Biomarker for diagnosing and treating pancreatic cancer and application thereof

Abstract

The invention relates to a biomarker for diagnosing and treating pancreatic cancer and application of the biomarker. Through systematic clinical sample analysis, in-vitro cell function experiment and in-vivo animal model verification, the invention reveals the core molecular mechanism that RBMS2 protects pancreatic cancer cells through active inhibition of ferroptosis for the first time, and indicates that RBMS2 is an important functional protein in cells, has the potential of serving as a pancreatic cancer diagnosis and treatment integrated target, and can be used as a pancreatic cancer diagnosis and treatment integrated target. The kit is used for auxiliary diagnosis of pancreatic cancer, judgment of malignancy degree of pancreatic cancer, evaluation of treatment effect or prediction of patient prognosis, and treatment or prevention of pancreatic cancer by taking RBMS2 as a target spot, provides a basis for development of a synergistic treatment scheme, and can further expand application, screen and develop reagents for regulating and controlling related cellular mechanisms, and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to biomarkers for the diagnosis and treatment of pancreatic cancer and their applications. Background Technology

[0002] Pancreatic cancer is a malignant tumor originating from the epithelial cells of the pancreatic ducts or acinar cells. Because early symptoms are often atypical and specific diagnostic indicators are lacking, most patients are diagnosed at an advanced stage, missing the optimal time for surgery. Currently, clinical diagnosis of pancreatic cancer mainly relies on imaging examinations (such as CT and MRI), tumor marker testing (such as CA19-9), and endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNA). Treatment primarily involves a multidisciplinary approach combining surgery, chemotherapy, radiotherapy, and targeted therapy.

[0003] Although some progress has been made in the diagnosis and treatment of pancreatic cancer, there are still many bottlenecks and challenges, specifically in the following aspects: (1) Limitations of diagnostic markers: The serum tumor marker CA19-9, which is widely used in clinical practice, has unsatisfactory sensitivity and specificity. In patients with early pancreatic cancer, the positive rate of CA19-9 is low, and it may also be elevated in some benign pancreatobiliary diseases (such as cholangitis and pancreatitis), which can easily lead to misdiagnosis or missed diagnosis. Imaging examinations have limited ability to identify small lesions, while puncture biopsy, as the gold standard, is an invasive procedure with certain risks and complications, and is not suitable for all patients. Therefore, there is an urgent need in clinical practice to develop new, highly sensitive, and highly specific non-invasive diagnostic biomarkers to achieve early detection and accurate diagnosis of pancreatic cancer. (2) Lack of therapeutic targets and drug resistance: The poor treatment effect of pancreatic cancer is largely due to its complex tumor microenvironment and genetic heterogeneity. Pancreatic cancer tumor tissue is often accompanied by dense fibrotic matrix, which forms a physical barrier that hinders the effective penetration of chemotherapy drugs. At the same time, there is still a lack of effective targeted drugs for key gene mutations that drive pancreatic cancer (such as KRAS mutations). Although existing chemotherapy regimens can prolong the survival of patients to a certain extent, they generally have serious toxic side effects and drug resistance problems. The application of immunotherapy in pancreatic cancer is also limited by the characteristics of "cold" tumors. Therefore, finding new therapeutic targets that can accurately target tumor cells and overcome drug resistance is the key to improving the prognosis of pancreatic cancer.

[0004] RNA-binding proteins (RBPs) play a crucial role in tumorigenesis and development. They influence gene expression by regulating RNA splicing, stabilization, localization, and translation, thereby participating in various life processes such as cell proliferation, differentiation, and apoptosis. Among numerous RNA-binding proteins, RNA-binding motif single-stranded-interacting protein 2 (RBMS2), a newly discovered RNA-binding protein, has been shown to play a role in certain tumors such as breast cancer and clear cell renal cell carcinoma. However, systematic and in-depth research reports on the specific function, expression pattern, and potential as a diagnostic biomarker and therapeutic target of RBMS2 in pancreatic cancer are still lacking. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides biomarkers for the diagnosis and treatment of pancreatic cancer and their applications, and conducts an in-depth analysis of the specific functions and expression patterns of RBMS2 in pancreatic cancer, with the aim of developing new applications related to RBMS2.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides the use of a detection reagent for RBMS2 protein in any of the following aspects:

