Use of a marker in the manufacture of a test product for the prognosis of cholangiocarcinoma

Abstract

The application belongs to the technical field of biology and medicine, and particularly relates to a use of a marker in preparation of a detection product for prognosis of cholangiocarcinoma. The marker is spermidine and / or GPRC5A. Based on the marker spermidine and / or GPRC5A provided in the application, the prognosis of cholangiocarcinoma is detected, and the detection accuracy of the prognosis of cholangiocarcinoma is high.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and pharmaceutical technology, and specifically relates to the use of a biomarker in the preparation of a detection product for the prognosis of cholangiocarcinoma. Background Technology

[0002] Cholangiocarcinoma (CCA) is a highly heterogeneous tumor with an extremely high degree of malignancy. Originating from intrahepatic or extrahepatic bile duct epithelial cells, CCA can be classified into intrahepatic cholangiocarcinoma (iCAA), hilar cholangiocarcinoma (pCCA), and distal cholangiocarcinoma (dCCA). While they exhibit different molecular and clinical phenotypes, they all share a common origin in the bile duct epithelium.

[0003] Because cholangiocarcinoma is highly invasive, has an insidious onset, and is often diagnosed at an advanced stage, the 5-year survival rate is still less than 10%. Cholangiocarcinoma is a malignant tumor originating from the bile duct epithelium. The biliary system is the first point of contact with cholangiocarcinoma. During the progression of cholangiocarcinoma, the composition of bile within the biliary system often undergoes significant changes.

[0004] Therefore, new diagnostic biomarkers for the prognosis of cholangiocarcinoma urgently need to be discovered in order to improve the early prediction of the prognosis of cholangiocarcinoma patients and prolong their survival. Summary of the Invention

[0005] This invention has discovered that spermidine can serve as a prognostic marker for cholangiocarcinoma, and its prognostic accuracy in cholangiocarcinoma is high.

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

[0007] In one aspect, this invention provides the use of a biomarker in the preparation of a detection product for the prognosis of cholangiocarcinoma, wherein the biomarker is spermidine.

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

[0009] Preferably, the marker also includes GPRC5A.

[0010] Preferably, the GPRC5A is derived from cancerous tissue.

[0011] Preferably, the above-mentioned detection products are reagents, kits, test strips, or diagnostic chips.

[0012] Preferably, the above reagents or kits are reagents or kits based on ELISA detection or PCR amplification detection.

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

[0014] The beneficial effects of this invention include:

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

[0016] (2) Based on the biomarker spermidine provided by the present invention, the combination of GPRC5A is used to detect the prognosis of cholangiocarcinoma. The detection is more accurate and better than using spermidine alone as a biomarker. Attached Figure Description

[0017] Figure 1 Standard curve for ELISA detection of spermidine;

[0018] Figure 2a The TGF-α shedding experiment was used to verify the activation effect of spermidine on GPRC5A;

[0019] Figure 2b To verify the activation effect of spermidine on GPRC5A in the Glosensor cAMP experiment;

[0020] Figure 3 The histochemical staining of GPRC5A microarray data for cholangiocarcinoma tissue;

[0021] Figure 4 To detect bile content in patients with cholangiocarcinoma and cholelithiasis using ELISA;

[0022] Figure 5a The relationship between GPRC5A expression and spermine content in bile and prognosis of intrahepatic cholangiocarcinoma (iCCA) patients with survival curves.

[0023] Figure 5b The relationship between GPRC5A expression and spermine content in bile in patients with bile duct cancer and prognosis (pCCA, gene mutation of propionyl-CoA carboxylase α associated with propionic acidemia) survival curves.

[0024] Figure 5c The relationship between GPRC5A expression and spermine content in bile and prognosis (dCCA, distal bile duct carcinoma) in patients with cholangiocarcinoma is shown in the survival curve. Detailed Implementation

[0025] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0026] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0027] This invention provides the use of a biomarker in the preparation of a detection product for the prognosis of cholangiocarcinoma, wherein the biomarker is spermidine.

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

[0029] In some specific examples, the aforementioned spermidine is derived from bile.

