Application of CTSV protein in preparation of sorafenib curative effect prediction kit for liver cancer

Abstract

The invention relates to the technical field of biological medicine, in particular to application of CTSV protein in preparation of a sorafenib curative effect prediction kit for liver cancer. The expression quantity of CTSV protein in liver cancer tissues is detected by adopting an immunohistochemical method, and the sensitivity of liver cancer postoperative recurrence patients to sorafenib treatment can be judged. The CTSV protein is used as a protein molecular marker for judging the curative effect of the sorafenib on the postoperative recurrent patient of the liver cancer, and has important guiding significance on whether the postoperative recurrent patient of the liver cancer is treated by the sorafenib or not.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an application of CTSV protein, in particular to an application of CTSV protein as a protein molecular marker for predicting the efficacy of sorafenib in liver cancer. Background Art

[0003] The judgment of liver cancer prognosis is the key to controlling liver cancer recurrence and metastasis. In recent years, some studies have found some molecules that may predict liver cancer prognosis, but the sensitivity and specificity of these indicators are still difficult to meet the requirements of clinical prediction. In recent years, the research on the use of molecular targeted drugs to treat liver cancer has gradually received attention and is becoming a new hot spot. The multi-target receptor tyrosine kinase inhibitor sorafenib (Nexavar) is one of the main drugs currently approved by the FDA for molecular targeted therapy of liver cancer, but recent studies have found that the response rate of sorafenib in patients is relatively low, and only a small number of patients can really benefit. A considerable number of patients not only have no effect on sorafenib, but also have significant side effects. It is an urgent problem to find biomarkers that can effectively predict the efficacy of sorafenib to guide the clinical use of sorafenib.

[0004] Therefore, it is important to find markers that can predict the prognosis of liver cancer and target-related genes or proteins that can predict the efficacy of sorafenib in the adjuvant treatment of postoperative recurrence of liver cancer. This research is of great significance for the clinical treatment of liver cancer and guiding the clinical use of sorafenib.

[0005] CTSV is a lysosomal cysteine ​​protease that belongs to the peptidase C1 family and plays an important role in thymus and corneal physiology. Previous studies have shown that CTSV is differentially expressed in a variety of tumors, including bladder cancer, breast ductal carcinoma, colorectal cancer, pancreatic cancer, and skin squamous cell carcinoma. Abnormal expression of CTSV is often closely related to tumor progression and indicates a poor prognosis for patients. In addition, in some tumors, CTSV can enhance inflammatory responses by activating the NF-κB pathway, thereby promoting tumorigenesis. All of these indicate that CTSV is an important prognostic marker in a variety of tumors and plays an important biological role.

[0006] Current studies have shown that the expression of CTSV is generally elevated in liver cancer, and patients with high CTSV expression often have a worse prognosis, suggesting that CTSV may be an important prognostic marker in liver cancer. However, there is no literature report on the efficacy of CTSV in predicting the efficacy of postoperative sorafenib adjuvant therapy.

[0007] So far, there are no research reports on the application of CTSV protein in the preparation of a sorafenib efficacy prediction kit for liver cancer. Summary of the invention

[0008] The purpose of the present invention is to provide a new application of CTSV protein, especially an application in preparing a kit for predicting the efficacy of sorafenib for recurrent liver cancer.

[0009] The present invention provides the use of CTSV protein in preparing a reagent or a kit for predicting the effect of sorafenib treatment on postoperative recurrence of liver cancer patients.

[0010] After in-depth and systematic research, the inventors of this application discovered for the first time that the relative expression of CTSV protein in liver cancer tissue detected by immunohistochemistry can predict the responsiveness of liver cancer patients to sorafenib; and found that among patients with liver cancer recurrence after surgery and treated with sorafenib, patients with high expression of CTSV had a longer overall survival compared with liver cancer patients with low expression of CTSV. Based on this correlation between the relative expression of CTSV protein and the efficacy of sorafenib, the expression of this protein as a molecular marker can be used to predict the efficacy of sorafenib treatment in patients with liver cancer recurrence after surgery.

[0011] The present invention provides an application of CTSV protein in the preparation of a kit for predicting the efficacy of sorafenib for liver cancer. The application refers to using CTSV protein as a molecular marker, using CTSV monoclonal antibody or polyclonal antibody, combined with immunohistochemical experimental reagents, detecting the expression of CTSV in liver cancer tissues of patients who have relapsed after liver cancer surgery and received sorafenib treatment, and analyzing the overall survival of patients with low and high expression of CTSV.

[0012] Furthermore, the reagent or kit detects the relative expression level of CTSV protein in liver cancer tissue.