[0008] (1) Develop pancreatic cancer prognostic risk assessment products;

[0009] (2) Reagents for screening agents that regulate the sensitivity of cells to iron death;

[0010] (3) Reagents for screening formulations that regulate the expression levels of antioxidant proteins;

[0011] (4) Reagents for screening agents that regulate and promote the expression level of ferroptosis protein;

[0012] (5) Preparation of screening reagents for agents that regulate mitochondrial damage;

[0013] (6) Reagents for screening agents to prepare formulations that regulate the expression levels of stress proteins;

[0014] (7) Reagents for screening agents to regulate the expression levels of pro-apoptotic / death factors;

[0015] (8) Reagents for screening preparations of markers that regulate the expression level of DNA double-strand breaks;

[0016] (9) Reagents for screening agents to prepare formulations that regulate the stability of NRF2 mRNA;

[0017] (10) Preparation of screening reagents for drugs to treat pancreatic cancer.

[0018] This invention revealed that RBMS2 is highly expressed in pancreatic cancer. Further analysis of RBMS2 protein expression levels in human pancreatic cancer cell lines, including knockdown, and in-depth cellular changes, showed that inhibiting RBMS2 significantly enhances the sensitivity of pancreatic cancer cells to ferroptosis. Specifically, this includes inhibiting the expression of antioxidant proteins, upregulating ferroptosis-promoting factors, inducing mitochondrial damage, and inducing endoplasmic reticulum stress and DNA damage. Furthermore, it was found that RBMS2 upregulates NRF2 protein expression by binding to the 3'-untranslated region (3'-UTR) of NRF2 mRNA, thereby enhancing NRF2 mRNA stability. These findings indicate that RBMS2 is an important functional protein in cells, powerfully inhibiting ferroptosis by maintaining the function of the antioxidant system and simultaneously suppressing the expression of ferroptosis-promoting factors, thus enabling cells to survive and proliferate under high oxidative stress. It has the potential to serve as an integrated target for the diagnosis and treatment of pancreatic cancer, and can be used to assist in the diagnosis of pancreatic cancer, determine its malignancy, evaluate the treatment effect or predict the patient's prognosis. RBMS2 can be used to treat or prevent pancreatic cancer. In addition, its applications can be further expanded to screen reagents that regulate related cell mechanisms (specifically, it can be used for therapeutic purposes or non-therapeutic purposes, such as basic mechanism research, preparation of cells with specific traits, etc.).

[0019] Preferably, the detection reagent includes a reagent for detecting the expression level of RBMS2 protein.

[0020] It is understood that reagents commonly used in the field for detecting RBMS2 protein expression levels are theoretically applicable to this invention.

[0021] Preferably, the detection reagent includes an ELISA kit, immunohistochemistry kit, or chemiluminescence detection reagent containing RBMS2 protein-specific antibodies, or a PCR kit containing specific primers and probes for detecting RBMS2 mRNA or DNA.

[0022] Preferably, the prognostic risk assessment product includes a prognostic risk assessment kit.

[0023] Preferably, the antioxidant protein includes at least one of NRF2, NQO1, or HO-1.

[0024] Preferably, the ferroptosis-promoting protein includes ACSL4 and / or NCOA4.

[0025] Preferably, the mitochondrial damage includes a decrease in mitochondrial membrane potential.

[0026] Preferably, the stress protein includes at least one of p-PERK, GADD34, or GRP75.

[0027] Preferably, the pro-apoptotic / death factor includes CHOP.

[0028] Preferably, the marker of DNA double-strand breakage includes p-H2AX.

[0029] Secondly, the present invention provides a screening method, the screening method comprising:

[0030] The expression level of RBMS2 protein in cells was detected in the presence of the substances to be screened. Based on the expression level, the following reagents were obtained:

[0031] (a) Agents that regulate the sensitivity of cells to iron death;

[0032] (b) Pharmacological agents that regulate the expression levels of antioxidant proteins;

[0033] (c) Pharmacological agents that regulate and promote the expression level of ferroptosis protein;

[0034] (d) Agents that regulate mitochondrial damage;

[0035] (e) Pharmacists that regulate stress protein expression levels;

[0036] (f) Pharmacists that regulate the expression levels of pro-apoptotic / death factors;

[0037] (g) Developed formulations that regulate the expression levels of markers for DNA double-strand breaks;

[0038] (h) Regulators of NRF2 mRNA stability;

[0039] (i) Drugs for treating pancreatic cancer.

