A kit for detecting ferroptosis sensitivity in lung adenocarcinoma

Abstract

The present invention discloses a kit for detecting the ferroptosis sensitivity of lung adenocarcinoma, comprising: an mRNA extraction and separation system for extracting and separating mRNA from tumor tissue of a lung adenocarcinoma patient; a MAFF gene detection system for detecting the mRNA expression level of the MAFF gene; and a mathematical model vector for recording a high / low cutoff standard for determining the MAFF expression level. When the MAFF expression level in the tumor tissue of a lung adenocarcinoma patient is higher than the cutoff standard, it indicates that the patient is sensitive to drug treatment with a ferroptosis inducer; conversely, when the MAFF expression level is lower than the cutoff standard, it indicates that the patient is insensitive to drug treatment with the ferroptosis inducer or has developed treatment resistance.

Description

Technical Field

[0001] The invention belongs to the field of biological detection and relates to a kit for detecting the ferroptosis sensitivity of lung adenocarcinoma. Background Art

[0002] Lung adenocarcinoma is one of the most common and most lethal tumors, with both morbidity and mortality ranking among the highest among all tumors. Chemotherapy and radiotherapy are very important treatments for patients with postoperative lung adenocarcinoma with local lymph node metastasis and advanced stage. However, the development of chemotherapy resistance and radiotherapy resistance greatly limits their efficacy, leading to treatment failure and tumor recurrence and metastasis. The emerging mechanism of ferroptosis can alleviate this treatment resistance. By inducing ferroptosis in tumors, especially in drug-resistant cells, whether as a single therapy or in combination with other chemotherapy drugs or radiotherapy, it has important clinical guidance significance for the treatment effect and reducing the economic burden on patients.

[0003] However, there is currently a lack of effective diagnostic methods in clinical practice to determine which lung adenocarcinoma patients are sensitive to ferroptosis-inducing drug treatment, resulting in the inability to implement precision medicine for them. Summary of the Invention

[0004] In order to find a biomarker that can be used to diagnose lung adenocarcinoma ferroptosis sensitivity, the inventors conducted extensive screening for high and low expression proteins or genes related to lung adenocarcinoma, especially lung adenocarcinoma ferroptosis, based on bioinformatics statistical data. They unexpectedly found that individual genes were highly expressed or underexpressed in lung adenocarcinoma ferroptosis events. After experimental verification, they confirmed that high expression of the MAFF gene (NM_012323.4) can be used as an indication of lung adenocarcinoma ferroptosis sensitivity. Therefore, the present invention includes the following technical solutions:

[0005] A kit for detecting ferroptosis sensitivity in lung adenocarcinoma comprises: an mRNA extraction and separation system for extracting and isolating mRNA from tumor tissue of a lung adenocarcinoma patient; a MAFF gene detection system for detecting the mRNA expression level of the MAFF gene (NM_012323.4); and a mathematical model vector for recording a high / low cutoff standard for determining MAFF expression level. When the MAFF expression level in the tumor tissue of a lung adenocarcinoma patient is higher than the cutoff standard, it indicates that the patient is sensitive to ferroptosis inducer drug treatment; conversely, when the MAFF expression level is lower than the cutoff standard, it indicates that the patient is insensitive to ferroptosis inducer drug treatment or has developed treatment resistance.

[0006] In one embodiment, the mathematical model carrier is an instruction manual, which can be written on a bottle, test tube, or the like, or on a separate piece of paper, or on the outside or inside of a container, such as a paper with an operation demonstration video APP download window such as a QR code. The instruction manual can also be in the form of multimedia, such as a CD, USB flash drive, network disk, etc.

[0007] The above mathematical model is: the expression level of gene MAFF mRNA is expressed as qPCR.Ct=qPCR.Ct.MAFF-qPCR.Ct.internal reference, that is, the difference between the Ct value of quantitative PCR of MAFF extracted from tumor tissue of lung adenocarcinoma patients and the Ct value of the internal reference.

