Cytoplasmic isoform of pglyrp2 and uses thereof

Abstract

PGLYRP2 cytoplasmic isoform and its application relate to the field of biological medicine, and aim to solve the problem that existing drug treatment of hepatocellular carcinoma is prone to drug resistance and immune escape. The cytoplasmic isoform is PGLYRP2 beta, which, compared with PGLYRP2, lacks a nuclear localization signal sequence, retains an unordered domain IDR1 and an intact PGRP domain, and the C-terminal of PGLYRP2 beta increases an intrinsic disordered domain IDR2. PGLYRP2 beta can form a membrane-free condensate in the cytoplasm through liquid-liquid phase separation, and endow it with a new function different from the nuclear type PGLYRP2. PGLYRP2 beta induces ferroptosis, activates antigen-presenting cells and CD8 + T cells, forming a self-amplifying positive feedback mechanism of "ferroptosis-immune activation". Significantly inhibits tumor growth and reverses PD-1 immunotherapy resistance. PGLYRP2 beta has a clear prognostic predictive value, showing its potential application in precision medicine.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a PGLYRP2 cytoplasmic isoform and its applications. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a common malignant tumor with high morbidity and mortality rates worldwide. Its progression is primarily driven by factors such as abnormal lipid metabolism and chronic hepatitis virus infection. Furthermore, by constructing an immunosuppressive tumor microenvironment, HCC cells can effectively evade traditional treatments. During disease development, HCC cells often exhibit significantly enhanced lipid antioxidant capacity and adaptive regulation of mitochondrial oxidative stress. In addition, the enrichment of regulatory T cells (Treg cells) and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment, along with the overexpression of immune checkpoint molecules such as PD-L1 and CTLA-4, collectively contribute to the immune system's inability to effectively recognize and eliminate tumor cells.

[0003] Currently, the clinical efficacy of treatments for patients with advanced HCC is generally limited, with high drug resistance and frequent immune escape remaining major challenges. Therefore, there is an urgent need to explore new regulatory mechanisms at the molecular level to achieve more precise and effective therapeutic interventions.

[0004] In recent years, ferroptosis, a programmed cell death mechanism characterized by iron-dependent lipid peroxidation, has gradually become a research hotspot in cancer treatment. Unlike traditional apoptosis and necrosis, ferroptosis is regulated by multiple metabolic pathways and is particularly suitable for HCC cells with highly active lipid metabolism. Studies have shown that ferroptosis can not only directly induce tumor cell death, but also activate anti-tumor immune responses and enhance dendritic cell maturation and T cell function by releasing damage-associated molecular patterns (DAMPs) such as calreticulin (CRT), high-mobility group box 1 (HMGB1), and mitochondrial DNA (mtDNA), thereby establishing a "ferroptosis-immune activation" linkage pathway.

[0005] However, current drugs used to induce ferroptosis (such as erastin and RSL3) have limitations such as poor specificity, high toxicity, and difficulty in eliciting immune responses, which restrict their widespread application in HCC. Therefore, developing a novel therapeutic strategy that can induce ferroptosis under endogenous conditions in tumor cells and synergistically activate anti-tumor immune responses has become a key pathway to overcome drug resistance and immune escape in HCC. Summary of the Invention

[0006] The present invention aims to address the problems of drug resistance and immune escape in existing drug treatments for hepatocellular carcinoma, and provides a PGLYRP2 cytoplasmic isoform and its applications.

[0007] This invention provides a cytoplasmic isoform of PGLYRP2, PGLYRP2β, which, compared to PGLYRP2, lacks the nuclear localization signal sequence (NLS), retains the disordered domain IDR1 and the complete PGRP domain, and adds the intrinsically disordered domain IDR2 at the C-terminus of PGLYRP2β. The protein sequence of PGLYRP2β is shown in SEQ ID NO: 1 of the sequence listing.

[0008] The amino acid sequence of the disordered domain IDR2 of the cytoplasmic isoform PGLYRP2β of PGLYRP2 is shown in the sequence listing SEQ ID NO: 2.

[0009] The amino acid sequence of the disordered domain IDR2 of PGLYRP2β:

[0010] VSLRSLHYTARRPSVYTSSTRPLPPACNSCARTASARPPTSRRHVYSGNLGPAFAGHSAGNIPDPVTSAYAASAQPQTQPACPFPSS.

[0011] The nucleotide sequence of PGLYRP2β is shown in SEQ ID NO: 3 in the sequence listing, which is the full-length sequence of PGLYRP2β.

[0012] This invention provides the application of the cytoplasmic isoform PGLYRP2β of PGLYRP2 in the preparation of drugs for treating hepatocellular carcinoma.

[0013] This invention provides the application of the cytoplasmic isoform of PGLYRP2, PGLYRP2β, as a biomarker in the preparation of products for prognostic assessment of hepatocellular carcinoma.

[0014] Furthermore, the products include testing reagents and kits.

[0015] This invention provides a gene therapy vector into which the coding region of the PGLYRP2β gene is inserted into the pAAV-EnII-EnCMV-M vector. The promoter used is a hepatocyte-specific promoter.

[0016] This invention provides the application of the above-mentioned gene therapy vector in the preparation of drugs for treating hepatocellular carcinoma.

[0017] This invention provides the application of the above-mentioned gene therapy vector combined with PD-1 monoclonal antibody in the preparation of drugs for treating hepatocellular carcinoma.

[0018] The present invention provides a pharmaceutical composition for treating hepatocellular carcinoma, comprising the above-mentioned gene therapy vector and PD-1 monoclonal antibody.

[0019] The beneficial effects of this invention are:

[0020] This invention is the first to clearly identify PGLYRP2β (NP_001350475.1) as a cytoplasmic isoform of PGLYRP2. This protein contains a C-terminal intrinsic disordered domain (IDR2) and can form membraneless aggregates in the cytoplasm via liquid-liquid phase separation (LLPS). This endows it with a novel function distinct from the nuclear PGLYRP2.

