Application of peripheral blood leukocyte RNA combined markers in the preparation of a kit for detecting or diagnosing early primary gallbladder cancer

Abstract

The present invention discloses the use of peripheral blood leukocyte RNA combined markers in the preparation of a kit for detecting or diagnosing early primary gallbladder cancer. The present invention is based on peripheral blood leukocyte RNA markers for detection. The marker is a liquid biopsy biomarker that is more sensitive, stable, non-invasive, and requires less sample volume, which is more acceptable to patients. Its successful development will help the diagnosis of gallbladder cancer. The etiology of primary gallbladder cancer is complex. Compared with a single molecular marker, the present invention evaluates the accuracy of the test sample from two dimensions of abnormally high expression and low expression, which can reduce the error caused by individual expression differences of a single indicator to a certain extent, making the result more accurate.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and medical diagnosis, and specifically relates to the application of peripheral blood leukocyte RNA combined markers in the preparation of a kit for detecting or diagnosing early primary gallbladder cancer. Background Art

[0002] Primary gallbladder cancer (GBC) is a type of malignant tumor that originates from the gallbladder and is highly heterogeneous and invasive. GBC is also a common malignant tumor of the biliary system, ranking sixth in the incidence of digestive tract malignancies. It is highly malignant and has a poor prognosis, with a 5-year survival rate of less than 5%. Due to the particularity of the anatomical location of the gallbladder and the ambiguity and non-specificity of gallbladder cancer symptoms, GBC patients are usually in the late stage when first diagnosed and miss the best time for treatment; while GBC patients who undergo surgical intervention at an early stage have a 5-year survival rate of up to 90%; therefore, early detection of GBC is very necessary. At present, the detection of GBC mainly relies on imaging, serum CEA, CA19-9, etc., with limited sensitivity and specificity. Therefore, it is urgent to develop a new and rapid biomarker for early detection of gallbladder cancer in clinical practice.

[0003] Peripheral blood leukocytes (WBCs) are a type of colorless spherical blood cells that participate in the body's immunity and eliminate endogenous mutant cells and exogenous pathogenic microorganisms by phagocytosis and antibody production. The occurrence and development of tumors are related to changes in the body's immune system. Tumor cells induce the immune system to produce immune tolerance, thereby breaking the balance between tumors and immunity, allowing tumor cells to break free from immune control, which also changes the gene expression changes and functions of immune cells in tumor patients. Therefore, the early screening model constructed based on the detection of changes in peripheral blood leukocyte RNA levels can perform specific detection in the early stages of tumor formation.

[0004] The present invention is based on the detection of peripheral blood leukocyte combined markers for the early screening and diagnosis kit of primary gallbladder cancer, which has the significant advantages of good specificity, high sensitivity and low cost. Summary of the invention

[0005] The first object of the present invention is to provide a peripheral blood leukocyte RNA composition for use as a primary gallbladder cancer marker in view of the deficiencies in the prior art. The peripheral blood leukocyte RNA composition includes KATNBL1, PTTG2, ATP6V1B2, RESF1 and CSRNP1.

[0006] Preferably, KATNBL1, PTTG2, ATP6V1B2, and RESF1 are highly expressed in primary gallbladder cancer;

[0007] Preferably, CSRNP1 is lowly expressed in primary gallbladder cancer.

[0008] The second object of the present invention is to provide the use of peripheral blood leukocyte RNA combined markers in the preparation of an early primary gallbladder cancer detection kit, wherein the peripheral blood leukocyte RNA composition includes KATNBL1, PTTG2, ATP6V1B2, RESF1 and CSRNP1.

[0009] The third object of the present invention is to provide a GBC detection kit for detecting or diagnosing patients with primary gallbladder cancer, wherein the kit comprises a detection reagent for specifically detecting a human peripheral blood leukocyte RNA composition; the peripheral blood leukocyte RNA composition comprises KATNBL1, PTTG2, ATP6V1B2, RESF1 and CSRNP1.

[0010] Preferably, the detection reagent is a probe or primer for specifically detecting the peripheral blood leukocyte RNA composition.

[0011] The fourth object of the present invention is to provide a diagnostic preparation for primary gallbladder cancer, comprising peripheral blood leukocyte joint markers KATNBL1, PTTG2, ATP6V1B2, RESF1 and CSRNP1.

[0012] The beneficial effects of the present invention are as follows:

[0013] 1. The present invention adopts liquid biopsy technology and proposes a specific peripheral blood leukocyte RNA composition as a marker to detect or diagnose early primary gallbladder cancer. The method is relatively sensitive, stable, and non-invasive, and requires a small amount of sample, which is more easily accepted by patients. Its successful development will contribute to the diagnosis of gallbladder cancer.

