Application of Ghana seed lectin I in the preparation of liver cancer diagnostic kits

Abstract

This invention provides the application of Ghana seed lectin I (GSL-I) in the preparation of liver cancer diagnostic kits. This invention is the first to determine that GSL-I can differentially bind to α-1,3-galactosidase on the surface of liver cancer and adjacent tissues. Therefore, the GSL-I obtained by screening in this invention can distinguish between hepatocellular carcinoma and adjacent tissues with a sensitivity of 71.43% and a specificity of 85.71%, and can be used as a potential means for clinical diagnosis of liver cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to the application of Ghana seed lectin I in the preparation of liver cancer diagnostic kits. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common primary liver cancers and a deadly malignant tumor in humans. It is the third leading cause of cancer death worldwide, with an overall 5-year survival rate of as low as 18%. The incidence and mortality rates of HCC have been rising rapidly globally in recent years, placing a significant public health burden on society. When HCC is diagnosed in its early stages, it can be treated with methods such as surgical resection. However, over 60% of patients are diagnosed at an advanced stage where the tumor is unresectable, making it difficult to apply the conventional treatments described above. Besides liver ultrasound imaging, the routine clinical screening strategy for liver cancer is serum alpha-fetoprotein (AFP) testing; however, its sensitivity and specificity are low, making it crucial for doctors and patients to use more sensitive and specific diagnostic markers for HCC.

[0003] Therefore, it is necessary to develop a new biomarker with high sensitivity and specificity for the diagnosis of liver cancer. Summary of the Invention

[0004] The purpose of this invention is to provide the application of Griffonia Simplicifolia Lectin I (GSL-I) in the preparation of a liver cancer diagnostic kit. Using GSL-I as a detection reagent for early liver cancer, the kit can detect GSL-I, distinguish between liver cancer patients and adjacent normal controls, with a sensitivity of 71.43% and a specificity of 85.71%.

[0005] To achieve the above objectives, the present invention provides the application of Ghana seed lectin I in the preparation of a liver cancer diagnostic kit.

[0006] Furthermore, the Ghana seed lectin I is used to distinguish between liver cancer patients and adjacent normal controls.

[0007] Furthermore, the liver cancer diagnostic kit includes a detection reagent or kit for Ghana seed lectin I.

[0008] Furthermore, the detection reagent or kit for Ghana seed lectin I includes Ghana seed lectin I immunohistochemical detection reagents: biotin-labeled Ghana seed lectin I primary antibody and horseradish peroxidase-labeled streptavidin.

[0009] GSL-I is an important glycoside conjugate on the cell membrane surface of hepatocellular carcinoma (HCC) cells. GSL-I can screen and differentially bind to α-1,3-galactosidase on the cell membrane surface of adjacent HCC tissues, thus serving as a potential means for the detection, clinical diagnosis, and targeted therapy of HCC.

[0010] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0011] The application of Ghana seed lectin I provided by this invention in the preparation of liver cancer diagnostic kits. This invention screened and obtained Ghana seed lectin I, which can distinguish hepatocellular carcinoma from adjacent tissues (GSL-I). The sensitivity of Ghana seed lectin I in binding to liver cancer and adjacent tissues is 71.43% and the specificity is 85.71%, which can be used as a potential means for clinical diagnosis of liver cancer. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 This is a graph showing the average fluorescence intensity of GSL-I binding in hepatocellular carcinoma and adjacent non-tumor tissues. In the graph, HCC represents hepatocellular carcinoma; adjacent non-tumor represents adjacent non-tumor tissue; and average fluorescence intensity represents the mean fluorescence intensity.

[0014] Figure 2 To verify the binding of GSL-I to human hepatocellular carcinoma (HCC) and adjacent normal tissue sections using immunohistochemistry. Figure A: Immunohistochemical results; Figure B: H-score quantitative analysis; In the figures, HCC Stage I: TNM stage I hepatocellular carcinoma; HCC Stage II: TNM stage II hepatocellular carcinoma; HCC Stage III: TNM stage III hepatocellular carcinoma; GSL-I: Ghana seed lectin I.

[0015] Figure 3 To verify the binding of Ghana seed lectin I to the cell membranes of human hepatocellular carcinoma and adjacent normal tissues using Western blotting. In the figure, T: hepatocellular carcinoma tissue; N: adjacent normal tissue of hepatocellular carcinoma; Biotin-GSL I: biotin-labeled GSL-I.

