Application of miR-1246 inhibitor in preparation of medicine for preventing or treating postoperative metastasis of hepatocellular carcinoma

Abstract

The invention discloses an application of a miR-1246 inhibitor in preparation of a medicine for preventing or treating postoperative metastasis of hepatocellular carcinoma (HCC). According to the application disclosed by the invention, the expression of miR-1246 in the serum exosome of an HCC patient after operation is obviously up-regulated, the expression of BMP9 protein is inhibited by directly targeting a GDF2 gene, and a downstream SMAD7-mediated metastasis inhibition signal channel is blocked, so that the HCC cell metastasis is driven, that is, the metastasis promoting process can be effectively reversed by inhibiting the function of miR-1246. In-vivo and in-vitro experiments prove that by using the miR-1246 inhibitor, the HCC cell anoikis apoptosis resistance, migration, invasion and in-vivo lung metastasis induced by the serum exosome after operation can be remarkably inhibited. The miR-1246 inhibitor can also be combined with a recombinant protein BMP9 to generate a synergistic anti-metastasis effect. Therefore, the miR-1246 inhibitor provides a new target and strategy for developing a novel anti-HCC postoperative metastasis drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of miR-1246 inhibitors in the preparation of drugs for the prevention or treatment of postoperative metastasis of hepatocellular carcinoma. Background Technology

[0002] Surgery remains one of the most effective strategies for treating solid tumors. However, a significant number of patients experience recurrence and metastasis shortly after primary tumor resection. Early recurrence is characterized by its synchronicity and suddenness, contrasting sharply with the low incidence of long-term, widespread recurrence and significant metastasis at initial diagnosis. This suggests the existence of an independent triggering event, likely occurring perioperatively. Indeed, a growing body of research indicates that unavoidable tissue damage during surgery, along with the resection of the primary tumor and its associated vascular system, increases the risk of tumor cells detaching and entering the bloodstream and lymphatic circulation. Simultaneously, alterations in local and systemic cytokine expression profiles and immune system remodeling promote the proliferation and metastasis of residual malignant cells. These events, acting synergistically within a short perioperative window, theoretically make cancer patients particularly susceptible to metastasis and recurrence.

[0003] Surgery-related systemic changes create favorable conditions for metastasis, and the intrinsic metastatic potential of tumor cells, regulated by multiple genetic factors, is equally crucial. The TGF-β superfamily-related signaling pathway is a classic pathway regulating tumor metastasis. This family includes more than 30 cytokines, such as TGF-β, activin, and the bone morphogenetic protein (BMP) subfamily. Studies have shown that inhibiting TGF-β / activin signaling by expressing dominant-negative TGFβRII or silencing downstream classical SMAD2 / 3 can effectively suppress metastasis in various cancers. Similarly, activating a classically signaling-induced inhibitory SMAD (SMAD7) in lung cancer antagonizes tumor metastasis induced by TGF-β receptor activation. Conversely, attenuating BMP signaling by inhibiting its homologous receptor BMPRII or knocking out classical SMAD1 / 5 proteins promotes spontaneous metastasis in lung and breast cancer. Overall, these studies highlight the intrinsic metastatic potential of epithelial tumor cells and the profound impact of specific SMAD pathway regulation on tumor metastasis.

[0004] Circulating body fluids, such as peripheral blood, are readily available clinical samples used for the diagnosis and treatment of various diseases, including cancer. Exosomes (Exosomes) are extracellular vesicles (EVs) ranging from 30 to 150 nm in size, ubiquitously present in these fluids. Because they carry a wide range of biological information, including DNA, messenger RNA (mRNA), non-coding RNA (ncRNA), proteins, and lipids, they are considered important mediators of intercellular communication. Exosomes and their contents can reflect the physiological state of their source tissues, especially when homeostasis is disrupted. For example, exercise-induced serum extracellular vesicles can mediate crosstalk between pancreatic islets and skeletal muscle by transmitting miRNAs, thereby coordinating metabolic adaptation. Studies have also demonstrated that ischemic preconditioning in remote limbs effectively alleviates steatohepatitis by secreting extracellular vesicles to transfer miRNAs from muscle to the liver. Furthermore, the protective nature of the exosome membrane prevents encapsulated biomolecules from being degraded by enzymes in body fluids, thus ensuring the stability of the transmitted biological information. For example, in the early detection of colorectal cancer, exosome-derived miRNAs exhibit a higher area under the curve (AUC) value than free miRNAs in plasma. Therefore, decoding the changes in bioinformatics in blood exosomes holds promise for revealing the potential mechanisms of tumor recurrence after surgery and improving diagnostic insight into postoperative metastasis. Summary of the Invention

[0005] In this invention, we collected preoperative and postoperative serum samples from patients with hepatocellular carcinoma (HCC) and found that postoperative serum or its derived exosomes significantly promoted HCC cell metastasis both in vivo and in vitro compared to preoperative samples. Through high-throughput miRNA sequencing, we further revealed the differential miRNA expression profiles in serum exosomes before and after HCC surgery, identifying a significant upregulation of miR-1246 in postoperative serum exosomes. Mechanistic studies showed that miR-1246 directly targets BMP9 mRNA, inhibiting its expression, thereby inactivating the SMAD7-mediated metastasis inhibition pathway and accelerating HCC metastasis. Inhibition of miR-1246 and addition of recombinant BMP9 protein both effectively inhibited HCC metastasis in vivo and in vitro. These findings reveal the molecular mechanism by which postoperative exosomal miR-1246 promotes HCC metastasis through the BMP9-SMAD7 axis, providing a theoretical basis for early postoperative therapeutic interventions targeting this pathway to control the dissemination of residual malignant cells.