[0008] The internal reference is selected from ACTB (β-Actin), GAPDH, 18sRNA, etc. More preferably, ACTB is used as the internal reference.

[0009] When the internal reference is ACTB, the expression level of gene MAFF mRNA is expressed as qPCR.Ct=qPCR.Ct.MAFF-qPCR.Ct.ACTB. The demarcation standard is: qPCR.Ct takes 13.0 as the critical value. If it is <13.0, the MAFF gene is highly expressed, indicating that the patient is sensitive to ferroptosis-inducing drug treatment; if it is ≥13.0, the MAFF gene is lowly expressed, indicating that the patient is insensitive to ferroptosis-inducing drug treatment or has treatment resistance.

[0010] Alternatively, when the internal reference gene is other than ACTB, such as GAPDH or 18sRNA, the qPCR.Ct cutoff value and high / low cutoff criteria need to be appropriately changed.

[0011] Preferably, the above-mentioned kit further comprises an mRNA reverse transcription system for reverse transcribing mRNA into cDNA.

[0012] For example, the mRNA reverse transcription system can be the II1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) reagent.

[0013] Preferably, the above-mentioned kit further comprises a qPCR reagent for performing a qPCR experiment to detect the expression level of the MAFF gene, namely, a MAFF gene detection system.

[0014] For example, the qPCR reagents can be from Yisheng Biotechnology (Shanghai) Co., Ltd. qPCRSYBR Green Master Mix (Low Rox Plus).

[0015] In one embodiment, the qPCR amplification primer sequences of the MAFF gene in the above qPCR reagent include:

[0016] Forward primer: 5′-GCGAGAGCTGAGCGAGAACA-3′;

[0017] Reverse primer: 5′-TGCTTGAGCCGTGTCACCTC-3′.

[0018] The qPCR primer sequence of the internal reference ACTB gene in the above qPCR reagent can be:

[0019] Forward primer: 5′–TGACGTGGACATCCGCAAAG-3′;

[0020] Reverse primer: 5′–CTGGAAGGTGGACAGCGAGG-3′.

[0021] The ferroptosis inducing drug can be selected from Erastin, sulfasalazine, glutamate, RSL3, and DP17. For example, the ferroptosis inducing drug is RSL3 or Erastin.

[0022] The kit of the present invention can be used to detect the expression level of the MAFF gene in tumor tissue of patients with lung adenocarcinoma, and determine the patient's sensitivity to ferroptosis-inducing drug treatment, thereby helping physicians select individualized treatment plans for patients with lung adenocarcinoma, especially chemotherapy plans, solve the problem of lung adenocarcinoma patients' resistance to chemotherapy and radiotherapy, and achieve precision medicine for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Figure 2. Changes in MAFF mRNA (left) and protein (right) expression levels in A549 cells after lentiviral overexpression of MAFF. GAPDH serves as the reference gene and protein. *** indicates a significant P value (less than 0.01).

[0024] Figure 2 Figure 2 shows the cytotoxicity curves of A549 cells overexpressing MAFF and normal A549 cells (control group) after treatment with two ferroptosis-inducing drugs (RSL3 and Erastin). Overexpressing MAFF significantly enhanced the cell-killing effect of these drugs at the same concentration, indicating increased sensitivity to treatment. *** indicates a significant P value (less than 0.01).

[0025] Figure 3The figure shows the levels of intracellular lipid peroxidation in A549 cells overexpressing MAFF and control cells after treatment with a ferroptosis-inducing agent. The vertical axis represents cell counts, while the horizontal axis represents FITC-A (intracellular and membrane reactive oxygen species detected by the FITC channel (green fluorescence) of a flow cytometer), representing the level of cellular lipid peroxidation. Overexpression of MAFF in A549 cells increased cellular lipid peroxidation, indicating that MAFF overexpression promotes ferroptosis in cells.