[0021] PGLYRP2β condensates induce lipid peroxidation and mitochondrial dysfunction by specifically binding to APOA1 and interfering with its antioxidant synergy with PON1, thereby inducing ferroptosis and releasing DAMPs (such as CRT, HMGB1, and mtDNA), activating antigen-presenting cells and CD8+. + T cells, thus forming a self-amplifying positive feedback mechanism of "ferroptosis-immune activation".

[0022] This invention constructs an AAV-based PGLYRP2β gene therapy vector that can achieve targeted expression and aggregate formation in liver cancer cells, significantly inhibiting tumor growth and reversing PD-1 immunotherapy resistance, demonstrating excellent therapeutic efficacy and safety, and achieving the first effective in vivo translation of this molecular mechanism.

[0023] Through validation using clinical tissue samples, this invention found that a positive PGLYRP2β agglutinate is significantly associated with prolonged patient survival, demonstrating clear prognostic value and showcasing its potential application in precision medicine. Attached Figure Description

[0024] Figure 1 To predict the disordered structural domains of PGLYRP2β using the PONDR online tool;

[0025] Figure 2 A comparison diagram of the structural domains of PGLYRP2 and PGLYRP2β is shown; PGLYRP2β is missing NLS, has a new C-terminal IDR2, and retains IDR1 and PGRP structural domains;

[0026] Figure 3 Results of PGLYRP2β subcellular localization assay;

[0027] Figure 4 To verify the FRAP experiment that the PGLYRP2β condensate has a liquid-liquid phase separation structure;

[0028] Figure 5 FRAP data statistics for PGLYRP2β condensates;

[0029] Figure 6 Analysis of the aggregate-forming ability of the PGLYRP2β-ΔIDR mutant;

[0030] Figure 7 The results show the immunoprecipitation of PGLYRP2β and APOA1.

[0031] Figure 8 Colocalization analysis of PGLYRP2β and APOA1 in Huh7 / PGLYRP2β stable transgenic cells;

[0032] Figure 9 Co-localization analysis of PGLYRP2β and APOA1 in liver cancer tissue samples;

[0033] Figure 10 Competitive binding experiments revealed that PGLYRP2β can block the interaction between APOA1 and PON1;

[0034] Figure 11 JC-1 staining method was used to analyze the decrease in mitochondrial membrane potential (ΔΨm) induced by PGLYRP2β in liver cancer cells;

[0035] Figure 12 Quantitative analysis of the decrease in mitochondrial membrane potential (ΔΨm) and statistical analysis of the red / green fluorescence ratio of JC-1 staining;

[0036] Figure 13 Seahorse cell efflux analysis showed that PGLYRP2β expression induced mitochondrial dysfunction in liver cancer cells;

[0037] Figure 14 Results of basal respiration in Seahorse cell efflux analysis;

[0038] Figure 15 Results of ATP production in Seahorse cell efflux analysis;

[0039] Figure 16 The results represent the maximum respiration in the Seahorse cell efflux analysis.

[0040] Figure 17 To observe the morphological characteristics of ferroptosis in PGLYRP2β-induced hepatocellular carcinoma cells using transmission electron microscopy;

[0041] Figure 18 The effect of PGLYRP2β overexpression on serum calreticulin (CRT) levels in an orthotopic hepatocellular carcinoma model; data are expressed as mean ± standard deviation. <0.001;

[0042] Figure 19 The effect of PGLYRP2β overexpression on serum high-mobility group box 1 (HMGB1) levels in an orthotopic hepatocellular carcinoma model; data are expressed as mean ± standard deviation. <0.001;

[0043] Figure 20 The effect of PGLYRP2β overexpression on serum mitochondrial DNA (mtDNA) levels in an orthotopic hepatocellular carcinoma model. <0.001;

[0044] Figure 21 To analyze the effect of PGLYRP2β overexpression on the expression of CD80 and CD86 on the surface of tumor-infiltrating dendritic cells (DCs) using flow cytometry;

[0045] Figure 22 PGLYRP2β overexpression against CD8 + The effect of T cell-secreted IFN-γ levels; data are expressed as mean ± standard deviation. <0.001;

[0046] Figure 23 HE staining and Flag immunohistochemistry were used to analyze the expression of AAV-PGLYRP2β in mouse hepatocellular carcinoma in situ and adjacent tissues.

[0047] Figure 24 In Vivo Imaging for a mouse orthotopic liver cancer model.

[0048] Figure 25 Quantitative analysis of relative luminescence intensity (RLI) in mouse tumors.

[0049] Figure 26 Differential expression analysis of PGLYRP2β condensates in pathological stages of hepatocellular carcinoma;

[0050] Figure 27 Analysis of PGLYRP2β aggregate levels and patient survival (Kaplan-Meier survival curves).

[0051] Figure 28 Prognostic predictive efficacy of PGLYRP2β aggregates (ROC curve analysis). Detailed Implementation

[0052] This invention is the first to clearly identify PGLYRP2β (NP_001350475.1) as a cytoplasmic isomer of PGLYRP2. Although highly conserved in its N-terminal sequence with PGLYRP2, PGLYRP2β lacks its C-terminal nuclear localization signal (NLS) and instead contains an intrinsically disordered domain (IDR2) closely associated with liquid-liquid phase separation (LLPS). This structure determines its ability to form membraneless condensates in the cytoplasm, thus endowing it with a novel function distinct from the nuclear PGLYRP2.

[0053] PGLYRP2β specifically binds to the lipid metabolism-related protein APOA1 through its conserved PGRP domain and forms a stable aggregate driven by phase separation, thereby "capturing" APOA1 and blocking its antioxidant synergy with PON1. This mechanism disrupts the APOA1-PON1 antioxidant axis, leading to the accumulation of lipid peroxides, mitochondrial dysfunction (a 34.2% reduction in ATP synthesis and a 65.0% reduction in basal respiration), and ultimately inducing ferroptosis.

