Lactate modification of NBS1 protein and its application

Abstract

The present invention relates to the field of tumor radiotherapy technology, and in particular to lactylated modified NBS1 protein and its application. The lactylated modification site of the present invention is lysine 388 of the NBS1 protein. The lactylated modification of the K388 site of the NBS1 protein activates the DNA repair pathway, and has multiple uses such as promoting tumor cell radiotherapy and chemotherapy resistance, enhancing DNA homologous recombination repair, etc. Tumor radiotherapy or chemotherapy resistance can be reversed by targeted inhibition of lactylated modified NBS1 protein. The present invention provides a treatment strategy for tumor radiotherapy resistance, and provides a potential target for reversing tumor radiotherapy or reversing radiotherapy resistance.

Description

Technical Field

[0001] The present invention relates to the field of tumor radiotherapy, and in particular to lactic acid-modified NBS1 protein and application thereof. Background Art

[0002] DNA double-strand breaks are considered the most severe form of DNA damage, as one unrepaired double-strand break is sufficient to cause permanent growth arrest or cell death. When a cell has a double-strand break in DNA, it immediately initiates DNA repair to maintain DNA integrity in order to survive. The MRN (MRE11-RAD50-NBS1) complex plays a crucial role in the perception and repair of DNA damage. When a double-strand break occurs in DNA, the MRN complex is immediately formed and rapidly recruited to the site of DNA damage to initiate DNA repair. The MRN complex is composed of MRE11, RAD50, and NBS1 proteins. MRE11 and RAD50 proteins are responsible for cutting broken DNA, and NBS1 protein is responsible for regulating the functions of MRE11 and RAD50. Therefore, NBS1 protein is a regulator of the MRN complex. The MRN complex can maintain genome stability and prevent malignant transformation of normal cells. Its function in cancer development and its potential as an anticancer target have been widely explored in various types of cancer. Analysis of gastric cancer specimens surgically resected after chemotherapy showed that low expression levels of the MRN complex were associated with a robust response to chemotherapy and surgical resection. In vitro experiments showed that disruption of the MRN complex or its components could confer sensitivity to cisplatin in cancer cells, with increased DNA damage and cytotoxicity.

[0003] NBS1 is a member of the MRN complex and a regulator of the MRN complex, responsible for regulating the function of the MRN complex. NBS1 mutations are the main cause of Nijmegen breakage syndrome (NBS). The symptoms of patients with Nijmegen breakage syndrome are slow development and high susceptibility to cancer. In tumorigenesis studies, NBS1 mutations have been observed in breast cancer, prostate cancer, lung cancer, liver cancer, and intrahepatic bile duct carcinoma and are associated with increased susceptibility to cancer. In the study of chemotherapy resistance mechanisms, downregulated expression of NBS1 is associated with hypoxia-induced chemotherapy resistance, inhibition of double-strand break repair pathways, and p53 activation in medulloblastoma cells. In the study of radiotherapy resistance mechanisms, NBS1 can stabilize hypoxia-inducible factors and promote cancer cell migration and invasion under ionizing radiation.

[0004] Protein lactylation plays a key role in human physiological and pathological processes. Given the important role of NBS1 in tumorigenesis and progression, further research on the lactylation of NBS1 protein is of great significance. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a lactic acid modified NBS1 protein and its application. The lactic acid modification of NBS1 protein plays an important role in DNA damage repair. Targeted inhibition of lactic acid modified NBS1 protein can reverse tumor radiotherapy or chemotherapy resistance.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides the use of a reagent for detecting the expression level of lactic acid modification of NBS1 protein in the preparation of a kit for predicting the efficacy of tumor radiotherapy or chemotherapy, wherein the lactic acid modification site is lysine 388 of the NBS1 protein.

[0008] The present invention discovered through lactic acid modified proteomics that the 388th lysine (Lysine, K) in the NBS1 protein is a lactic acid modified amino acid; the obtained lactic acid modified NBS1 protein promotes the formation of the MRN complex, thereby promoting tumor radiotherapy or chemotherapy resistance. The statistical results of immunohistochemical data show that compared with the chemotherapy-sensitive tumor tissue of gastric cancer, the lactic acid modification level of NBS1 protein is significantly higher in the drug-resistant tumor tissue of gastric cancer. Therefore, the higher the expression of lactic acid modified NBS1 protein, the worse the chemotherapy effect of tumor patients.

[0009] Preferably, the lactylation modification level of the NBS1 protein is regulated by lysine lactoyltransferase TIP60.

