (including early pancreatic ductal adenocarcinoma, pancreatitis, intraductal papillary mucinous tumor of the pancreas) and non-case serum samples.

[0141] According to the case group, the serum samples were collected before the first diagnosis of pancreatic cancer that met the diagnostic criteria of the Pancreatic Cancer Diagnosis and Treatment Guidelines (2018 edition) and before receiving surgical treatment, radiotherapy and chemotherapy measures. A total of 120 cases of pancreatic cancer in stage I, 128 cases in stage II, 67 cases in stage III, and 54 cases in stage IV were collected. The non-case group was other pancreatic disease subjects such as pancreatitis, and other malignant tumor subjects such as gastric cancer, liver cancer, intestinal cancer, urological tumors (prostate cancer), breast cancer, lung cancer, and hematological tumors (multiple myeloma), a total of 589 cases.

[0142] 2. Main experimental reagents

[0143] TRIzol TM LS Reagent (Invitrogen, 10296028),

[0144] TaqManTM MicroRNA Reverse Transcription Kit (ABI, 4366596),

[0145] Premix Ex TaqTM (Probe qPCR) (TAKARA, RR390);

[0146] Isopropyl alcohol, chloroform, anhydrous ethanol.

[0147] Primers and probes were synthesized by a biological company, and the sequences are shown in Table 1:

[0148] Table 1 Primer and probe sequences

[0149]

[0150]

[0151]

[0152]

[0153] II. Experimental methods

[0154] 1. RNA extraction

[0155] The serum sample was taken, and serum RNA was extracted with the RNA extraction reagent TRIzol TM LS Reagent.

[0156] 2. Reverse transcription

[0157] Any one of the aforementioned miRNAs as markers was combined with U6, and a primer working solution was prepared at a concentration of 5 μM of a single primer. Reverse transcription was performed using a reverse transcription kit TaqMan™ MicroRNA Reverse Transcription Kit (ABI, 4366596), and the reverse transcription system was 15 μL, which contained: RNA 50 ng, 10x Buffer 1.5 μL, dNTPmix 0.15 μL, RT enzyme 1 μL, RNase inhibitor 0.19 μL, U6 RT primer (5 μM) 1 μL, miRNA RT primer (5 μM) 1 μL, and ddH2O supplemented to 15 μL. The following program was performed on a PCR amplifier: 16 °C for 30 min, 42 °C for 30 min, 85 °C for 5 min, and 4 °C storage, and after completion, instantaneous centrifugation was performed to the bottom of the tube to obtain cDNA.

[0158] 3. qPCR

[0159] The kit Premix Ex Taq™ (Probe qPCR) (TAKARA, RR390) was used.

[0160] The reaction system was prepared: cDNA obtained by reverse transcription 3 μL, Premix Ex Taq (Probe qPCR) (2x) 5 μL, U6 forward primer (10 μM) 0.2 μL, U6 reverse primer (10 μM) 0.2 μL, miRNA forward primer (10 μM) 0.2 μL, miRNA reverse primer (10 μM) 0.2 μL, U6 probe (10 μM) 0.4 μL, miRNA probe (10 μM) 0.4 μL, and ddH2O supplemented to 10 μL.

[0161] PCR amplification was performed on a quantitative PCR instrument, and the reaction conditions were: pre-denaturation, 1 cycle, 95 °C for 30 seconds; PCR reaction, 40 cycles, 95 °C for 5 seconds and 60 °C for 30 seconds; annealing 50 °C for 30 seconds, 1 cycle.

[0162] 4. Data processing and analysis

[0163] The miRNAs involved in the five combinations were subjected to quantitative PCR analysis, respectively, and the results were counted according to the five combinations, respectively. Based on the miRNA expression amount in each combination combined with the clinical information of the patients, a logistic regression model was used to determine the regression coefficient, and the model parameters were optimized through cross-validation, and a comprehensive evaluation value was output to judge the pathological positive. Then, the evaluation threshold was optimized by the ROC curve Youden index, and finally the diagnostic index calculation formula based on the miRNA expression amount was obtained to judge the pathological state of the patients.

[0164] III. Result analysis

[0165] The Ct values ​​of each miRNA and the internal control obtained were summarized and organized according to five different combinations. Combined with the clinical diagnostic information of the samples, the sensitivity and specificity results of samples at different stages were calculated separately.