[0040] In this invention, based on the exploration of the functions of RBMS2 protein in cells, preparations with different functions can be screened. By detecting the expression level of RBMS2 protein in cells in the presence of the substance to be screened, the ability of the substance to be screened to regulate (inhibit or promote) ferroptosis sensitivity, regulate the expression level of antioxidant proteins, regulate the expression level of ferroptosis-promoting proteins, regulate mitochondrial damage, regulate the expression level of stress proteins, regulate the expression level of pro-apoptotic / death factors, regulate the expression level of DNA double-strand break markers, regulate NRF2 mRNA stability, and treat pancreatic cancer can be evaluated. These effects can be therapeutic or non-therapeutic.

[0041] Thirdly, the present invention provides the use of an inhibitor of the RBMS2 protein in any of the following aspects:

[0042] 1) Improve the sensitivity of cells to ferroptosis or prepare related formulations;

[0043] 2) Reduce the expression level of antioxidant proteins or prepare related formulations;

[0044] 3) Upregulate the expression level of ferroptosis-promoting proteins or prepare related formulations;

[0045] 4) Inducing mitochondrial damage or preparing related formulations;

[0046] 5) Upregulate stress protein expression levels or prepare related formulations;

[0047] 6) Upregulate the expression levels of pro-apoptosis / death factors or prepare related formulations;

[0048] 7) Upregulate the expression levels of markers of DNA double-strand breaks or prepare related formulations;

[0049] 8) Reduce the stability of NRF2 mRNA or prepare related formulations.

[0050] Based on the exploration of the function of RBMS2 protein in cells, its inhibitors can be further developed for various applications, including therapeutic and non-therapeutic purposes, such as basic mechanism research and preparation of cells with specific traits.

[0051] Preferably, the inhibitor of the RBMS2 protein includes any one or a combination of at least two of the following: nucleic acid molecules, nucleic acid constructs, lentiviruses, antibodies, protein degrading agents (such as PROTAC molecules that induce the degradation of RBMS2 protein), or small molecule compounds.

[0052] Preferably, the nucleic acid molecule is any one or a combination of at least two of the following: double-stranded RNA, siRNA, antisense oligonucleotide (ASO), or shRNA.

[0053] Preferably, the antioxidant protein includes at least one of NRF2, NQO1, or HO-1.

[0054] Preferably, the ferroptosis-promoting protein includes ACSL4 and / or NCOA4.

[0055] Preferably, the mitochondrial damage includes a decrease in mitochondrial membrane potential.

[0056] Preferably, the stress protein includes at least one of p-PERK, GADD34, or GRP75.

[0057] Preferably, the pro-apoptotic / death factor includes CHOP.

[0058] Preferably, the marker of DNA double-strand breakage includes p-H2AX.

[0059] Fourthly, the present invention provides a pharmaceutical composition comprising an inhibitor of the RBMS2 protein described in the third aspect and a second formulation, the second formulation comprising a ferroptosis inducer and / or a chemotherapeutic agent.

[0060] This invention not only identifies RBMS2 as a target but also clearly elucidates its mechanism of action through detailed data from various embodiments: RBMS2 inhibits ferroptosis by regulating the expression of key ferroptosis molecules such as the NRF2 antioxidant pathway and ACSL4 / NCOA4. This clear molecular mechanism provides a precise "navigation map" for drug development. Developers can highly target and design small molecule inhibitors or PROTAC degraders to specifically block RBMS2 function or degrade the protein. Simultaneously, it provides a foundation for developing synergistic treatment regimens (such as combining with ferroptosis inducers). This drug development model based on a clearly defined mechanism significantly improves the success rate of research and development and shortens the development cycle.

[0061] Preferably, the ferroptosis inducer includes RSL3, erastin, etc., and the chemotherapy drug includes gemcitabine, etc.

[0062] Preferably, the pharmaceutical composition is in the form of a standalone formulation or a combination formulation.