[0010] The present invention uses immunoprecipitation technology and immunoblotting technology to confirm the relationship between lysine lactoyltransferase TIP60 and NBS1 protein. Overexpression of lysine lactoyltransferase TIP60 increases the expression of NBS1 protein lactic acid modification; knocking down lysine lactoyltransferase TIP60 reduces the expression of NBS1 protein lactic acid modification. This indicates that lysine lactoyltransferase TIP60 interacts with NBS1 protein, and TIP60 regulates NBS1 protein lactic acid modification.

[0011] Preferably, the higher the expression level of the lactic acid-modified NBS1 protein, the worse the chemotherapy efficacy of tumor patients.

[0012] In a second aspect, the present invention provides the use of lactylated NBS1 protein as a target in the preparation of drugs for reversing tumor radiotherapy or reversing radiotherapy resistance, wherein the lactylated modification site is lysine 388 of the NBS1 protein.

[0013] In a third aspect, the present invention provides a combination of preparations for reversing tumor radiotherapy or reversing radiotherapy resistance, including a preparation that specifically inhibits lactic acid modification of NBS1 protein.

[0014] The present invention proves through CCK8 drug sensitivity experiment and flow cytometry apoptosis experiment that abolishing NBS1 protein lactic acid modification can reverse the radiotherapy and chemotherapy resistance of tumor cells.

[0015] Preferably, the agent comprises small molecule inhibitors, nucleic acids, polypeptides.

[0016] Preferably, the preparation further comprises a pharmaceutically acceptable carrier.

[0017] In a fourth aspect, the present invention provides the use of the above-mentioned combination of preparations for reversing tumor radiotherapy or reversing radiotherapy resistance in the preparation of drugs for reversing tumor radiotherapy or reversing radiotherapy resistance.

[0018] The beneficial effects of the present invention are:

[0019] The present invention uses lactic acid modification proteomics to find that the 388th lysine in the NBS1 amino acid sequence is lactic acid modified, and the lactic acid modification of NBS1 protein plays an important role in DNA damage repair. After lactic acid modification, NBS1 protein can promote the formation of MRN complex, activate DNA repair pathway, and significantly enhance tumor radiotherapy and chemotherapy resistance.

[0020] Lactic acid-modified NBS1 protein can be used as a biomarker to predict the efficacy of radiotherapy or chemotherapy in cancer patients; by targeted inhibition of lactic acid-modified NBS1 protein, tumor radiotherapy or chemotherapy resistance can be reversed. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a structural diagram of the NBS1 protein.

[0022] Figure 2 This is the mass spectrometry result of NBS1 protein.

[0023] Figure 3 The results of the interaction between lysine lactyltransferase TIP60 and NBS1 protein are shown in Figure 1; A, the results of endogenous immunoprecipitation experiment; B, the results of Western blot of overexpression of TIP60; C, the results of Western blot of knockdown of TIP60; where Flag represents the tag protein, NBS1-K388la represents the lactylation of NBS1 protein at K388, β-tubulin and H4 represent internal controls, and H4K8ac represents the positive control.

[0024] Figure 4Figure 1 is the result diagram of lactylation modification of NBS1 protein promoting the formation of MRN complex; A, the result diagram of first-generation sequencing; B, the result diagram of Western blot experiment; among them, NBS1-K388la represents the lactylation of NBS1 protein at position K388, β-tubulin represents the internal reference, and ATM and p-ATM (S1981) represent the positive control.

[0025] Figure 5 The results of abolishing the lactic acid modification of NBS1 protein and reversing the radiotherapy and chemotherapy resistance of tumor cells; A, CCK8 experimental results; Cell viability represents cell survival rate; B, flow cytometry apoptosis experimental results; C, CCK8 experimental results.

[0026] Figure 6 A graph showing the relationship between the lactic acid modification of the highly expressed NBS1 protein and the chemotherapy efficacy in tumor patients; A, immunohistochemical staining results; B, statistical graph of immunohistochemical data results. DETAILED DESCRIPTION

[0027] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0028] Embodiment 1:

[0029] One million AGS gastric cancer cells were collected and lysed using RIPA lysis buffer to extract intracellular proteins. The proteins were then hydrolyzed into peptides using trypsin. Lactate modification was enriched using the lactate antibody Anti-L-Lactyl Lysine Rabbit pAb (purchased from Jingjie Company, catalog number: PTM-1401), followed by mass spectrometry detection.

[0030] NBS1 protein structure Figure 1 The mass spectrometry results are shown in Figure 2 The results showed that the 388th lysine of NBS1 protein was lactylated.

[0031] Embodiment 2:

[0032] 1. Use immunoprecipitation and immunoblotting techniques to confirm the relationship between lysine lactoyltransferase TIP60 and NBS1 protein.