[0006] This invention provides the use of miR-1246 inhibitors in the preparation of medicaments for the prevention or treatment of postoperative metastasis of hepatocellular carcinoma.

[0007] In the application described in this invention, the drug further comprises recombinant BMP9 protein. That is, the miR-1246 inhibitor and the recombinant BMP9 protein are used together to synergistically inhibit postoperative metastasis.

[0008] Preferably, the miR-1246 inhibitor is at least one of antagomir-1246, locked nucleic acid modified antisense oligonucleotide, small interfering RNA, or microRNA; The sequence of antagomir-1246 used in this invention is 5'-CCUGCUCCAAAAAUCCAUU-3'.

[0009] Preferably, the BMP9 recombinant protein is a human BMP9 recombinant protein. The BMP9 recombinant protein used in this invention was purchased from MCE (MedChemExpress) with product number HY-P700530.

[0010] Preferably, the postoperative metastasis is lung metastasis. This invention has found that, compared to preoperative patients, postoperative serum exosomes accelerate the progression of lung metastasis, and miR-1246 is a key factor in postoperative exosome-driven HCC metastasis. miR-1246 inhibitors significantly inhibit postoperative exosome-driven lung metastasis, and this inhibition is further enhanced upon combined administration of recombinant BMP9 protein.

[0011] Preferably, when used, the drug is administered to patients with hepatocellular carcinoma within one week after surgery.

[0012] Furthermore, the miR-1246 inhibitor and the BMP9 recombinant protein are configured in the drug to be administered simultaneously, separately, or sequentially.

[0013] The present invention also provides a pharmaceutical combination for the prevention or treatment of postoperative metastasis of hepatocellular carcinoma, the pharmaceutical combination comprising a therapeutically effective amount of a miR-1246 inhibitor and a therapeutically effective amount of BMP9 recombinant protein.

[0014] Preferably, the miR-1246 inhibitor is at least one of antagomir-1246, locked nucleic acid modified antisense oligonucleotide, small interfering RNA, or microRNA; The sequence of the antagomir-1246 is 5'-CCUGCUCCAAAAAUCCAUU-3'; The BMP9 recombinant protein is a human BMP9 recombinant protein; The miR-1246 inhibitor and the BMP9 recombinant protein were configured to jointly inhibit postoperative metastasis.

[0015] The beneficial effects of this invention are: This invention reveals a novel mechanism of postoperative metastasis and provides a precise intervention target: For the first time, this invention discovers and confirms that miR-1246, specifically highly expressed in the serum exosomes of liver cancer patients after surgery, is a key molecule driving distant metastasis (such as lung metastasis) of tumor cells. This provides a novel, highly specific molecular target (miR-1246) and its pathway (BMP9 / SMAD7) for the clinical prevention and treatment of postoperative recurrence and metastasis.

[0016] This invention pioneers a proactive treatment strategy targeting the "postoperative high-risk window for metastasis": Unlike the traditional passive approach of waiting for recurrence or using broad-spectrum chemotherapy, the proposed treatment regimen (using miR-1246 inhibitors and / or BMP9 protein) aims to proactively intervene in this proven high-risk period for metastasis, lasting several days to weeks post-surgery. By neutralizing the abnormally elevated pro-metastatic factor (exosomal miR-1246) in the blood during this period, it is hoped that the risk of metastasis can be reduced at its source.

[0017] A synergistic combination therapy regimen was proposed, demonstrating significantly superior efficacy compared to monotherapy. One of the core findings of this invention is that while inhibiting miR-1246 is effective in suppressing metastasis, supplementing its downstream target protein BMP9 produces a synergistically enhanced inhibitory effect. This combination regimen not only exerts its therapeutic effect by "inhibiting the harmful factor" (miR-1246) but also positively activates the tumor suppressor pathway by "supplementing the beneficial factor" (BMP9), achieving a more comprehensive and potent blockade of the metastasis process from a mechanistic perspective.

[0018] The treatment regimen has the potential for high safety and good translational prospects: In animal models, intervention strategies targeting this axis "showed promising safety," histological examination "revealed no significant tissue damage," and serum biochemical indicators were not significantly abnormal, indicating a good therapeutic window.

[0019] This invention not only scientifically reveals a key driving mechanism of postoperative metastasis in liver cancer, but also transforms this into an innovative treatment plan that is highly targeted, has a novel mechanism, and shows synergistic effects. It provides a new drug development approach and a potential effective treatment tool for improving the postoperative prognosis of liver cancer patients and reducing the metastasis rate. Attached Figure Description

[0020] Figure 1 Postoperative serum induces resistance to anodic apoptosis and metastasis in HCC.

[0021] A: Schematic diagram of the experimental procedure. BC: Flow cytometry analysis and quantification of anodic apoptosis in Mahlavu and MHCC-97H cells treated with pre- or post-operative serum. DE: Representative images and quantification of cell numbers in Transwell migration and invasion assays. FG: Representative images and quantification of metastatic area of ​​lung metastases in a tail vein injection model.

[0022] Figure 2 Postoperative serum-derived exosome characterization.

[0023] A: Electron micrographs of serum exosomes before / after surgery. B: Exosome particle size and concentration analysis. C: Confocal image of cellular uptake of DiO-labeled exosomes.

[0024] Figure 3 Postoperative serum-derived exosomes promote HCC metastasis.

[0025] AB: Flow cytometry and quantification of anodic apoptosis in cells of different treatment groups. CD: Representative images and quantification of Transwell migration and invasion assays. EF: Representative images and quantification of lung metastasis in mice pretreated with exosomes.

