Application and application of miR-145 as pancreatic cancer diagnosis marker

Abstract

The invention belongs to the technical field of biological detection and diagnosis, and particularly relates to application and application of miR-145 serving as a pancreatic cancer diagnosis marker, especially a pancreatic cancer KRAS G12D gene mutation diagnosis marker. The invention provides application of miR-145 in serum as a detection marker for pancreatic cancer, especially KRAS G12D, and KRAS G12D mutation can be accurately detected by detecting the expression level of miR-145 in serum, so that diagnosis and screening of pancreatic cancer based on a liquid biopsy technology are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection and diagnosis, and particularly relates to the use and application of miR-145 as a diagnostic marker for pancreatic cancer, especially as a diagnostic marker for the KRAS G12D gene mutation in pancreatic cancer. Background Art

[0002] Pancreatic cancer is the third leading cause of cancer-related deaths globally, and most patients with pancreatic cancer are diagnosed at an advanced stage once diagnosed. Therefore, novel biomarkers with high sensitivity and specificity for early diagnosis and prognosis are currently considered the best strategy to improve the treatment effect of pancreatic cancer.

[0003] Studies have shown that KRAS is one of the common oncogenes in humans, which is involved in key processes such as cell proliferation, differentiation, and survival. Gene mutations in KRAS are one of the most common genetic alterations in pancreatic cancer, and G12D is the most common mutation type. This mutation leads to an increase in the GTPase activity of the KRAS protein, making it in an active state, and then continuously activating downstream signaling pathways (such as the MAPK and PI3K pathways), promoting cell proliferation and survival. Therefore, the KRAS G12D mutation is closely related to the malignancy, prognosis, and drug treatment sensitivity of tumors.

[0004] Clinically, KRAS mutations have become important molecular markers for guiding tumor treatment strategies. Currently, the commonly used methods for detecting KRAS G12D mutations in clinical practice require tissue biopsies to obtain tissue samples, usually through surgical operations or punctures to obtain tumor tissues. This is an invasive process for patients, which may bring complications such as pain, infection, and bleeding. In addition, tumors may be heterogeneous among different regions. Therefore, the information obtained from a local tissue sample may not comprehensively reflect the characteristics of the entire tumor. Moreover, tissue biopsies usually take a certain amount of time to complete, including surgical waiting time, laboratory processing, and pathological analysis, etc., which may lead to delays in diagnosis and treatment decisions.

[0005] Liquid biopsy is a non-invasive method for tumor diagnosis and monitoring. By analyzing tumor-related markers in body fluids (such as serum), such as exosomes and circulating tumor RNA (ctRNA), etc., tumor information can be obtained. Compared with traditional tissue biopsies, liquid biopsy has the following advantages:

[0006] Non-invasiveness: Liquid biopsy can collect routine body fluid samples such as blood, urine, and saliva without invasive surgeries or tissue biopsies, reducing patient discomfort and risks and providing a more convenient way of sample collection;

[0007] Systemic: Liquid biopsy can provide systemic tumor information, not limited to a specific tumor site. It can detect circulating tumor markers throughout the body, reflecting the systemic characteristics and changes of tumors.

[0008] Dynamic monitoring: Since liquid biopsy can repeatedly collect and analyze samples, it can achieve dynamic monitoring of tumors. It can be used to detect residual lesions after treatment, monitor tumor recurrence and metastasis, and evaluate treatment effects and drug resistance, etc.

[0009] Consideration of spatial heterogeneity: Liquid biopsy can overcome the limitations of local heterogeneity in tissue biopsy because circulating tumor markers can reflect the genetic and expression characteristics of tumors throughout the body.

[0010] Provide individualized treatment strategies: Liquid biopsy can help select the best treatment plan and formulate individualized treatment strategies based on the molecular characteristics and variability of tumors.