[0054] The cell membrane rupture and release of DAMPs (such as HMGB1, CRT, and mitochondrial DNA) that accompany ferroptosis can promote the maturation of antigen-presenting cells and enhance CD8. + The immune functions of T cells and NK cells. The PGLYRP2β condensate of the present invention constructs a positive feedback loop of "ferroptosis-immune activation" within tumor cells through this mechanism, forming a sustained immune response.

[0055] This invention utilizes the recombinant adeno-associated virus (AAV) pAAV-EnII-EnCMV-M vector to construct a PGLYRP2β gene therapy system. This system can efficiently express PGLYRP2β in hepatocellular carcinoma cells with low PGLYRP2 expression and form functional aggregates in the cytoplasm. In a mouse model of hepatocellular carcinoma, this system significantly inhibited tumor growth (reducing tumor volume by 36.9%), effectively reversed PD-1 immunotherapy resistance, and increased the treatment response rate to 86.6%. Simultaneously, it exhibited highly selective tumor cytotoxicity and low systemic toxicity, demonstrating good therapeutic efficacy and safety.

[0056] This invention, through analysis of human hepatocellular carcinoma tissue samples, found that patients positive for PGLYRP2β had a median survival 54.7% longer than those negative, demonstrating its clear prognostic value and therapeutic potential. This finding provides a theoretical basis for establishing patient subtyping and individualized treatment strategies based on PGLYRP2β in clinical practice.

[0057] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0058] Example 1: Screening and verification of the structural-functional characteristics of the PGLYRP2β gene

[0059] 1. Analysis of the structural domain of the isomer PGLYRP2β

[0060] The hepatocyte isoform of the PGLYRP2 gene (NP_001350475.1) is named PGLYRP2β, and its protein sequence is shown in SEQ ID NO: 1. Using the online tool PONDR (Predictor of Natural Disordered Regions), the disordered domains of PGLYRP2β were predicted. The disordered domain IDR1 was found to be approximately 53 amino acids in the middle, while the disordered domain IDR2 was located at the C-terminus, with a length of 87 amino acids, rich in easily separable amino acid residues such as Pro, Gln, Ser, and Arg (e.g., Pro, Gln, Ser, Arg). Figure 1 ).

[0061] The amino acid sequence of the disordered domain IDR2 of PGLYRP2β is as follows:

[0062] VSLRSLHYTARRPSVYTSSTRPLPPACNSCARTASARPPTSRRHVYSGNLGPAFAGHSAGNIPDPVTSAYAASAQPQTQPACPFPSS.

[0063] Compared to PGLYRP2, which lacks the nuclear localization signal sequence (NLS) at the C-terminus, PGLYRP2β retains the disordered structural domain IDR1 and the complete PGRP structural domain, and adds an intrinsic disordered structural domain IDR2 at the C-terminus (such as...). Figure 2 ).

[0064] The full-length nucleotide sequence of PGLYRP2β is shown in the sequence listing SEQ ID NO: 3.

[0065] 2. PGLYRP2β subcellular localization assay

[0066] The target gene expression vectors Plvsin-PGLYRP2 and Plvsin-PGLYRP2β were constructed using the following methods:

[0067] The vector used was a pLVSIN-CMV Pur Vector, which was purchased. PGLYRP2 and PGLYRP2β were both inserted between XhoI and NotI in the MCS region of the vector.

[0068] PGLYRP2 primers:

[0069] Primer F1: 5'-CTCGAGATGGCCCAGGGCGTG-3'

[0070] Primer R1: 5'-GCGGCCGCTTACTGCAGGTCGGTGGCGGG-3'

[0071] PGLYRP2β primers:

[0072] Primer F2: 5'-CTCGAGATGGCCCAGGGCGTG-3'

[0073] Primer R2: 5'-GCGGCCGCTCAGGAGCTGGGGAAAGGACAGG-3'

[0074] Using the full-length nucleotide sequence of PGLYRP2β as a template, the target gene fragment was amplified by PCR. The reaction mixture consisted of 50 μL of PCR mixture, 25 μL of PCR mix, 10 μM primers, 80 ng template, and deionized water to make up the remainder. The reaction program was 35 cycles: denaturation at 92℃ for 30 s, annealing at 58℃ for 30 s, and extension at 72℃ for 2 min. The PCR product was electrophoresed on a nucleic acid gel to check the correct band positions, and the target fragment was recovered from the gel. The target fragment and vector (XhoI and NotI) were digested using a double enzyme digestion method. The mixture consisted of 50 μL of digestion buffer, 1 μL of enzyme, 2 μg of target fragment or vector, and deionized water to make up the remainder. The digestion was carried out at 37℃ for 2 h. The target fragment and vector were ligated using a ligase mixture of 20 μL:10 μL ligation buffer, 10 μL of the digested target fragment, vector, and ligase (4:1 ratio), and 1 μL of ligase. Ligation was carried out at room temperature for 5 min. The ligation product was added to competent E. coli, placed on ice for 30 min, then treated at 42℃ for 1 min, plated, and after growth, single clones were picked for expansion culture to extract plasmids, sequenced for verification, and the target gene expression vectors Plvsin-PGLYRP2 and Plvsin-PGLYRP2β were constructed.

[0075] Lentiviral packaging was used, with lentiviral packaging plasmids pCMV-VSV-G and pPACKH1-GAG mixed with the recombinant vector Plvsin-PGLYRP2 / Plvsin-PGLYRP2β in a ratio of 1:9:10 (20 μg). The transfection complex was added to 293T cells. The medium was completely changed on the second day after transfection, and the viral supernatant was collected after 72 hours. The collected virus was concentrated using PEG-8000. The concentrated virus was used to infect Huh cells, and puromycin was added for selection after 48 hours. Thereafter, the medium was changed and puromycin was added every two days until the cells stopped dying. The cells were then expanded to obtain stable transfected cell lines Huh7 / PGLYRP2 and Huh7 / PGLYRP2β.

