Application of SUMO2-K11la in preparation of product for detecting lung adenocarcinoma ferroptosis and chemotherapy sensitivity

Abstract

The invention relates to the technical field of biology, in particular to application of SUMO2 protein 11th lysine site lactic acid in preparation of products for detecting lung adenocarcinoma ferroptosis and chemotherapy sensitivity. Based on a series of molecular biological experiment results, the invention reveals that the lactic acid (SUMO2-K11la) of the SUMO2-K11 site inhibits the ferroptosis of the lung adenocarcinoma and increases the chemotherapy resistance, the sensitivity of the lung adenocarcinoma to the ferroptosis and cis-platinum is judged and the prognosis of a patient is predicted by detecting the level of the SUMO2-K11la, and the application prospect is relatively good.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to the application of SUMO2 protein lysine 11 lactylation (SUMO2-K11la) in the preparation of products for detecting ferroptosis and chemotherapy sensitivity of lung adenocarcinoma. Background Art

[0002] Lung cancer is one of the most common malignancies and the leading cause of cancer death. Non-small cell lung cancer accounts for the majority of lung cancer cases (approximately 85%), of which lung adenocarcinoma is the most common pathological subtype. Despite many advances in the treatment of malignant tumors, the prognosis of lung adenocarcinoma remains poor, with a five-year survival rate of less than 26%. For patients with lung adenocarcinoma without known driver mutations, platinum-based doublet chemotherapy ± immunotherapy is the first-line treatment option. However, the efficacy of chemotherapy is often hampered by the emergence of drug resistance in cancer cells. Some cancer cells are able to survive multiple cycles of chemotherapy and resume growth after treatment is stopped. Interestingly, these cells show increased sensitivity to lipid peroxidation and can be killed by ferroptosis inducers, which provides a promising strategy to overcome platinum drug resistance.

[0003] Ferroptosis is a regulated cell death characterized by iron-dependent lipid peroxidation and subsequent cell membrane disruption. The biochemical characteristics of ferroptosis are the production of reactive oxygen species, increased lipid peroxides, accumulation of intracellular iron, and inhibition of the antioxidant system. Inducing ferroptosis can inhibit the progression of lung cancer and restore drug resistance to a certain extent.

[0004] Cellular metabolic reprogramming is one of the hallmark features of malignant tumors. Cancer cells are able to shift glucose metabolism primarily to glycolysis even under aerobic conditions, a phenomenon known as the "Warburg effect". In recent years, in addition to being a metabolite in the glycolysis process, lactate has been identified as a key player in a novel post-translational modification of lysine residues, lactylation (KLA), thereby regulating protein function. However, the biological role of lactylation of non-histone proteins in cancer cells needs further exploration. Summary of the invention

[0005] The present invention provides a new biomarker SUMO2-K11la as a diagnostic marker for evaluating ferroptosis and chemotherapy sensitivity of lung adenocarcinoma. By detecting the level of SUMO2-K11la in lung adenocarcinoma, it is used to judge the sensitivity of lung adenocarcinoma patients to ferroptosis inducers and cisplatin chemotherapy. The method has the advantages of simple operation, high sensitivity, specificity, high cost performance and application value, and is helpful to realize precision medicine for lung adenocarcinoma patients.

[0006] In order to achieve the above-mentioned purpose, the present invention provides an application of a detection reagent in the preparation of a kit for evaluating the sensitivity of lung adenocarcinoma to ferroptosis inducers and chemotherapy. The detection reagent is a reagent for detecting the expression level of SUMO2-K11la in lung adenocarcinoma samples. The detection reagent serves as the only key component of the kit for determining the sensitivity of lung adenocarcinoma to ferroptosis and chemotherapy.