[0063] Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0064] Preferably, the excipients include any one or a combination of at least two of the following: carrier, cosolvent, osmotic pressure regulator, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0065] Compared with the prior art, the present invention has at least the following beneficial effects:

[0066] This invention, through systematic clinical sample analysis, in vitro cell function experiments, and in vivo animal model verification, reveals for the first time the core molecular mechanism by which RBMS2 protects pancreatic cancer cells by actively inhibiting ferroptosis. It demonstrates that RBMS2 is an important functional protein in cells and has the potential to serve as a target for integrated diagnosis and treatment of pancreatic cancer. This could be used to assist in the diagnosis of pancreatic cancer, assess its malignancy, evaluate treatment efficacy, or predict patient prognosis. Targeting RBMS2 for the treatment or prevention of pancreatic cancer provides a foundation for developing synergistic treatment regimens. Furthermore, its applications can be further expanded to include the screening and development of reagents that regulate related cell mechanisms, etc. Attached Figure Description

[0067] Figure 1Figure A shows the high expression of RBMS2 in pancreatic cancer tissues and its predictive effect on poor prognosis. Figure A shows the expression of RBMS2 protein in adjacent and tumor tissues of pancreatic cancer patients, while Figure B shows the positive rate and strong positive rate of RBMS2 protein in tumor tissues and adjacent tissues of pancreatic cancer patients.

[0068] Figure 2 The figure shows the results of Western blot (WB) assays to detect RBMS2 protein expression levels in the control and experimental groups.

[0069] Figure 3 The graph shows the IC50 values ​​of Erastin and RSL3, the ferroptosis inducers, in the control and experimental groups in the CCK8 experiment.

[0070] Figure 4 The figure shows the results of Western blot analysis to detect the antioxidant capacity of the NRF2 signaling pathway damaged by knockdown of RBMS2.

[0071] Figure 5 The figure shows the results of Western blotting (WB) experiments detecting the upregulation of ferroptosis protein expression by knocking down RBMS2.

[0072] Figure 6 Figure showing the results of flow cytometry analysis using the JC-1 probe to detect a significant decrease in mitochondrial membrane potential in the sh-RBMS2 group of cells.

[0073] Figure 7 The figure shows the results of Western blot analysis to detect the effects of RBMS2 knockdown on endoplasmic reticulum stress and DNA damage.

[0074] Figure 8 The figures show the results of RT-qPCR and SPR experiments demonstrating the binding interaction between RBMS2 protein and Nrf2 mRNA. Figure A shows the results of RT-qPCR detection of RBMS2 knockdown reducing Nrf2 RNA levels, and Figure B shows the results of SPR experiments demonstrating the direct binding interaction between RBMS2 protein and Nrf2 mRNA.

[0075] Figure 9 The results of silencing RBMS2 significantly inhibiting tumor growth in a nude mouse model of subcutaneous pancreatic cancer xenografts are shown in Figure A, which shows the in vitro results of subcutaneous xenografts in the control and experimental groups; Figure B shows the weight statistics of subcutaneous xenografts in the control and experimental groups; and Figure C shows the immunohistochemical detection results of RBMS2 and Ki67 protein expression in the control and experimental groups. Detailed Implementation

[0076] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0077] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0078] Unless otherwise defined, scientific and technical terms and their abbreviations used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Some of the terms and abbreviations used in this invention are listed below.

[0079] Ferroplasty is a novel form of programmed cell death characterized by abnormal intracellular iron metabolism leading to lipid peroxidation, which in turn causes cell membrane rupture and cell death. Unlike other types of cell death (such as apoptosis, necrosis, and autophagy), ferroptosis depends on intracellular iron ion levels and lipid peroxidation, and is usually accompanied by a decrease in glutathione (GSH) levels and the accumulation of lipid peroxides.

[0080] NRF2: Nuclear factor-erythroid 2 related factor 2 is a ubiquitous master transcription factor that can upregulate the expression of antioxidant enzymes and cytoprotective proteins mediated by antioxidant response elements (AREs).

[0081] NQO1: NAD(P)H quinone dehydrogenase 1 is a flavin-containing quinone reductase that can use NADH or NADPH as electron donors to catalyze the two-electron reduction reaction of quinones to produce hydroquinones.

[0082] HO-1: Heme Oxygenase-1 (HO-1) mainly catalyzes the breakdown and metabolism of heme into ferrous iron, carbon monoxide and biliverdin, and is an important antioxidant enzyme.