[0166] 1. The results obtained from five different combinations of miRNAs were compared with clinical diagnostic information, and ROC curves were plotted. A more convex ROC curve, closer to the upper left corner, indicates greater diagnostic value and facilitates comparison between different indicators. The area under the curve (AUC) evaluates diagnostic accuracy; a larger AUC indicates greater diagnostic efficacy. The AUC value is between 1.0 and 0.5. When AUC > 0.5, the closer the AUC is to 1, the better the diagnostic effect. AUC between 0.5 and 0.7 indicates lower accuracy, AUC between 0.7 and 0.9 indicates some accuracy, and AUC above 0.9 indicates high accuracy. Results are as follows: Figure 1 As shown, the AUC (area under the curve) of the aforementioned five different miRNA combinations—combinations 1, 2, 3, 4, and 5—are 0.976, 0.969, 0.964, 0.958, and 0.964, respectively, all exhibiting high accuracy.

[0167] 2. Stratify and statistically analyze pancreatic cancer samples from different stages in each combination result, and calculate sensitivity and specificity separately. The results are as follows: Figures 2-6 As shown, the sensitivity and specificity of different stages are all greater than 90%, with the sensitivity of stage I being 91.74-93.58%, which is much higher than other detection methods. Among them, combination 1 has the highest sensitivity of stage I, reaching 93.58%, and the specificity is 97.12%.

[0168] 3. The detection results of five different combinations of miRNAs were categorized into normal individuals and pancreatic cancer patients, and the cumulative gene amplification fold of miRNAs in each combination was statistically analyzed (*p<0.05). The results are as follows: Figure 7 As shown, in each combination, pancreatic cancer patients had a significant difference in gene amplification fold compared to normal individuals.

[0169] 4. Taking combination 1 as an example, the differences between different target combinations are statistically analyzed, and the results are as follows: Figure 8 Table 2 shows that ROC curve analysis was performed on all combinations of single-target, dual-target, triple-target, quadruple-target, and five-target targets. The AUC area was compared, and the target combination with the largest AUC area is the optimal combination. The analysis results show that the five-target combination has the largest AUC area, meaning that the five-target combination is the optimal combination. For single-target, dual-target, triple-target, and quadruple-target combinations, the AUC values ​​show an upward trend as the number of targets increases. Table 2 also includes the AUC areas for five-target combinations 1-5.

[0170] Table 2

[0171]

[0172]

[0173] Example 2

[0174] I. Sample source

[0175] A total of 1124 clinical serum samples (serum samples from patients of Shanghai Changhai Hospital and Xiangya Second Hospital of Central South University and serum samples of non-case group): (1) According to the case group, the serum samples were collected before receiving surgical treatment, radiotherapy and chemotherapy treatment measures, and 268 and 332 clinical samples of I and II stage pancreatic cancer patients with CA19-9 detection results were selected respectively, a total of 600 cases; (2) 524 non-case group pancreatic cancer negative clinical samples: other pancreatic disease subjects such as pancreatitis, and other malignant tumor subjects such as gastric cancer, liver cancer, intestinal cancer, urological tumor (prostate cancer), breast cancer, lung cancer, hematological tumor (multiple myeloma) and the like.

[0176] II. Experimental method

[0177] The above 1124 serum samples were used as experimental samples, and the miRNA fluorescence probe detection technology in Example 1 (RNA extraction, reverse transcription, qPCR, data analysis method and Example 1) was used for detection. The detection results of the 5 miRNAs combination in Example 1 and CA19-9 were compared.

[0178] At the same time, since the inventors did not disclose the detection sensitivity and specificity results for early pancreatic cancer patients in the prior patent CN2020103564980 (authorized announcement number: CN111575374B; invention name: molecular marker for early pancreatic tumor detection, its detection method and application), the molecular marker (combination of hsa-miR-30c-5p, hsa-miR-24-3p, hsa-miR-23a-3p and hsa-miR-132-3p) in CN2020103564980 was used to detect all 1124 clinical samples in this embodiment, in order to compare the sensitivity and specificity of the detection with the present application.