[0033] The specific steps are as follows:

[0034] (1) Collect 1 million 293T cells, add 1 mL of cell lysis buffer for lysis, and place on ice for 30 min. Vortex and shake once every 15 min, then centrifuge at 12,000 g for 20 min at 4°C, discard the precipitate, and take the supernatant;

[0035] (2) Add 2 μg of the corresponding NBS1 antibody to the supernatant and incubate at 4°C in a shaker for 2 h;

[0036] (3) Take 20 μL of protein A / G-beads (magnetic beads) and add them to a 1.5 mL centrifuge tube. Wash the magnetic beads with 800 μL of RIPA lysis buffer, discard the supernatant, repeat three times, add the magnetic beads to the incubation solution treated in step (2), and incubate overnight in a shaker at 4°C;

[0037] (4) On the next day, place the centrifuge tube on a magnetic rack. After the magnetic beads aggregate on the tube wall, carefully remove the supernatant and wash with 800 μL RIPA lysis buffer. Repeat the washing five times. Add 80 μL RIPA lysis buffer and 20 μL 5× SDS Loading Buffer, shake and mix, heat at 99°C for 10 min, and perform Western blot to detect the relationship between TIP60 protein and NBS1 protein. The results are as follows: Figure 3 As shown in A.

[0038] The results of endogenous immunoprecipitation experiments proved that lysine lactoyltransferase TIP60 interacted with NBS1 protein.

[0039] 2. In order to further confirm the relationship between lysine lactoyltransferase TIP60 and NBS1 protein lactylation modification, lysine lactoyltransferase TIP60 was overexpressed and knocked down respectively.

[0040] The specific experimental method is:

[0041] (1) Overexpression of lysine lactyltransferase TIP60: The overexpression plasmid PLVX-Flag-TIP60 (constructed by Guangzhou Aiji Biotechnology Co., Ltd.) was transfected into AGS cells. 24 hours after transfection, the protein was collected and subjected to Western blot experiment. The results are as follows Figure 3 As shown in B;

[0042] (2) Knockdown of lysine lactoyltransferase TIP60: Si-TIP60 primers (primer 1: ACGGAAGGUGGAGGUGGUU; primer 2: AAGAAGAUCCAGUUCCCCAAGTT) were transfected into AGS cells to knockdown TIP60, and then Western blot experiments were performed. The results are shown in Figure 3 As shown in C.

[0043] Experimental results showed that overexpression of lysine lactoyltransferase TIP60 increased the expression level of NBS1 protein lactic modification; knockdown of lysine lactoyltransferase TIP60 reduced the expression level of NBS1 protein lactic modification.

[0044] In summary, lysine lactoyltransferase TIP60 interacts with NBS1 protein, and TIP60 regulates the lactylation modification of NBS1 protein.

[0045] Embodiment 3:

[0046] 1. Use gene editing technology and immunoprecipitation technology to confirm whether lactylated NBS1 protein can promote the formation of MRN complex.

[0047] The experimental process is as follows:

[0048] The AGS-NBS1-K388R gene-edited cell line was constructed, and the lysine 388 on the NBS1 gene was mutated to arginine, thereby abolishing the lactic acid modification of the NBS1 protein.

[0049] The construction method of AGS-NBS1-K388R gene-edited cell line is as follows:

[0050] (1) First, synthesize a spacer DNA sequence and an extension DNA sequence, wherein the spacer DNA sequence is: 5'-GAAATCAAAGTCTCCAAAA-3'; and the extension DNA sequence is: 5'-TTTTTGTTCCATTCTGGAGACTTTGAT-3';

[0051] (2) Using the Golden Gate assembly method, the pU6-pegRNA-GG plasmid (purchased from Addgene, #132777), the spacer DNA sequence, and the extension DNA sequence were connected into a pU6-pegRNA-GG-NBS1-K388R plasmid;

[0052] (3) pCMV-PE2 plasmid (purchased from Addgene, #132775) and pU6-pegRNA-GG-NBS1-K388R plasmid were transfected into the gastric cancer cell line AGS (purchased from Shanghai Cell Bank, Chinese Academy of Sciences, #TCHu232);

[0053] (4) Monoclonal bacteria were selected from the transfected AGS cells for sequencing. The sequencing results were as follows: Figure 4 As shown in A.

[0054] The first-generation sequencing results showed that the present invention successfully constructed the AGS-NBS1-K388R gene-edited cell line.

[0055] 2. Explore the relationship between lactic acid modification of NBS1 protein and the formation of MRN complex

[0056] Through immunoprecipitation technology (experimental steps as shown in Example 2) and wertern blot experiments, it was verified that lactic acid modification of NBS1 protein promoted the formation of MRN complex, while abolition of lactic acid modification of NBS1 protein significantly reduced the formation of MRN complex ( Figure 4 B).