[0076] Stable cell lines Huh7 / PGLYRP2 and Huh7 / PGLYRP2β were constructed to transform PGLYRP2 and PGLYRP2β. Subcellular localization of PGLYRP2 and PGLYRP2β was analyzed using anti-PGLYRP2 (54-160aa) antibody and immunofluorescence. Figure 3 As shown, PGLYRP2 was primarily localized in the nucleus, while PGLYRP2β was localized in the cytoplasm, forming granular structures within the cytoplasm. Unlike the nuclear localization of PGLYRP2, the subcellular localization of PGLYRP2β in Huh7 cells showed that it formed aggregated granules in the cytoplasm.

[0077] 3. Verification of phase separation / condensate properties

[0078] Sample preparation and fluorescent labeling process: construct pEGFP-C1-PGLYRP2β recombinant, i.e., PGLYRP2β can emit green fluorescence in cells.

[0079] The method for constructing the pEGFP-C1-PGLYRP2β recombinant is as follows:

[0080] The vector used was PEGFP-C1 (purchased), and the PGLYRP2β sequence was inserted into the XhoI and KpnI regions of the vector's MCS region. The PGLYRP2β primers were:

[0081] Primer F3: 5'-CCGCTCGAGCGGCGGCCCAGGGCGTGCTCTGG-3'

[0082] Primer R3: 5'-CGGGGTACCCCGCTAGCTGGAAGGGAAAGG-3'

[0083] Using the full-length PGLYRP2β nucleotide sequence as a template, the target gene fragment was amplified by PCR. The PCR system consisted of 50 μL: 25 μL PCR mix, 10 μM primers each, 80 ng template, and the remainder was made up with deionized water. The reaction program was: 35 cycles, denaturation at 92℃ for 30 s, annealing at 58℃ for 30 s, and extension at 72℃ for 2 min. The PCR product was electrophoresed in a nucleic acid gel to observe the correct band positions, and the target fragment was recovered from the gel. The target fragment and vector (XhoI and KpnI) were digested using a double enzyme digestion method. The system consisted of 50 μL of enzyme digestion buffer, 1 μL of enzyme, 2 μg of target fragment or vector, and the remainder was made up with deionized water. The digestion was carried out at 37℃ for 2 h. The target fragment and vector were ligated using a ligase mixture of 20 μL: 10 μL ligation buffer, with a total volume of 10 μL of digested target fragment, vector, and ligase (target fragment to vector ratio 4:1). The ligase was added at 1 μL and the ligase was added at room temperature for 5 min. The ligation product was added to competent E. coli, placed on ice for 30 min, then treated at 42℃ for 1 min, plated, and after growth, single clones were picked for expansion culture to extract plasmids, and sequenced to verify the pEGFP-C1-PGLYRP2β recombinant.

[0084] Photobleaching process: Huh7 cells transfected with pEGFP-C1-PGLYRP2β plasmid were placed under a confocal microscope. The software was opened, and the 405nm and 488nm laser channels were first activated for laser preheating. Then, Smartsetup was opened, and the DAPI and EGFP channels were selected. The excitation light intensity and time were set for the fluorescence bleaching recovery experiment. 100% strong light was used for quenching for 1 second, and images were taken at 3-second intervals after quenching to 20% of the original intensity, for a total of 150 seconds. Data were collected and plotted using GraphPad.

[0085] The methods for constructing single-segment deletion mutants and double-segment deletion mutants are as follows:

[0086] Using the full-length PGLYRP2β nucleotide sequence as a template, PCR amplification was performed using primers F4 and R4; PCR amplification was also performed using the full-length PGLYRP2β nucleotide sequence as a template, using primers F5 and R5; the products from both PCR amplifications were then used as templates for PCR amplification using primers F4 and R5 to obtain the PGLYRP2β-ΔIDR1 target fragment.

[0087] PGLYRP2β-ΔIDR1 primers:

[0088] Primer F4: 5'-CCGCTCGAGCGGCGGCCCAGGGCGTGCTCTGG-3'

[0089] Primer R4: 5'-CAGGCTAGGGGCGATGGCC-3'

[0090] Primer F5: 5'-GCCCCTAGCCTGCTCGCTGT-3'

[0091] Primer R5: 5'-CGGGGTACCCCGCTAGCTGGAAGGGAAAGG-3'

[0092] Using the full-length nucleotide sequence of PGLYRP2β as a template, PCR amplification was performed using F6 and R6 primers to obtain the target fragment PGLYRP2β-ΔIDR2.

[0093] PGLYRP2β-ΔIDR2 primers:

[0094] Primer F6: 5'-CCGCTCGAGCGGCGGCCCAGGGCGTGCTCTGG-3'

[0095] Primer R6: 5'-CGGGGTACCCCGCTAGGCGGTGAAATGGGGCCATGT-3'

[0096] Using PGLYRP2β-ΔIDR1 as a template and F7 and R7 as primers, PCR amplification was performed to obtain the target fragments PGLYRP2β-ΔIDR1 & ΔIDR2.

[0097] PGLYRP2β-ΔIDR1 & ΔIDR2 primers:

[0098] Primer F7: 5'-CCGCTCGAGCGGCGGCCCAGGGCGTGCTCTGG-3'

[0099] Primer R7: 5'-CGGGGTACCCCGCTAGGCGGTGAAATGGGGCCATGT-3'

[0100] The PCR amplification system (50 μL) consisted of: 25 μL PCR mix, 10 μM primers each, 80 ng template, and the remainder was made up with deionized water. The reaction program was: 35 cycles, denaturation at 92℃ for 30 s, annealing at 58℃ for 30 s, and extension at 72℃ for 2 min.