[0026] Figure 4 Removing exosomes eliminates the metastatic ability of postoperative serum.

[0027] AB: Representative images and quantifications of Transwell migration and invasion experiments.

[0028] Figure 5 Sequencing and analysis of miRNAs derived from serum exosomes.

[0029] A: Volcano plot of differentially expressed miRNAs. B: Heatmap of differentially expressed miRNAs. C: Bar chart of KEGG pathway enrichment analysis. D: LASSO regression analysis plot.

[0030] Figure 6 Serum exosome-derived miR-1246 expression was upregulated postoperatively.

[0031] A: List of four candidate miRNAs. B: Validation of candidate miR-1246 in independent cohorts. C: Survival curves of high miR-1246 expression and poor prognosis in HCC patients from the ENCORI database.

[0032] Figure 7 Postoperative serum exosome-derived miR-1246 drives HCC to acquire metastatic potential.

[0033] AC: Flow cytometry and quantification of anodic apoptosis in exosome-treated cells after miR-1246 regulation. DE: Transwell images and quantification of cell migration and invasion after miR-1246 regulation.

[0034] Figure 8 Postoperative serum exosome-derived miR-1246 drives HCC lung metastasis.

[0035] AB: Representative images and quantitative data on lung metastasis in mice after regulation of miR-1246 in exosomes.

[0036] Figure 9 Postoperative serum exosome-derived miR-1246 directly binds GDF2 .

[0037] A: Venn diagram of miR-1246 target gene prediction. B: RT-qPCR verification of miR-1246 regulation of GDF2 mRNA and TET2 mRNA. C: Schematic diagram of GDF2 3'UTR wild-type / mutant reporter gene construction and dual-luciferase assay results.

[0038] Figure 10 Targeting the miR-1246-BMP9 signaling axis activates the SMAD pathway.

[0039] AD: Immunohistochemical images and quantification of BMP9 in mouse lung metastases treated with different sera or exosomes. EF: Immunohistochemical images and quantification of BMP9 in mouse lung metastases after miR-1246 regulation.

[0040] Figure 11 Postoperative serum exosome-derived miR-1246 targets GDF2 Regulates key proteins in the BMP9-SMAD pathway.

[0041] Figure 12 Postoperative serum exosome-derived miR-1246 targets GDF2 Regulates the subcellular localization of key proteins in the BMP9-SMAD pathway.

[0042] AD: Representative images and quantitative fluorescence intensity of p-SMAD1 / 5 nuclear translocation detected by immunofluorescence.

[0043] Figure 13 The miR-1246-BMP9 signaling axis regulates the expression of transfer-related genes.

[0044] AB: RT-qPCR results of multiple transfer-related genes (Cxcr4, Itgb3, Plcg1, Bmp4, Smad4, Smad7) after regulation of miR-1246 and BMP9.

[0045] Figure 14 Targeting the miR-1246-BMP9 signaling axis reverses resistance to anodic apoptosis after HCC surgery.

[0046] AC: Flow cytometry and quantification of anodic apoptosis after regulation of miR-1246 and BMP9.

[0047] Figure 15 Targeting the miR-1246-BMP9 signaling axis to inhibit postoperative metastasis of HCC.

[0048] AC: Transwell images and quantification of cell migration and invasion after regulation of miR-1246 and BMP9. DE: Images and quantification of the inhibitory effect of combined use of AntagonomiR-1246 and recombinant BMP9 protein on mouse lung metastasis.

[0049] Figure 16 Immunohistochemical detection of lung metastases in mice after targeting the miR-1246-BMP9 signaling axis.

[0050] AB: Immunohistochemical images and quantification of p-SMAD1 / 5 in lung metastases of mice in the treatment group. CD: Immunohistochemical images and quantification of SMAD4 in lung metastases of mice in the treatment group. EF: Immunohistochemical images and quantification of SMAD7 in lung metastases of mice in the treatment group.

[0051] Figure 17 The strategy of targeting the miR-1246-BMP9 signal axis has a good biosafety margin.

[0052] A: Detection of serum liver function (ALT, AST, ALP, T-Bil), kidney function (CREA), and LDH levels in mice in the treatment group. B: Stained images of liver and kidney tissues in mice in the treatment group, showing no significant damage.

[0053] Figure 18 This is a schematic diagram of the process of the present invention. Detailed Implementation

[0054] General experimental materials 1. Cell Lines and Culture: Human hepatocellular carcinoma cell lines Mahlavu and MHCC-97H were cultured under standard conditions of 37°C and 5% CO2. Mahlavu and MHCC-97H cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; Cytiva, Marlborough, USA) supplemented with 10% fetal bovine serum (FBS; newzerum, Christchurch, New Zealand) and 1% penicillin / streptomycin (PS; Cienry, Zhejiang, China). To investigate the effect of postoperative serum on cancer cells compared to preoperative serum, Mahlavu or MHCC-97H cells were initially cultured under standard conditions. After cell attachment, the medium was replaced with DMEM containing 10% human serum and cultured for another 2 days. Before changing the medium, the cells were washed three times with phosphate-buffered saline (PBS).

[0055] 2. Clinical Samples: All human serum samples were obtained from HCC patients who underwent surgical resection at the First Affiliated Hospital of Zhejiang University School of Medicine (sample details are shown in Tables 1 and 2). Sample collection took place from January 2024 to June 2025. Preoperative serum was collected intraoperatively, and postoperative serum was collected on the morning of the first postoperative day. Each blood sample was 5 mL. This study was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine (Approval No.: [2025B] IIT Ethics Approval No. 0391).