[0013] Furthermore, the reagent or kit uses an immunohistochemical method to detect the relative expression of FOXA3 protein in liver cancer tissue, thereby determining the risk of recurrence and survival status of liver cancer patients.

[0014] Furthermore, the reagent or kit uses an immunohistochemical method to detect the relative expression of CTSV protein in liver cancer tissue, thereby predicting the efficacy of sorafenib treatment in patients with postoperative recurrence of liver cancer.

[0015] Furthermore, the CTSV polyclonal antibody is a commercial antibody, such as rabbit CTSV polyclonal antibody (Proteintech, catalog number 18442-1-AP).

[0016] Furthermore, the immunohistochemical reagents include xylene, ethanol, 3% H 2 O 2 solution, 1% BSA blocking solution, DAB colorimetric reagent, hematoxylin and horseradish peroxidase-labeled goat anti-rabbit IgG.

[0017] The immunohistochemical method was used to detect the expression of CTSV protein, which could predict the efficacy of sorafenib treatment in patients with postoperative recurrent liver cancer. In patients with postoperative recurrent liver cancer who received sorafenib treatment, when the immunohistochemical score was lower than 0.122 points, the overall survival of liver cancer patients was shorter and the prognosis was poorer.

[0018] The method for detecting the expression level of CTSV protein in liver cancer tissue comprises the following steps: (1) Prepare paraffin sections of liver cancer tissue and oven-warm them at 60°C overnight; (2) Dewaxing the sections to water; (Xylene ①10min →Xylene ②10min →Xylene ③10min →100% ethanol 5min →95% ethanol 5min →85% ethanol 5min →75% ethanol 5min →double distilled water 5min) (3) 3%H 2 O 2 Methanol solution, placed at room temperature for 20 min; (4) Wash with double distilled water for 5 min × 3; (5) Antigen retrieval: Boil the sections in 0.01 M citrate buffer (pH 6.0) for 5 min, stop boiling for 10 min, and boil again for 5 min. (6) Cool naturally to room temperature and wash with double distilled water for 5 min × 3; (7) Block with 1% BSA for 30 min at 37°C; (8) Remove the blocking solution and add the primary antibody (the above rabbit CTSV polyclonal antibody, diluted 1:100) without washing. Place in a humidified chamber for 30 min at 37°C, then in a refrigerator at 4°C overnight for 16 hours; (9) Remove from the well at 4°C, warm to room temperature for 15 min, and then wash with 0.01 M PBS for 5 min × 4; (10) Add secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG, purchased from DAKO, Denmark, ready-to-use, no dilution required) for 45 min at 37°C; (11) Wash with 0.01 M PBS for 5 min × 4, develop with DAB for 2-10 min, and observe under a microscope; (12) Color development was terminated with double distilled water and counterstained with hematoxylin for 10 seconds; (13) After differentiation, tap water turns blue and is soaked in distilled water; (14) Dehydrate and make transparent, then cover with coverslip; (15) Observe positive staining under a microscope and randomly select three fields of view from each of the liver cancer tissue and adjacent tissue and take photos; (16) The tissue samples were scanned using Image Scope (Aperio) software. After scanning, immunohistochemical scoring was performed according to the cytoplasmic positive ratio and staining intensity using the following formula:

[0019] The immunohistochemical score of the tissue was obtained for subsequent analysis. A CTSV staining score of hepatocellular carcinoma tissue <0.122 was considered low expression, and a tissue staining score ≥0.122 was considered high expression.

[0020] The advantages of the present invention are: 1. The present invention uses CTSV protein as a protein molecular marker for judging the efficacy of sorafenib in patients with postoperative recurrent liver cancer, which has important guiding significance for whether to use sorafenib for patients with postoperative recurrent liver cancer.

[0021] 2. The discovery of the correlation between the CTSV protein of the present invention and the efficacy of sorafenib for recurrent liver cancer has important guiding significance for postoperative monitoring and sequential treatment of liver cancer patients. In patients with postoperative recurrence of liver cancer and receiving sorafenib treatment, when the immunohistochemistry score is lower than 0.122 points, the patients have a shorter overall survival. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a picture of immunohistochemical staining of CTSV in liver cancer tissue, showing that CTSV polyclonal antibody can specifically recognize CTSV protein in liver cancer tissue. (The left picture is liver cancer tissue, and the right picture is paired paracancerous tissue) Figure 2 This is a survival analysis of 97 patients with liver cancer who relapsed after surgery and received sorafenib treatment. The liver cancer patients were divided into groups according to the level of CTSV expression, and the results of the survival analysis of the patients' prognosis were shown in the figure. It can be seen that patients with low CTSV expression have a relatively poor prognosis.

