Kit for detecting lung adenocarcinoma apoptosis and ferroptosis sensitivity

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a kit for detecting lung adenocarcinoma apoptosis and ferroptosis sensitivity. The kit comprises: a ZNF146 detection system, which is used for detecting ZNF146 immunohistochemical scores in lung adenocarcinoma tumor tissues; a vector recording a mathematical model for determining a high / low boundary standard for the expression level of ZNF146, when the expression level of ZNF146 in the tumor tissue of a lung adenocarcinoma patient is lower than the standard, prompting that the patient is sensitive to apoptosis and ferroptosis drug treatment and is suitable for a treatment scheme for inducing apoptosis and ferroptosis; on the contrary, when the value is not lower than the standard value, the patient is prompted to be insensitive to apoptosis and ferroptosis drug treatment and is not suitable for a treatment scheme for inducing apoptosis and ferroptosis. The sensitivity of the ZNF146 to apoptosis and ferroptosis is judged by detecting the expression of the ZNF146 in tumors of lung adenocarcinoma patients, and then whether the lung adenocarcinoma patients are suitable for adopting apoptosis and ferroptosis drug treatment schemes or not is judged, so that precise medical treatment is realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a kit for detecting the sensitivity of lung adenocarcinoma to apoptosis and ferroptosis. Background Art

[0002] Lung cancer has always been one of the malignant tumors with the highest incidence and mortality rates globally, and lung adenocarcinoma is the most common histological subtype among them. Due to the high proliferative ability of lung adenocarcinoma and its resistance to existing chemotherapy and targeted drugs, the treatment effects of some patients are still not ideal. Therefore, it is of great significance to deeply study the molecular mechanisms of the occurrence and development of lung adenocarcinoma.

[0003] Apoptosis is a form of programmed cell death, involving complex molecular mechanisms, including the activation of the Caspase family, the regulation of the Bcl-2 family, etc.; while ferroptosis is a way of cell death caused by iron-dependent lipid peroxidation. Inducing apoptosis and ferroptosis in tumor cells are molecular mechanisms that have attracted much attention in modern tumor treatment, and there are also many related clinical trials. Among them, the apoptosis-related marketed drug is venetoclax targeting Bcl-2, and Erastin and its derivatives that mainly induce ferroptosis have also shown potential in clinical trials. However, considering the strong heterogeneity of lung adenocarcinoma, not all patients can benefit from the above treatments. Therefore, accurately predicting the sensitivity of lung adenocarcinoma patients to apoptosis and ferroptosis drugs has practical significance for improving the treatment and economic benefits of patients. Summary of the Invention

[0004] To solve the above problems, one of the objectives of the present invention is to provide a kit for detecting the sensitivity of lung adenocarcinoma to apoptosis and ferroptosis. The kit determines the sensitivity of lung adenocarcinoma patients to apoptosis and ferroptosis by detecting the expression of ZNF146 in the tumors of lung adenocarcinoma patients, and further determines whether lung adenocarcinoma patients are suitable for the treatment regimens of apoptosis and ferroptosis drugs, so as to achieve precision medicine.

[0005] To achieve the above objective, the specific technical solution adopted by the present invention is as follows:

[0006] A kit for detecting the sensitivity of lung adenocarcinoma to apoptosis and ferroptosis, comprising: a ZNF146 detection system for detecting the immunohistochemical score of ZNF146 in lung adenocarcinoma tumor tissues; a carrier recording a mathematical model for determining the high / low expression level boundary standard of ZNF146. When the expression level of ZNF146 in the tumor tissues of lung adenocarcinoma patients is lower than the standard, it indicates that the patients are sensitive to the treatment with apoptosis and ferroptosis drugs and are suitable for the treatment regimens of inducing apoptosis and ferroptosis; on the contrary, when it is not lower than the standard, it indicates that the patients are insensitive to the treatment with apoptosis and ferroptosis drugs and are not suitable for the treatment regimens of inducing apoptosis and ferroptosis. The nucleotide sequence number of ZNF263 is: NM_007145.3.

[0007] The above mathematical model is as follows: The immunohistochemical staining score of ZNF146 in the tumor tissue of lung adenocarcinoma patients is expressed as Score = 3 × percentage of strongly positive cells + 2 × percentage of moderately positive cells + percentage of weakly positive cells, and the obtained score ranges from 0 to 300. The standard is: Taking 150 as the critical value for Score. If < 150, then ZNF263 is lowly expressed, indicating that the patient is sensitive to apoptosis and ferroptosis drug therapy and is suitable for the treatment regimen of inducing apoptosis and ferroptosis; if ≥ 150, then ZNF146 is highly expressed, indicating that the patient is insensitive to apoptosis and ferroptosis drug therapy and is not suitable for the treatment regimen of inducing apoptosis and ferroptosis.

