Use of FBXO9 protein in preparation of drugs for resisting pancreatic cancer

Abstract

The application relates to the biomedical field and particularly relates to application of FBXO9 protein in preparation of anti-pancreatic cancer drugs. The application finds for the first time that FBXO9 can inhibit the growth of pancreatic cancer cells and the occurrence of tumors, FBXO9 can inhibit the occurrence of tumors by regulating the immune response, and low expression of FBXO9 indicates the poor prognosis of a pancreatic cancer patient, so that the FBXO9 protein can be used as a marker to judge the prognosis of the pancreatic cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of FBXO9 protein in the preparation of drugs for treating pancreatic cancer. Background Technology

[0002] Immune checkpoint blockade (ICB), especially immunotherapy targeting PD-1 / PD-L1, has opened up a new paradigm for anti-tumor treatment. This therapy specifically activates anti-tumor immunity and inhibits the immune escape function of cancer cells. PD-L1, a membrane protein, is abnormally expressed in various tumor cells, including melanoma, acute myeloid leukemia, and colorectal cancer. PD-L1 can participate in inhibiting the activation of CD8+ T cells and the immune response, thereby promoting cancer cells' evasion of immune surveillance. Studies have shown that anti-PD-L1 immunotherapy can generate strong anti-tumor immunity and induce tumor regression. However, in clinical applications for various cancers, including pancreatic cancer, ICB therapy is effective in only one-third of patients. Therefore, thoroughly elucidating the regulatory mechanism of PD-L1 provides a basis for predicting the prognosis of pancreatic cancer immunotherapy.

[0003] A growing body of research indicates that posttranslational modifications (PTMs) play a crucial role in anti-PD-L1 tumor suppression, particularly protein ubiquitination. Ubiquitination is key in various cellular processes, including regulating protein stability and interactions, which to some extent constrain cell viability and differentiation, as well as innate and adaptive immunity. Studies have shown that posttranslational ubiquitination and deubiquitination processes play a critical role in regulating PD-L1 protein levels and tumor immunosuppression. For example, TMUB1 reduces PD-L1 stability by inhibiting polyubiquitination of PD-L1 at K281 in the endoplasmic reticulum, thereby promoting PD-L1 degradation and tumor immune evasion.

[0004] FBXO9, also known as FBX9, is a member of the F-box protein family and is the adaptor subunit of the SCF (SKP1-cullin-F-box complex) E3 ligase. It has been reported that FBXO9 can regulate the expression of various proteins, such as FBXW7, Neurog2, DPPA5, PPARγ, and PRMT4. Currently, FBXO9 is often studied as a potential biomarker or therapeutic target for sepsis. Further research is needed to expand the application scope of FBXO9 protein to include other potential applications. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides the application of FBXO9 protein in the preparation of drugs for treating pancreatic cancer.

[0006] The first aspect of this invention provides the use of FBXO9 protein in the preparation of drugs for treating pancreatic cancer, wherein the NCBI accession number of the mRNA of the FBXO9 protein is NM_012347.

[0007] This invention investigated the expression, biological effects, and tumor inhibition of FBXO9 protein. The results showed that the expression level of FBXO9 in pancreatic cancer tissue was significantly lower than that in normal tissue. FBXO9 overexpression inhibited cell viability, while FBXO9 silencing enhanced cell viability. Furthermore, FBXO9 was closely associated with CD8+ T cells in PAAD (Palm Alternative Anti-cancer Acid). These results indicate that low FBXO9 expression is associated with a poor prognosis in pancreatic cancer patients, and that FBXO9 protein can inhibit the occurrence of pancreatic tumors and promote anti-tumor immune responses. This suggests that FBXO9 protein can be used to prepare anti-pancreatic cancer drugs, providing a new application direction for FBXO9 protein.

[0008] In another preferred embodiment, the drug is a drug that inhibits the proliferation and migration of pancreatic cancer cells.

[0009] In another preferred embodiment, the drug contains the FBXO9 protein or its encoding gene.

