Application of marker in preparation of detection product for prognosis of bile duct cancer

Abstract

The invention belongs to the technical field of biology and medicine, and particularly relates to application of a marker in preparation of a detection product for prognosis of bile duct cancer. The marker is spermidine and / or GPRC5A (Glutathione Protein Receptor 5A). On the basis of the marker spermidine and / or GPRC5A provided by the invention, the prognosis of the cholangiocarcinoma is detected, and the detection accuracy of the prognosis of the cholangiocarcinoma is high.

Description

Technical Field

[0001] The present invention belongs to the technical field of biology and medicine, and particularly relates to the use of a biomarker in the preparation of a detection product for cholangiocarcinoma prognosis. Background Art

[0002] Cholangiocarcinoma (CCA) is a highly heterogeneous tumor with extremely high malignancy. Cholangiocarcinoma (CCA) originates from intrahepatic or extrahepatic bile duct epithelial cells and can be divided into intrahepatic cholangiocarcinoma (iCAA), hilar cholangiocarcinoma (pCCA), and distal cholangiocarcinoma (dCCA). They have different molecular and clinical phenotypes but share a common origin from biliary epithelium.

[0003] Due to the strong invasiveness, insidious onset, and often advanced stage of the disease at the time of diagnosis of cholangiocarcinoma, the 5-year survival rate of cholangiocarcinoma is still less than 10%. Cholangiocarcinoma is a malignant tumor originating from biliary epithelium. The biliary system is the first to contact cholangiocarcinoma, and during the progression of cholangiocarcinoma, the content of bile components in the biliary system often changes significantly, and the components in bile.

[0004] Therefore, new diagnostic markers for cholangiocarcinoma prognosis are urgently needed to be studied and discovered to improve the early prediction of the prognosis of cholangiocarcinoma patients and extend their survival period. Summary of the Invention

[0005] The present invention has discovered that spermidine can be used as a biomarker for cholangiocarcinoma prognosis, with high detection accuracy for cholangiocarcinoma prognosis.

[0006] To achieve the above object, the present invention can adopt the following technical solutions:

[0007] On the one hand, the present invention provides the use of a biomarker in the preparation of a detection product for cholangiocarcinoma prognosis, and the biomarker is spermidine.

[0008] Preferably, the above-mentioned spermidine is derived from bile.

[0009] Preferably, the above-mentioned biomarker further includes GPRC5A.

[0010] Preferably, the above-mentioned GPRC5A is derived from cancer tissue.

[0011] Preferably, the above-mentioned detection product is a reagent, a kit, a test strip, or a diagnostic chip.

[0012] Preferably, the above-mentioned reagent or kit is a reagent or kit based on ELISA detection or PCR amplification detection.

[0013] Preferably, the above-mentioned cholangiocarcinoma prognosis is intrahepatic cholangiocarcinoma, a gene mutation of propionyl-CoA carboxylase α related to propionic acidemia, or distal cholangiocarcinoma.

[0014] The beneficial effects of the present invention include:

[0015] (1) Based on the biomarker spermidine provided by the present invention for detecting the prognosis of cholangiocarcinoma, the higher the content of spermidine, the worse the prognosis of cholangiocarcinoma, and the detection accuracy of cholangiocarcinoma prognosis is high.

[0016] (2) Based on the biomarker spermidine provided by the present invention in combination with GPRC5A for detecting the prognosis of cholangiocarcinoma, the detection is more accurate, which is superior to using spermidine alone as a biomarker for detection. Brief Description of the Drawings

[0017] Figure 1 is the standard curve for detecting spermidine by Elisa;

[0018] Figure 2a is the verification of the activation effect of spermidine on GPRC5A by TGF-α shedding experiment;

[0019] Figure 2b is the verification of the activation effect of spermidine on GPRC5A by Glosensor cAMP experiment;

[0020] Figure 3 is the immunohistochemical staining of GPRC5A in the cholangiocarcinoma tissue microarray;

[0021] Figure 4 is the detection of the bile content in cholangiocarcinoma patients and cholelithiasis patients by ELISA;