[0008] The above carrier can be a paper instruction manual, such as an instruction manual with an APP download window like a QR code, or a computer program storage device or system, such as a USB flash drive, a network disk, etc.

[0009] In a preferred embodiment, the above kit includes an immunohistochemical staining detection system for detecting the immunohistochemical staining score in the tumor tissue of lung adenocarcinoma patients. For example, the above immunohistochemical staining detection system can be the immunochromogenic reagent GTVisionTMⅢ Detection System / Mo&Rb (Including DAB) secondary antibody kit of GeneTech (Shanghai) Co., Ltd., and operate according to the kit instruction manual. At the same time, the above kit also contains an anti-ZNF146 antibody, which can be the ZNF146 polyclonal antibody (1:200, 20385-1-AP) of Wuhan Sanying Biotechnology Co., Ltd. The kit of the present invention can detect the immunohistochemical staining score of ZNF146 in patient samples, judge whether lung adenocarcinoma patients are sensitive to apoptosis and ferroptosis drugs, and whether they are suitable for the treatment regimen of inducing apoptosis and ferroptosis, so as to achieve the purpose of precision medicine.

[0010] In the second aspect, the present invention provides the application of a reagent for detecting the expression level of ZNF146 in the preparation of a kit for detecting the sensitivity of lung adenocarcinoma to apoptosis and ferroptosis. The reagent for detecting the expression level of ZNF146 is the above ZNF146 detection system.

[0011] The present invention has the following beneficial effects:

[0012] 1. The present invention first proposes a method for judging whether a patient is sensitive to apoptosis and ferroptosis drugs by detecting the expression of ZNF146, which can effectively guide the individualized treatment of lung adenocarcinoma patients, improve clinical benefits, and at the same time avoid unnecessary waste of medical resources.

[0013] 2. The present invention has the advantages of simple operation, low cost, high accuracy, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : In Example 1, western blot was used to verify the knockout and overexpression effects of ZNF146 in A549 and PC-9 cells. In the figure, Vector: control group; KO: ZNF146 knockout group; OE: overexpression group after knockout.

[0015] Figure 2 : The apoptosis ratios of A549 and PC-9 cells with knockout and overexpression of ZNF146 after knockout in Example 1.

[0016] Figure 3 : The cell viability of A549 and PC-9 cells with knockout and overexpression of ZNF146 after knockout in Example 2, and then treated with ferroptosis inducer RSL-3.

[0017] Figure 4 : The cell viability of A549 and PC-9 cells with knockout and overexpression of ZNF146 after knockout in Example 2, and then treated with ferroptosis inducer IKE. Detailed implementation manners

[0018] Through comprehensive analysis of the expression data from more than a thousand LUAD samples, the inventors determined that ZNF146 is a transcription factor significantly overexpressed in lung adenocarcinoma and is closely related to poor patient prognosis. Through in vitro experiments, the inventors discovered two key downstream effector molecules of ZNF146 - MDM2 and PHGDH. The research results of the inventors further confirmed that overexpression of ZNF146 can accelerate the progression of lung adenocarcinoma by inhibiting the apoptotic effect of the MDM2 / p53 pathway and promoting the ferroptosis inhibitory effect mediated by PHGDH. The research of the inventors shows that detecting the expression of ZNF146 in the tumor tissues of lung adenocarcinoma patients can effectively judge the sensitivity of patients to apoptotic and ferroptosis drugs, and can be effectively applied to the formulation of individualized chemotherapy regimens for lung adenocarcinoma patients to achieve precision medicine for patients.

[0019] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0020] In the following examples, the human lung adenocarcinoma cell lines A549 and PC-9 were provided by Zhongshan Hospital, Fudan University, and the culture conditions were as follows: in DMEM (high glucose) medium (Jiangsu Kaygee Biotechnology Co., Ltd.) containing 10% FBS and 1% double antibody, at 37°C and 5% CO 2 incubator (Thermo Fisher Scientific, USA).