[0056] Table 1 Table 2 3. Laboratory Animals: All 6-week-old male nude mice were purchased from Hangzhou Ziyuan Laboratory Animal Co., Ltd., and housed at the Laboratory Animal Center of the First Affiliated Hospital of Zhejiang University School of Medicine. Conditions were maintained at 20-22℃, 61-65% relative humidity, with a 12-hour light / dark cycle, and free access to food and water. All animal experiments were approved by the Institutional Animal Care and Use Committee of Zhejiang University (Reference No.: 2025-SD-92).

[0057] 4. General reagents and sequences: RNA oligonucleotide sequence (purchased from Genema Gene): Table 3 qPCR primer sequences (purchased from Sangon, Shanghai, China): Table 4 miRNA qPCR primer sequences: Table 5 5. General experimental methods: RNA oligonucleotide transfection: Select cells with approximately 50% confluence for transfection, following the manufacturer's instructions. Add the specified amount of si... GDF2 The miR-1246 mimic or inhibitor was diluted in jetPRIME buffer and then mixed with jetPRIME transfection reagent (Polyplus, Ilkhich, France) in another tube. The mixture was incubated at room temperature for 10 minutes to allow the formation of positively charged jetPRIME-RNA complexes, which electrostatically adsorbed onto the cell membrane. After incubation, the complex was added dropwise evenly to the plated cells. The culture plate was gently shaken to ensure uniform distribution. The cells were then maintained in a 37°C, 5% CO2 incubator for 4–6 hours. Subsequently, the medium containing the transfection complex was removed and replaced with fresh, complete growth medium to reduce cytotoxicity. To assess transfection efficiency and functional effect, cells were harvested for analysis 48 hours post-transfection.

[0058] Apox resistance assay: treated HCC cells were administered at a concentration of 1×10⁻⁶ cells / cells. 6 Cells were reseeded in Costa 6-well plates (Corning) and kept in suspension for 24 hours. Cell suspensions were collected by centrifugation and stained in the dark using the Annexin V:FITC / PI Apoptosis Detection Kit (Multi Sciences). After staining, samples were filtered through a 0.2 μm cell sieve and analyzed immediately using flow cytometry (Beckman Coulter).

[0059] Transwell assay: Mahlavu cells (1×10⁻⁶) were used. 4 / well) or MHCC-97H cells (1×10⁶) 5Cells were seeded in 8 μm pore size polycarbonate membrane Transwell chambers (NEST). For invasion assays, the chambers were pre-coated with 60 μL of 1:8 diluted Matrigel (Mogengel-Bio); uncoated chambers were used for migration assays. 200 μL of serum-free medium resuspended in the upper chamber and 600 μL of complete medium were added to the lower chamber. After incubation at 37°C and 5% CO2 for 72 hours, the cells were washed twice with PBS, fixed with 4% paraformaldehyde (Tongsheng bio-technology) at room temperature for 30 minutes, and stained with crystal violet for 30 minutes. Unpenetrated cells were gently wiped away from the inner membrane with a cotton swab, and the cells that penetrated to the lower membrane surface were observed and counted under a microscope (Leica).

[0060] Establishment of an animal lung metastasis model: Mahlavu cells pretreated with patient serum were collected, resuspended in sterile PBS, and injected into mice via tail vein, with each mouse receiving 5 × 10⁶ cells. 5 Each cell.

[0061] Tissue processing and hematoxylin-eosin (H&E) staining: At the experimental endpoint, mice were euthanized by intraperitoneal injection of an excessive amount of sodium pentobarbital (150 mg / kg). After confirming respiratory and cardiac arrest, cervical dislocation was performed. Lung tissue from the mice was collected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (4 μm thick). After dewaxing and hydration, the sections were stained with H&E. Lung metastases were scanned and analyzed using a panoramic multispectral tissue scanner (Akoya Biosciences), and the metastatic area was quantified.

[0062] Statistical analysis: Statistical analysis and graphing were performed using GraphPad Prism software (version 9, La Jolla, CA, USA). All quantitative data represent at least three independent experiments and are reported as mean ± standard error (SEM). After validation for normality, comparisons between two groups were performed using unpaired or paired Student's tests, and comparisons among multiple groups were performed using one-way ANOVA. P < 0.05 was considered statistically significant.

[0063] A schematic diagram of the process of this invention is shown below. Figure 18 As shown.

[0064] Example 1: Postoperative serum-induced resistance to anointing apoptosis and metastasis phenotype in HCC cells I. Experimental Methods Serum-treated cells: Mahlavu or MHCC-97H cells were seeded and cultured. After cell attachment, the culture medium was replaced with DMEM containing 10% preoperative or postoperative human serum, and cultured for another 2 days. The cells were washed three times with PBS before replacement.

[0065] Functional testing: Apoptosis resistance, Transwell migration and invasion assays were performed according to the "General Experimental Methods".

[0066] In vivo experiments: An animal lung metastasis model was established and analyzed according to the "General Experimental Methods".

[0067] II. Experimental Results To investigate whether the perioperative period is associated with an increased risk of metastasis, we collected paired serum samples from HCC patients before and after surgery (information is shown in Table 1). We cultured Mahlavu and MHCC-97H cells using 10% human serum derived from patient samples. Figure 1 A). After 4 days of culture, the cells were suspended in a specially designed culture dish for 24 hours, and apoptosis was detected by flow cytometry. The results showed that HCC cells cultured with postoperative serum exhibited significant anointing apoptosis resistance compared with cells cultured with preoperative serum, which is a key phenotype for cancer cell survival during metastasis. Figure 1 To determine whether exposure to postoperative serum conferred enhanced metastatic potential on HCC cells, we examined their migration and invasion abilities after treating HCC cells with different sera. Notably, HCC cells treated with postoperative serum exhibited enhanced metastatic potential in vitro compared to those treated with preoperative serum. Figure 1 DE). Furthermore, we injected HCC cells treated with different serums into the tail vein of nude mice. The results showed that, compared with preoperative serum, postoperative serum significantly enhanced the ability of HCC cells to colonize the lungs and form metastatic lesions. Figure 1 (FG). In summary, these data suggest that serum changes induced by surgery or related events are an important factor promoting HCC metastasis.