[0010] In another preferred embodiment, the drug further includes a pharmaceutically acceptable carrier.

[0011] A second aspect of the invention provides a recombinant vector containing the coding gene of the FBXO9 protein, obtained by inserting the coding gene between BamHI and XhoI in the pcDNA3.1-3xFlag-C plasmid.

[0012] A third aspect of the present invention provides the use of the recombinant vector in the preparation of a medicament for treating pancreatic cancer.

[0013] The fourth aspect of this invention provides the application of the FBXO9 protein expression detection reagent in the preparation of pancreatic cancer prognostic products.

[0014] In another preferred embodiment, the product is a reagent kit.

[0015] In another preferred embodiment, the detection reagent comprises an antibody that specifically binds to the FBXO9 protein.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention is the first to discover through experiments that FBXO9 can inhibit the growth of pancreatic cancer cells and the occurrence of tumors. FBXO9 inhibits tumor occurrence by regulating the immune response, and low expression of FBXO9 can serve as a biomarker to predict the prognosis of pancreatic cancer.

[0018] This invention utilizes bioinformatics analysis of the TCGA and GEO databases, specifically the GSE183795 cohort, to reveal that FBXO9 expression levels in pancreatic cancer tissues are significantly lower than in normal tissues. Furthermore, low FBXO9 expression is associated with decreased overall survival, suggesting a link between low FBXO9 expression and poor prognosis in pancreatic cancer patients. Panc-1 and Patu-8988 cell experiments showed that FBXO9 overexpression inhibits cell viability, while FBXO9 silencing enhances cell viability, indicating that FBXO9 inhibits pancreatic tumor development. Analysis of FBXO9 using the TIMER database revealed that FBXO9 interacts with CD8 in PAAD. + The close relationship with T cells suggests that FBXO9 plays an immunomodulatory role in the tumor microenvironment of pancreatic cancer and can promote anti-tumor immune effects. Attached Figure Description

[0019] Figure 1 The figures shown are related to the low expression of FBXO9 and poor prognosis in pancreatic cancer patients in this embodiment of the invention. Specifically, A represents the results of FBXO9 mRNA expression analysis in pancreatic cancer and normal control tissues using TCGA and GTEx databases; B represents the results of FBXO9 mRNA expression analysis in pancreatic cancer and control tissues using GSE183795; C represents the survival analysis results of FBXO9 expression in 177 pancreatic cancer tissues analyzed by TCGA; D represents the survival analysis results of FBXO9 expression in 139 pancreatic cancer tissues analyzed by GSE183795; E represents the results of IHC detection of FBXO9 expression in pancreatic cancer tissues and adjacent normal tissues; F represents the results of FBXO9 expression analysis in pancreatic cancer tissues of 112 pancreatic cancer patients and 82 adjacent normal tissues; G represents the results of FBXO9 expression in pancreatic cancer tissues of 82 pancreatic cancer patients and paired adjacent normal tissues; and H represents the survival analysis results of FBXO9 expression in 112 pancreatic cancer tissues.

[0020] Figure 2 The graph shows the results of CCK8 detection of the proliferation ability of FBXO9 overexpression plasmids Panc-1 and Patu-8988; where A is the result of Panc-1 and B is the result of Patu-8988.

[0021] Figure 3The graph shows the changes in the proliferation capacity of Panc-1 and Patu-8988 after transfection with FBXO9 sgRNA by CCK8 assay; where A is the result of Panc-1 and B is the result of Patu-8988.

[0022] Figure 4 The results of the scratch assay were used to analyze the changes in the migration ability of Patu-8988 cells after transfection with the FBXO9 overexpression plasmid; where A is the result of the migration ability of Patu-8988 cells, and B is the quantitative result of A.

[0023] Figure 5 The results of the scratch assay were used to analyze the changes in the migration ability of Patu-8988 cells after transfection with FBXO9 sgRNA; where A is the result of the migration ability of Patu-8988 cells, and B is the quantitative result of A.