[0016] Figure 4The figure shows the ROC curves of the binding affinity between Ghana seed lectin I and liver cancer and adjacent non-cancerous tissue. In the figure, Sensitivity, Specificity, and ROC curve represent the receiver operating characteristic curve. Detailed Implementation

[0017] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0018] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0019] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0020] The technical solution of this application embodiment is to solve the above-mentioned technical problems, and the general idea is as follows:

[0021] The technical solution of this invention is as follows:

[0022] The first step involved screening Ghana seed lectin I-GSL-1 from 35 candidate lectins using lectin chip technology. GSL-1 showed a significant difference in binding to α-1,3-galactoside on the cell membrane surface of liver cancer and its paired adjacent normal tissues. The fold difference in binding between GSL-1 and liver cancer and adjacent normal tissues was then calculated.

[0023] The second step involved using immunohistochemistry to investigate the binding of GSL-I to human liver cancer and adjacent tissue sections, and to calculate the sensitivity and specificity of GSL-I binding to liver cancer tissue.

[0024] This invention utilizes lectin chip technology to prepare a chip composed of 35 lectins. Analysis of glycosides on the cell membrane surface of 10 human hepatocellular carcinoma (HCC) tissues and their paired adjacent normal tissues revealed that the number of glycosides recognized by GSL-1 (which specifically binds to α1,3-galactose) was significantly lower than that in adjacent normal tissues. Immunohistochemistry was then used to further examine the binding of GSL-1 to HCC tissues (TNM stages I, II, and III) and their paired adjacent normal tissues, and the binding was scored using H-scores and statistically analyzed. We found that GSL-1 bound to more glycosides on the surface of adjacent normal tissues compared to HCC tissues, and the results were statistically significant. Therefore, GSL-1 capable of distinguishing hepatocellular carcinoma from adjacent normal tissues was identified. Next, we further calculated the sensitivity and specificity of GSL-1 binding to HCC tissues to explore the potential of GSL-1 for clinical HCC diagnosis. The calculated sensitivity reached 71.43%; the specificity reached 85.71%, indicating its potential as a clinical diagnostic tool for HCC.

[0025] This invention explores a diagnostic method for hepatocellular carcinoma, providing new tools and ideas for tumor detection and clinical diagnosis, and has broad prospects in the development and clinical application of cancer detection technologies. This invention can be directly applied to the scientific research field or guide the development of novel liver cancer detection technologies, and also has important theoretical significance for finding new detection targets and screening new tumor markers.

[0026] The application of Ghana seed lectin I in the preparation of liver cancer diagnostic kits will be described in detail below with reference to examples and experimental data.

[0027] Example 1: Investigating the difference in fluorescence intensity between hepatocellular carcinoma and adjacent non-cancerous tissues bound to GSL-I using lectin microarrays.

[0028] Cancer tissues and their paired adjacent normal tissues were collected from 10 patients with hepatocellular carcinoma. Membrane proteins were extracted and their concentrations determined. These proteins were then conjugated with a Cy7 fluorescent group for lectin microarray screening. After data normalization, the results showed that among numerous lectins, the fluorescence intensity of Ghana seed lectin I bound to cancer tissues was significantly lower than that bound to adjacent normal tissues. Subsequently, using the average fluorescence intensity of adjacent normal tissues bound to the lectin as a baseline, the fold difference was calculated according to the following formula:

[0029] Fold change = Average fluorescence intensity of hepatocellular carcinoma tissue bound to lectin / Average fluorescence intensity of adjacent normal tissue bound to lectin. The calculated fold change between GSL-I and the binding of hepatocellular carcinoma tissue to adjacent normal tissue was 0.307.

[0030] Example 2: Immunohistochemistry and lectin immunoblotting techniques were used to verify the binding affinity of GSL-I to liver cancer and adjacent tissue sections.