[0068] Example 2: Postoperative serum exosomes are key mediators promoting HCC metastasis I. Experimental Methods Exosome isolation: Exosomes were isolated from serum by ultracentrifugation. First, the collected serum was centrifuged at 3500 ×g for 15 minutes at 4°C, followed by centrifugation at 10,000 ×g for 45 minutes to remove cell debris. Finally, the supernatant was centrifuged at 100,000 ×g for 70 minutes at 4°C to precipitate the exosomes. The precipitate was resuspended in PBS and washed, and the ultracentrifugation steps were repeated to purify the exosomes (Beckman Coulter). All exosomes were resuspended in PBS and stored at -80°C.

[0069] Exosome characterization: Exosome morphology was observed using transmission electron microscopy (Hitachi). The particle size distribution and concentration of exosomes were determined by nanoparticle tracking analysis (Particle Metrix).

[0070] Exosome processing and uptake validation: Cells were co-cultured with 20 μg of exosomes for 48 hours. To validate uptake, exosomes were labeled with DiO dye (Beyotime), co-incubated with cells, and then the cytoskeleton was stained with phalloidin, the nucleus was stained with DAPI, and observed under a confocal microscope (Leica STELLARIS 5).

[0071] Functional testing: Apoptosis resistance, Transwell migration and invasion assays were performed according to the "General Experimental Methods".

[0072] In vivo experiments: Mice were "initiated" by receiving four injections (every two days) of 20 μg of preoperative or postoperative serum exosomes via tail vein. Subsequently, a lung metastasis model was established by tail vein injection of Mahlavu cells according to the "general experimental method" and the results were analyzed.

[0073] II. Experimental Results Exosomes can carry protected and functionally important biomolecules. Therefore, to identify specific components in serum that enhance HCC metastasis, we focused on serum-derived exosomes. We first isolated exosomes from serum samples and observed them using electron microscopy. The results showed the presence of exosomes with particle sizes ranging from 30 to 150 nm, and they exhibited a unique cup-shaped morphology. Figure 2 A). Furthermore, the number and size distribution of these exosomes were also verified through nanoparticle tracking analysis ( Figure 2 B). We also examined the uptake of exosomes by HCC cells and found little difference in the ability of HCC cells to internalize exosomes before and after surgery. Figure 2 C). Subsequently, we exposed HCC cells to these exosomes and surprisingly found that postoperative serum-derived exosomes significantly enhanced cellular resistance to anodic apoptosis compared to exosomes derived from preoperative serum. Figure 3 AB). However, this effect was eliminated when we used exosome-free serum. Figure 3 AB). We also examined the migration and invasion potential of HCC cells after exposure to different exosomes. Consistently, exosomes derived from postoperative serum significantly increased the migration and invasion capabilities of HCC cells compared to exosomes from preoperative serum. Figure 3 CD). In contrast, exosome-free serum had no significant effect on the function of either cell type. Figure 4 (AB). To assess the impact of postoperative serum exosomes on metastasis in vivo, we constructed a lung metastasis model. Mice were administered patient-derived exosomes via tail vein injection before or after surgery. Figure 3As shown in the EF, postoperative serum exosomes accelerated the progression of HCC cell lung metastasis compared to exosomes from preoperative patients. In summary, these results highlight the pro-metastatic role of postoperative serum exosomes, suggesting that certain perioperative events in at least a subset of the HCC population lead to changes in serum exosomes, thereby increasing the risk of postoperative cancer metastasis.

[0074] Example 3: Upregulation of miR-1246 expression in postoperative serum exosomes and its validation as a metastasis biomarker I. Experimental Methods Exosomal miRNA sequencing: Serum exosomal samples from preoperative and postoperative HCC patients were sent to Lianchuan Biotechnology Co., Ltd. for high-throughput sequencing analysis.

[0075] Sequencing data analysis: Raw miRNA expression data were normalized and logarithmically transformed before analysis. Subsequently, differential expression analysis between group A and group B was performed using the limma R package (version 4.3.0) to identify significantly dysregulated miRNAs, with thresholds set at P < 0.05 and |log2FC| > 1. Since there were only two groups, a t-test was used. Scatter plots were used to visualize the correlation of normalized expression levels between the two groups, and the number of upregulated, downregulated, and no significantly different miRNAs was summarized.

[0076] Hierarchical clustering analysis was performed on differentially expressed miRNAs based on Euclidean distance and complete linkage. The results were visualized as a heatmap using the pheatmap R package, where color bars represent normalized expression values ​​and circle sizes indicate the magnitude of log2 fold change (|log2FC|). The statistical significance of each miRNA was indicated by its corresponding p-value.

[0077] Predicted target genes of differentially expressed miRNAs were obtained using the miRTarBase and TarBase (v9.0) databases, retaining only overlapping targets identified by at least two algorithms. Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using the clusterProfiler R package. Pathways with a corrected p-value <0.05 were considered significantly enriched.

[0078] LASSO logistic regression analysis was performed on candidate miRNAs using the glmnet R package to identify key miRNAs with the strongest discriminative power. The optimal penalty parameter (λ) was determined by 10-fold cross-validation based on least-binary bias.

