CN113588951B - Application of ECA as molecular marker in preparation of kit for diagnosing and/or prognosis evaluation of liver cancer - Google Patents

Application of ECA as molecular marker in preparation of kit for diagnosing and/or prognosis evaluation of liver cancer Download PDF

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CN113588951B
CN113588951B CN202110765251.9A CN202110765251A CN113588951B CN 113588951 B CN113588951 B CN 113588951B CN 202110765251 A CN202110765251 A CN 202110765251A CN 113588951 B CN113588951 B CN 113588951B
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CN113588951A (en
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章晓联
孔颖
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Wuhan University WHU
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Abstract

The invention provides application of ECA as a molecular marker in preparing a kit for diagnosing and/or evaluating liver cancer in a prognosis way, and the ECA is found by immunofluorescence that the binding degree of ECA and beta 1, 3/beta 1,4 galactose glycoside on the cell membrane surface of liver cancer tissue is obviously reduced compared with that of a paracancerous tissue, and the binding strength of ECA and beta 1, 3/beta 1,4 galactose glycoside on the liver cancer tissue surface of stage I is obviously stronger than that of stage II and stage III, so that the ECA can be used as a molecular marker for distinguishing TNM I liver cancer patients, TNM II stage and stage III liver cancer patients. ECA is adopted as an analysis marker, and is singly detected to distinguish liver cancer patients from paracancerous controls, and the sensitivity reaches 75%; the specificity reaches 60%; ECA and AFP combined diagnosis distinguishes liver cancer patients and cancer side control persons, the sensitivity reaches 85%, and the specificity reaches 85%.

Description

Application of ECA as molecular marker in preparation of kit for diagnosing and/or prognosis evaluation of liver cancer
Technical Field
The invention relates to the technical field of biomedical detection, in particular to application of ECA serving as a molecular marker in preparation of a kit for diagnosing and/or prognosis evaluation of liver cancer.
Background
Primary liver cancer (Primary Liver Cancer, PLC) is the fifth most common malignancy worldwide, and is also the second most cancer-related fatal disease. Liver cancer (Hepatocellular Carcinoma, HCC) is about 70% -85% of the major pathological types of primary liver cancer, with 90% of HCC patients undergoing malignant transformation of chronic hepatitis-liver fibrosis-cirrhosis, and more than 60% of patients have advanced or metastasized when they are diagnosed due to rapid onset and lack of significant features. At present, the survival prognosis rate of HCC in China is only 12%, however, if patients are diagnosed in early stage, the five-year survival rate can be improved to more than 70%.
According to the integrated recommendations of the american liver disease institute (AASLD), european liver research institute (EASL) and asian pacific liver research institute (APASL) guidelines for liver cancer, the 2018 primary liver cancer diagnosis and treatment guideline (CSCO) established by the chinese clinical society for oncology indicates that the current routine screening and diagnosis strategies for clinical HCC are serum alpha-fetoprotein (AFP) level detection and liver ultrasound imaging examinations, at least once every 6 months, if necessary with Computed Tomography (CT) scanning and/or Magnetic Resonance Imaging (MRI) scanning and in combination with hepatic angiography (DSA). However, the sensitivity and specificity of the above method are not high, so that the patients can easily lose the optimal treatment time. Therefore, development of new liver cancer markers to assist in early warning, disease staging and prognosis of treatment of HCC is urgent to improve the accuracy of HCC diagnosis.
Therefore, the identification of novel biomarkers is very important for the timely and early diagnosis of liver cancer, and it is necessary to develop a novel marker with high sensitivity and specificity.
Disclosure of Invention
The invention aims to provide application of ECA as a molecular marker in preparing a kit for diagnosing and/or evaluating liver cancer in advance, and the combination level of ECA and liver cancer cell membrane proteins beta 1, 3/ beta 1,4 galactose glycoside of a patient can be detected by using the liver cancer early diagnosis kit, and the ECA is independently detected to distinguish the liver cancer patient from a cancer side contrast patient, so that the sensitivity reaches 75%; the specificity reaches 60%, and the sensitivity reaches 40% when the AFP is detected singly to distinguish liver cancer patients from healthy people; ECA and AFP combined diagnosis distinguishes liver cancer patients and cancer side control persons, the sensitivity reaches 85%, and the specificity reaches 85%.
In a first aspect of the invention there is provided the use of ECA as a molecular marker in the manufacture of a kit for diagnosing and/or prognosis evaluation of liver cancer.