[0024] Figure 6 The image shows the results of a Transwell assay to detect changes in cell migration and invasion of PATU-8988 cells caused by transfection with the FBXO9 overexpression plasmid. In the image, A shows the results of migration and invasion of PATU-8988 cells, and B is the quantitative graph of A.

[0025] Figure 7 The image shows the results of Transwell assays to detect changes in cell migration and invasion of PATU-8988 cells after transfection with FBXO9 sgRNA. In the image, A shows the results of migration and invasion of PATU-8988 cells, and B is the quantitative graph of Figure A.

[0026] Figure 8 Figures showing the results of constructing a mouse subcutaneous xenograft model overexpressing FBXO9: A shows the growth of the subcutaneous xenograft tissue, B shows the weight of the mouse subcutaneous xenograft tissue, and C shows the volume of the mouse subcutaneous xenograft tissue.

[0027] Figure 9 The flowchart shows the subcutaneous injection of EV and FBXO9-overexpressing Panc-02 cells into C57BL / 6 mice, respectively; where EV stands for empty vector.

[0028] Figure 10 The images show the analysis results of tumor tissue from C57BL / 6 mice; where A is the tumor size graph, B is the tumor weight curve graph, and C is the tumor growth curve graph.

[0029] Figure 11The images show the results of CD8+ T cell infiltration in tumors detected by flow cytometry. In the images, A shows the infiltration results of CD8+ T cells in the EV group tumors, B shows the infiltration results of CD8+ T cells in the FBXO9 group tumors, and C shows the quantitative results of the infiltration.

[0030] Figure 12 The results of apoptosis in Panc-02 cells after co-culturing spleen cells from healthy C57BL / 6 mice are shown in Figure A, which shows the apoptosis of Panc-02 cells in the EV group, Figure B shows the apoptosis of Panc-02 cells in the FBXO9 group, and Figure C shows the quantitative apoptosis. Detailed Implementation

[0031] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0032] The following embodiments of the present invention disclose the application of FBXO9 protein in the preparation of drugs for treating pancreatic cancer. The NCBI accession number of the FBXO9 protein mRNA is NM_012347, and the sequence of the sgRNA targeting the FBXO9 gene is shown in SEQ ID NO.1:

[0033] SEQ ID NO. 1:GATGTACATCTTCCGATGGG.

[0034] In the following examples, all cell lines used for cell culture and transfection were obtained from the Cell Bank of the Chinese Academy of Sciences.

[0035] The experimental methods include:

[0036] Construction of the overexpression vector plasmid: The FBXO9 gene was inserted between BamHI and XhoI in the pcDNA3.1-3xFlag-C overexpression vector plasmid to obtain the FBXO9 overexpression plasmid.

[0037] Cell culture and transfection: Human pancreatic cancer cells Panc-1, PaTu-8988, and mouse pancreatic cancer cells Panc-02 were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Specific overexpression or sgRNA plasmids were transfected using Lipofectamine 3000 when cells reached 80% confluence. Transfection efficiency was determined by immunoblotting and quantitative real-time PCR analysis of protein and mRNA expression levels.

[0038] CCK8 assay: Cells in logarithmic growth phase were digested, centrifuged, collected, resuspended in culture medium, and counted. Then, 3000 cells were seeded into each well of a 96-well plate and cultured for 24, 48, or 72 hours. 10 μL of CCK8 reagent solution was added to each well, and incubation continued for 4 hours. The absorbance at 450 nm was then measured using a microplate reader.

[0039] Scratch assay: Cells were seeded into 6-well plates and cultured until the cell density reached 80%–90%. Using a 20 μl pipette tip, a straight scratch was gently made along the center of each well. Under a microscope, the scratched area appeared as a straight line. Floating and scraped cells were washed away with PBS, and culture medium was added for further experiments. Wound healing was observed at 0 and 18 hours after treatment, and photographs were taken under a microscope to record the results.