[0011] In summary, as a non-invasive method for tumor diagnosis and monitoring, liquid biopsy has the advantages of non-invasiveness, systemic nature, dynamic monitoring, and individualized treatment, providing new opportunities and options for the diagnosis and treatment of tumor patients. Therefore, there is an expectation in this field to develop a simple and non-invasive method for detecting pancreatic cancer, especially KRAS G12D mutations, based on liquid biopsy technology, which is of great significance for the formulation of clinical diagnosis and treatment strategies. Summary of the Invention

[0012] To this end, the technical problem to be solved by the present invention is to provide the use of miR-145 as a diagnostic marker for pancreatic cancer, especially as a diagnostic marker for KRAS G12D gene mutations in pancreatic cancer, which can detect KRAS G12D mutations by detecting the expression level of miR-145 in serum, thus solving the defects of traditional tissue biopsy methods such as invasiveness, local heterogeneity, and time lag.

[0013] The second technical problem to be solved by the present invention is to provide the application of miR-145 as a diagnostic marker for pancreatic cancer, especially as a diagnostic marker for KRAS G12D gene mutations in pancreatic cancer, and further provide a rapid, non-invasive, and systemic method for diagnosing and detecting pancreatic cancer based on liquid biopsy technology.

[0014] To solve the above technical problems, the present invention relates to the use of miR-145 as a diagnostic marker for pancreatic cancer.

[0015] Specifically, the pancreatic cancer includes KRAS G12D gene mutations.

[0016] Specifically, the miR-145 is derived from serum.

[0017] The present invention also discloses the use of miR-145 as a diagnostic marker for KRAS G12D gene mutation in pancreatic cancer.

[0018] The present invention also discloses the use of a substance and / or reagent for detecting the level of miR-145 in the preparation of a product for diagnosing or assisting in the diagnosis, screening or assisting in the screening of pancreatic cancer.

[0019] The present invention also discloses the use of a substance and / or reagent for detecting the expression level of miR-145 in the preparation of a product for detecting the correlation and / or mutation expression level of KRAS G12D mutation in pancreatic cancer.

[0020] The present invention also discloses a product for diagnosing or assisting in the diagnosis, screening or assisting in the screening of pancreatic cancer, characterized in that the product comprises a reagent for detecting the correlation and / or mutation level of KRAS G12D mutation in pancreatic cancer based on RT-qPCR method.

[0021] Specifically, the product for diagnosing or assisting in the diagnosis, screening or assisting in the screening of pancreatic cancer comprises a plasma kit.

[0022] Specifically, the reagent of the product for diagnosing or assisting in the diagnosis, screening or assisting in the screening of pancreatic cancer comprises a stem-loop fluorescence quantitative PCR kit.

[0023] Specifically, for the optional stem-loop fluorescence quantitative PCR kit of the present invention, the stem-loop primers of Thermo Fisher Scientific company can be used for reverse transcription, and its detection system is as shown in Table 1 below, and the PCR reaction program is as shown in Table 2 below.

[0024] Table 1 Stem-loop fluorescence quantitative PCR system

[0025] Reagent Volume (μL) AMV 0.5 5×AMV Buffer 2 dNTP Mix (2.5 mM) 4 DEPC 1.5 Stem-loop RT Primer 1 RNA 1 Total 10

[0026] Table 2 Reaction program

[0027] Temperature (℃) Time (min) 16 30 42 30 85 5

[0028] After the above reverse transcription is completed, quantitative detection is carried out using the corresponding qPCR probe. Specifically, the q-PCR system is as shown in Table 3 below, and the qPCR reaction program is as shown in Table 4 below.