[0023] Figure 3 This is the result of immunohistochemical staining of CTSV in liver cancer tissue. It can be seen that the staining of CTSV in tumor tissue is relatively strong. The staining score >0.122 is considered high expression. Follow-up found that the patient has a good prognosis.

[0024] Figure 4 This is the result of immunohistochemical staining of CTSV in liver cancer tissue. It can be seen that the staining of CTSV in tumor tissue is relatively weak, and the staining score <0.122 is considered low expression. Follow-up found that the patient had a poor prognosis.

[0025] Figure 5 This is the result of immunohistochemical staining of CTSV in liver cancer tissue. It can be seen that the staining of CTSV in tumor tissue is relatively strong. The staining score >0.122 indicates high expression. Follow-up found that the patient had a good prognosis.

[0026] Figure 6 This is the result of immunohistochemical staining of CTSV in liver cancer tissue. It can be seen that the staining of CTSV in tumor tissue is relatively weak, and the staining score <0.122 is considered low expression. Follow-up found that the patient had a poor prognosis. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0028] In the following examples, tumor tissue sections of liver cancer patients were all from Oriental Hepatobiliary Surgery Hospital and were diagnosed as hepatocellular carcinoma by two pathologists. Example 1

[0029] The present invention uses immunohistochemistry to detect the relative expression of CTSV protein in liver cancer tissues, and the specific steps are: (1) Prepare paraffin sections of liver cancer tissue and oven-warm them at 60°C overnight; (2) Dewaxing the sections to water; (Xylene ①10min →Xylene ②10min →Xylene ③10min →100% ethanol 5min →95% ethanol 5min →85% ethanol 5min →75% ethanol 5min →double distilled water 5min) (3) 3%H 2 O 2 Methanol solution, placed at room temperature for 20 min; (4) Wash with double distilled water for 5 min × 3; (5) Antigen retrieval: Boil the sections in 0.01 M citrate buffer (pH 6.0) for 5 min, stop boiling for 10 min, and boil again for 5 min. (6) Cool naturally to room temperature and wash with double distilled water for 5 min × 3; (7) Block with 1% BSA for 30 min at 37°C; (8) Remove the blocking solution, do not wash, and directly add the primary antibody (1:100). Place in a humidified box for 30 min at 37°C, then in a refrigerator at 4°C overnight for 16 hours; (9) Remove from the well at 4°C, warm to room temperature for 15 min, and then wash with 0.01 M PBS for 5 min × 4; (10) Add DAKO secondary antibody for 45 min at 37°C; (11) Wash with 0.01 M PBS for 5 min × 4, develop with DAB for 2-10 min, and observe under a microscope; (12) Color development was terminated with double distilled water and counterstained with hematoxylin for 10 seconds; (13) After differentiation, tap water turns blue and is soaked in distilled water; (14) Dehydrate and make transparent, then cover with coverslip; (15) Observe positive staining under a microscope and randomly select three fields of view from each of the liver cancer tissue and adjacent tissue and take photos; (16) The tissue samples were scanned using Image Scope (Aperio) software. After scanning, immunohistochemical scoring was performed according to the cytoplasmic positive ratio and staining intensity using the following formula:

[0030] The immunohistochemical score of the tissue was obtained for subsequent analysis. A CTSV staining score of hepatocellular carcinoma tissue <0.122 was considered low expression, and a tissue staining score ≥0.122 was considered high expression.

[0031] Two patients with liver cancer were randomly selected to undergo surgical resection of tumor tissues, and the above immunohistochemical method was used to detect the expression of CTSV protein in liver cancer tissues. The results showed that CTSV polyclonal antibodies could specifically recognize CTSV protein in liver cancer tissues. Figure 1 . Example 2

[0032] A total of 97 patients with hepatocellular carcinoma who had recurrence after surgery and received sorafenib were divided into two groups according to the expression of CTSV in their hepatocellular carcinoma tissues (high expression when the immunohistochemical score was higher than or equal to 0.122 points, and low expression when the score was lower than 0.122 points). Survival analysis was performed based on the postoperative follow-up information, and a 4-year survival curve was drawn (see Figure 2 The results showed that the overall survival of patients with low CTSV expression in their tumors who had recurrence after surgery and received sorafenib treatment was significantly shorter than that of patients with high CTSV expression. Example 3

[0033] Sample: Tissue sections of a patient with liver cancer who had recurrence after surgery and received sorafenib treatment. The above immunohistochemical method was used to detect the expression of CTSV protein in liver cancer tissue. Figure 3 The immunohistochemical score of the tissue was calculated to be 0.19, which is higher than 0.122.