Further, the ECA is used as a molecular marker for distinguishing TNM stage I liver cancer patients, TNM stage II and stage III patients.
In the technical scheme, ECA expression on the surface of a liver cancer cell membrane is lower than that of a tissue beside the cancer, and the ECA is reduced along with the increase of the malignancy degree of the liver cancer, so that the ECA can be used for distinguishing TNM I liver cancer patients, TNM II patients and III patients. In addition, the applicant finds that the expression of ECA in liver cancer tissues is obviously down-regulated, and the binding strength of ECA and beta 1, 3/ beta 1,4 galactose glycoside on the liver cancer tissue surface of stage I is obviously stronger than that of stage II and stage III.
Further, the kit for diagnosing and/or prognosis evaluation of liver cancer comprises a detection reagent or a detection kit of an ECA molecular marker.
Further, the detection reagent or the detection kit of the ECA molecular marker comprises an ECA immunohistochemical experiment detection reagent.
Further, the ECA immunohistochemical assay detection reagent includes: biotin-labeled ECA and horseradish peroxidase-labeled streptavidin.
Further, the kit for diagnosing and/or prognosis evaluating liver cancer further comprises: AFP detection reagent.
In a second aspect of the invention, there is provided the use of a biotin-labelled ECA in the manufacture of an immunohistochemical detection kit for the diagnosis of early liver cancer and/or post-operative prognosis of liver cancer.
Further, the immunohistochemical detection kit further comprises horseradish peroxidase-labeled streptavidin.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
1. the application of the ECA provided by the invention as a molecular marker in preparing a kit for diagnosing and/or evaluating liver cancer in a prognosis way, the lectin ECA obtained by screening is lower than that of a tissue beside a cancer on the surface of a liver cancer cell membrane in different TNM stages (tumor-lymph node-metastasis stages), and the binding strength of beta 1, 3/ beta 1,4 galactose glycoside on the surface of the ECA and the liver cancer cell membrane is inversely related to the serum alpha fetoprotein concentration of a liver cancer patient along with the increase of the malignancy degree of the liver cancer. Can be used as a marker for diagnosis or prognosis of liver cancer; can also provide a new potential drug target for treating liver cancer.
2. The invention can detect the combination level of ECA and liver cancer cell membrane protein beta 1, 3/ beta 1,4 galactose glycoside of a patient by using the liver cancer early diagnosis kit, and independently detect ECA to distinguish liver cancer patients from paracancer contrast patients, wherein the sensitivity reaches 75 percent, and the specificity reaches 60 percent; the AFP is detected independently to distinguish liver cancer patients from healthy people, and the sensitivity reaches 40%; ECA and AFP combined diagnosis distinguishes liver cancer patients and cancer side control persons, the sensitivity reaches 85%, and the specificity reaches 85%.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of screening for differential expression of beta 1, 3/ beta 1,4 galactosides for binding of ECA to the cell membrane surface of liver cancer tissue and paracancestral tissue; wherein FIG. 1A is a heat map showing the difference in expression of beta 1, 3/ beta 1,4 galactosides recognized by 35 lectins on the cell membrane surface of liver cancer tissue and paracancestral tissue; FIG. 1B is a graph showing the statistics of fold changes; FIG. 1C shows the result of P-value analysis;
FIG. 2 shows the results of the identification of differential expression beta 1, 3/ beta 1,4 galactosides of ECA bound to the cell membrane surface of liver cancer tissue and paracancestral tissue; FIG. 2A shows the fluorescence intensity of immunofluorescence detection lectin ECA combined with the cell membrane surface of the paracancerous tissue and the liver cancer tissue; FIG. 2B shows the staining intensity of immunohistochemical detection of lectin ECA binding to the cell membrane surface of paracancerous tissue and liver cancer tissue with beta 1, 3/ beta 1,4 galactosyl glycoside; FIG. 2C is a graph showing the comparison of staining intensity of ECA with beta 1, 3/ beta 1,4 galactosides bound to cell membrane surface of different TNM-staged liver cancer tissues;
FIG. 3A is a receiver operating profile (ROC) curve for the detection of ECA alone to distinguish between liver cancer patients and paracancerous controls; FIG. 3B is a correlation analysis of ECA and liver cancer tissue beta 1, 3/ beta 1,4 galactose glycoside binding difference and serum Alpha Fetoprotein (AFP).
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, 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 belongs. In case of conflict, the present specification will control.
Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc., used in the present invention are commercially available or may be obtained by existing methods.