[0022] Figure 5a is the survival curve of the relationship between the expression of GPRC5A and the content of spermidine in bile and the prognosis of cholangiocarcinoma patients (iCCA, intrahepatic cholangiocarcinoma);

[0023] Figure 5b is the survival curve of the relationship between the expression of GPRC5A and the content of spermidine in bile and the prognosis of cholangiocarcinoma patients (pCCA, gene mutation of propionyl-CoA carboxylase α related to propionic acidemia);

[0024] Figure 5c is the survival curve of the relationship between the expression of GPRC5A and the content of spermidine in bile and the prognosis of cholangiocarcinoma patients (dCCA, distal cholangiocarcinoma). Detailed Embodiments

[0025] The examples given are for better illustration of the present invention, but the content of the present invention is not limited to the examples given. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation scheme according to the above invention content still fall within the protection scope of the present invention.

[0026] The terms used in this document are only for describing specific embodiments and are not intended to limit the present disclosure. Unless there is an obviously different meaning in the context, the expressions in the singular form include those in the plural form. As used herein, it should be understood that terms such as "including", "having", "containing" are intended to indicate the existence of features, numbers, operations, components, parts, elements, materials or combinations. Terms of the present invention are disclosed in the specification, and it is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or their combinations may exist or can be added. As used herein, depending on the circumstances, " / " can be interpreted as "and" or "or".

[0027] An embodiment of the present invention provides the use of a biomarker in the preparation of a detection product for the prognosis of cholangiocarcinoma, and the biomarker is spermidine.

[0028] It should be noted that based on the biomarker spermidine provided by the present invention for detecting the prognosis of cholangiocarcinoma, the higher the content of spermidine, the worse the prognosis of cholangiocarcinoma, and the detection accuracy of the prognosis of cholangiocarcinoma is high.

[0029] In some specific examples, the above-mentioned spermidine is derived from bile.

[0030] It should be noted that in the use of the present invention, the test sample can be bile, that is, the spermidine in the present invention can be derived from bile. In addition, spermidine (SPD), a low molecular weight aliphatic carbide containing three amino groups, is one of the natural polyamines present in all organisms. The content of spermidine in cells is the final result of the uptake of polyamines from the extracellular space, endogenous biosynthesis, catabolism and excretion. The precursor ornithine forms putrescine, spermidine and spermine in strictly regulated stepwise reactions to achieve biosynthesis. In current research, spermidine has potential carcinogenic characteristics.

[0031] In some specific examples, the above-mentioned biomarker further includes GPRC5A.

[0032] It should be noted that G protein-coupled receptors are the largest membrane protein superfamily in the human genome, with more than 800 family members. Among the drugs approved by the FDA, more than 40% of the drugs target GPCRs. G protein-coupled receptor, family C, group 5, member A (GPRC5A), is located on chromosome 12p13-p12.3. According to existing research, GPRC5A can promote tumor progression through the G protein signaling pathway and recruitment of β-arrestin. Based on the biomarker spermidine provided by the present invention in combination with GPRC5A for detecting the prognosis of cholangiocarcinoma, the detection is more accurate, which is better than using spermidine alone as a biomarker for detection.

[0033] In some specific examples, the above-mentioned GPRC5A is derived from cancer tissue.

[0034] In some specific examples, the above detection product is a reagent, a kit, a test strip or a diagnostic chip.

[0035] In some specific examples, the above reagent or kit is a reagent or kit based on ELISA detection or PCR amplification detection.

[0036] It should be noted that the present invention can detect the prognosis of cholangiocarcinoma by detecting the expression of spermidine and / or GPRC5A, and the detection product for detecting the expression of spermidine and / or GPRC5A is a reagent, a kit, a test strip or a diagnostic chip.

[0037] In some specific examples, the above cholangiocarcinoma prognosis is intrahepatic cholangiocarcinoma, gene mutation of propionyl-CoA carboxylase α related to propionic acidemia or distal cholangiocarcinoma.

[0038] To better understand the present invention, the content of the present invention will be further clarified below in combination with specific examples, but the content of the present invention is not limited to the following examples.