[0007] Preferably, the kit also includes instructions, which record the evaluation cutoff value of SUMO2-K11la gene expression, and the SUMO2-K11la gene expression is calculated by tissue immunohistochemical scoring, and the expression of SUMO2-K11la in the sample is scored according to immunohistochemical staining: four fields of view are randomly selected from each immunohistochemical staining slide to evaluate its staining intensity and coverage; the staining intensity is divided into 0 (no staining), 1 (weak staining), 2 (moderate staining) or 3 (strong staining); the staining area is divided into 0 points (≤5%), 1 point (6%-25%), 2 points (26%-50%), 3 points (51%-75%) and 4 points (>75%) according to the proportion of positive cells. The histological score is obtained by multiplying the staining intensity and coverage area scores, and is divided into low expression (0-4 points) and high expression (5-12 points) accordingly. Patients with high expression are resistant to ferroptosis and cisplatin chemotherapy, and have poor survival; while patients with low SUMO2-K11la expression are sensitive to ferroptosis and cisplatin chemotherapy, and have better survival.

[0008] Preferably, the lung adenocarcinoma sample is a lung adenocarcinoma cell line or a fresh lung adenocarcinoma tumor sample.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] The present invention proposes for the first time that SUMO2-K11la can be used as a biomarker to determine the sensitivity of lung adenocarcinoma to ferroptosis and chemotherapy; the present invention reveals through a series of biological experiments that SUMO2-K11la inhibits ferroptosis of lung adenocarcinoma and leads to cisplatin chemotherapy resistance, and has the advantages of simple operation, high sensitivity and specificity, and provides a new method for clinically evaluating the sensitivity of lung adenocarcinoma patients to cisplatin treatment and prognosis evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1:Feroptosis induces SUMO2 lactylation at lysine (K) 11. (A) SUMO2-K11la levels increased after 24 h of treatment with ferroptosis inducers RSL3 (2 μM), IKE (10 μM) or cisplatin (CDDP, 10 μM); (B) Flowchart illustrating the construction of SUMO2-K11R lactylation-deficient lung adenocarcinoma (LUAD) cells by homology-directed repair (HDR) mediated by the CRISPR-Cas9 system; (C) Sanger sequencing verified the successful introduction of K11R mutations in A549 and PC9 cells, respectively.

[0012] Figure 2 :SUMO-K11 site lactylation inhibits ferroptosis. Among them, (AB) apoptosis inhibitor Z-VAD-FMK (Z-VAD, 10μM), necroptosis inhibitor Necrosulfonamide (Necro, 10μM) and ferroptosis inhibitor Ferrstatin-1 (Ferr-1, 10μM) and Deferoxamine Mesylate (DFO, 10μM) rescued the K11R mutation-enhanced death of lung adenocarcinoma cells A549 and PC9; (CD) relative cell survival rates of A549 and PC9 cells after treatment with different concentrations of RSL3 (48 hours), IKE (72 hours) or cisplatin (CDDP, 72 hours) in the presence or absence of lactate (5mM); (EF) flow cytometry showed that K11R mutation significantly increased the level of lipid peroxidation in A549 and PC9 cells.

[0013] Figure 3 :SUMO-K11 site lactylation is associated with poor prognosis in patients. Among them, (AB) Immunohistochemical images (A) and quantitative analysis (B) of SUMO2-K11la and 4-hydroxynonenal (4-HNE) expression in lung adenocarcinoma tissues; scale bar, 200μm. (CD) Kaplan-Meier survival analysis shows the overall survival (OS) of 140 lung adenocarcinoma patients (C) and a subset of 45 lung adenocarcinoma patients who received cisplatin-based chemotherapy (D). Patients were divided into high and low groups according to their SUMO2-K11la levels. Survival analysis was performed using the log-rank test. DETAILED DESCRIPTION

[0014] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0015] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the specific implementation methods will be briefly introduced below. Obviously, the embodiments described below are some implementation methods of the present invention. For ordinary technicians in this field, other implementation methods can be obtained based on these embodiments without paying creative work.