[0101] The PCR products are subjected to electrophoresis in a nucleic acid gel. The band positions are observed to ensure they are correct. The target fragment is then recovered from the gel.

[0102] The pEGFP-C1 vector used was purchased. The target fragment and vector (XhoI and KpnI) were digested using a double enzyme digestion method in a 50 μL system: 5 μL of digestion buffer, 1 μL of enzyme, 2 μg of target fragment or vector, and the remainder was made up with deionized water. Digestion was carried out at 37°C for 2 h. The target fragment and vector were then ligated using a ligase mixture of 20 μL:10 μL of ligation buffer, with a total volume of 10 μL of digested target fragment, vector, and ligase (target fragment to vector ratio 4:1). Ligation was carried out at room temperature for 5 min. The ligation product was added to competent *E. coli* cells, incubated on ice for 30 min, then treated at 42°C for 1 min. The cells were plated, and after growth, single colonies were picked for expansion culture, plasmid extraction, and sequencing verification. Single-segment deletion mutants PGLYRP2β-ΔIDR1 and PGLYRP2β-ΔIDR2, and double-segment deletion mutants PGLYRP2β-ΔIDR1&ΔIDR2 were obtained.

[0103] The structure was observed using FRAP (fluorescence recovery photobleaching) experiments, as shown in the figure. Figure 4 and Figure 5 As shown, PGLYRP2β particles exhibited rapid fluorescence recovery characteristics, suggesting that they possess dynamic phase separation features and rapidly recover fluorescence signals.

[0104] The constructed dual-segment deletion mutants PGLYRP2β-ΔIDR1&ΔIDR2 lost their ability to form aggregates, indicating that the IDR region is crucial for phase separation. Further constructed single-segment deletion mutants (PGLYRP2β-ΔIDR1 and PGLYRP2β-ΔIDR2) could still form aggregates, but their particles were significantly smaller; while the dual-segment deletion mutants PGLYRP2β-ΔIDR1&ΔIDR2 almost completely failed to form aggregates, existing in a diffusely distributed state within the cell, such as... Figure 6 As shown, Figure 6 FL in the text represents pEGFP-C1-PGLYRP2β.

[0105] Example 2: PGLYRP2β-mediated ferroptosis mechanism and immune activation effect

[0106] 1. Validation of the binding of PGLYRP2β to APOA1

[0107] Immunoprecipitation: Cells (containing protease inhibitors) were lysed on ice, centrifuged, and the supernatant was collected. Magnetic beads were added to the supernatant, and the mixture was incubated at 4°C with shaking for 2 hours. The precipitate was then centrifuged again, and the precipitate was collected. The precipitate was washed three times with pre-cooled wash buffer, and the supernatant was discarded after centrifugation. SDS loading buffer was added to the precipitate, and the mixture was incubated in a boiling water bath for 10 minutes. The supernatant was then centrifuged, and the target protein and interacting proteins were detected by Western blot.

[0108] Immunofluorescence assay: Cell slides were washed with PBS to remove impurities. Fixed with 4% paraformaldehyde for 20 minutes, followed by 3 washes with PBS; permeabilized with 0.1% Triton X-100 for 10 minutes, followed by washes with PBS. Blocked with 5% bovine serum albumin (BSA) at room temperature for 30 minutes to block non-specific binding. Diluted specific primary antibody was added, and the slides were incubated overnight at 4°C, followed by 3 washes with PBS. Fluorescently labeled secondary antibody was added, and the slides were incubated at room temperature in the dark for 1 hour, followed by 3 washes with PBS. Paraffin sections were dewaxed sequentially in xylene (twice, 10 minutes each time), then hydrated with a gradient of ethanol (100%, 95%, 80%, 70%) for 5 minutes each, and finally washed 3 times with PBS (5 minutes each time). Depending on the antigen characteristics, citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) was selected, and the sections were repaired by microwave heating or autoclaving, cooled, and washed with PBS. Blocked with PBS containing 5% bovine serum albumin (BSA) at room temperature for 30 minutes to block non-specific binding. Discard the blocking solution, add diluted specific primary antibody (diluted according to the antibody instructions), and incubate overnight at 4°C; wash three times with PBS (5 minutes each time) the next day. Add fluorescently labeled secondary antibody (e.g., FITC, Cy3 labeled), incubate at room temperature in the dark for 1 hour, and wash three times with PBS. Add DAPI to stain the nucleus (5 minutes), wash with PBS, and mount with an anti-fluorescence quencher.

[0109] Competitive binding experiment: Set up different groups, with different plasmid transfection ratios between groups, for example... Figure 10 As shown, the remaining steps are consistent with immunoprecipitation.

[0110] The results of the immunoprecipitation experiment are as follows: Figure 7 As shown in the figure, the experimental group expressed both PGLYRP2β-Flag and APOA1-HA in 293 cells, while the control group expressed only PGLYRP2β. Cells from both groups were lysed to obtain protein samples. One portion of the protein samples was directly used to prepare Western blotting samples, while the other portion was incubated with Flag beads. After eluting the Flag beads, the beads carried both Flag-containing proteins and proteins interacting with them. This was then used to prepare Western blotting samples. The input consisted of protein samples not incubated with Flag beads. Western blotting experiments showed that both proteins were detectable in the experimental group, while only PGLYRP2β was detected in the control group. This was consistent with the experimental system, indicating no errors. In the IP group, both proteins were detected simultaneously in the experimental group, demonstrating their interaction, while only PGLYRP2β was detected in the control group; no band was observed for APOA1.