[0014] 2. The present invention is based on the detection of multiple peripheral blood leukocyte combined markers. Compared with a single molecular marker, it evaluates the accuracy of the test samples from two dimensions: abnormally high expression and low expression. It can reduce the error caused by individual expression differences of a single indicator to a certain extent, make the results more accurate, and overcome the problem of difficulty in detection or diagnosis due to the complex causes of primary gallbladder cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The detection results of peripheral blood leukocyte marker KATNBL1 for identifying primary gallbladder cancer patients were analyzed for ROC curve analysis.

[0016] Figure 2 ROC curve analysis was performed to identify the detection results of patients with primary gallbladder cancer using the peripheral blood leukocyte marker PTTG2.

[0017] Figure 3ROC curve analysis was performed to identify the detection results of patients with primary gallbladder cancer using the peripheral blood leukocyte marker ATP6V1B2.

[0018] Figure 4 ROC curve analysis was performed to identify the detection results of primary gallbladder cancer patients using the peripheral blood leukocyte marker RESF1.

[0019] Figure 5 The detection results of peripheral blood leukocyte marker CSRNP1 for identifying primary gallbladder cancer patients were analyzed for ROC curve analysis.

[0020] Figure 6 ROC curve analysis was performed to identify the detection results of primary gallbladder cancer patients using peripheral blood leukocyte combined markers.

[0021] Figure 7 The ROC curve was used to analyze the test results of using the traditional gallbladder cancer serum marker carbohydrate antigen 19-9 (CA19-9) to identify patients with primary gallbladder cancer. DETAILED DESCRIPTION

[0022] The present invention is further analyzed below in conjunction with specific embodiments.

[0023] The kit models used in the embodiments of the present invention are as follows:

[0024] Human peripheral blood leukocyte separation kit: PGB2001, Beyotime, China;

[0025] Human peripheral blood leukocyte RNA extraction kit: PGB2002, Beyotime, China;

[0026] RNA reverse transcription kit: 4368813, ABI, United States;

[0027] Q-PCR kit: 11202ES08, Yeasen, China.

[0028] In the embodiment of the present invention, the instrument for detecting the human peripheral blood leukocyte joint markers is a Q-PCR instrument.

[0029] The KATNBL1, PTTG2, ATP6V1B2, RESF1, and CSRNP1 gene sequences of the present invention can be obtained through the website https: / / www.ncbi.nlm.nih.gov / .

[0030] The molecular detection primers and internal reference primers used in the present invention for detecting peripheral blood leukocyte RNA joint markers KATNBL1, PTTG2, ATP6V1B2, RESF1, CSRNP1 have the following sequences:

[0031] KATNBL1 F: 5'-GATATTTCCACACTGGTTTCCC-3'

[0032] KATNBL1 R: 5'-ACTCTTCCATGTATCATCCC-3'

[0033] PTTG2 F: 5'-CCCTCTGCAAGTTTCAGG-3'

[0034] PTTG2 R: 5'-TGTAGCATCTCAAATCCATCTG-3'

[0035] ATP6V1B2 F: 5'-ACTTCGTAAAGTGATCTATCGCAG-3'

[0036] ATP6V1B2 R: 5'-TCATGGTCCTGAGAAACCTC-3'

[0037] RESF1 F: 5'-CGTTGCTGTGATTCTGTTGG-3'

[0038] RESF1 R: 5'-GTAGGGAAGGAAACAAACAAGG-3'

[0039] CSRNP1 F: 5'-GAAAGACGAGGAGTACGAGC-3'

[0040] CSRNP1 R:5'-CAAGTGGAGTGGAGTCTGC-3'

[0041] Internal reference primer sequence:

[0042] HPRT1 F: 5'-GCCATCACATTGTAGCCCTC-3'

[0043] HPRT1 R: 5'-CGTCATTACAATAGCTCTTCAGTC-3'

[0044] Example 1 Subjects and blood sample extraction

[0045] The samples were provided by the Affiliated Hospital of Hangzhou Normal University (Ethics Approval No.: 2022(E2)-KS-068). Among them, there were 38 patients with primary gallbladder cancer and 13 healthy controls. 5 ml of peripheral blood was collected from the subjects with a disposable syringe, quickly transferred into an EDTA anticoagulant tube, and mixed.