[0039] In the following examples, an Elisa reagent kit (the Elisa kit was purchased from Keyan Cloud Company) was used to detect the concentration of spermidine in bile, specifically as follows:

[0040] (1) Sampling: Standard wells, blank wells (no sample and enzyme-labeled reagent are added to the blank control wells, and the remaining operations are the same), and wells for samples to be tested were set up respectively. Add 50 μl to the standard wells on the enzyme-labeled coated plate. First add 40 μl of sample diluent to the wells for samples to be tested, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). When sampling, add the sample to the bottom of the wells on the enzyme-labeled plate, try not to touch the well walls, and gently shake and mix;

[0041] (2) Adding enzyme: Add 50 μl of enzyme-labeled reagent to each well, except for the blank wells;

[0042] (3) Incubation: Seal the plate with a sealing film and incubate at 37 °C for 60 minutes;

[0043] (4) Preparation of solution: Dilute the 30-fold concentrated washing solution 30-fold with distilled water and set aside;

[0044] (5) Washing: Carefully remove the sealing film, discard the liquid, drain, fill each well with washing solution, let stand for 30 seconds and then discard, repeat this 5 times, and pat dry;

[0045] (6) Color development: First add 50 μl of chromogenic agent A to each well, then add 50 μl of chromogenic agent B, gently shake and mix, and develop color at 37 °C in the dark for 15 minutes;

[0046] (7) Termination: Add 50 μl of termination solution to each well to terminate the reaction (at this time, the blue immediately turns yellow);

[0047] (8) Determination: Zero the blank wells, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm (the determination should be carried out within 15 minutes after adding the stop solution).

[0048] The ELISA standard curve constructed according to the above method is as Figure 1 shown, where Y = 0.1684X^2 - 1.2483X + 2.3256R 2 = 0.999.

[0049] In the following examples, the specific steps for making tissue chips are as follows:

[0050] (1) Paraffin block preparation: Put a part of the patient's cancer tissue into 4% paraformaldehyde for fixation, embed it into a paraffin block with a paraffin embedding machine, perform HE staining after sectioning, and find the location of the cancer tissue and mark the scope of the tumor tissue under the microscope. Collect 1 well for each tissue paraffin block;

[0051] (2) Chip design: Design the tissue types (CCA tissue, normal tissue) and arrangement patterns of the tissue microarray according to the experimental purpose;

[0052] (3) Punching paraffin blocks: According to the positioning results in step 1, collect tissue cores on the tissue paraffin blocks of each patient with a fine needle punching machine, and the pore diameter is 1.5 mm;

[0053] (4) Assembly of tissue array blocks: Select qualified tissue cores and number them; prepare a suitable blank receptor paraffin block with a tissue embedding machine, and arrange the tissue cores regularly on the blank receptor paraffin block according to the array design in step (2) with a tissue array instrument; heat and fuse the tissue array block in a 52°C constant temperature oven to make the tissue cores tightly connected to the receptor paraffin block.

[0054] (5) Tissue sectioning: Trim the tissue array block with a fully automatic tissue slicer at a feed speed of 20 microns / rotation until 80% of the tissue cores are completely exposed; slice the tissue array block with a fully automatic tissue slicer at a feed speed of 4 microns / rotation, attach the slices to imported high-absorbency glass slides; place the array slices in a 60°C constant temperature oven for baking for 12 hours;

[0055] (6) Quality inspection: After the tissue chip is made, first perform HE staining, and a professional pathologist conducts quality inspection on each tissue sample in the tissue chip;

[0056] (7) Preservation: The blank slides of the tissue chip experiment are stored in a slide box and placed in the 4°C refrigerator for preservation; perform wax sealing treatment on the used pathological tissue paraffin blocks and tissue array blocks, and return them to the wax block cabinet and slide box together with the corresponding HE-stained sections for storage at room temperature.