[0040] Transwell assay: First, collect the required cells, wash and centrifuge with PBS, then resuspend the cells in serum-free medium and count them. Next, add 800 μl of serum-containing medium to the bottom of a 24-well plate, and add 2 × 10⁶ cells to the Transwell chamber. 4 Cells were cultured for 18 hours. To assess cell invasion, 100 μl of pre-cooled, diluted Matrigel was added to the chamber before adding the cell suspension. After 18 hours of culturing, the chamber was removed, and any remaining non-migrating cells were wiped away with a cotton swab. Cells were fixed in 0.3% paraformaldehyde for 30 minutes, stained with crystal violet for 15 minutes, and then air-dried before microscopic observation of cell migration and invasion.

[0041] Animal experiments: All animal experiments were approved by the Animal Ethics Committee of Bengbu Medical College. First, a subcutaneous xenograft model was established in nude mice or C57BL / 6 mice. Mice were subcutaneously injected with 5×10⁻⁶ tumor-bearing agents. 6 We used either human pancreatic cancer cells (Panc-1) or mouse pancreatic cancer cells (Panc-02) that stably overexpress FBXO9. After tumor formation in mice, we performed the following procedures: observed the mice's living conditions, weighed and recorded changes in body weight; measured changes in tumor size weekly; 4-6 weeks after tumor formation, we removed the tumor fragments and weighed them; and performed flow cytometry analysis on the tumors from C57BL / 6 mice.

[0042] Methods for feeding and euthanizing laboratory animals: Provide animals with standard animal feed and water, anesthetize with isoflurane, and euthanize experimental rats by decapitation or cervical dislocation. Animal carcasses are disposed of by the school.

[0043] Flow cytometry: To detect apoptosis, cells were first collected by centrifugation at 1000 rpm for 5 minutes. After washing twice with PBS, the cells were resuspended in 200 μL of PBS, and PE-annexin V and 7-AAD antibody were added. After incubation for 15 minutes, the apoptotic cell population was analyzed by flow cytometry. To detect CD8+ in tumor tissue... + At the T cell level, tumor tissue was first ground, then Ficoll solution was added, and density gradient centrifugation was performed to collect PBMCs. PBMCs were resuspended in 100 μL PBS, CD3, CD4, and CD8 antibodies were added, and the cells were incubated in the dark for 30 minutes. The cells were then washed with PBS and resuspended. Flow cytometry parameters were set, and the cells were analyzed.

[0044] Clinical data and immunohistochemistry were collected from patients: Clinical data of pancreatic cancer patients were collected from the Department of Hepatobiliary Surgery and the Department of Oncology Surgery at Bengbu Medical College, and statistical analysis was performed. Pathological paraffin blocks were collected from patients at the Department of Pathology at Bengbu Medical College and cut into 4μm sections. IHC staining was performed using an automated staining system. After dewaxing, hydration, antigen retrieval, and blocking, the sections were incubated with primary and secondary antibodies, developed with DAB, blocked, observed under a microscope, and photographed. IHC scores were performed on each section.

[0045] Bioinformatics analysis: Clinical data of pancreatic cancer were downloaded and extracted from the UCSC XENA database (https: / / xenabrowser.net / datapages / ) and the GEO database (www.ncbi.nlm.nih.gov / geo / GSE183795) to analyze the expression differences of FBXO9 between pancreatic cancer and normal tissues. Kaplan-Meier survival analysis was performed; the correlation between FBXO9 and immune effector cells was analyzed using TIMER (http: / / timer.cistrome.org / ).

[0046] Statistical analysis: GraphPad Prism 8 software was used for statistical analysis. Unpaired t-tests and paired t-tests were used for comparisons between the two groups. Patient survival analysis was performed using Kaplan-Meier analysis and log-rank Mantel-Cox test. P < 0.05 was considered statistically significant.

[0047] Results Analysis

[0048] 1. Low expression of FBXO9 is closely related to poor prognosis in pancreatic cancer patients.

[0049] First, bioinformatics analysis was performed using the TCGA and GEO databases, specifically the GSE183795 cohort. The results showed that the expression level of FBXO9 in pancreatic cancer tissue was significantly lower than that in normal tissue. Figure 1 As shown in A and B in the diagram.