[0029] Table 3 q-PCR system

[0030] Reagent Volume (μL) <![CDATA[ddH2O]]> 14.77 10×buffer 2 <![CDATA[MgCl2]]> 1.2 dNTP (10 mM) 0.4 rTaq 0.3 TM Probe 0.33 cDNA 1 Total 20

[0031] Table 4 The qPCR reaction program is as follows:

[0032]

[0033] The present invention also discloses a system for diagnosing or assisting in the diagnosis, screening or assisting in the screening of pancreatic cancer, comprising:

[0034] a detection device for determining the pancreatic cancer KRAS G12D mutation correlation and / or mutation level in a biological sample;

[0035] a judgment device for diagnosing or assisting in the diagnosis, screening or assisting in the screening of whether a person to be tested has pancreatic cancer based on whether the pancreatic cancer KRAS G12D in the biological sample is mutated or the mutation expression level.

[0036] The present invention provides the use of miR-145 in serum as a detection biomarker for pancreatic cancer, especially KRAS G12D, which can accurately detect the KRAS G12D mutation by detecting the expression level of miR-145 in serum, effectively solving the problems of difficult early diagnosis and treatment selection for highly lethal pancreatic cancer, thereby realizing the diagnosis and screening of pancreatic cancer based on liquid biopsy technology and solving the defects such as invasiveness, local heterogeneity and time lag existing in traditional tissue biopsy methods.

[0037] The present invention further provides the application of miR-145 in serum as a detection biomarker for pancreatic cancer, especially KRAS G12D, and further provides a rapid, non-invasive and systemic method for diagnosing and detecting pancreatic cancer based on liquid biopsy technology, which is of great significance for the diagnosis and treatment of pancreatic cancer.

[0038] Experiments of the present invention have confirmed that the down-regulation of miR-145 is closely related to the occurrence, development and prognosis of pancreatic cancer. In particular, the down-regulation of miR-145 is considered to be a promoting factor for the proliferation, invasion and metastasis of pancreatic cancer cells, and miR-145 is involved in a series of key biological processes, such as cell cycle regulation, epithelial-mesenchymal transition, angiogenesis and anti-apoptosis, by regulating the expression of multiple target genes. More importantly, the down-regulation of miR-145 is related to the poor prognosis of pancreatic cancer patients. The research finds that the expression level of miR-145 is closely related to the clinicopathological features of pancreatic cancer, such as pathological type, grade and lymph node metastasis. In addition, the expression level of miR-145 is also related to the survival rate and recurrence rate of pancreatic cancer patients, and the low expression of miR-145 is often associated with poor prognosis and increased recurrence risk.

[0039] By detecting miR-145 in the serum of patients, the present method establishes a liquid biopsy method for the KRAS G12D mutation of pancreatic cancer. The present method has the following advantages:

[0040] Non-invasive: The detection of miR-145 can be performed by collecting a blood sample from the patient without invasive surgery or tissue biopsy. This non-invasive detection method is more convenient and acceptable for patients;

[0041] KRAS mutation specificity: The downregulation of miR-145 has a high correlation with KRAS mutations in pancreatic cancer. Therefore, by detecting the expression level of miR-145, the presence or absence of KRAS mutations can be indirectly inferred;

[0042] High sensitivity: The downregulation of miR-145 is prevalent in pancreatic cancer and is associated with KRAS mutations. Therefore, the detection of miR-145 can provide high-sensitivity screening and diagnosis of KRAS mutations in pancreatic cancer;

[0043] Dynamic monitoring: Due to the characteristics of liquid biopsy, the detection of miR-145 can achieve dynamic monitoring of KRAS mutations in pancreatic cancer. By repeatedly collecting samples, changes in KRAS mutations can be monitored in a timely manner, including residual lesions, recurrence, and metastasis after treatment, etc.;

[0044] Potential prognostic value: Some studies have shown that the downregulation of miR-145 is associated with poor prognosis in pancreatic cancer patients. Therefore, the detection of miR-145 may help evaluate the prognostic risk of patients and provide a reference for the formulation of individualized treatment plans. Brief Description of the Drawings

[0045] To make the content of the present invention easier to understand clearly, the following further details the present invention according to specific embodiments of the present invention in combination with the drawings, wherein,

[0046] Figure 1 Results of comparing the levels of miR-145 in the sera of healthy volunteers, pancreatic cancer patients with other mutations, and pancreatic cancer patients with KRAS G12D mutations in Example 1;