[0034] Postoperative follow-up showed that the patient's overall survival time after postoperative recurrence was 26 months, and the prognosis was good. Example 4

[0035] Sample: Tissue sections of a patient with liver cancer who had recurrence after surgery and received sorafenib treatment. The above immunohistochemical method was used to detect the expression of CTSV protein in liver cancer tissue. Figure 4After calculation, the immunohistochemical score of the tissue was 0.036, which was lower than 0.122.

[0036] Postoperative follow-up showed that the patient's overall survival time after postoperative recurrence was 5 months, and the prognosis was poor. Example 5

[0037] Sample: Tissue sections of a patient with liver cancer who had recurrence after surgery and received sorafenib treatment. The above immunohistochemical method was used to detect the expression of CTSV protein in liver cancer tissue. Figure 5 After calculation, the immunohistochemical score of the tissue was 0.187, which was higher than 0.122.

[0038] Postoperative follow-up showed that the patient's overall survival time after postoperative recurrence was 28 months, and the prognosis was good. Example 6

[0039] Sample: Tissue sections of a patient with liver cancer who had recurrence after surgery and received sorafenib treatment. The above immunohistochemical method was used to detect the expression of CTSV protein in liver cancer tissue. Figure 6 After calculation, the immunohistochemical score of the tissue was 0.083, which was lower than 0.122.

[0040] Postoperative follow-up showed that the patient's overall survival time after postoperative recurrence was 3 months, and the prognosis was poor.

[0041] From the above experimental results, it can be seen that the efficacy of sorafenib in patients with postoperative recurrence of hepatocellular carcinoma can be predicted by detecting the expression of CTSV protein by immunohistochemistry. When the immunohistochemistry score is lower than 0.122 points, the effect of sorafenib in patients with postoperative recurrence of liver cancer is poor. Obviously, the CTSV protein level has a certain correlation with the responsiveness of patients with postoperative recurrence of liver cancer to sorafenib treatment. Therefore, using CTSV protein as a protein molecular marker to detect its expression can predict the effect of postoperative recurrence of liver cancer patients treated with sorafenib. Correspondingly, antibodies specific to CTSV protein, including monoclonal antibodies and polyclonal antibodies, can be used to prepare preparations or kits for predicting the responsiveness of patients with postoperative recurrence of liver cancer to sorafenib, which is obvious to those skilled in the art. The preferred embodiments of the invention have been specifically described above, but the invention is not limited to the embodiments. Those familiar with the art can also make various equivalent modifications or substitutions without violating the spirit of the invention, such as detecting the DNA and mRNA levels of CTSV, etc. These equivalent modifications or substitutions are all included in the scope defined by the claims of the present application.

Claims

1. Application of CTSV protein in the preparation of reagents or kits for predicting the effect of sorafenib treatment on postoperative recurrence of liver cancer patients.

2. The use of the CTSV protein according to claim 1 in the preparation of a reagent or a kit for predicting the effect of sorafenib treatment on postoperative recurrence of liver cancer patients, characterized in that: The reagent or kit detects the relative expression level of CTSV protein in liver cancer tissue.

3. Use of the CTSV protein according to claim 2 in the preparation of a reagent or kit for predicting the effect of sorafenib treatment on postoperative recurrence of liver cancer patients, characterized in that: The reagent or kit uses an immunohistochemical method to detect the relative expression of CTSV protein in liver cancer tissue, thereby predicting the efficacy of sorafenib treatment for patients with postoperative recurrent liver cancer.

4. Use of the CTSV protein according to claim 3 in the preparation of a reagent or kit for predicting the effect of sorafenib treatment on postoperative recurrence in patients with liver cancer, characterized in that: The application refers to using CTSV protein as a molecular marker, using CTSV monoclonal antibodies or polyclonal antibodies, combined with immunohistochemical reagents, to detect the relative expression of CTSV protein in liver cancer tissue, so as to predict the efficacy of sorafenib treatment for patients with postoperative recurrence of liver cancer.

5. Use of the CTSV protein according to claim 4 in the preparation of a reagent or kit for predicting the effect of sorafenib treatment on postoperative recurrence in patients with liver cancer, characterized in that: The immunohistochemical reagents include xylene, ethanol, 3% H2O2 solution, 1% BSA blocking solution, DAB color developing reagent, hematoxylin and horseradish peroxidase labeled goat anti-rabbit IgG.

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