[0046] Example 2 Isolation of peripheral blood leukocytes

[0047] Leukocytes were separated according to the operating instructions of the human peripheral blood leukocyte separation kit (PGB2001, Beyotime, China), with the following specific steps: 1) add 10 mL of reagent A to a 15 mL centrifuge tube for later use, use a 1 mL pipette tip with a filter to draw 1 mL of anticoagulated blood, and slowly inject it into the 15 mL centrifuge tube along the tube wall; 2) cover the tube, turn it upside down 5 times, let it stand for 5 minutes, then turn it upside down 5 times again, let it stand for 5 minutes, and centrifuge it at room temperature and 500 g for 5 minutes; 3) remove the waste liquid, add 10 ml of reagent B to resuspend, and centrifuge it at room temperature and 500 g for 5 minutes; 4) remove the waste liquid, add 700 μL of reagent C to the white cell precipitate at the bottom of the centrifuge tube, and repeatedly blow and beat 10 times until the precipitate is completely dissolved; 5) store at -80°C.

[0048] Example 3 Extraction of RNA from peripheral blood leukocytes

[0049] Human peripheral blood RNA was extracted according to the operating instructions of the Human Peripheral Blood Leukocyte RNA Extraction Kit (PGB2002, Beyotime, China). The specific steps are: 1) Take the white blood cell sample in Example 2 out of the -80°C refrigerator and completely thaw it at room temperature; 2) Add 280μL DEPC water, vortex mix for 15 seconds, let stand at room temperature for 10 minutes, and centrifuge at 12000g for 15 minutes; 3) After centrifugation, draw 850μL of supernatant into a new 1.5mL centrifuge tube, add 5μL of 4-bromoanisole, vortex mix for 15 seconds, let stand at room temperature for 5 minutes, and centrifuge at 12000g for 10 minutes; 4) After centrifugation, Transfer 700 μL of supernatant to a new 1.5 mL low-absorption centrifuge tube, add an equal volume of isopropanol and mix by inversion 20 times, and place in a -80°C refrigerator for 30 minutes; 5) Take out and centrifuge at 12000g for 30 minutes after complete thawing; 6) Remove the supernatant, add 1 mL of 75% alcohol to wash once, and centrifuge at 12000g for 4 minutes; 7) Discard the supernatant, dry in a fume hood for 1 minute, add 20 μL of DEPC water and blow 10-20 times to dissolve the RNA, and immediately place on ice. The extracted RNA was tested for concentration using NanoDrop2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE).

[0050] Example 4 Early screening prediction of primary gallbladder cancer using peripheral blood leukocyte combined markers

[0051] The extracted RNA was reverse transcribed using an RNA reverse transcription kit (4368813, ABI, United States). The Q-PCR kit (11202ES08, Yeasen, China) and specific primers were used to detect the gene expression levels of KATNBL1, PTTG2, ATP6V1B2, RESF1, CSRNP1 and the internal reference gene HPRT1 in 38 gallbladder cancer samples and 13 healthy samples, and the ROC curve was drawn. The ROC curve analysis showed that the detection results of peripheral blood leukocyte markers KATNBL1, PTTG2, ATP6V1B2, RESF1 and CSRNP1 alone in identifying primary gallbladder cancer patients were as follows: Figure 1-5 As shown in the figure; ROC curve analysis of the detection results of peripheral blood leukocyte combined markers and traditional gallbladder cancer serum marker carbohydrate antigen 19-9 (CA19-9) in identifying primary gallbladder cancer patients are shown in the figure. Figure 6 and Figure 7 shown.

[0052] The analysis showed that when KATNBL1, PTTG2, ATP6V1B2, RESF1, and CSRNP1 were used as combined markers in peripheral blood leukocytes, the AUC value was 1.00 ( Figure 6 ). The AUC of the serological marker carbohydrate antigen 19-9 (CA19-9) for distinguishing GBC group from non-GBC patients was only 0.77 ( Figure 7 ).

[0053] The above embodiments are not limitations of the present invention, and the present invention is not limited to the above embodiments. As long as the requirements of the present invention are met, they belong to the protection scope of the present invention.

Claims

1. Use of a peripheral blood leukocyte RNA composition in the preparation of a kit for diagnosing or detecting early primary gallbladder cancer, characterized in that: The peripheral blood leukocyte RNA composition consists of KATNBL1, PTTG2, ATP6V1B2, RESF1 and CSRNP1.

2. A kit for detecting or diagnosing early primary gallbladder cancer, characterized in that: The kit comprises a detection reagent for specifically detecting a human peripheral blood leukocyte RNA composition; the peripheral blood leukocyte RNA composition consists of KATNBL1, PTTG2, ATP6V1B2, RESF1 and CSRNP1.

3. The kit according to claim 2, characterized in that The detection reagent includes a probe or primer for specifically detecting the RNA composition of peripheral blood leukocytes.

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