[0057] In the following examples, the steps for immunohistochemical staining and scoring of tissue microarrays are as follows:

[0058] (1) Deparaffinization: Place the blank tissue microarray slides on the slide rack, bake them in an incubator at 60 °C for 1 hour, then immerse them in xylene. After 10 minutes, transfer them to another xylene-filled staining jar and soak for another 10 minutes;

[0059] (2) Hydration: Immerse the microarray in gradient alcohol solutions of 100%, 95%, 85%, and 75% for 5 minutes each in sequence, and rinse with running water for 5 minutes;

[0060] (3) Antigen retrieval: Place the microarray in an antigen retrieval solution containing EDTA (pH = 9.0), and perform heat antigen retrieval in a pressure cooker at 100 °C for 10 minutes in a water bath; after cooling to room temperature, wash with PBS 3 times, 5 minutes each time;

[0061] (4) Enzyme inactivation: Drop 3% H2O2 and incubate at room temperature for 15 minutes to eliminate the activity of endogenous peroxidase in the tissue, then wash with PBS 3 times, 5 minutes each time;

[0062] (5) Primary antibody incubation: Drop 5% imported goat serum and let it stand at room temperature for 30 minutes, then remove the blocking solution. Drop the diluted WDRS primary antibody working solution at a dilution ratio of 1:10, place it in a wet box, and incubate overnight at 4 °C;

[0063] (6) Secondary antibody incubation: Take out the microarray incubated overnight at 4 °C, rinse it with PBS 3 times, 5 minutes each time; wipe off the liquid around the section, drop about 50 μl of the secondary antibody working solution, incubate at room temperature for about 30 min, discard the secondary antibody, and wash with PBS 3 times, 5 minutes each time.

[0064] I. Biomarker screening

[0065] In previous studies, it was found that the expression of GPRC5A can be used as an independent prognostic biomarker for cholangiocarcinoma (patent application ZL202210610026.2 has been filed), and spermidine can be used as an effective activating ligand for GPRC5A, which can activate the Gαi and Gαq pathways of GPRC5A ( Figure 2a and Figure 2b ).

[0066] In addition, by detecting the expression of GPRC5A in cancer tissues through the above immunohistochemical staining method, the results showed that the expression of GPRC5A was abnormally elevated in cancer tissues ( Figure 3 ).

[0067] Furthermore, by detecting the content of spermidine in the bile of cholangiocarcinoma patients through the above ELISA method, it was found that the content of spermidine was abnormally elevated in the bile of cholangiocarcinoma patients ( Figure 4 , where C is the tumor group and N is the biliary stone group).

[0068] All of the above indicate that spermidine and GPRC5A predict a poor prognosis in patients with cholangiocarcinoma.

[0069] II. Marker Verification

[0070] Tissue microarrays were prepared from partial cancer tissues of 307 cholangiocarcinoma patients. The patients with cholangiocarcinoma in the tissue microarrays were grouped into high-expression and low-expression groups according to the immunohistochemical staining of GPRC5A, and the spermidine in the paired bile was grouped into high-content and low-content groups. Prognostic analysis was performed on this, and it was found that the high-content spermidine and high-expression GPRC5A group indicated a worse prognosis of cholangiocarcinoma ( Figure 5a , Figure 5b and Figure 5c , where the black curve represents low expression of GPRC5A and low content of spermidine, the blue represents the group with only low expression of GPRC5A or only low content of spermidine, and the red represents the group with high expression of GPRC5A and high content of spermidine), which had a better prediction effect than the single high-expression or high-content group.

[0071] In addition, the receiver operating characteristic curves of spermidine, GPRC5A, and their combination were plotted respectively. Among them, the AUC value of spermidine alone was 0.7763, the AUC value of GPRC5A alone was 0.7658, and the AUC value of their combination was 0.8698.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. Use of a marker in the preparation of a detection product for the prognosis of cholangiocarcinoma, wherein the marker is spermidine.

2. The use according to claim 1, characterized in that Spermidine is derived from bile.

3. The use according to claim 1 or 2, characterized in that Markers also include GPRC5A.

4. The use according to claim 3, characterized in that GPRC5A is derived from cancer tissues.

5. The use according to any one of claims 1 to 4, characterized in that The detection products are reagents, test kits, test strips or diagnostic chips.

6. The use according to claim 5, characterized in that The reagent or kit is a reagent or kit based on ELISA detection or PCR amplification detection.

7. The use according to any one of claims 1 to 6, characterized in that The prognosis of cholangiocarcinoma is intrahepatic cholangiocarcinoma, propionyl-CoA carboxylase α gene mutation associated with propionic acidemia, or distal cholangiocarcinoma.

Images