[0079] Quantitative real-time PCR (RT-qPCR) validation: Total exosome RNA was extracted using TRIzol reagent (Takara) and quantified using a UV spectrophotometer (Thermo Fisher). cDNA was synthesized using the miRcute Plus miRNA First-Strand cDNA Kit (Tiangen). Detection was performed using the miRcute Plus miRNA qPCR Kit (Tiangen) and a real-time PCR system (Thermo Fisher). Primer sequences are listed in "General Experimental Materials".

[0080] Database analysis: The correlation between miR-1246 expression and overall survival (OS) in HCC patients was analyzed using the ENCORI database.

[0081] II. Experimental Results Exosomes can transmit a range of biological information to regulate the behavior of recipient cells, among which miRNAs are the most abundant and widely studied. To elucidate the relationship between miRNA characteristics in serum exosomes and their interaction with HCC metastasis, we performed miRNA sequencing on serum exosomes from HCC patients before and after surgery. Analysis showed that, based on statistical significance (|log2FC|>1 and P<0.05), 39 differentially expressed miRNAs were identified (15 upregulated and 24 downregulated). Figure 5 AB). Furthermore, based on the target genes of 39 differentially expressed miRNAs, a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed, revealing biological processes and pathways significantly enriched in cancer metastasis and colonization, including the PI3K-Akt signaling pathway, MAPK signaling pathway, actin cytoskeleton regulation, focal adhesion, Wnt signaling pathway, and Hippo signaling pathway, etc. Figure 5 C). Subsequently, we used Least Absolute Contraction and Selection Operator (LASSO) regression analysis to identify key miRNAs associated with HCC metastasis (C). Figure 5 D). Ultimately, we identified four candidate miRNAs: miR-1246, miR-17-3p, miR-339-3p, and miR-3187-3p. Figure 6 A).

[0082] To validate these findings, we analyzed a new cohort comprising 65 HCC patients (information in Table 2). Only miR-1246 showed significantly differential expression in this independent cohort, consistent with sequencing data. Figure 6 B). To assess the prognostic value of miR-1246, we analyzed its association with HCC patient survival using the ENCORI pan-cancer analysis platform. We found that high expression of miR-1246 was significantly associated with poorer overall survival in HCC patients (B). Figure 6 (C) This supports its potential as a metastasis predictor. In summary, these data suggest a potential link between elevated serum miR-1246 levels in exosomes post-surgery and HCC metastasis.

[0083] Example 4: Validating the key role of postoperative exosomal miR-1246 in driving HCC metastasis I. Experimental Methods RNA oligonucleotide transfection: Performed according to "General Experimental Methods". HCC cells treated with exosomes before surgery were transfected with miR-1246 mimics or their negative controls (mimic-NC); cells treated with exosomes after surgery were transfected with miR-1246 inhibitors or their negative controls (inhibitor-NC). Sequences are available in "General Experimental Materials".

[0084] Functional testing: Apoptosis resistance, Transwell migration and invasion assays were performed according to the "General Experimental Methods".

[0085] In vivo animal experiments: Mice were pretreated with four tail vein injections before Mahlavu cell injection: mice injected with exosomes before the procedure were simultaneously injected with AgomiR-1246 or AgomiR-NC; mice injected with exosomes after the procedure were simultaneously injected with AntagonomiR-1246 or AntagonomiR-NC. Sequences are shown in "General Experimental Materials". Mice were sacrificed at the experimental endpoint, and lung tissue was collected for H&E staining and quantitative analysis of metastatic lesions.

[0086] II. Experimental Results To determine the role of postoperative serum exosome miR-1246 in driving HCC metastasis, we transfected preoperatively exosome-treated HCC cells with a miR-1246 mimic or its negative control (mimic-NC). Conversely, postoperatively serum-exosome-treated HCC cells were transfected with a miR-1246 inhibitor or its negative control (inhibitor-NC). We then assessed the anti-anorepopulation capacity of these cells by flow cytometry. Figure 7 As shown in AC, overexpression of miR-1246 in cells treated with preoperative serum exosomes significantly enhanced anodic apoptosis resistance. Conversely, knockdown of miR-1246 weakened postoperative serum exosome-induced anodic apoptosis resistance. Consistently, cell migration and invasion assays demonstrated that miR-1246 mimics conferred preoperative serum exosomes with the ability to promote cell migration and invasion, while their inhibitors eliminated these effects of postoperative serum exosomes. Figure 7Finally, an in vivo lung metastasis model confirmed that, in the context of preoperative serum exosome treatment, miR-1246 significantly enhanced the metastatic burden in mice, while its inhibition attenuated the pro-metastatic effect of postoperative serum exosomes. Figure 8 (AB). In summary, these results indicate that miR-1246 is a key factor in postoperative serum exosomes promoting HCC metastasis.

[0087] Example 5: Elucidating that miR-1246 inhibits the SMAD7 signaling pathway by directly targeting GDF2 / BMP9. I. Experimental Methods Bioinformatics target prediction: Potential downstream target genes of miR-1246 were predicted using the TargetScan, miRWalk, and miRDB databases, and their intersection with the anointing apoptosis-related gene set was taken.

[0088] Dual-luciferase reporter gene assay: The GDF2 3'UTR fragment containing the predicted miR-1246 wild-type (WT) or mutant (MUT) binding site was cloned into the pmiR-GLO reporter vector (GenaPharma). The reporter plasmid was co-transfected into cells with the miR-1246 mimic. Forty-eight hours after transfection, luciferase activity was measured using the dual-luciferase reporter gene assay system (Promega), and relative activity was calculated.