[0179] III. Result analysis

[0180] (1) The miRNA detection results were compared with the detection values of CA19-9 and patent CN2020103564980, and the results were as follows Figure 9As shown, the miRNA combinations of the present invention have significantly better detection accuracy in early pancreatic cancer cases than CA19-9 and CN2020103564980. The AUC (area under the curve) of miRNA combination 1 of the present invention is 0.977, the AUC of combinations 2-5 is 0.949-0.970, while the AUC of CA19-9 is 0.82 and the AUC of CN2020103564980 is 0.910.

[0181] (2) The clinical sample detection data in this embodiment (Table 3) were used to calculate the sensitivity and specificity of the miRNA combinations 1-5 and CA19-9 of the present invention and the detection results of patent CN2020103564980. The results are shown in Table 4. The miRNA combinations of the present invention have a sensitivity and specificity of more than 95% in the detection results of stage I clinical samples of pancreatic cancer, which is much higher than CA19-9 and also higher than patent CN2020103564980.

[0182] Table 3

[0183]

[0184]

[0185] Table 4

[0186] Marker Sensitivity Specificity Inventive miRNA combination 1 94.50％ 93.89％ Inventive miRNA combination 2 94.00％ 93.51％ Inventive miRNA combination 3 93.67％ 93.32％ Inventive miRNA combination 4 92.83％ 93.13％ Inventive miRNA combination 5 92.17％ 92.94％ CA19-9 61.17％ 82.44％ Patent CN2020103564980 85.33％ 87.02％

[0187] The experimental data above show that the five miRNA combinations of this invention all exhibit high detection accuracy for pancreatic cancer. Figure 1 Combination 1 was optimal, and in the stratified statistics of each case stage, early cases also showed high sensitivity and specificity. Figure 2 Combination 1 showed the highest sensitivity in detecting stage 1 pancreatic cancer patients. Statistical analysis of fold change in the results of different combinations revealed significant fold change differences in both positive and negative samples across all five combinations. Figure 7 Taking combination 1 as an example, comparing the differences between combinations with different numbers of targets, the results show that the AUC area increases in the order of single-target, dual-target, triple-target, quadruple-target, and five-target combinations, with the five-target combination being the optimal one. Figure 8 In comparative analyses with other biomarkers, the miRNA combinations of this invention exhibited significant advantages. The AUC (area under the curve) of the five miRNA combinations of this invention was 0.958-0.976, while the AUC of CA19-9 was only 0.82. Figure 9 The sensitivity and specificity of the Phase I clinical samples are superior to CA19-9 and also superior to the comparative patent CN2020103564980.

[0188] It should be noted that the above-mentioned specific embodiments are exemplary, and those skilled in the art can think of various solutions under the inspiration of the disclosure of the present application, and these solutions also belong to the disclosed range of the present application and fall within the protection scope of the present application. Those skilled in the art should understand that the present application specification and its drawings are illustrative and do not constitute a limitation on the claims. The protection scope of the present application is defined by the claims and their equivalents. The present application specification contains a plurality of inventive concepts, such as "preferably", "according to a preferred embodiment" or "optionally", which all indicate that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application according to each inventive concept.

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Claims

1. A molecular biomarker for early detection of pancreatic cancer, characterized in that: The molecular marker is any combination of the following miRNAs: Combination 1: Composed of miR-21, miR-30c, miR-141, miR-205, and miR-224; Combination 2: Composed of miR-24, miR-141, miR-155, miR-210, and miR-224; Combination 3: Composed of miR-24, miR-132, miR-146b, miR-210, and miR-221; Combination 4: Composed of miR-24, miR-30c, miR-146b, miR-197 and miR-501; Combination 5: Composed of miR-23a, miR-197, miR-205, miR-222 and miR-301.

2. The molecular marker according to claim 1, characterized in that: The molecular marker is miRNA in serum or plasma.

3. The use of the reagent for detecting the molecular markers of claim 1 or 2 in the preparation of products for early pancreatic cancer diagnosis or auxiliary diagnosis.

4. The application according to claim 3, wherein the product comprises a reagent, a chip, a test strip, or a reagent kit.

5. The application according to claim 4, wherein the product includes portable test strips, digital test strips, digital test cards, and a testing instrument.