[0021] Example 1: Changes in apoptosis degree of lung adenocarcinoma cells after knockout and overexpression of ZNF146

[0022] Lentivirus was used to knockout or overexpress ZNF146 in A549 and PC-9 lung adenocarcinoma cells, and the knockout and overexpression effects after knockout were verified by western blot experiment. The specific methods are as follows: The cell proteins of A549 and PC-9 control groups, knockout and overexpression of ZNF146 after knockout were extracted by RIPA lysis method. After electrophoresis using 12.5% polyacrylamide gel and membrane transfer using 0.2 μm PVDF membrane, the membrane was blocked in 5% BSA. The primary antibody incubation was performed using ZNF146 polyclonal antibody (1:2000, 20385-1-AP), and the secondary antibody incubation was performed using horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) (1:2000, A0208) from Shanghai Beyotime Biotechnology Co., Ltd. Subsequently, the signal was detected using ECL luminescent solution. The results are as 6 shown. A549 and PC-9 cells with ZNF146 knockout did not express ZNF146, while the expression level of ZNF146 in A549 and PC-9 cells with re-overexpression of ZNF146 was significantly higher than that of the control group. Figure 1 Annexin V-FITC was used to label phosphatidylserine on the cell surface, and PI was used to label necrotic cells to detect the apoptosis level of the above-mentioned A549 and PC-9 lung adenocarcinoma cells with ZNF146 knockout and overexpression after knockout. Annexin V-FITC single-positive cells were considered early apoptotic cells, and Annexin V-FITC and PI double-positive cells were considered late apoptotic cells. The sum of their proportions in the whole cells was called the apoptosis rate (%). The results are as

[0023] shown. The apoptosis rate of A549 and PC-9 cells with ZNF146 knockout increased, while the apoptosis rate of A549 and PC-9 cells with re-overexpression of ZNF146 decreased significantly. It can be inferred that after the increase in ZNF146 expression level, the apoptosis ratio of lung adenocarcinoma cells decreased, while a low ZNF146 expression level indicated a higher sensitivity to apoptosis drugs. Figure 2 Example 2: Detection of the sensitivity of lung adenocarcinoma cells to ferroptosis inducers after knockout and overexpression of ZNF146

[0024] A549 and PC-9 lung adenocarcinoma cells with ZNF146 knockout or overexpression after knockout were treated with gradient concentrations of ferroptosis inducers (RSL-3 and IKE) for 120 h, and then the cell viability was detected to calculate the half-inhibitory concentration (IC50) of different cell lines to the two ferroptosis inducers.

[0025] The experimental results are as

[0026] shown. Figures 3 - 4As shown. The study found that, compared with the control, knocking out ZNF146 significantly increased the sensitivity of A549 and PC-9 cells to two ferroptosis inducers, while re-overexpressing ZNF146 significantly decreased the sensitivity of A549 and PC-9 cells to the two ferroptosis inducers. It can be inferred from this that after the expression level of ZNF146 increases, the sensitivity of lung adenocarcinoma cells to ferroptosis drugs decreases, while a low expression level of ZNF146 indicates a higher sensitivity to ferroptosis drugs.

[0027] Example 3: Effect test of a kit for detecting apoptosis and ferroptosis sensitivity of lung adenocarcinoma

[0028] This example provides a kit for detecting apoptosis and ferroptosis sensitivity of lung adenocarcinoma, which includes a ZNF146 detection system for detecting the immunohistochemical score of ZNF146 in lung adenocarcinoma tumor tissues, and a carrier recording a mathematical model for determining the high / low expression level boundary standard of ZNF146.

[0029] The ZNF146 detection system includes an immunohistochemical staining detection system and a ZNF146 antibody. The immunohistochemical staining detection system is the immune chromogenic reagent GTVisionTMⅢ Detection System / Mo&Rb (Including DAB) secondary antibody kit of GeneTech (Shanghai) Co., Ltd., and the operation is carried out according to the kit instructions. The ZNF146 antibody is the ZNF146 polyclonal antibody (1:200, 20385-1-AP) of Wuhan Sanying Biotechnology Co., Ltd.

[0030] The scoring / mathematical model uses the H-score immunohistochemical staining scoring system to semi-quantitatively analyze the cell protein expression by integrating the cell staining intensity and staining area. The calculation formula is Score = 3×percentage of strongly positive cells + 2×percentage of moderately positive cells + percentage of weakly positive cells, and the obtained score range is between 0 and 300. This kit takes the median score as the cut-off value to judge the high / low expression of ZNF146, that is, 150 is taken as the critical value. If <150, then ZNF146 is lowly expressed, indicating that the patient is sensitive to apoptosis and ferroptosis drug treatment and is suitable for the treatment plan of inducing apoptosis and ferroptosis; if ≥150, then ZNF146 is highly expressed, indicating that the patient is insensitive to apoptosis and ferroptosis drug treatment and is not suitable for the treatment plan of inducing apoptosis and ferroptosis.