[0089] Western Blot Analysis: Total cellular protein was extracted using RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime). Concentration was determined by BCA method. Equal volumes of protein were subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 1 hour and then incubated overnight at 4°C with primary antibodies (BMP9: Santa Cruz, sc-514211, 1:100; SMAD4: CST, #46535, 1:1000; SMAD7: Santa Cruz, sc-101152, 1:100; p-SMAD1 / 5: CST, #9516, 1:1000; GAPDH: Beyotime, #AF0006, 1:5000). After washing, the membrane was incubated at room temperature for 1 hour with HRP-labeled secondary antibody (Beyotime, 1:1000). ECL development was performed, and the image was captured using an imaging system. Quantification was performed using ImageJ.

[0090] Immunofluorescence: Cells were seeded on coverslips, fixed, permeabilized, and blocked after 48 hours of treatment. They were incubated overnight at 4°C with rabbit anti-p-SMAD1 / 5 primary antibody (Invitrogen, 700047, 2 µg / mL). After washing, they were incubated at room temperature in the dark with FITC-labeled secondary antibody (Abclonal, AS011, 1:500) for 1 hour. Phalloidin was used to stain actin, and DAPI was used to stain the nucleus. Confocal microscopy was used for imaging, and ImageJ was used to quantify the nuclear fluorescence intensity.

[0091] Immunohistochemistry: Paraffin sections were dewaxed, rehydrated, and antigen-retrieved, then blocked with 3% H2O2 for 30 minutes and 5% BSA for 1 hour. They were incubated overnight at 4°C with primary antibodies (BMP9:1:50; SMAD4:1:100; SMAD7:1:50; p-SMAD1 / 5:1:100). After washing, they were incubated at room temperature for 1 hour with biotin-labeled secondary antibody (Beyotime, 1:50). DAB staining was performed, followed by hematoxylin counterstaining. Scanning was performed. Quantitative scoring was calculated using the following formula: H-score (<300%) = A × B, where A is the percentage of positively stained cells, and B represents the immunostaining intensity (0: negative; 1: weakly positive; 2: positive; 3: strongly positive).

[0092] II. Experimental Results Next, we used the TargetScan, miRWalk, and miRDB databases, along with a set of genes associated with anointing apoptosis, to predict potential downstream genes for miR-1246. The intersection of these datasets yielded two candidate target genes. GDF2 and TET2 ( Figure 9 A). GDF2 encodes BMP9, a ligand that activates classical SMAD signaling via BMPRs, while TET2 is a key enzyme in DNA demethylation, catalyzing the oxidation of 5mC to 5hmC. To verify the regulatory role of miR-1246 in GDF2 and TET2 expression, we transfected miR-1246 mimics or inhibitors into HCC cells treated with serum exosomes before or after surgery, and then measured gene expression. Figure 9 As shown in B, overexpression of miR-1246 inhibited GDF2 and TET2 The expression of mRNA in Mahlavu cells was reduced. Knocking down miR-1246 enhanced their expression. Nevertheless, considering that postoperative serum exosomes had a greater effect on Mahlavu cells compared to preoperative serum exosomes... TET2 The expression has very little impact ( Figure 9 B), we decided to prioritize research GDF2 Subsequently, we used analysis of the TargetScan database to reveal... GDF2A putative miR-1246 binding site within the 3'UTR ( Figure 9 C). To verify this direct interaction, we performed a dual-luciferase reporter gene experiment. We used wild-type... GDF2 The 3' UTR sequence or a mutant version was cloned into a reporter vector. Co-transfection of the wild-type construct with miR-1246 significantly inhibited luciferase activity in HCC cells, while this inhibition was eliminated when the mutant construct was used. Figure 9 C). We then examined the BMP9 protein levels in tissue samples from the animal model. (For example...) Figure 10 As shown in AD, BMP9 expression was significantly downregulated in postoperative serum or exosome-treated mouse metastatic lesions compared to the preoperative group. In another animal model, miR-1246 mimics significantly reduced BMP9 protein expression in preoperative serum exosome-treated mice, while miR-1246 inhibitors partially restored BMP9 levels in the postoperative serum exosome-treated group. Figure 10 In summary, these findings suggest that postoperative serum exosomal miR-1246 directly targets... GDF2 Inhibition of BMP9 may promote HCC metastasis.

[0093] The classic BMP-SMAD pathway is activated by cytokine-induced receptor pairing, leading to transphosphorylation. This event promotes the assembly of R-SMADs (SMADs 1 / 5 / 8) with SMAD4 into a transcriptional complex that enters the nucleus to regulate target genes. To determine whether this pathway is disrupted by miR-1246-mediated BMP9 inhibition, we assessed its impact on downstream signaling by regulating miR-1246 or BMP9 expression. Western blot analysis showed that in HCC cells treated with preoperative serum exosomes, miR-1246 mimics significantly reduced the levels of phosphorylated SMAD1 / 5 (p-SMAD1 / 5), SMAD4, and SMAD7. This inhibition was partially rescued upon exposure to recombinant protein BMP9. Figure 11 Conversely, in HCC cells treated with postoperative serum exosomes, inhibition of miR-1246 increased the expression of these proteins, an effect that can be detected by si... GDF2 Partially weakened ( Figure 11 Consistent with these findings, immunofluorescence staining showed that miR-1246 overexpression reduced nuclear localization of p-SMAD1 / 5, while BMP9 supplementation partially reversed this phenomenon. Figure 12 In addition, we examined several downstream target genes that may be regulated by the SMAD pathway. For example... Figure 13 As shown in AB, in the metastasis-promoting gene ( Cxcr4 , Itgb3 , Plcg1 ) and transfer repressor genes ( Bmp4 , Smad4 , Smad7 Similar expression patterns were observed in [the samples]. Overall, these data suggest that postoperative serum exosome-derived miR-1246 expresses miR-1246 through direct targeting... GDF2 It suppressed the classic BMP9-SMAD signaling pathway.