[0083] ACSL4: Acyl-CoA synthetase long chain family member 4. As an important enzyme involved in lipid metabolism, it participates in ferroptosis by converting free amino acids into arachidonic acid-CoA to generate lipid peroxides.

[0084] NCOA4: nuclear receptor coactivator 4, as a selective substrate receptor, can bind to ferritin, a cytoplasmic iron storage complex, mediate ferritinophagy, release a large amount of free iron, and regulate the iron metabolism balance in the body.

[0085] p-PERK: PERK (PKR-like ER kinase) is a transmembrane protein located in the endoplasmic reticulum (ER) with eIF2α kinase activity, which couples ER stress signals with protein translation inhibition. During ER stress, PERK activity is upregulated, and phosphorylated PERK (p-PERK) subsequently phosphorylates eukaryotic translation initiation factor 2α (eif2α) to attenuate global protein translation.

[0086] CHOP: C / EBP homologous protein is a protein closely related to cellular stress and apoptosis. It belongs to the C / EBP transcription factor family and is encoded by the DDIT3 gene. It is mainly activated under conditions such as endoplasmic reticulum stress, oxidative stress, or nutrient deficiency. It participates in cell survival or death decisions by regulating the expression of downstream genes and is associated with various diseases such as cancer and diabetic complications.

[0087] p-H2AX: Phosphorylated histone H2AX (Phospho-Histone H2A.X), specifically histone H2AX phosphorylated at serine residue 139, is a molecular marker of DNA double-strand breaks. As the initiation signal molecule for DNA double-strand break damage sensing, p-H2AX recruits a series of DNA damage response proteins to DNA damage sites, forming a DNA damage response functional complex that initiates and activates cellular DNA damage responses such as DNA repair and cell cycle checkpoints.

[0088] GADD34, also known as Protein Phosphatase 1 Regulatory Subunit 15A, recruits serine / threonine protein phosphatase PP1 to dephosphorylate translation initiation factors eIF-2A / EIF2S1, thereby reversing the shutdown of stress-induced kinase-initiated protein synthesis and promoting cellular recovery from stress.

[0089] GRP75: Glucose-regulated protein 75 is a member of the highly conserved heat shock protein family. In the cell, it mainly functions as a chaperone protein, helping unfolded or misfolded proteins to fold correctly. It also binds to multiple intracellular factors and participates in several important intracellular biological processes.

[0090] Example 1

[0091] This embodiment performs omics analysis on RBMS2 and uses it for prognostic risk assessment.

[0092] Through high-throughput proteomics screening and bioinformatics analysis of tumor tissue samples and adjacent normal tissues from 10 pairs of pancreatic cancer patients, we identified RBMS2 as a significantly upregulated RNA-binding protein in pancreatic cancer for the first time. Furthermore, using large-scale clinical tissue microarrays for immunohistochemical (IHC) staining, we achieved, for the first time, a precise depiction of the expression pattern of RBMS2 protein levels in pancreatic cancer tissues. Figure 1 More importantly, through correlation analysis of patients' clinicopathological parameters, we found that high RBMS2 expression predicted worse tumor stage, lymph node metastasis, and tumor recurrence (Table 1). Furthermore, through univariate and multivariate Cox regression analyses, Table 2 shows the results of the univariate Cox regression analysis of clinicopathological parameters in pancreatic cancer patients, and Table 3 shows the results of the multivariate Cox regression analysis of clinicopathological parameters in pancreatic cancer patients. These results confirm that RBMS2 can serve as an independent risk prognostic factor for pancreatic cancer patients, thus providing solid clinical evidence for RBMS2 as a prognostic indicator.

[0093] Table 1

[0094]

[0095] Table 2

[0096]

[0097] Table 3

[0098]

[0099] Example 2

[0100] This embodiment analyzes the specific functions and expression patterns of RBMS2 in pancreatic cancer, as well as its potential as a diagnostic biomarker and therapeutic target.

[0101] (1) Experimental materials and methods:

[0102] Cell lines: human pancreatic cancer cell lines BxPC-3 and MIA PaCa-2.