[0111] Colocalization analysis of PGLYRP2β and APOA1 in Huh7 / PGLYRP2β stable cells is as follows: Figure 8 As shown in the figure. Immunofluorescence experiments were performed, staining PGLYRP2β green and APOA1 red. Confocal microscopy revealed co-localization of the two proteins of different colors, providing evidence of protein-protein interaction.

[0112] like Figure 9 As shown, in the tumor tissue of liver cancer patients, endogenous PGLYRP2β was stained green and endogenous APOA1 was stained red by immunofluorescence experiments. Observation under a confocal microscope showed that the two proteins of different colors co-localized, indicating that the interaction and co-localization of these two proteins also exist in the tumors of liver cancer patients.

[0113] Competitive binding experiments such as Figure 10 As shown in the figure, 293 cells were divided into four groups: Group 1 expressed only APOA1-HA; Group 2 expressed both APOA1-HA and EGFP-PON1; Group 3 expressed APOA1-HA, EGFP-PON1, and PGLYRP2β-Flag (normal transfection level); and Group 4 expressed APOA1-HA, EGFP-PON1, and PGLYRP2β-Flag (twice the transfection level). Immunoprecipitation experiments were performed. Western blotting results showed that in the Input group, Group 1 expressed only APOA1; Group 2 expressed both APOA1 and PON1; Group 3 expressed APOA1, PON1, and PGLYRP2β; and Group 4 expressed APOA1, PON1, and PGLYRP2β. Furthermore, the expression level of PGLYRP2β was 1.96 times that of Group 3, which is consistent with the experimental system and indicates that the experimental system was functioning correctly. In the IP groups, only APOA1 was detected in the first group; APOA1 and PON1 were detected in the second group; APOA1, PON1, and PGLYRP2β were detected in the third group, indicating that PGLYRP2β interacts with APOA1, but the gray value of the PON1 protein band was only 0.14 times that of the second group, indicating that the interaction between PGLYRP2β and APOA1 blocked the interaction between APOA1 and PON1; APOA1 and PGLYRP2β were detected in the fourth group, and the gray value of PGLYRP2β was 1.63 times that of the third group, indicating that there was more interaction between PGLYRP2β and APOA1 in the third group, but PON1 was no longer detected, indicating that the increased expression level of PGLYRP2β further blocked the interaction between APOA1 and PON1 by interacting with APOA1.

[0114] In summary, immunoprecipitation and immunofluorescence experiments showed that PGLYRP2β can form a complex with APOA1 in cells and hepatocellular carcinoma tissues, and further competitive binding experiments showed that PGLYRP2β blocked the interaction between APOA1 and PON1.

[0115] 2. Lipid peroxidation and mitochondrial damage

[0116] The Beyotime JC-1 staining kit (C2006) was used. The required amount of JC-1 staining working solution per well in a six-well plate was 1 ml. For cell suspensions, 0.5 ml of JC-1 staining working solution was required for every 500,000-1,000,000 cells. Take an appropriate amount of JC-1 (200×) and dilute it with 8 ml of ultrapure water per 50 µL of JC-1. Dissolve and mix the JC-1 thoroughly with Vortex. Then add 2 ml of JC-1 staining buffer (5×) and mix well to obtain the JC-1 staining working solution. Add the CCCP (10 mM) provided in the kit to the cell culture medium at a ratio of 1:1000, diluting to 10 µM, and treat the cells for 20 minutes. Then, load JC-1 according to the following method and perform mitochondrial membrane potential detection. For most cells, the mitochondrial membrane potential is usually completely lost after treatment with 10µM CCCP for 20 minutes, and the cells should show green fluorescence after JC-1 staining; while normal cells should show red fluorescence after JC-1 staining.

[0117] a. Experiments were conducted using six-well plates, with three groups: Huh7 / Con, Huh7 / PGLYRP2β, and Huh7 CCCP. Each group had three replicate wells. For the Huh7 CCCP group, 10 µM CCCP was added to Huh7 cells 20 minutes before the start of the experiment.

[0118] b. Aspirate the culture medium, wash the cells once with PBS, and add 1 ml of cell culture medium. The cell culture medium may contain serum and phenol red.

[0119] c. Add 1 ml of JC-1 staining working solution and mix thoroughly. Incubate at 37°C for 20 minutes in a cell culture incubator.

[0120] d. During incubation, prepare JC-1 staining buffer (1×) by adding 4 ml of distilled water to every 1 ml of JC-1 staining buffer (5×) and place it in an ice bath.

[0121] e. After incubation at 37℃, remove the supernatant and wash twice with JC-1 staining buffer (1×).

[0122] f. Use a cell scraper to scrape off the cells and resuspend them in 1 ml of JC-1 staining buffer.

[0123] g. Use flow cytometry for analysis, with the channels set to PE and FITC.

[0124] JC-1 staining analysis of PGLYRP2β-induced mitochondrial membrane potential in liver cancer cells showed the following results: Figure 11 As shown, the statistical analysis of the red / green fluorescence ratio of JC-1 staining is as follows: Figure 12 As shown in the figure. The results showed that, compared with Huh7 / Con cells, the mitochondrial membrane potential (ΔΨm) in Huh7 / PGLYRP2β hepatocellular carcinoma stable cells was significantly decreased. The ratio of red fluorescence (JC-1 dimer form) to green fluorescence (JC-1 monomer form) calculated by the JC-1 staining method showed that the mitochondrial membrane potential of PGLYRP2β expressing cells was significantly lower than that of the control group.