[0016] (I) SUMO2-K11la increases the sensitivity of lung adenocarcinoma cells to ferroptosis and cisplatin chemotherapy:

[0017] The lung adenocarcinoma cell lines A549 and PC9 were treated with ferroptosis inducers RSL3 (2 μM), IKE (10 μM) and cisplatin (CDDP, 10 μM), respectively. Using a specific polyclonal antibody that can detect SUMO2-K11la, it was found that the level of SUMO2-K11la was increased ( Figure 1 A). To investigate the effect of SUMO2-K11la on ferroptosis in lung adenocarcinoma, we used CRISPER / Cas9-mediated gene mutation knock-in technology to generate a cell line with the K11R mutation [lysine (K) at position 11 was replaced by arginine (R), making it unable to undergo lactylation] ( Figure 1 B&C). To examine the effect of SUMO2-K11la on ferroptosis, we performed a cytotoxicity assay. The results showed that the introduction of the K11R mutation enhanced the sensitivity of cells to ferroptosis compared with the control group (Ctrl). Figure 2 A&B). More importantly, neither apoptosis inhibitors (Z-VAD-FMK, Z-VAD) nor necroptosis inhibitors (Necrosulfonamide, Necro-1) could rescue cells that died due to K11R promotion. In contrast, ferroptosis-specific inhibitors deferoxamine mesylate (DFO) and ferrostatin-1 (Ferr-1) were effective in restoring cell viability ( Figure 2 A&B). The half inhibitory concentration (IC50) assay also confirmed that K11R made lung adenocarcinoma cells more sensitive to ferroptosis, and the addition of lactate further exacerbated this difference ( Figure 2 C&D). Lipid peroxidation is the most important biological process in ferroptosis. We used flow cytometry and Thermo Fisher's BODIPY-C11 lipid peroxidation probe to detect lipid peroxidation levels in cells. The results showed that K11R significantly enhanced lipid peroxidation levels in cells ( Figure 2 E&F).

[0018] (II) SUMO2-K11la levels are associated with poor prognosis of patients:

[0019] The analysis was conducted using a lung adenocarcinoma patient cohort (140 people) from Zhongshan Hospital Affiliated to Fudan University. The Zhongshan Hospital cohort detected the level of SUMO2-K11la in the tumor tissues of lung adenocarcinoma patients by immunohistochemistry, and divided the expression of SUMO2-K11la into high and low expression groups according to a 40% positive threshold. The results showed that SUMO2-K11la levels were negatively correlated with the ferroptosis marker tetrahydroxynonenal ( Figure 3 A&B). Further Kaplan-Meier survival analysis showed that lung adenocarcinoma patients with low SUMO2-K11la levels had a better prognosis, while patients with high levels had a worse prognosis ( Figure 3 C&D). In the cohort of 140 people, 45 received platinum-based chemotherapy after surgery, and Kaplan-Meier survival analysis showed that lung adenocarcinoma patients with low SUMO2-K11la levels had a better prognosis and better response to cisplatin treatment.

[0020] The above embodiments are only preferred embodiments of the present invention and are not any formal or substantial limitations of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a detection reagent in the preparation of a kit for evaluating the sensitivity of lung adenocarcinoma to ferroptosis and chemotherapy, characterized in that: The detection reagent is a reagent for detecting the level of SUMO2-K11 site lactylation (SUMO2-K11la) in lung adenocarcinoma samples. The detection reagent is the only key component of a kit for determining the sensitivity of lung adenocarcinoma to ferroptosis and cisplatin.

2. The use according to claim 1, characterized in that The kit also includes instructions, which record the evaluation threshold of SUMO2-K11la level. The SUMO2-K11la level is calculated by tissue immunohistochemistry scoring, and the expression of SUMO2-K11la in the sample is scored according to immunohistochemical staining: four fields of view are randomly selected from each immunohistochemical staining slide to evaluate their staining intensity and coverage; the staining intensity is divided into level 0 (no staining), level 1 (weak staining), level 2 (moderate staining) or level 3 (strong staining); the staining area According to the proportion of positive cells, the scores were divided into 0 points (≤5%), 1 point (6%-25%), 2 points (26%-50%), 3 points (51%-75%) and 4 points (>75%). The histological score was obtained by multiplying the staining intensity and the coverage area score, and was divided into low expression (0-4 points) and high expression (5-12 points). Patients with high expression were resistant to ferroptosis and cisplatin chemotherapy, and had poor survival. Patients with low SUMO2-K11la expression were sensitive to ferroptosis and cisplatin chemotherapy, and had better survival.

3. The use according to claim 2, characterized in that The lung adenocarcinoma sample is a lung adenocarcinoma cell line or a fresh lung adenocarcinoma tumor sample.

Images