[0103] Main reagents: ferroptosis inducers RSL3 (MCE, HY-100218A) and erastin (MCE, HY-15763); specific primary antibodies for detecting various proteins [RBMS2 (proteintech, 67395-1-Ig), NCOA4 (CST, #66849T), GADD34 (proteintech, 10449-1-AP), GRP75 (proteintech, 14887-1-AP), p-PERK (proteintech, 82534-1-RR), ACSL4 (proteintech, 22401-1-AP), CHOP (proteintech, 15204-1-AP), p-H2AX (proteintech, 29380-1-AP), NRF2 (proteintech, 16396-1-AP), NQO1 (proteintech, [11451-1-AP), HO-1 (proteintech, 10701-1-AP), GAPDH (proteintech, 60004-1-Ig), etc.]; JC-1 probe (Beyotime, C2003S) for detecting mitochondrial membrane potential.

[0104] Experimental Groups:

[0105] Control group (sh-con): pancreatic cancer cells transfected with lentiviral shRNA targeting non-specific targets.

[0106] Experimental group (sh-RBMS2): Pancreatic cancer cells transfected with lentiviral shRNA that specifically targets the RBMS2 gene to achieve stable knockdown of RBMS2.

[0107] Detection method:

[0108] Protein expression detection: Western blotting was used to detect changes in the expression levels of relevant proteins in each group of cells.

[0109] Drug sensitivity assay: The CCK-8 assay was used to detect changes in cell viability in different groups after treatment with ferroptosis inducers RSL3 or erastin, in order to assess their sensitivity to ferroptosis.

[0110] The specific treatment method includes: seeding control and experimental group cells into 96-well plates and allowing them to adhere overnight; diluting RSL3 (0, 0.5, 1, 2, 4, 8 μM) or erastin (0, 2, 4, 8, 16, 32 μM) with DMEM medium; incubating for 24 h; adding CCK-8 reagent and incubating for another 2 h; and finally measuring the absorbance at 450 nm using a microplate reader and calculating the IC50 value.

[0111] Mitochondrial function assay: Changes in mitochondrial membrane potential (MMP) were detected using the JC-1 fluorescent probe combined with flow cytometry or fluorescence microscopy.

[0112] (2) Experimental results:

[0113] 1) Changes in RBMS2 expression levels

[0114] Western blot results of cells in the control and experimental groups are as follows: Figure 2 As shown, sh-con represents the control group results, and sh-RBMS2#1, sh-RBMS2#2 and sh-RBMS2#3 represent the results of three biological replicate experiments in the experimental group. The results indicate that the stable knockdown of RBMS2 in the experimental group cells was successfully achieved.

[0115] 2) Inhibition of RBMS2 significantly enhances the sensitivity of pancreatic cancer cells to ferroptosis.

[0116] Drug sensitivity testing, such as Figure 3 As shown, compared with the control group (sh-NC), the growth of cells in the experimental group (sh-RBMS2) with stable RBMS2 knockdown was inhibited to some extent. More importantly, when cells were treated with low doses of the ferroptosis inducers RSL3 or erastin, the cell viability of the experimental group decreased sharply, and its IC50 value (half-maximal inhibitory concentration) was significantly lower than that of the control group. This result shows that the absence of RBMS2 makes pancreatic cancer cells extremely sensitive to ferroptosis inducers, proving that RBMS2 plays a key protective role against ferroptosis in pancreatic cancer cells.

[0117] 3) RBMS2 inhibits ferroptosis through a dual mechanism.

[0118] To investigate the molecular mechanism of the above phenomenon, a series of key proteins in the ferroptosis regulatory pathway were further examined, and the following results were obtained:

[0119] a) Inhibition of the antioxidant system, undermining cellular defense: Ferroprelation is fundamentally caused by iron-dependent lipid peroxidation. NRF2 is the most important transcription factor for cellular resistance to oxidative stress, and its downstream NQO1 and HO-1 are key antioxidant proteins. Western Blot results ( Figure 4The study showed that the protein expression levels of NRF2, NQO1, and HO-1 were significantly decreased in the sh-RBMS2 group. This indicates that a key function of RBMS2 is to maintain the activity of the NRF2 signaling pathway, thereby protecting cancer cells from oxidative damage. Knockdown of RBMS2 directly led to the collapse of this core defense system.