[0026] Figure 4 Photographs of mitochondrial structure in A549 cells overexpressing MAFF and control cells are shown. Transmission electron microscopy ultrastructure of MAFF-overexpressing A549 cells revealed altered mitochondrial morphology, indicating that MAFF overexpression promotes ferroptosis. DETAILED DESCRIPTION

[0027] There are many proteins or genes whose expression levels significantly change (either upregulated or downregulated) with the development and progression of lung adenocarcinoma. We conducted an extensive bioinformatics screening of these proteins or genes, focusing on ANLN, ASPM, CDCA4, CHI3L1, ERRFI1, FURIN, GOLGA8A, ITGA6, JAG1, LRP12, MAFF, MASP2, MRPS17, NOB1, PLK1, PNP, PPP1R13L, PRKCA, PTTG1, PYGB, RIOK2, RPP25, SCPEP1, SLC7A11, SLC46A3, SNX7, TPBG, and XBP1. We unexpectedly discovered that the expression of the MAFF gene (NM_012323.4) was significantly elevated in tumor tissues, and experimentally confirmed its potential as a biomarker for ferroptosis sensitivity in lung adenocarcinoma.

[0028] MAFF (Maf F) is a member of the protein family encoded by the avian musculoaponeurot-icfibrosarcoma virus (Maf) oncogene. It is a transcription factor, but lacks a transcriptional activation domain (it belongs to the small Maf protein). It can only bind to proteins containing a b-Zip motif as a chaperone protein to promote gene transcription. When overexpressed in cells, it can also form homodimers and act as a transcriptional repressor, inhibiting gene transcription. Maf F has a wide range of cytoprotective functions, including anti-tumor, neuroprotective, and anti-inflammatory responses.

[0029] MAFF, as a transcription factor, plays an important role in the development and progression of lung adenocarcinoma. Our study found that MAFF can increase the cell-killing effect of lung adenocarcinoma cells on ferroptosis-inducing drugs. Specifically, it can increase the level of intracellular lipid peroxidation, change mitochondrial morphology, and weaken its function, thereby promoting the therapeutic sensitivity of lung adenocarcinoma cells to ferroptosis-inducing drugs. The results showed that detecting the expression of the MAFF gene in tumor tissues of patients with lung adenocarcinoma can effectively determine the patient's sensitivity to ferroptosis-inducing drugs before and during treatment, solve the problem of lung adenocarcinoma patients' resistance to chemotherapy and radiotherapy, and can be effectively used in the formulation of personalized treatment plans for patients with lung adenocarcinoma to achieve precision medicine for patients.

[0030] In this article, for the sake of simplicity, the names of certain proteins, such as MAF, and their encoding genes (DNA) are sometimes used interchangeably. Those skilled in the art should understand that they represent different substances in different descriptions and their meanings can be easily understood based on the context.

[0031] It will be readily understood by those skilled in the art that the kit of the present invention may further include at least one of the following items: a carrying tool, the space of which is divided into a defined space that can accommodate one or more containers, 96-well plates or strips, such as reagent kits, medicine bottles, test tubes, and the like, each of which contains a separate component for the method of the present invention; instructions, which may be written on bottles, test tubes and the like, or on a separate piece of paper, or on the outside or inside of the container, such as a paper with an operation demonstration video APP download window such as a QR code, and the instructions may also be in the tangible or intangible form of multimedia, such as a USB flash drive, a network disk, etc.

[0032] When measuring the MAFF gene expression level of a patient, the detection target of the kit of the present invention is the mRNA expression level of MAFF in lung adenocarcinoma tumor tissue.

[0033] In the mathematical model used to determine the high / low cutoff standard for MAFF mRNA expression, Ct = -1 / lg(1+Ex)*lgX0+lgN / lg(1+Ex), where n is the number of amplification cycles, X0 is the initial template amount, Ex is the amplification efficiency, and N is the amount of amplified product when the fluorescence amplification signal reaches the threshold intensity.