[0030] It should be noted that, in the application of this invention, the test sample can be bile, meaning that spermidine in this invention can be derived from bile. Furthermore, spermidine (SPD), a low-molecular-weight aliphatic carbide containing three amino groups, is one of the naturally occurring polyamines found in all organisms. Intracellular spermidine levels represent the final result of polyamine uptake from the extracellular space, endogenous biosynthesis, catabolism, and excretion. The precursor ornithine undergoes a strictly regulated stepwise reaction to form putrescine, spermidine, and spermine, thus achieving biosynthesis. In current research, spermidine has potential carcinogenic properties.

[0031] In some specific examples, the aforementioned markers also include GPRC5A.

[0032] It should be noted that G protein-coupled receptors (GPCRs) constitute the largest superfamily of membrane proteins in the human genome, with over 800 members. More than 40% of FDA-approved drugs target GPCRs. GPCR A (GPRC5A), a member of the C family, group 5, located on chromosome 12p13-p12.3, has been shown in existing studies to promote tumor progression through G protein signaling pathways and by recruiting β-arrestin. The prognostic detection of cholangiocarcinoma based on the spermidine biomarker provided in this invention, combined with GPRC5A, is more accurate and superior to using spermidine alone as a biomarker.

[0033] In some specific examples, the aforementioned GPRC5A originates from cancerous tissue.

[0034] In some specific examples, the aforementioned testing products are reagents, test kits, test strips, or diagnostic chips.

[0035] In some specific examples, the reagents or kits mentioned above are reagents or kits based on ELISA detection or PCR amplification detection.

[0036] It should be noted that this invention can detect the prognosis of cholangiocarcinoma by detecting the expression of spermidine and / or GPRC5A. The detection products for detecting the expression of spermidine and / or GPRC5A are reagents, kits, test strips, or diagnostic chips.

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

[0038] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0039] In the following example, the concentration of spermidine in bile was detected using an ELISA kit (purchased from Scientific Cloud Company). Details are as follows:

[0040] (1) Sample addition: Set up standard wells, blank wells (blank control wells do not contain sample or enzyme-labeled reagent, all other steps are the same), and sample wells. Add 50 μL of sample diluent to the standard wells of the enzyme-labeled plate. Add 40 μL of sample diluent to the sample wells, and then add 10 μL of the sample to be tested (the final sample dilution is 5 times). Add the sample to the bottom of the wells of the enzyme-labeled plate, trying not to touch the well walls, and gently shake to mix.

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

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

[0043] (4) Solution preparation: Dilute the 30-fold concentrated washing solution with distilled water 30 times and set aside.

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

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

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

[0047] (8) Measurement: Zero the blank well and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm (the measurement should be performed within 15 minutes after adding the stop solution).

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

[0049] In the following example, the specific steps for tissue chip fabrication are as follows:

[0050] (1) Preparation of paraffin blocks: Part of the patient's cancerous tissue was fixed in 4% paraformaldehyde, embedded into paraffin blocks using a paraffin embedding machine, sectioned, and stained with hematoxylin and eosin (HE). The location of the cancerous tissue was located under a microscope, and the extent of the tumor tissue was marked. One well was collected from each tissue block;

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

[0052] (3) Punching the paraffin block: Based on the positioning results in step 1, the tissue core is collected from the paraffin block of each patient using a fine needle punch with a hole diameter of 1.5 mm.

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

[0054] (5) Tissue sectioning: The tissue array block was trimmed using a fully automatic tissue slicer at an infeed speed of 20 micrometers / revolution until 80% of the tissue core was fully exposed; the tissue array block was sectioned using a fully automatic tissue slicer at an infeed speed of 4 micrometers / revolution, and the sections were mounted on imported glass slides with high adsorption; the array sections were placed in a constant temperature oven at 60℃ and baked for 12 hours.

[0055] (6) Quality inspection: After the tissue microarray is fabricated, HE staining is performed first, and a pathologist performs quality inspection on each tissue sample in the tissue microarray.