[0120] b) Upregulation of ferroptosis-promoting factors, actively initiating the ferroptosis process: Simultaneously, we found a significant increase in the expression levels of several proteins that promote ferroptosis. ACSL4 (catalyzes the synthesis of long-chain polyunsaturated fatty acids, providing substrates for lipid peroxidation) and NCOA4 (mediates ferritin autophagy, releasing excess free iron) are two key positive regulators of ferroptosis initiation. Western Blot results ( Figure 5 The results showed that knocking down RBMS2 significantly increased the protein expression of both ACSL4 and NCOA4, revealing that RBMS2 inhibits the expression of these ferroptosis-promoting proteins.

[0121] 4) Inhibition of RBMS2 triggers widespread organelle stress and damage, ultimately leading to ferroptosis.

[0122] The combined effect of these two mechanisms ultimately led to catastrophic consequences at the cellular level:

[0123] Mitochondrial damage: JC-1 probe flow cytometry results showed ( Figure 6 The mitochondrial membrane potential of cells in the sh-RBMS2 group was significantly reduced, which is a typical marker of mitochondrial dysfunction and early apoptosis / death, and is also consistent with mitochondrial changes during ferroptosis.

[0124] Endoplasmic reticulum stress and DNA damage: Western Blot results ( Figure 7 This further confirmed that a severe stress response occurred within the cell. The expression levels of the endoplasmic reticulum stress marker p-PERK and its downstream pro-apoptotic / death factor CHOP were significantly upregulated. Simultaneously, the DNA double-strand break marker p-H2AX also increased dramatically. Furthermore, the elevated levels of stress proteins GADD34 and GRP75 further confirmed that the cells were in a state of irreversible damage.

[0125] In summary, this embodiment, through detailed and coherent experimental data, demonstrates for the first time, mechanistically, that RBMS2 plays a crucial role in pancreatic cancer cells. It powerfully inhibits ferroptosis by maintaining the function of the NRF2 antioxidant system and simultaneously suppressing the expression of ferroptosis-promoting factors such as ACSL4 and NCOA4, thereby enabling cancer cells to survive and proliferate under high oxidative stress. RBMS2 can serve as a key target for integrated diagnosis and treatment of pancreatic cancer.

[0126] Example 3

[0127] This embodiment analyzes the molecular mechanism by which RBMS2 regulates the progression of pancreatic cancer.

[0128] The downstream target messenger RNA (mRNA) molecules that RBMS2 binds to in pancreatic cancer cells were identified. Results are as follows: Figure 8 As shown in Figure A, the results of RT-qPCR detection show that knocking down RBMS2 reduces Nrf2 RNA levels. Figure B shows the SPR experiment demonstrating that RBMS2 protein directly binds to Nrf2 mRNA, confirming that RBMS2 protein can enhance Nrf2 mRNA stability, thereby upregulating its protein expression and ultimately promoting the malignant progression of tumors.

[0129] Example 4

[0130] A nude mouse model of subcutaneous pancreatic cancer xenografts was constructed, and the test results are as follows: Figure 9 As shown, Figure A shows the in vitro results of subcutaneous xenografts in nude mice in the control and experimental groups; Figure B shows the statistical results of the weight of subcutaneous xenografts in nude mice in the control and experimental groups; Figure C shows the immunohistochemical detection of RBMS2 and Ki67 protein expression in the control and experimental groups. The experiment verified that targeted silencing of RBMS2 significantly inhibited tumor growth.

[0131] In summary, this invention, through systematic clinical sample analysis, in vitro cell function experiments, and in vivo animal model verification, reveals for the first time the core molecular mechanism by which RBMS2 protects pancreatic cancer cells by actively inhibiting ferroptosis. This demonstrates that RBMS2 is an important functional protein in cells and possesses the potential to serve as a target for integrated diagnosis and treatment of pancreatic cancer. It can be used to assist in the diagnosis of pancreatic cancer, assess its malignancy, evaluate treatment efficacy, or predict patient prognosis. Targeting RBMS2 for the treatment or prevention of pancreatic cancer provides a foundation for developing synergistic treatment regimens (such as combined use with ferroptosis inducers). Furthermore, its applications can be further expanded to include the screening and development of reagents that regulate related cell mechanisms, etc.