[0031] Immunohistochemical experiments were performed using paraffin sections from two TNM stage I, II, and III liver cancer tissues and paired adjacent normal tissues. The specific steps were as follows: Paraffin sections were baked in a 65°C oven for 1 hour; immediately followed by dewaxing twice with 100% xylene, 15 minutes each time, until completely dewaxed; hydration was achieved by sequentially placing the sections in a gradient of 100%, 95%, 85%, and 75% ethanol for 2 minutes each, and finally in double-distilled water for 5 minutes; the prepared sections were placed on a slide holder in a beaker containing 500 mL of sodium citrate antigen retrieval solution and 0.05% Tween 20, heated at 100°C for 30 minutes, allowed to cool naturally to room temperature, and rinsed twice with PBS for 3 minutes each time; 100 μL of 3% hydrogen peroxide solution was added to each section, and incubation was performed at room temperature for 10 minutes, followed by rinsing twice with PBS for 3 minutes each time; 100 μL of 5% hydrogen peroxide solution was added to each section. Incubate with BSA solution at room temperature for 30 min, then wash twice with PBS for 3 min each time. Add 100 μL of biotin-labeled GSL-I lectin primary antibody (1:250) to each slide and incubate overnight at 4°C. Use PBS as a negative control instead of the primary antibody. Wash twice with PBS for 3 min each time. Add 100 μL of horseradish peroxidase-labeled streptavidin secondary antibody to each slide and incubate at 37°C for 30 min. Wash twice with PBS, 3 min each time; add 100 μL of freshly prepared DAB chromogenic solution to each slide, and immediately rinse with running water to stop the reaction when the slide turns brownish-yellow; then counterstain with hematoxylin for 2 min, add 75% hydrochloric acid alcohol for color separation, and rinse with tap water immediately after 2-3 seconds to restore blue color; place in a gradient of 75%, 85%, 95%, and 100% alcohol for dehydration for 2 min each, then immerse in xylene for 10 min for clearing, dry overnight, add neutral resin, and store at room temperature. Immunohistochemical sections were prepared using inForm 2.2. The software was used to perform H-score scoring, and the results are shown in Table 1. Subsequently, the unpaired T-test in Prism 9.0 software was applied to analyze the differences in binding affinity of Ghana seed lectin I between hepatocellular carcinoma tissues at different TNM stages and their paired adjacent normal tissues. Figure 2 As shown, the results indicate that the brown-yellow staining depth of GSL-I lectin is significantly lower than that of adjacent normal tissue, suggesting that GSL-I can bind fewer glycosides on the surface of liver cancer tissue compared to the surface of adjacent normal tissue.

[0032] Example 3: Protein immunoblotting verification of the binding affinity of GSL-I to human liver cancer and adjacent tissues.

[0033] The specific steps of the lectin immunoblotting technique are as follows: using Beyotime... Cell membrane proteins were extracted from liver cancer and adjacent normal tissues using a membrane separation kit. 1 mL of membrane protein extraction reagent A was added, and the cells were repeatedly pipetted to form a cell suspension. The suspension was then incubated on ice for 10 min. The cell suspension was repeatedly frozen and thawed twice in liquid nitrogen and at 37°C until the cell disruption was greater than 70%. Cell membrane proteins were collected by centrifugation. 5×SDS-PAGE protein loading buffer was added to the collected protein sample. The sample was heated at 100°C or in a boiling water bath for 3-5 minutes to fully denature the proteins. After cooling to room temperature, the protein sample was directly loaded into the wells of an SDS-PAGE gel. Electrophoresis was performed: stacking gel at 80V / 30 minutes, separating gel at 100V / 2 hours. The gel was then removed and electrophoresed at 200V using a PVDF membrane at 200mA / 2 hours. Blocking with 5% BSA at 37°C for 2 hours. 5% BSA was incubated overnight at 4°C with a 1:1000 dilution of Biotin-GSL-I lectin primary antibody. The membrane was washed with TBST for 3×10 min. Horseradish peroxidase-labeled streptavidin secondary antibody (1:5000) was incubated at room temperature for 1 h. The cells were then rinsed with TBST for 10 min × 3 times. ECL chromogenic buffer was added, and the colorimetric results were recorded using a chemiluminescence analyzer.

[0034] The results showed that GSL-I could bind fewer glycoproteins on the surface of liver cancer tissue compared to the surface of adjacent tissue, which was consistent with the immunohistochemical results.

[0035] Example 4: Calculation of the sensitivity and specificity of GSL-I binding to liver cancer and adjacent tissues.

[0036] Statistical analysis of the H-score of immunohistochemical scoring results was performed using Prism 9.0 software. Receiver operating characteristic (ROC) curves were plotted, and the sensitivity and specificity of GSL-I binding to liver cancer and adjacent normal tissues were calculated. The results showed that, within the 95% confidence interval, the specificity of GSL-I binding to liver cancer and adjacent normal tissues was 85.71%, and the sensitivity was 71.43%. The ROC curves are shown below. Figure 3 As shown, the area under the curve is 0.8611, the standard deviation is 0.1157, and the p-value is 0.0374, indicating that the result is significant.

[0037] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0038] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0039] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a reagent for detecting Ghana seed lectin I in the preparation of a liver cancer diagnostic kit, characterized in that, A reagent for detecting Ghana seed lectin I can be used as an early diagnostic kit for liver cancer. Ghana seed lectin I can screen and differentially bind to α-1,3-galactosidase on the cell membrane surface of adjacent tissues in hepatocellular carcinoma, thereby distinguishing liver cancer patients from adjacent controls. The sensitivity reaches 71.43%, and the specificity reaches 85.71%. The liver cancer diagnostic kit includes a reagent or kit for detecting Ghana seed lectin I.

2. The application according to claim 1, characterized in that, The detection reagent or kit for Ghana seed lectin I includes Ghana seed lectin I immunohistochemical detection reagents: biotin-labeled Ghana seed lectin I primary antibody and horseradish peroxidase-labeled streptavidin.

Images