6. A kit for the diagnosis or auxiliary diagnosis of early pancreatic cancer, characterized in that: The kit includes reagents for detecting the expression level of the molecular marker of claim 1.

7. The reagent kit according to claim 6, characterized in that: The reagents include miRNA probes.

8. The reagent kit according to claim 7, characterized in that: The probe sequences corresponding to the molecular markers described in claim 1 are as follows: The probe sequence corresponding to miR-21 is shown in SEQ ID NO.

1. The probe sequence corresponding to miR-30c is shown in SEQ ID NO.

13. The probe sequence corresponding to miR-141 is shown in SEQ ID NO.

21. The probe sequence corresponding to miR-205 is shown in SEQ ID NO.

37. The probe sequence corresponding to miR-224 is shown in SEQ ID NO.

53. The probe sequence corresponding to miR-24 is shown in SEQ ID NO.

9. The probe sequence corresponding to miR-155 is shown in SEQ ID NO.

29. The probe sequence corresponding to miR-210 is shown in SEQ ID NO.

41. The probe sequence corresponding to miR-132 is shown in SEQ ID NO.

17. The probe sequence corresponding to miR-146b is shown in SEQ ID NO.

25. The probe sequence corresponding to miR-221 is shown in SEQ ID NO.

45. The probe sequence corresponding to miR-197 is shown in SEQ ID NO.

33. The probe sequence corresponding to miR-501 is shown in SEQ ID NO.

57. The probe sequence corresponding to miR-23a is shown in SEQ ID NO.

5. The probe sequence corresponding to miR-222 is shown in SEQ ID NO.

49. The probe sequence corresponding to miR-301 is shown in SEQ ID NO.

61.

9. The reagent kit according to claim 7, characterized in that: The reagents also include miRNA primers, which include reverse transcription primers, quantitative PCR forward primers, and reverse primers.

10. The reagent kit according to claim 9, characterized in that: The miRNA primer sequences corresponding to the molecular markers described in claim 1 are as follows: The sequences of the forward and reverse primers and reverse transcription primers for quantitative PCR corresponding to miR-21 are shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. The sequences of the forward and reverse primers and reverse transcription primers for quantitative PCR corresponding to miR-30c are shown in SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively. The sequences of the forward and reverse primers and reverse transcription primers for quantitative PCR corresponding to miR-141 are shown in SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.24, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to miR-205 are shown in SEQ ID NO.38, SEQ ID NO.39, and SEQ ID NO.40, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to miR-224 are shown in SEQ ID NO. 54, SEQ ID NO. 55, and SEQ ID NO. 56, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to miR-24 are shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to miR-155 are shown in SEQ ID NO.30, SEQ ID NO.31, and SEQ ID NO.32, respectively. The sequences of the forward and reverse primers and reverse transcription primers for quantitative PCR corresponding to miR-210 are shown in SEQ ID NO.42, SEQ ID NO.43, and SEQ ID NO.44, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to miR-132 are shown in SEQ ID NO.18, SEQ ID NO.19, and SEQ ID NO.20, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to miR-146b are shown in SEQ ID NO.26, SEQ ID NO.27, and SEQ ID NO.28, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to miR-221 are shown in SEQ ID NO.46, SEQ ID NO.47, and SEQ ID NO.48, respectively. The sequences of the forward and reverse primers and reverse transcription primers for quantitative PCR corresponding to miR-197 are shown in SEQ ID NO.34, SEQ ID NO.35, and SEQ ID NO.36, respectively. The sequences of the forward and reverse primers and reverse transcription primers for quantitative PCR corresponding to miR-501 are shown in SEQ ID NO.58, SEQ ID NO.59, and SEQ ID NO.60, respectively. The sequences of the forward and reverse primers and reverse transcription primers for quantitative PCR corresponding to miR-23a are shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively. The sequences of the forward and reverse primers and reverse transcription primers for quantitative PCR corresponding to miR-222 are shown in SEQ ID NO.50, SEQ ID NO.51, and SEQ ID NO.52, respectively. The sequences of the forward and reverse primers and reverse transcription primers for quantitative PCR corresponding to miR-301 are shown in SEQ ID NO. 62, SEQ ID NO. 63, and SEQ ID NO. 64, respectively.

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