[0034] The higher the starting copy number, the smaller the Ct value. Using a standard with a known starting copy number, a standard curve can be constructed, where the horizontal axis represents the logarithm of the starting copy number and the vertical axis represents the Ct value. Therefore, once the Ct value of an unknown sample is obtained, the starting copy number of that sample can be calculated from the standard curve.

[0035] When this formula is used in the present invention, the target gene is MAFF, and the internal reference is preferably ACTB (β-Actin). In this mathematical model, qPCR.Ct takes 13.0 as the critical value. If it is <13.0, the MAFF gene is highly expressed, indicating that the patient is sensitive to ferroptosis-inducing drug treatment; if it is ≥13.0, the MAFF gene is underexpressed, indicating that the patient is insensitive to ferroptosis-inducing drug treatment or has treatment resistance. It should be understood that when the internal reference gene is other internal reference genes such as GAPDH and 18sRNA, the qPCR.Ct critical value and high / low dividing line need to be appropriately changed.

[0036] Ferroptosis inducers are chemotherapy drugs. There are two main categories of ferroptosis inducers: the first category includes erastin, sulfasalazine, and glutamate, which can act through system xc-; the second category includes RSL3, DP17, and others, which can directly inhibit the activity of glutathione peroxidase (GPX). Erastin, unlike other ferroptosis inducers that typically mediate a single pathway, can mediate multiple molecules, and its effects are highly effective, rapid, and long-lasting. Currently, the most commonly used ferroptosis inducers include RSL3 and Erastin.

[0037] Ferroptosis was first proposed by Brent R. Stockwell in 2012. Studies have shown that erastin can specifically induce the death of Ras mutant cells, but without typical apoptotic features. Iron chelators can inhibit this process, and another compound, RSL3, also exhibits a similar cell death phenotype. Ferroptosis is a regulated cell death that relies on iron ions and reactive oxygen species to induce lipid peroxidation. It differs significantly from other forms of regulated cell death, such as apoptosis, necrosis, and autophagy, at the morphological, biological, and genetic levels. The two main hallmarks of ferroptosis are iron accumulation and lipid peroxidation.

[0038] Unlike classic apoptosis, there is no cell shrinkage, chromatin condensation and other phenomena during ferroptosis, but mitochondrial shrinkage and increased lipid peroxidation will occur.

[0039] Ferroptosis-related characteristics include the following: 1. Morphological characteristics: Ultrastructurally, ferroptosis involves cell membrane rupture and blebbing, mitochondrial atrophy, decreased or even absent mitochondrial cristae, increased membrane density, and normal nuclear morphology, but lacks chromatin condensation. Electron microscopy reveals reduced mitochondrial size and increased double membrane density. 2. Biological characteristics: Increased reactive oxygen species (ROS) and iron accumulation activate the mitogen-activated protein kinase (MAPK) system, which reduces cystine uptake, depletes glutathione, inhibits systemXc-, and increases reduced acyl adenine dinucleotide phosphate oxidase, releasing mediators such as arachidonic acid. 3. Immunological characteristics include the release of pro-inflammatory mediators (such as high-mobility group protein B1) through damage-associated molecular patterns (DAMPs). 4. Gene level: It is mainly regulated by ribosomal protein L8 (RPL8), iron response element binding protein 2 (IREB2), ATP synthase F0 complex subunit C3 (ATP5G3), tetratricopeptide repeat domain 35 (TTC35), citrate synthase (CS), acyl-CoA synthetase family member 2 (ACSF2), as well as metabolic and storage genes TFRC, ISCU, FTH1, FTL, and SLC11A2.

[0040] The essence of ferroptosis is the disorder of lipid oxide metabolism in cells. Under the catalysis of iron ions, metabolism becomes abnormal. When the antioxidant capacity of cells is weakened, lipid reactive oxygen accumulates, causing redox imbalance in cells and inducing cell death.