[0132] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. Application of RBMS2 protein detection reagents in any of the following aspects: (1) Develop pancreatic cancer prognostic risk assessment products; (2) Reagents for screening agents that regulate the sensitivity of cells to iron death; (3) Reagents for screening formulations that regulate the expression levels of antioxidant proteins; (4) Reagents for screening agents that regulate and promote the expression level of ferroptosis protein; (5) Preparation of screening reagents for agents that regulate mitochondrial damage; (6) Reagents for screening agents to prepare formulations that regulate the expression levels of stress proteins; (7) Reagents for screening agents to regulate the expression levels of pro-apoptotic / death factors; (8) Reagents for screening preparations of markers that regulate the expression level of DNA double-strand breaks; (9) Reagents for screening agents to prepare formulations that regulate the stability of NRF2 mRNA; (10) Preparation of screening reagents for drugs to treat pancreatic cancer.

2. The application according to claim 1, characterized in that, The detection reagents include reagents for detecting RBMS2 protein expression levels; Preferably, the detection reagent includes an ELISA kit, immunohistochemistry kit, or chemiluminescence detection reagent containing RBMS2 protein-specific antibodies, or a PCR kit containing specific primers and probes for detecting RBMS2 mRNA or DNA.

3. The application according to claim 1 or 2, characterized in that, The prognostic risk assessment product includes a prognostic risk assessment kit.

4. The application according to any one of claims 1-3, characterized in that, The antioxidant protein includes at least one of NRF2, NQO1, or HO-1; Preferably, the ferroptosis-promoting protein includes ACSL4 and / or NCOA4; Preferably, the mitochondrial damage includes a decrease in mitochondrial membrane potential; Preferably, the stress protein includes at least one of p-PERK, GADD34, or GRP75; Preferably, the pro-apoptotic / death factor includes CHOP; Preferably, the marker of DNA double-strand breakage includes p-H2AX.

5. A screening method, characterized in that, The screening method includes: The expression level of RBMS2 protein in cells was detected in the presence of the substance to be screened. Based on the expression level, the following formulations were obtained: (a) Agents that regulate the sensitivity of cells to iron death; (b) Pharmacological agents that regulate the expression levels of antioxidant proteins; (c) Pharmacological agents that regulate and promote the expression level of ferroptosis protein; (d) Agents that regulate mitochondrial damage; (e) Pharmacists that regulate stress protein expression levels; (f) Pharmacists that regulate the expression levels of pro-apoptotic / death factors; (g) Developed formulations that regulate the expression levels of markers for DNA double-strand breaks; (h) Regulators of NRF2 mRNA stability; (i) Drugs for treating pancreatic cancer.

6. The application of RBMS2 protein inhibitors in any of the following aspects: 1) Improve the sensitivity of cells to ferroptosis or prepare related formulations; 2) Reduce the expression level of antioxidant proteins or prepare related formulations; 3) Upregulate the expression level of ferroptosis-promoting proteins or prepare related formulations; 4) Inducing mitochondrial damage or preparing related formulations; 5) Upregulate stress protein expression levels or prepare related formulations; 6) Upregulate the expression levels of pro-apoptosis / death factors or prepare related formulations; 7) Upregulate the expression levels of markers of DNA double-strand breaks or prepare related formulations; 8) Reduce the stability of NRF2 mRNA or prepare related formulations.

7. The application according to claim 6, characterized in that, The inhibitors of the RBMS2 protein include any one or a combination of at least two of the following: nucleic acid molecules, nucleic acid constructs, lentiviruses, antibodies, protein degraders, or small molecule compounds. Preferably, the nucleic acid molecule is any one or a combination of at least two of double-stranded RNA, siRNA, or shRNA.

8. The application according to claim 6, characterized in that, The antioxidant protein includes at least one of NRF2, NQO1, or HO-1; Preferably, the ferroptosis-promoting protein includes ACSL4 and / or NCOA4; Preferably, the mitochondrial damage includes a decrease in mitochondrial membrane potential; Preferably, the stress protein includes at least one of p-PERK, GADD34, or GRP75; Preferably, the pro-apoptotic / death factor includes CHOP; Preferably, the marker of DNA double-strand breakage includes p-H2AX.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an inhibitor of the RBMS2 protein as described in claim 6 or 7 and a second formulation, the second formulation comprising a ferroptosis inducer and / or a chemotherapeutic agent.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition may be in the form of a standalone formulation or a combination formulation; Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients; Preferably, the excipients include any one or a combination of at least two of the following: carrier, cosolvent, osmotic pressure regulator, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

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