The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:
the lectin ECA which is obviously down-regulated by combining with beta 1, 3/ beta 1,4 galactose glycoside on the cell membrane surface of liver cancer tissue is obtained through screening by a lectin chip technology, and the combined degree of ECA on the cell membrane surface of liver cancer tissue and beta 1, 3/ beta 1,4 galactose glycoside is found to be obviously down-regulated by immunofluorescence compared with that of a tissue beside cancer, so that the ECA can be used as a molecular marker to prepare a kit for diagnosing liver cancer.
Subsequently, the immunohistochemical technology shows that ECA expression in different TNM stage liver cancer tissues is significantly down-regulated compared with other tissues, and the binding strength of ECA and beta 1, 3/ beta 1,4 galactose glycoside on the cell membrane surface of stage I liver cancer tissues is significantly stronger than that of stage II and stage III, namely, modification and change of beta 1, 3/ beta 1,4 galactose glycoside can be related to HCC early differentiation. The ECA can be used as a molecular marker for distinguishing TNM I liver cancer patients, TNM II and III patients.
The application adopts ECA as an analysis marker to carry out immunohistochemical detection experiments, and the ECA immunohistochemical detection kit comprises: biotin-labeled ECA and horseradish peroxidase-labeled streptavidin.
The ECA immunohistochemical experiment detection kit is used for singly detecting ECA to distinguish liver cancer patients from paracancerous controls, and the sensitivity reaches 75%; the specificity reaches 60%, and the sensitivity reaches 40% when the AFP is detected singly to distinguish liver cancer patients from healthy people;
furthermore, the present application also found that ECA binding strength to β 1,3/β1,4 galactosides on the cell membrane surface of liver cancer tissue was inversely related to AFP at the overall level. The ECA and AFP combined diagnosis is adopted to distinguish liver cancer patients from cancer side contrast patients, the sensitivity reaches 85%, and the specificity reaches 85%.
The application of ECA of the present application as a molecular marker in the preparation of a kit for diagnosing and/or prognosis evaluation of liver cancer will be described in detail with reference to examples and experimental data.
EXAMPLE 1 lectin chip screening of liver cancer tissue cell membrane surface specific expression Down-regulated beta 1, 3/ beta 1,4 galactoside
Firstly, searching beta 1, 3/ beta 1,4 galactose glycoside specifically expressed on the surface of a liver cancer cell membrane compared with a paracancerous tissue.
1. Extraction of cell membrane proteins: cell membrane protein extraction was performed on 10 liver cancer tissues and paired cancer tissues according to the specification of a cell membrane and cytoplasmic protein extraction kit (Jiangsu Biyun biotechnology institute), and protein concentration was measured by BCA method and adjusted to be consistent.
2. Beta 1, 3/ beta 1,4 galactose glycoside difference analysis is carried out on the above cell membrane proteins by using a lectin chip consisting of 35 lectins, fold change (fold change) analysis is carried out according to the normalized value of the measured average fluorescence intensity, fold difference of each lectin in liver cancer groups/paracancer groups is calculated, and a heat map, a Wen map and a volcanic map are drawn to screen out beta 1, 4/ beta 1,4 galactose glycoside with down-regulated specific expression on the cell membrane surface of liver cancer tissues and lectin ECA combined with the beta 1,4 galactose glycoside.
As can be seen from FIGS. 1A-C, the cell membrane proteins of 10 liver cell hepatoma patients and paired cancer tissues are screened by using lectin chips containing 35 lectins, and the result shows that 15 total lectins show down-regulation (multiple < 0.76) of binding to beta 1, 3/ beta 1,4 galactose glucosides on the cell membrane surfaces of the liver cancer tissues, and the difference of 11 lectins in the down-regulated lectins is significant in statistical significance, wherein the ECA (multiple=0.307, p < 0.0001) is obviously lower than the beta 1, 3/ beta 1,4 galactose glucosides recognized on the cell membrane surfaces of the liver cancer tissues.
Example 2 immunofluorescence comparison of binding differences between ECA and cell membrane surface beta 1, 3/ beta 1,4 galactosides in liver cancer tissue and paired paracancerous tissue of different TNM stages
1. Experimental objects
The tissue specimens and the paired cancer side tissue specimens of 9 TNM III liver cancer patients in the south-middle-arm university hospital were collected and clinically information was collected. All patients incorporated were diagnosed based on their pathology report, and the judgment of tumor stage was referred to the united states joint committee for cancer (AJCC) seventh edition of handbook of stage cancer.
All experimental designs and procedures were approved by the ethics committee of the university of martial arts, south China hospital.