[0125] The Seahorse assay was used, with two groups: Huh7 / Con and Huh7 / PGLYRP2β, each with six replicate wells. Cells were seeded into Seahorse XF microplates. The probe plates were hydrated overnight in a CO2-free incubator at 37°C. The assay solution was prepared aseptically by adding 10 mmol / L XF glucose, 1 mmol / L XF sodium pyruvate, and 2 mmol / L XF glutamine to 100 ml of Seahorse XF DMEM medium (pH 7.4). The assay solution was then preheated to 37°C and incubated at 37°C until ready for use. The Seahorse XF cell culture microplates were then removed from the 37°C CO2 incubator, the cell growth medium was discarded, and the cells were washed once with the preheated assay solution. The plates were then incubated in a CO2-free incubator at 37°C for 60 minutes with the assay solution. Before starting the XF assay, the assay solution was discarded again, and fresh, preheated assay solution was added to each well. Prepare the compound stock solutions: Using compounds prepared on the same day, remove the oligomycin and rotenone / antimycin A tubes from the kit. Gently tap the tubes to ensure the powder is at the bottom. Resuspend each component with an appropriate volume of test solution according to the instructions, vortexing for about 1 minute to ensure complete resuscitation. Prepare the compounds to be added to the probe plate wells: Prepare 3 ml of each compound with test solution, using 1.5 µmol / L oligomycin and 0.5 µmol / L rotenone + antimycin A (final concentration). Add the compounds to the probe plate wells.

[0126] The template was loaded onto the Seahorse XFe analyzer, and the XF real-time ATP rate assay was run.

[0127] Seahorse cell efflux analysis results are as follows: Figure 13-16As shown. Seahorse extracellular respiration analysis revealed that PGLYRP2β-expressing cells exhibited significant mitochondrial dysfunction, with key respiratory parameters significantly reduced compared to the control group: basal respiration decreased by 65.0%, ATP production decreased by 34.2%, and maximum respiratory capacity decreased by 27.7%. Transmission electron microscopy further revealed typical morphological features of ferroptosis, such as... Figure 17 As shown, mitochondria exhibit shrinkage, disappearance of cristae, and increased membrane density.

[0128] 3. DAMPs release and immune activation

[0129] A mouse orthotopic liver cancer model was established by surgically inoculating Hep1-6 / Con and Hep1-6 / PGLYRP2β cells into the liver of mice.

[0130] At 25 days after tumor transplantation, fresh blood was obtained from mice by enucleation, and mouse serum was obtained by centrifugation. CRT and HMGB1 in the serum of Hep1-6 / Con and Hep1-6 / PGLYRP2β mice were detected by ELISA. Simultaneously, mtDNA was detected by qPCR using serum as a template.

[0131] In serum analysis of an orthotopic hepatocellular carcinoma model (day 25 of tumor transplantation), PGLYRP2β overexpression significantly upregulated the release levels of damage-associated molecular patterns (DAMPs), including calreticulin (CRT), high-mobility group box 1 (HMGB1), and mitochondrial DNA (mtDNA) (such as...). Figure 18-20 Simultaneously, the expression levels of CD80 and CD86 infiltrating dendritic cells (DCs) in tumor tissue were significantly increased (e.g., Figure 21 This indicates that DCs are effectively activated. Furthermore, CD8... + In T cell killing assays, IFN-γ secretion levels were significantly increased (e.g., Figure 22 These results suggest that PGLYRP2β overexpression may promote DAMPs release, activate DCs, and enhance CD8 activation. + T cell function, thereby inducing a specific anti-tumor immune response.

[0132] Example 3: In vivo expression of AAV-mediated PGLYRP2β (AAV-PGLYRP2β vector) and its tumor-suppressive effect

[0133] 1. Construction of AAV-PGLYRP2β vector

[0134] Primer design:

[0135] Primer F8: 5'-GAATTCATGGCCCAGGGCGTGCTCTGGATT-3'

[0136] Primer R8: 5'-AGATCTTCAGGAGCTGGGGAAAGGACAGGCT-3'

[0137] Using the full-length PGLYRP2β nucleotide sequence as a template, the target gene fragment was amplified by PCR. The reaction mixture consisted of 50 μL of PCR mixture (25 μL), 10 μM primers, 80 ng template, and deionized water to make up the remainder. The reaction program was: 35 cycles of denaturation at 92℃ for 30 s, annealing at 58℃ for 30 s, and extension at 72℃ for 2 min. The PCR products were electrophoresed on a nucleic acid gel to check the correct band positions, and the target fragment was recovered from the gel. The target fragment and the pAAV-EnII-EnCMV-M vector (EcoRI and BglII) were digested using a double enzyme digestion method. The digestion mixture consisted of 50 μL of digestion buffer, 1 μL of enzyme, 2 μg of the target fragment or vector, and deionized water to make up the remainder. The digestion was carried out at 37℃ for 2 h. The target fragment and vector were ligated using ligase in a 20 μL:10 μL ligation buffer mixture. The target fragment, vector, and ligase were combined in a 10 μL volume (4:1 ratio), and ligase was added. Ligation was carried out at room temperature for 5 min. The ligation product was added to competent *E. coli* cells, incubated on ice for 30 min, then treated at 42 °C for 1 min. The cells were plated and allowed to grow. Single colonies were then picked for expansion culture, plasmid extraction was performed, and sequencing confirmed the presence of the AAV-PGLYRP2β vector.

[0138] The pAAV-EnII-EnCMV-M carrier has been disclosed in patent CN117899238A.

[0139] HE staining and Flag immunohistochemical analysis were used to analyze the expression of AAV-PGLYRP2β in mouse hepatocellular carcinoma in situ and adjacent tissues. Figure 23 As shown in the figure. The results confirmed that by using a hepatocyte-specific promoter, its efficient expression in hepatocellular carcinoma cells was achieved, while its efficient expression in normal liver tissue cells was restricted, thus achieving tumor cell specificity and safety.

[0140] 2. Validation of treatment in mouse liver cancer model

[0141] Hep1-6 cells were surgically inoculated into the livers of C57BL / 6 mice to establish a Hepa1-6 xenograft model.