[0041] Research on the relationship between ferroptosis and cancer has become a hot topic in recent years. By improving the solubility, isolation, and potency of erastin derivatives, some have shown initial success in xenograft tumors. Nanoparticle-induced ferroptosis has also been demonstrated in xenograft studies. The tumor suppressor gene p53 can inhibit SLC7A11 (a component of systemXC), which can also induce ferroptosis in certain conditions. The nuclear transcription factor Nrf2 can inhibit ferroptosis in hepatocellular carcinoma, and inhibiting Nrf2 expression can enhance ferroptosis.

[0042] The degree of lipid peroxidation in cells can reflect the level of cellular ferroptosis in one aspect. By combining whether the mitochondrial structure in the cells has changed or not, the level of ferroptosis can basically be determined.

[0043] By using the kit of the present invention to detect MAFF gene expression, comparing it with an internal reference gene such as ACTB, and calculating the difference between the Ct value of MAFF quantitative PCR and the Ct value of the internal reference, the patient's sensitivity to ferroptosis inducers can be determined, effectively guiding the individualized treatment of lung adenocarcinoma patients, especially those with treatment resistance, improving clinical benefits, and avoiding unnecessary waste of medical resources.

[0044] As a specific application of the above mathematical model, it allows it to be input into the information processing module of genetic testing equipment such as a gene sequencer, or into the information processing module of a PCR instrument in the form of computer software (computer program). Moreover, the above mathematical model also allows it to be input into an intelligent medical system or into a doctor's computer in the form of computer software (computer program), helping doctors determine whether patients are sensitive to ferroptosis-inducing drugs and implement corresponding clinical treatment plans.

[0045] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only for illustrative purposes and are not intended to limit the present invention.

[0046] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned therein, unless otherwise specified, refer to the percentage by mass.

[0047] Example

[0048] The mRNA extraction and separation system used in the embodiment is TRNzolUniversal total RNA extraction reagent (DP424) of Tiangen Biochemical Technology (Beijing) Co., Ltd., and the mRNA reverse transcription system can be the TRNzolUniversal total RNA extraction reagent (DP424) of Yisheng Biotechnology (Shanghai) Co., Ltd. II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digesterplus) reagent. MAFF gene qPCR reagent is a product of Yisheng Biotechnology (Shanghai) Co., Ltd. qPCR SYBRGreen Master Mix (Low Rox Plus).

[0049] The operation was carried out according to the kit instructions. The primers were synthesized and provided by Sangon Biotech (Shanghai) Co., Ltd.

[0050] Human lung adenocarcinoma cell line A549 was provided by Zhongshan Hospital Affiliated to Fudan University. The cells were cultured in DMEM (high glucose) medium (Jiangsu KeyGen Biotechnology Co., Ltd.) containing 10% FBS and 1% double-antibody in a 37°C, 5% CO2 incubator (Thermo Fisher Scientific, USA).

[0051] The fluorescent labeling kit used in lipid peroxidation experiments was the BODIPY TM 581 / 591C11 (lipid peroxidation sensor) kit (D3861) D3861, the detection instrument is BD AriaIII (355) flow cytometer.

[0052] The culture plate used in the in vitro 3D culture is a 96-well round (U) bottom plate Sphera low adhesion surface (Cat. No. 174925) from ThermoFisherScientific.