2. Experimental method
1. Preparation of frozen sections: taking out the tissue block from the liquid nitrogen by using forceps, placing the tissue block on dry ice, then dripping a plurality of O.C.T embedding agents on the tissue block, keeping the cross section of one side of the tissue block to be flat, and forming the embedded block. The power precooling of the frozen microtome is started half an hour in advance, the box body temperature is set to be minus 20 ℃, the embedded tissue blocks are gently clamped on the carrying disc for fixing, the thickness of the slice is set to be 8 mu m, the angle and the distance between the blade and the tissue blocks are adjusted, then the slice is cut, and the slice is stored at minus 20 ℃.
2. Immunofluorescent staining: the slices are taken out from the temperature of minus 20 ℃ and slowly restored to room temperature, and are fixed for 10 minutes at room temperature in 4% paraformaldehyde precooled at 4 ℃; rinsing 2 times in 1 XPBS pre-chilled at 4deg.C for about 5min each; the sections were blocked for 30min at 5% BSA room temperature; adding biotin-labeled lectin ECA, and incubating at 4deg.C overnight; rinsing 2 times in 1 XPBS pre-chilled at 4deg.C for about 5min each; FITC-streptavidin (1:500) and DAPI (1:5000) were added and incubated at room temperature for 30min in the absence of light; rinsing 2 times in 1 XPBS pre-chilled at 4deg.C for about 5min each; sealing with anti-fluorescence quenching sealing agent, and storing at 4deg.C.
3. Experimental results
FIG. 2A shows that ECA expression is significantly down-regulated in liver cancer tissue; specifically: the fluorescence intensity of ECA combined with beta 1, 3/ beta 1,4 galactose glycoside on the cell membrane surface of liver cancer tissue and paired paracancerous tissue is subjected to significance analysis by adopting unpaired t test. The immunofluorescence experiment is used for verifying the binding degree of ECA and beta 1, 3/ beta 1,4 galactose glycoside on the cell membrane surface of 9 liver cancer patients and paired cancer side tissues, and the result shows that the binding degree of ECA and beta 1, 3/ beta 1,4 galactose glycoside on the cell membrane surface of liver cancer tissues is obviously reduced.
EXAMPLE 3 immunohistochemical comparison of binding differences between ECA and cell membrane surface beta 1, 3/ beta 1,4 galactosides in liver cancer tissue and paired paracancerous tissue of different TNM stages
1. Experimental objects
Paraffin embedded sections and clinical information of 5 TNM stage I liver cancer patients, 10 TNM stage II liver cancer patients and 5 TNM stage III liver cancer patients who were diagnosed in the middle and south hospitals of the university of martial arts were collected. All patients incorporated were diagnosed based on their pathology report, and the judgment of tumor stage was referred to the united states joint committee for cancer (AJCC) seventh edition of handbook of stage cancer.
All experimental designs and procedures were approved by the ethics committee of the university of martial arts, south China hospital.
2. Experimental method
1. Placing the paraffin tissue in a 65 ℃ oven to bake the slices for 1h;
2. dewaxing with 100% xylene twice for 15min each time until completely dewaxed;
3. sequentially placing into 100%, 95%, 85%, 75% gradient alcohol for 2min for hydration, and finally placing into double distilled water for 5min;
4. placing the processed slice in a slice rack, placing the slice in a beaker containing 500mL of sodium citrate antigen retrieval liquid and 0.05% Tween 20, heating at 100 ℃ for 30min, naturally cooling to room temperature, and flushing with Phosphate Buffered Saline (PBS) for 2 times each for 3min; a step of
5. 100 mu L of 3% catalase solution is dripped into each slice, incubated for 10min at room temperature, and washed with PBS for 2 times and 3min each time;
6. adding a primary antibody labeled ECA (1:100 dilution), incubating overnight at 4 ℃, and using PBS instead of the primary antibody as a negative control;
PBS rinse 2 times, 3min each; 100 mu L of horseradish peroxidase-labeled streptavidin (1:500) was added dropwise to each slice, and incubated at 37℃for 30min;
PBS rinse 2 times, 3min each; 100 mu L of freshly prepared DAB color development liquid is dripped into each slice, and the reaction is stopped by flushing with flowing water immediately when the slice appears brown yellow;
9. then counterstaining with hematoxylin for 2min, adding hydrochloric acid alcohol for color separation, and immediately flushing with tap water to turn blue after 2-3 s;
10. sequentially dehydrating in 75%, 85%, 95% and 100% gradient alcohol for 2min, adding xylene for 10min for transparency, drying overnight, and dripping neutral gum for room temperature storage.