[0142] Hepa1-6 xenograft mouse models were divided into four groups: negative control group (CON), AAV-PGLYRP2β group, Anti-PD-1 group, and combined drug administration group. The administration methods for each group are as follows:

[0143] Negative control group (CON): Seven days after the in situ tumor began to grow, PBS was injected via the tail vein.

[0144] AAV-PGLYRP2β group: Seven days after the in situ tumors began to grow, AAV-PGLYRP2β was injected via the tail vein at a dose of 1×10¹¹vg.

[0145] Anti-PD-1 group: Anti-mousePD-1 (CD279)-InVivo was injected intraperitoneally at 10 mg / kg per mouse on days 11, 15 and 19 of in situ tumor growth.

[0146] Combined administration group: On day 7 of in situ tumor growth, AAV-PGLYRP2β (1×10¹¹vg) was injected via the tail vein, and Anti-mousePD-1 (CD279)-InVivo was injected intraperitoneally on days 11, 15 and 19 of in situ tumor growth, at a dose of 10 mg / kg per mouse.

[0147] 3. Evaluation of treatment effectiveness

[0148] In vivo imaging of mouse orthotopic liver cancer model, such as Figure 24 As shown, quantitative analysis of fluorescence signal intensity in mouse tumors is as follows: Figure 25 As shown. After 21 days, the tumors in the negative control Con group mice continued to grow, and the mortality rate was 40% (2 / 5 mice); the tumor growth in the Anti-PD-1 treatment group mice was not significantly inhibited, and the mortality rate was 40% (2 / 5 mice); the tumor size in the AAV-PGLYRP2β treatment group mice shrank by 36.9% (from day 14 to day 21), and there were no deaths (0 / 5 mice); the tumor shrank by 86.6% (from day 14 to day 21) in the AAV-PGLYRP2β and Anti-PD-1 combined treatment group mice, and there were no deaths (0 / 5 mice).

[0149] Example 4: Correlation between PGLYRP2β aggregate expression and prognosis in patient tissues

[0150] Tissue samples from 90 hepatocellular carcinoma patients were analyzed using a liver cancer tissue microarray (patient data from ChipSuperBio's liver cancer tissue microarray: HLivH180Su31-M-062). Immunofluorescence staining was performed using PGLYRP2 antibody, and scores were assigned based on granular cytoplasmic expression patterns. The results were then combined with clinical follow-up data for analysis.

[0151] Immunofluorescence staining procedure: Paraffin sections are dewaxed sequentially by immersing them in xylene (twice, 10 minutes each), followed by hydration with a gradient of ethanol (100%, 95%, 80%, 70%) for 5 minutes each, and finally washed three times with PBS (5 minutes each time). Depending on the antigen characteristics, citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) is selected, and the sections are repaired by microwave heating or autoclaving. After cooling, they are washed with PBS. Blocking is performed for 30 minutes at room temperature with PBS containing 5% bovine serum albumin (BSA) or goat serum to block non-specific binding. The blocking solution is discarded, and diluted specific primary antibody (diluted according to the antibody instructions) is added, and incubated overnight at 4°C; the next day, the sections are washed three times with PBS (5 minutes each time). Fluorescently labeled secondary antibody (e.g., FITC, Cy3-labeled) is added, and incubated at room temperature in the dark for 1 hour, followed by washing three times with PBS. DAPI is added to stain the nuclei (5 minutes), and after washing with PBS, the sections are mounted with an anti-fluorescence quencher.

[0152] Scoring Criteria: ImageJ was used to quantitatively detect the number of PGLYRP2β aggregates in the tissues, obtaining the specific number of PGLYRP2β aggregates in each tissue. The tissues were scored based on these numbers, divided into three levels (1, 2, and 3 points): 0–355 PGLYRP2β phase separation points in the tissues: 0–355, 1 point; 356–648, 2 points; 649–904, 3 points. Simultaneously, ImageJ was used to score PGLYRP2β expression in the tissues, divided into two levels (1 and 2 points): low PGLYRP2β expression was 1 point, and high expression was 2 points. Figure 26 Based solely on the PGLYRP2β aggregate score, Figure 27 The total score of PGLYRP2β and expression score was used as the standard.

[0153] The results showed that the level of PGLYRP2β aggregates was significantly increased in hepatocellular carcinoma tissues with lower pathological stages (Stage I vs. Stage II, p=0.002; Stage I vs. Stage III, p<0.001; Stage II vs. Stage III, p<0.001). Figure 26 Furthermore, the median survival of patients in the high-level PGLYRP2β condensate group was 49.5 months, significantly higher than the 32 months in the low-level group, with a median survival extension of 54.7% (p=0.007, R=0.516). Figure 27 ROC curve analysis of PGLYRP2β aggregates in hepatocellular carcinoma tissue showed that PGLYRP2β aggregates had independent prognostic predictive value (AUC=0.816, p<0.001). Figure 28 ).

Claims

1. The application of the cytoplasmic isoform PGLYRP2β in the preparation of drugs for treating hepatocellular carcinoma, wherein the protein sequence of PGLYRP2β is shown in SEQ ID NO: 1 of the sequence listing.

2. The application of gene therapy vectors in the preparation of drugs for treating hepatocellular carcinoma, wherein the gene therapy vector is a pAAV-EnII-EnCMV-M vector in which the PGLYRP2β gene coding region as described in claim 1 is inserted.

3. The application of gene therapy vector combined with PD-1 monoclonal antibody in the preparation of drugs for treating hepatocellular carcinoma, wherein the gene therapy vector is a pAAV-EnII-EnCMV-M vector with the PGLYRP2β gene coding region as described in claim 1 inserted into it.

4. A pharmaceutical composition for treating hepatocellular carcinoma, comprising a gene therapy vector and a PD-1 monoclonal antibody, wherein the gene therapy vector is a pAAV-EnII-EnCMV-M vector containing the PGLYRP2β gene coding region as described in claim 1.

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