[0053] Example 1: Overexpression of MAFF in lung adenocarcinoma cells

[0054] Lentivirus vectors are gene therapy vectors developed based on the human immunodeficiency virus (HIV). They are capable of infecting both dividing and non-dividing cells, allowing for long-term expression in vivo with high safety. To enhance MAFF gene expression in lung adenocarcinoma cells, we first obtained and synthesized the MAFF protein coding region (CDS) sequence (NCBI: NM_012323.4) and cloned it into a GeneCare Genetics tool vector (accession number GV208). We then used the GeneCare Genetics lentiviral vector system (comprising the aforementioned GV lentiviral vector, pHelper 1.0 vector, and pHelper 2.0 vector) to transduce the amplified and extracted lentiviral vector plasmids, along with the two pHelper plasmids, into 293T cells for lentiviral packaging. Cell supernatants were collected 48 hours after transfection for lentiviral concentration and purification. Finally, we transfected the purified lentivirus and control virus into the lung adenocarcinoma A549 cell line to construct an A549 stable cell line overexpressing MAFF, and used real-time fluorescence quantitative PCR and western blot to detect the overexpression effect. Figure 1 The study found that in A549 cells overexpressing MAFF, the mRNA and protein expression levels of the MAFF gene were significantly increased compared with the control group, indicating that the MAFF gene was successfully overexpressed in A549 cells.

[0055] Example 2: Overexpression of MAFF in lung adenocarcinoma cells increases the tumor-killing effect of ferroptosis inducers

[0056] We treated MAFF-overexpressing cells and control cells with two ferroptosis inducers, RSL3 and Erastin, respectively. Cell viability was assessed using Alamar Blue 48 hours after treatment. Figure 2 As shown in Figure 2, the study found that compared with the control group, A549 cells overexpressing MAFF showed significantly increased sensitivity to treatment with both ferroptosis-inducing agents. This suggests that increased MAFF expression significantly enhances the cytotoxicity of ferroptosis-inducing agents on tumor cells, increasing their sensitivity to treatment.

[0057] Example 3: Ferropoptosis inducers increase the degree of ferroptosis in lung adenocarcinoma cells overexpressing MAFF

[0058] We further tested the lipid peroxidation level in A549 cells after overexpression of MAFF under the action of ferroptosis inducer RSL3 (concentration 1uM). Figure 3 and Figure 4 As shown. The study found that after 6 hours of RSL3 drug treatment, the lipid peroxidation level of A549 cells overexpressing MAFF was higher than that of the control cells. At the same time, we took pictures of the intracellular mitochondrial structure of MAFF-overexpressing cells and control cells after RSL3 drug treatment. The experimental results are shown in Figure 3 The study found that the mitochondrial membrane structure of A549 cells overexpressing MAFF was blurred compared with that of control cells. Both of these results indicate that MAFF can enhance the level of ferroptosis promoted by ferroptosis inducers.

[0059] Example 4: MAFF expression in lung adenocarcinoma tissues and the therapeutic effect of ferroptosis inducers

[0060] We further collected tissue from 50 patients with stage IIIA lung adenocarcinoma who received first-line platinum plus pemetrexed chemotherapy and / or radiotherapy after surgery (the patients did not receive other postoperative adjuvant treatments other than chemoradiotherapy). We detected the mRNA expression level of MAFF in these 50 original tumor tissue samples, isolated the tumor cells in the samples, and seeded them into 96-well plates (1000 cells / well) for 3D culture. After three days of culture, each tumor spheroid was treated with RSL3 drug (concentration 1uM) for 48 hours. By comparing the expression level of MAFF in the original tumor tissue and the volume change of in vitro 3D tumor spheroids before and after treatment, we preliminarily judged the potential effect of this kit in judging the therapeutic efficacy of ferroptosis inducers.

[0061] We used real-time quantitative PCR to detect the mRNA expression levels of the MAFF gene in lung adenocarcinoma patients after chemotherapy and radiotherapy and before and after treatment with two ferroptosis inducers, RSL3 and Erastin. Based on the statistical changes in MAFF gene expression, we summarized the following patterns:

[0062] Using the ACTB gene as the internal reference, the mRNA expression level of the MAFF gene was expressed as qPCR.Ct = qPCR.Ct.MAFF - qPCR.Ct.ACTB. If qPCR.Ct < 13.0, the MAFF gene was highly expressed and the patient was sensitive to ferroptosis-inducing drug treatment; if qPCR.Ct ≥ 13.0, the MAFF gene was lowly expressed and the patient was insensitive to ferroptosis-inducing drug treatment.