[0047] Figure 2 Results of survival analysis (survival curves) of pancreatic cancer patients with KRAS G12D mutations with low expression of miR-145 in serum and pancreatic cancer patients with other mutation types with high expression of miR-145 in serum in Example 2;

[0048] Figure 3 Based on the results of miRNA target prediction software in Example 3. Detailed Description of the Specific Embodiments

[0049] Example 1

[0050] In this example, serum samples of pancreatic cancer patients were collected, and miRNAs in the serum were extracted by appropriate technical means (such as the TRIzol method). The specific method is as follows.

[0051] Separate plasma: Collect anticoagulated blood samples (EDTA anticoagulant) from normal people and colorectal cancer patients. Centrifuge the blood samples at 3000 rpm for 10 minutes. Carefully aspirate the supernatant plasma (avoid aspirating blood cells) into a 1.5 mL EP tube. Store the plasma in an ultra-low temperature freezer at -80 °C.

[0052] The steps for extracting small RNAs from plasma are as follows:

[0053] (1) Aspirate 100 μL of plasma into a new 1.5 mL EP tube. Add 900 μL of TRIzol. Vortex for 30 s until completely homogenized, and invert to mix evenly.

[0054] (2) Let it stand at room temperature for 5 minutes to allow the sample to lyse completely. Add 200 μL of chloroform. Vortex vigorously for 15 s to mix the solution evenly, and then let it stand at room temperature for 5 minutes. Centrifuge at 12000 rpm at 4 °C for 15 minutes. After centrifugation, the sample will be divided into three layers: a yellow organic phase, a white intermediate layer, and a colorless aqueous phase. RNA is mainly in the aqueous phase. Transfer the top aqueous phase to a new 1.5 mL EP tube.

[0055] (3) Measure the volume when transferring the aqueous phase, and then slowly add an equal volume of isopropanol (for example, add 500 μL of isopropanol to 500 μL of the transfer solution, and slowly invert to mix evenly;

[0056] (4) Precipitate the resulting solution at -20 °C overnight (at least precipitate for 1 h);

[0057] (5) Centrifuge the overnight-precipitated mixture at 12000 rpm at 4 °C for 20 minutes.

[0058] (6) Discard the supernatant. Resuspend the precipitate with 75% DEPC ethanol, and then centrifuge at 12000 rpm at 4 °C for 20 minutes;

[0059] (7) Discard the supernatant. Invert the EP tube to air dry. Dissolve the precipitate with 20 μL of DEPC water. Store the RNA in an ultra-low temperature freezer at -80 °C.

[0060] In this example, the stem-loop RT-qPCR method described above was used to measure the expression level of miR-145. The levels of miR-145 in the sera of healthy volunteers, sera of other mutant pancreatic cancer patients, and sera of pancreatic cancer patients with KRAS G12D mutation were compared. The results are shown in the appendix Figure 1 .

[0061] The results showed that compared with healthy people and pancreatic cancer patients with other mutation types, the level of serum miR-145 in pancreatic cancer patients with KRAS G12D mutation was significantly decreased, indicating that the detection of serum miR-145 can better reflect the mutation of KRAS G12D in pancreatic cancer patients.

[0062] Example 2

[0063] In this example, the correlation between the expression level of miR-145 and the KRAS G12D mutation was analyzed, and the presence or absence of the KRAS G12D mutation in pancreatic cancer patients was determined based on the expression level of miR-145.

[0064] In this example, pancreatic cancer patients with KRAS G12D mutation and low expression of miR-145 in serum and pancreatic cancer patients with other mutation types and high expression of miR-145 in serum were selected for survival analysis.