[0056] (7) Preservation: The tissue microarray experimental white slides are stored in the slide box and kept in the refrigerator at 4°C; the used pathological tissue paraffin blocks and tissue array blocks are sealed with wax and returned to the paraffin block cabinet and slide box along with the corresponding HE stained slides, and stored at room temperature.

[0057] In the following example, the steps for tissue microarray immunohistochemical staining and scoring are as follows:

[0058] (1) Dewaxing: Place the tissue chip white slides on the slide rack and bake them in a constant temperature oven at 60°C for 1 hour. Then soak them in xylene for 10 minutes and then transfer them to another staining vat filled with xylene for another 10 minutes.

[0059] (2) Hydration: Immerse the chip in a gradient alcohol solution of 100%, 95%, 85%, and 75% for 5 minutes each, and rinse with running water for 5 minutes.

[0060] (3) Antigen retrieval: The chip was placed in an antigen retrieval solution containing EDTA (pH=9.0) and placed in a water bath at 100℃ for 10 minutes for heat antigen retrieval; after cooling to room temperature, it was washed 3 times with PBS for 5 minutes each time.

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

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

[0063] (6) Secondary antibody incubation: Take out the chip after overnight incubation at 4°C, rinse it with PBS 3 times for 5 minutes each time; wipe off the liquid around the slice, add about 50 μL of secondary antibody working solution, incubate at room temperature for about 30 minutes, remove the secondary antibody and wash with PBS 3 times for 5 minutes each time.

[0064] I. Biomarker Screening

[0065] Previous studies have found that GPRC5A expression can serve as an independent prognostic marker for cholangiocarcinoma (patent application ZL202210610026.2), and spermidine can act as an effective activating ligand for GPRC5A, activating the Gαi and Gαq pathways of GPRC5A. Figure 2a and Figure 2b ).

[0066] In addition, the expression of GPRC5A in cancer tissue was detected by the above-mentioned histochemical staining method. The results showed that GPRC5A was abnormally elevated in cancer tissue. Figure 3 ).

[0067] Furthermore, the spermidine content in the bile of patients with cholangiocarcinoma was detected using the aforementioned ELISA method, and the results showed that spermidine levels were abnormally elevated in the bile of these patients. Figure 4 (where C represents the tumor group and N represents the bile duct stone group).

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

[0069] II. Marker Validation

[0070] Tissue microarrays were prepared from cancerous tissue samples from 307 patients with cholangiocarcinoma. The GPRC5A expression levels in the tissue microarrays were grouped into high and low expression groups based on histochemical staining. Correspondingly, spermidine levels in the bile were grouped into high and low content groups. Prognostic analysis revealed that high spermidine levels and high GPRC5A expression indicated a worse prognosis for cholangiocarcinoma. Figure 5a , Figure 5b and Figure 5c The black curve represents low GPRC5A expression and low spermidine content, the blue curve represents only low GPRC5A expression or only low spermidine content, and the red curve represents high GPRC5A expression and high spermidine content. The black curve represents low GPRC5A expression and low spermidine content, which has a better predictive effect than the high expression or high content groups alone.

[0071] In addition, receiver operating characteristic (ROC) curves were plotted for spermidine, GPRC5A, and the combination of the two. The AUC value for spermidine alone was 0.7763, the AUC value for GPRC5A alone was 0.7658, and the AUC value for the combination of the two 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 not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The use of a biomarker in the preparation of a diagnostic product for the prognosis of cholangiocarcinoma, wherein the biomarker is spermidine derived from bile.

2. The use according to claim 1, characterized in that, The markers also include GPRC5A.

3. The use according to claim 2, characterized in that, GPRC5A originates from cancerous tissue.

4. The use according to any one of claims 1 to 3, characterized in that, The testing products are reagents, kits, test strips, or diagnostic chips.

5. The use according to claim 4, characterized in that, The reagents or kits are based on ELISA detection or PCR amplification detection.

6. The use according to claim 1, 2, 3 or 5, characterized in that, The prognosis for cholangiocarcinoma is intrahepatic cholangiocarcinoma, propionyl-CoA carboxylase α gene mutation associated with propionic acidemia, or distal cholangiocarcinoma.

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