[0057] Embodiment 4:

[0058] The CCK-8 kit (Brand: Fude Biotechnology, Catalog No.: FD378) and Annexin V-FITC flow apoptosis kit (Brand: abcam, Catalog No.: ab14085) were used to perform drug sensitivity tests and flow apoptosis tests on the AGS-NBS1-K388R gene-edited cell line and the AGS parental line. The results are shown in Figure 5 shown.

[0059] The results of CCK8 experiments showed that abolishing the lactic acid modification of NBS1 protein can reverse chemotherapy resistance ( Figure 5 A); Flow cytometry apoptosis results showed that abolishing NBS1 protein lactylation modification can reverse chemotherapy resistance ( Figure 5 B); CCK8 experimental results show that abolishing NBS1 protein lactylation modification can reverse radiotherapy resistance ( Figure 5 C).

[0060] Therefore, this example demonstrates that abolishing the lactic acid modification of NBS1 protein can reverse the radiotherapy and chemotherapy resistance of tumor cells through CCK8 drug sensitivity experiment and flow cytometry apoptosis experiment.

[0061] Embodiment 5:

[0062] In this example, 94 gastric cancer tumor tissue samples before neoadjuvant chemotherapy were collected from the Digestive Medicine Center of the Seventh Affiliated Hospital of Sun Yat-sen University. According to the principle of tumor regression grade (TRG), these 94 patient samples were divided into two groups: chemotherapy sensitive (Sensitive) and chemotherapy resistant (Resistant):

[0063] Chemosensitive group: TRG grade 0-2 was defined as chemotherapy sensitive;

[0064] Chemotherapy-resistant (Resistant) group: TRG grade 3 was defined as chemotherapy-resistant.

[0065] Subsequently, immunohistochemical staining was performed using the NBS1-K388ka antibody (which can specifically recognize the lactic acid modification of NBS1 protein). The immunohistochemical staining is shown in the figure. Figure 6 As shown in A.

[0066] The statistical results of immunohistochemical data showed that compared with chemotherapy-sensitive gastric cancer tumor tissues, the lactic acid modification level of NBS1 protein was significantly higher in gastric cancer resistant tumor tissues ( Figure 6 B). Therefore, the higher the expression of lactic acid modification of NBS1 protein, the worse the chemotherapy effect of tumor patients.

[0067] In summary, the present invention has found through lactic acid modified proteomics that lysine (K) at position 388 in NBS1 protein is a lactic acid modified amino acid. The present invention has experimentally confirmed that lactic acid modified NBS1 protein has multiple uses such as promoting the formation of MRN complex, promoting tumor cell radiotherapy and chemotherapy resistance, enhancing DNA homologous recombination repair, etc. Lactic acid modified NBS1 protein has the following application prospects: (1) It can regulate the function of NBS1 protein; (2) It can be used as a biomarker to predict the efficacy of radiotherapy or chemotherapy in tumor patients; (3) By targeted inhibition of lactic acid modified NBS1 protein, tumor radiotherapy or chemotherapy resistance can be reversed.

[0068] Lactic acid modification of NBS1 protein can be used as a predictor of the prognosis of tumor patients and the efficacy of neoadjuvant chemotherapy. Lactic acid modification is of great significance in regulating the function of NBS1 protein and predicting the therapeutic efficacy of tumor patients.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a reagent for detecting the lactic acid modification expression level of NBS1 protein in the preparation of a kit for predicting the efficacy of tumor radiotherapy or chemotherapy, characterized in that: The lactic acid modification site is lysine 388 of the NBS1 protein.

2. Use of the reagent for detecting the lactic acid modification expression level of NBS1 protein according to claim 1 in preparing a kit for predicting the efficacy of tumor radiotherapy or chemotherapy, characterized in that: The lactylation modification level of the NBS1 protein is regulated by the lysine lactoyltransferase TIP60.

3. Use of the reagent for detecting the lactic acid modification expression level of NBS1 protein according to claim 1 in preparing a kit for predicting the efficacy of tumor radiotherapy or chemotherapy, characterized in that: The higher the expression level of the lactic acid-modified NBS1 protein, the worse the chemotherapy efficacy of tumor patients.

4. A preparation combination for reversing tumor radiotherapy or reversing radiotherapy resistance, characterized in that: The invention comprises a preparation for reducing or abolishing the lactylation modification of NBS1 protein; the preparation is a pU6-pegRNA-GG-NBS1-K388R plasmid.

5. The preparation combination for reversing tumor radiotherapy or reversing radiotherapy resistance according to claim 4, characterized in that: Also included are pharmaceutically acceptable carriers.

6. Use of the combination of preparations for reversing tumor radiotherapy or reversing radiotherapy resistance as described in any one of claims 4 to 5 in the preparation of drugs for reversing tumor radiotherapy or reversing radiotherapy resistance.

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