[0063] Of these 50 samples, 20 showed a volume reduction of more than 50% after in vitro 3D culture and treatment with RSL3, while 30 showed a volume reduction of less than 50% (all samples were obtained from the Department of Thoracic Surgery, Zhongshan Hospital, Fudan University). Quantitative PCR results showed that 17 of the 20 samples with a volume reduction of more than 50% after RSL3 treatment had a qPCR Ct value less than 13.0, indicating high MAFF expression. However, only 6 of the 30 samples with a volume reduction of less than 50% had a qPCR Ct value less than 13.0. Therefore, the preliminary sensitivity of this kit is 85% and the specificity is 80%.

[0064] Based on the above research results, the present invention discovered that MAFF gene expression levels can be used to assess the sensitivity of lung adenocarcinoma patients to treatment with ferroptosis-inducing drugs, helping to formulate precise treatment plans for them. It should be understood that after reading the above content of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope of the present invention.

Claims

1. A kit for detecting ferroptosis sensitivity of lung adenocarcinoma, characterized in that: include: An mRNA extraction and separation system for extracting and isolating mRNA from tumor tissues of patients with lung adenocarcinoma; MAFF gene detection system for measuring the mRNA expression of MAFF gene; A mathematical model vector for determining the high / low dividing line standard for MAFF expression is recorded. When the MAFF expression level in the tumor tissue of a lung adenocarcinoma patient is higher than the dividing line standard, it indicates that the patient is sensitive to ferroptosis-inducing drug treatment; conversely, when it is lower than the dividing line standard, it indicates that the patient is insensitive to ferroptosis-inducing drug treatment or has treatment resistance, wherein the ferroptosis-inducing drug is RSL3 or Erastin, and the MAFF gene is NCBI: NM_012323.

4.

2. The kit according to claim 1, wherein The mathematical model is: the expression level of gene MAFF mRNA is expressed as qPCR.Ct=qPCR.Ct.MAFF-qPCR.Ct.internal reference, that is, the difference between the Ct value of quantitative PCR of MAFF extracted from tumor tissue of lung adenocarcinoma patients and the Ct value of the internal reference.

3. The kit according to claim 2, wherein The internal reference is selected from ACTB, GAPDH, and 18sRNA.

4. The kit according to claim 3, wherein When the internal reference is ACTB, the expression level of the MAFF gene mRNA is expressed as qPCR.Ct=qPCR.Ct.MAFF-qPCR.Ct.ACTB. The cutoff standard is: qPCR.Ct takes 13.0 as the critical value. If it is <13.0, the MAFF gene is highly expressed, indicating that the patient is sensitive to ferroptosis-inducing drug treatment; If ≥13.0, the MAFF gene is underexpressed, indicating that the patient is insensitive to ferroptosis-inducing drug treatment or has treatment resistance.

5. The kit according to claim 1, wherein Also included are mRNA reverse transcription systems for reverse transcribing mRNA into cDNA.

6. The kit according to claim 5, wherein It further includes a qPCR reagent for performing qPCR experiments to detect the expression level of the MAFF gene, namely a MAFF gene detection system.

7. The kit according to claim 6, wherein The qPCR amplification primer sequences of the MAFF gene in the qPCR reagent include: Forward primer: 5′-GCGAGAGCTGAGCGAGAACA-3′; Reverse primer: 5′-TGCTTGAGCCGTGTCACCTC-3′.

8. The kit according to claim 6, wherein The qPCR primer sequence of the internal reference ACTB gene in the qPCR reagent is: Forward primer: 5′–TGACGTGGACATCCGCAAAG-3′; Reverse primer: 5′-CTGGAAGGTGGACAGCGAGG-3′.

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