11. Scanning microscopy detects the staining intensity of ECA.
3. Experimental results
1. As can be seen from fig. 2B-C, ECA was significantly analyzed by the unpaired t test for the staining intensity of cell membrane surface binding to β1,3/β1,4 galactose glycoside in TNM stage I, stage II, and stage III liver cancer tissues and paired paracancerous tissues. The results of verifying the binding degree of ECA and beta 1, 3/ beta 1,4 galactose glycoside by using immunohistochemical experiments on tissues of 5 TNM I, 10 II and 5 III liver cancer patients and paired paracancerous tissues show that the binding degree of ECA and beta 1, 3/ beta 1,4 galactose glycoside on the cell membrane surface of liver cancer tissues is obviously reduced, and the binding strength of ECA and beta 1, 3/ beta 1,4 galactose glycoside on the liver cancer tissue surface of stage I is obviously stronger than that of stage II and stage III, namely that the modification change of beta 1, 3/ beta 1,4 galactose glycoside is possibly related to the early differentiation of HCC.
2. Calculating sensitivity and specificity of singly detecting ECA to distinguish liver cancer patients from paracancerous controls and combining ECA with AFP to distinguish liver cancer patients from paracancerous controls according to an immunohistochemical fraction (H score), and determining a cut off value through Graphpad Prism 9.0 software, wherein the sensitivity calculation formula is as follows: TP/(TP+FN) ×100%, TP is true positive for liver cancer patients, and FN liver cancer patients are false negative; the specificity calculation formula: TN/(TN+FP). Times.100%, TN was true negative for the paracancerous control and FP was false positive for the paracancerous control.
All experimental data are expressed as mean ± SD, the differences between the two groups were analyzed for significance using the unpaired student's t test, graphpad Prism 9.0 software, with the two-tailed test p <0.05 indicating that the differences were statistically significant, p <0.001 indicating that the differences were highly significant, and p <0.001 indicating that the differences were particularly highly significant.
As can be seen from FIG. 3A, the binding level of ECA of a patient and beta 1, 3/ beta 1,4 galactose glycoside on the surface of a liver cancer cell membrane can be detected by using the liver cancer early diagnosis kit, and the ECA is independently detected to distinguish liver cancer patients from paracancer controls, so that the sensitivity reaches 75%, and the specificity reaches 60%; the AFP is detected independently to distinguish liver cancer patients from healthy people, and the sensitivity reaches 40%; ECA and AFP combined diagnosis distinguishes liver cancer patients and cancer side control persons, the sensitivity reaches 85%, and the specificity reaches 85%.
As can be seen from FIG. 3B, by analyzing the correlation between ECA and the surface beta 1, 3/ beta 1,4 galactose glycoside of liver cancer tissue cell membrane and the concentration of AFP in serum of liver cancer patient, ECA immune component of the group with AFP equal to or greater than 20ng/L is significantly reduced compared with that of the group with AFP equal to or less than 20ng/L, i.e. the ECA and the surface beta 1, 3/ beta 1,4 galactose glycoside of liver cancer tissue cell membrane are inversely correlated with AFP on the whole level.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (6)

1. The application of the reagent for detecting the liver cancer tissue cell membrane surface protein beta 1, 3/beta 1,4 galactose glycoside in preparing the reagent kit for diagnosing liver cancer is characterized in that the reagent for detecting the liver cancer tissue cell membrane surface protein beta 1, 3/beta 1,4 galactose glycoside is lectin ECA.
2. The use according to claim 1, wherein the liver cancer tissue cell membrane surface protein beta 1, 3/beta 1,4 galactose glycoside is used for distinguishing between a TNM stage I liver cancer patient, a TNM stage II liver cancer patient and a TNMIII stage liver cancer patient.
3. The use according to claim 1, wherein the kit for diagnosing liver cancer comprises a reagent for detecting the liver cancer tissue cell membrane surface protein beta 1, 3/beta 1,4 galactoside, and the reagent for detecting the liver cancer tissue cell membrane surface protein beta 1, 3/beta 1,4 galactoside is lectin ECA.
4. The use according to claim 3, wherein the kit for diagnosing liver cancer comprises an immunohistochemical assay detection reagent.
5. The use according to claim 4, wherein the kit for diagnosing liver cancer comprises: biotin-labeled ECA and horseradish peroxidase-labeled streptavidin.
6. The use according to claim 3, wherein the kit for diagnosing liver cancer further comprises: AFP detection reagent.
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