[0031] The actual application of this kit is as follows:

[0032] Forty additional tumor tissues from patients with lung adenocarcinoma were collected, and immunohistochemical scoring was performed on some of the tissues using this kit. The method was as follows: The collected tumor tissues were fixed in 4% paraformaldehyde, then paraffin-embedded, and finally paraffin sections were prepared for immunohistochemical staining experiments. Antigen retrieval, blocking, primary antibody incubation (ZNF146 polyclonal antibody, 1:200, 20385-1-AP), and secondary antibody incubation were carried out according to the instructions of the GTVisionTMⅢ Detection System / Mo&Rb (Including DAB) secondary antibody kit. Finally, DAB chromogenic reagent was used to complete the staining. When necessary, hematoxylin counterstaining was performed, and stained section images of three fields of view were taken under a microscope. The immunohistochemical score was statistically analyzed using image j software and the IHC-Profiler plugin.

[0033] Some of the tissues were used to establish xenograft models in immunodeficient mice and evaluate the clinical efficacy of solid tumors. Two weeks after the model was established, the tumor size was measured as the baseline, and then the apoptosis-inducing drug Nutlin-3a (25 mg / kg body weight / week) + ferroptosis inducer IKE (25 mg / kg body weight / week) were intraperitoneally injected (injected once a week), and the tumor size was evaluated two weeks after the first injection.

[0034] According to the clinical efficacy evaluation criteria for solid tumors (RECIST 1.1), 20 models were evaluated as complete remission or partial remission, among which 6 cases had immunohistochemical staining scores ≥150 in the corresponding source tumor tissues. Twenty models were evaluated as disease progression or stable disease, among which 18 cases had immunohistochemical staining scores ≥150 in the corresponding source tumor tissues. Therefore, the sensitivity of this kit was judged to be 90%, and the specificity was 70%.

[0035] This specific implementation manner is only an interpretation of the present invention and not a limitation thereof. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A kit for detecting apoptosis and ferroptosis sensitivity of lung adenocarcinoma, characterized in that: It includes: ZNF146 detection system, used to detect the immunohistochemical score of ZNF146 in lung adenocarcinoma tumor tissue; A vector of a mathematical model for determining the high / low demarcation standard of ZNF146 expression level is recorded. When the ZNF146 expression level in the tumor tissue of a lung adenocarcinoma patient is lower than the standard, it indicates that the patient is sensitive to apoptosis and ferroptosis drug treatment and is suitable for a treatment regimen that induces apoptosis and ferroptosis; conversely, when it is not lower than the standard, it indicates that the patient is insensitive to apoptosis and ferroptosis drug treatment and is not suitable for a treatment regimen that induces apoptosis and ferroptosis.

2. The kit for detecting apoptosis and ferroptosis sensitivity of lung adenocarcinoma according to claim 1, characterized in that: The mathematical model is: the ZNF146 immunohistochemical staining score in the tumor tissue of patients with lung adenocarcinoma is expressed as Score = 3×strong positive cell percentage + 2×moderate positive cell percentage + weak positive cell percentage, and the obtained score ranges from 0 to 300; The standard is: the score takes 150 as the critical value. If it is <150, ZNF263 is lowly expressed, indicating that the patient is sensitive to apoptosis and ferroptosis drug treatment and is suitable for apoptosis and ferroptosis inducing treatment plans; if it is ≥150, ZNF146 is highly expressed, indicating that the patient is insensitive to apoptosis and ferroptosis drug treatment and is not suitable for apoptosis and ferroptosis inducing treatment plans.

3. The kit for detecting apoptosis and ferroptosis sensitivity of lung adenocarcinoma according to claim 1, characterized in that: The ZNF146 detection system includes an immunohistochemical staining detection system and a ZNF146 antibody.

4. The kit for detecting apoptosis and ferroptosis sensitivity of lung adenocarcinoma according to claim 3, characterized in that: The immunohistochemical staining detection system is an immunochromogenic reagent GT VisionTM III Detection System / Mo&Rb (Including DAB) secondary antibody kit.

5. The kit for detecting apoptosis and ferroptosis sensitivity of lung adenocarcinoma according to claim 3, characterized in that: The ZNF146 antibody is a ZNF146 polyclonal antibody (1:200, 20385-1-AP).

6. The kit for detecting apoptosis and ferroptosis sensitivity of lung adenocarcinoma according to claim 1, characterized in that: The carrier is a paper instruction manual or a computer program storage device or system.

7. Use of reagents for detecting ZNF146 expression levels in the preparation of a kit for detecting apoptosis and ferroptosis sensitivity of lung adenocarcinoma.

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