[0065] In this example, KRAS G12D mutation was diagnosed by histopathological examination, and the patient's survival time was defined as the period from the date of cancer diagnosis to the last follow-up or death. Whether and when the patient died was determined based on hospitalization and outpatient records, the patient's family, or follow-up calls. The data were analyzed using GraphPad Prism software. An independent sample t-test was used for comparison between groups, and repeated measurement data were used for comparison at each time point within the group, and repeated measurement analysis of variance was performed; Log-Rank test was used for survival analysis, and P<0.05 was considered statistically significant. The results are attached. Figure 2 .

[0066] The results showed that low expression of miR-145 in serum was significantly correlated with poor prognosis in patients with pancreatic cancer.

[0067] Example 3

[0068] In this example, the miRNA target prediction software miRanda v3.3a was used to verify the relationship between miR-145 and KRAS transcript NM_001369786.1. The Score Threshold was set to 120, the Energy Threshold was set to -1.000000 kcal / mol, and the binding with the lowest Energy Threshold, i.e., the lowest binding free energy, was selected. The specific operation and results are shown in the attached Figure 3 shown.

[0069] The results showed that there was a potential binding site between miR-145 and KRAS, and the binding free energy was -20.309999 kCal / Mol, which was relatively stable, suggesting the regulatory correlation between miR-145 and KRAS.

[0070] In summary, the above embodiments of the present invention verify that the down-regulation of miR-145 is closely related to the occurrence, development and prognosis of pancreatic cancer. Specifically, the down-regulation of miR-145 is considered to be a promoting factor for the proliferation, invasion and metastasis of pancreatic cancer cells. At the same time, miR-145 participates in a series of key biological processes, such as cell cycle regulation, epithelial-mesenchymal transition, angiogenesis and anti-apoptosis, by regulating the expression of multiple target genes. In particular, the down-regulation of miR-145 is associated with poor prognosis in patients with pancreatic cancer. The study found that the expression level of miR-145 is closely related to clinicopathological features such as the pathological type, grade and lymph node metastasis of pancreatic cancer. In addition, the expression level of miR-145 is also related to the survival rate and recurrence rate of patients with pancreatic cancer. Low expression of miR-145 is often associated with poor prognosis and increased recurrence risk.

[0071] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or variations derived therefrom still fall within the protection scope of the present invention.

Claims

1. Use of miR-145 as a diagnostic marker for pancreatic cancer.

2. The use according to claim 1, characterized in that, The pancreatic cancer includes KRAS G12D gene mutation.

3. The use according to claim 1 or 2, characterized in that, The miR-145 is derived from serum.

4. Use of miR-145 as a diagnostic marker for KRAS G12D gene mutation in pancreatic cancer.

5. Use of a substance and / or reagent for detecting miR-145 level in the preparation of a product for diagnosing or assisting in diagnosing, screening or assisting in screening pancreatic cancer.

6. Use of a substance and / or reagent for detecting miR-145 expression level in the preparation of a product for detecting the correlation and / or mutation expression level of KRAS G12D mutation in pancreatic cancer.

7. A product for the diagnosis or auxiliary diagnosis, screening or auxiliary screening of pancreatic cancer, characterized in that, The product includes a reagent for detecting the correlation and / or mutation level of KRAS G12D mutation in pancreatic cancer based on RT-qPCR method.

8. The product for the diagnosis or auxiliary diagnosis, screening or auxiliary screening of pancreatic cancer according to claim 7, characterized in that, The product includes a plasma kit.

9. The product for diagnosing pancreatic cancer or assisting in diagnosis, screening or assisting in screening according to claim 7 or 8, characterized in that, The reagent includes a stem-loop fluorescence quantitative PCR kit.

10. A system for diagnosing or assisting in the diagnosis, screening or assisting in the screening of pancreatic cancer, characterized in that, Comprising: A detection device for determining the correlation and / or mutation level of KRAS G12D mutation in pancreatic cancer in a biological sample; A judgment device for diagnosing or assisting in diagnosing, screening or assisting in screening whether a person to be tested has pancreatic cancer based on whether KRAS G12D in the biological sample is mutated or the mutation expression amount.

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