[0094] Example 6: Validation of the efficacy and biosafety of a combined therapy strategy targeting the miR-1246-BMP9 signaling axis I. Experimental Methods Cell function rescue experiment: Groups were set up as follows: (1) Preoperative serum exosomes + miR-1246 mimic / mimic-NC + recombinant protein BMP9 (human BMP9 recombinant protein, purchased from MCE (MedChemExpress), product number HY-P700530); (2) Postoperative serum exosomes + miR-1246 inhibitor / inhibitor-NC + si GDF2 Apoptosis, migration, and invasion experiments were conducted according to the "General Experimental Methods".

[0095] In vivo combined therapy experiment: A lung metastasis model driven by postoperative serum exosomes was established. Mice in the treatment group received alternating tail vein injections (postoperative serum exosomes + Antagonomi R-1246) and intraperitoneal injections (recombinant protein BMP9, 1 mg / kg / day for five consecutive days). The control group received the corresponding solvent. Finally, Mahlavu cells were injected via tail vein. The endpoint analysis was lung metastasis burden.

[0096] Serum biochemical analysis: Blood was collected from behind the eye socket and centrifuged at 3000 rpm for 10 minutes. Serum ALT, AST, ALP, T-Bil, CREA, and LDH levels were measured using a Mindray fully automated biochemical analyzer (BS200) and matching kits.

[0097] Histopathological examination: Liver and kidney tissues from mice were fixed, embedded, sectioned, and then evaluated by H&E staining.

[0098] II. Experimental Results We then investigated whether postoperative serum exosomes could promote HCC metastasis by inhibiting the BMP9-SMAD7 signaling pathway through miR-1246 transport. By regulating miR-1246 or BMP9 expression, we assessed the metastatic potential of HCC cells. Flow cytometry analysis showed that in cells treated with preoperative serum exosomes, the miR-1246 mimic conferred resistance to anodic apoptosis, while BMP9 supplementation reversed this effect. Figure 14Conversely, in cells exposed to postoperative serum exosomes, miR-1246 inhibitors sensitized them to anointing apoptosis, while knockdown of BMP9 partially restored resistance. Similar results were obtained in cell migration and invasion assays. Figure 15 AC). To evaluate the therapeutic potential targeting this signaling axis, we used a mouse lung metastasis model driven by postoperative serum exosomes to simulate the postoperative condition of HCC patients. We found that the miR-1246 inhibitor (AntagomiR-1246) significantly inhibited postoperative serum exosome-driven lung metastasis, and this inhibition was further enhanced upon combined administration of recombinant protein BMP9. Figure 15 DE). Immunohistochemical analysis confirmed that the levels of p-SMAD1 / 5, SMAD4, and SMAD7 were significantly increased in the treatment group. Figure 16 AF).

[0099] We also evaluated the biosafety of this treatment strategy. Apart from a decrease in lactate dehydrogenase (LDH) in the combination therapy group, other serum parameters, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine (CREA), and total bilirubin (T-Bil), showed no significant differences compared to the control group. Figure 17 A). Furthermore, H&E staining of the liver and kidneys showed no significant tissue damage, indicating that targeting the miR-1246-BMP9 signaling axis has good biocompatibility. Figure 17 B). In summary, these results indicate that targeting the miR-1246-BMP9 signaling axis can effectively inhibit postoperative metastasis of HCC and demonstrates good safety in vivo.

Claims

1. Application of miR-1246 inhibitors in the preparation of drugs for the prevention or treatment of postoperative metastasis of hepatocellular carcinoma.

2. The application according to claim 1, characterized in that, The drug also contains recombinant BMP9 protein.

3. The application according to claim 2, characterized in that, The miR-1246 inhibitor and the BMP9 recombinant protein are used together to synergistically inhibit postoperative metastasis.

4. The application according to any one of claims 1-3, characterized in that, The miR-1246 inhibitor is at least one of antagomir-1246, locked nucleic acid modified antisense oligonucleotide, small interfering RNA, or microRNA; The sequence of the antagomir-1246 is 5'-CCUGCUCCAAAAAUCCAUU-3'.

5. The application according to claim 2 or 3, characterized in that, The BMP9 recombinant protein is a human BMP9 recombinant protein.

6. The application according to any one of claims 1-5, characterized in that, The postoperative metastasis was a lung metastasis.

7. The application according to any one of claims 1-6, characterized in that, When used, the drug is intended to be administered to patients with hepatocellular carcinoma within one week after surgery.

8. The application according to any one of claims 2, 3, 5 or 6, characterized in that, The miR-1246 inhibitor and the BMP9 recombinant protein are configured in the drug to be administered simultaneously, separately, or sequentially.

9. A pharmaceutical combination for the prevention or treatment of postoperative metastasis of hepatocellular carcinoma, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of a miR-1246 inhibitor and a therapeutically effective amount of BMP9 recombinant protein.

10. The pharmaceutical combination according to claim 9, characterized in that, The miR-1246 inhibitor is at least one of antagomir-1246, locked nucleic acid modified antisense oligonucleotide, small interfering RNA, or microRNA; The sequence of the antagomir-1246 is 5'-CCUGCUCCAAAAAUCCAUU-3'; The BMP9 recombinant protein is a human BMP9 recombinant protein; The miR-1246 inhibitor and the BMP9 recombinant protein are configured to synergistically inhibit postoperative metastasis.

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