[0050] Further analysis of the relationship between FBXO9 and the clinical prognosis of pancreatic cancer patients revealed that low FBXO9 expression was associated with decreased overall survival. Figure 1 As shown in C and D in the figure. Subsequently, pathological data of pancreatic cancer patients were collected, and immunohistochemical detection of FBXO9 protein levels in pancreatic cancer and adjacent normal tissues was performed. The results showed that the expression level of FBXO9 in tumor tissues was lower than that in adjacent normal tissues. Figure 1 As shown in E, F, and G.

[0051] Overall survival analysis of pancreatic cancer patients showed that patients with low FBXO9 levels had significantly lower overall survival rates. Figure 1 Furthermore, a consistent association was found between lower FBXO9 expression and TNM stage and other clinical indicators, as shown in Table 1. These findings suggest that low FBXO9 expression is associated with a poorer prognosis in pancreatic cancer patients.

[0052] Table 1. Clinical indicators of FBXO9 in pancreatic cancer

[0053]

[0054]

[0055] Note: ρvalues ​​is x 2 The test values ​​are given, where n represents the number of cases, Low indicates low expression, and High indicates high expression.

[0056] 2. FBXO9 can inhibit cell growth and tumor development.

[0057] Knockout and overexpression experiments were performed in Panc-1 and Patu-8988 cells, respectively, and changes in cell proliferation were examined. Results showed that FBXO9 overexpression inhibited cell viability, while FBXO9 silencing enhanced cell viability. Figure 2 and Figure 3 As shown.

[0058] The knockout experiment was conducted as follows: Cells were seeded into six-well plates and transfected with FBXO9 sgRNA using Lipofectamine 3000 transfection reagent. After 48 hours, cells were collected and the expression level of FBXO9 was verified by qPCR and Western blotting.

[0059] The specific procedure for the overexpression experiment is as follows: Cells were seeded into six-well plates and transfected with FBXO9 overexpression plasmid using Lipofectamine 3000 transfection reagent. After 48 hours, cells were collected and the expression level of FBXO9 was verified by qPCR and Western blotting.

[0060] To assess the migration and invasion capabilities of cells, scratch assays and Transwell assays were performed on transfected pancreatic cancer cells.

[0061] First, human pancreatic cancer cells (PaTu-8988) were collected, washed with PBS, centrifuged, and resuspended in serum-free medium for cell counting. Then, 800 μL of serum-containing medium was added to the bottom of a 24-well plate, and 2 × 10⁶ cells were added to the Transwell chamber. 4 Cells were cultured for 18 hours. To assess cell invasion, 100 μL of pre-cooled, diluted Matrigel was added to the chamber before adding the cell suspension. After 18 hours of culture, the chambers were removed, and any remaining non-migrating cells were wiped away with cotton swabs. Cells were fixed in 0.3% paraformaldehyde for 30 minutes, stained with crystal violet for 15 minutes, and then air-dried before microscopic observation of cell migration and invasion. Results showed that overexpression of FBXO9 significantly reduced cell migration and invasion abilities. Figures 4-6 As shown.

[0062] To study the anti-tumor effects of FBXO9 in vivo, a subcutaneous xenograft tumor model was constructed. All animal experiments were approved by the Animal Ethics Committee of Bengbu Medical College.

[0063] Panc-1 cells overexpressing FBXO9 were subcutaneously inoculated into nude mice. Using a subcutaneous xenograft tumor model, the growth and size of tumor tissue in the FBXO9 overexpression group were significantly lower than those in the EV control group. Figure 7 and Figure 8 As shown in the above experimental results, FBXO9 inhibits the occurrence of pancreatic tumors.

[0064] 3. FBXO9 activates anti-tumor immune responses

[0065] To assess whether FBXO9 inhibits tumorigenesis by modulating the immune response, the correlation between FBXO9 and various immune effector cells was first analyzed using the TIMER database (http: / / timer.cistrome.org / ). The results showed that FBXO9 correlates with CD8+ in PAAD cells. + Closely related to T cells, FBXO9 may play an immunomodulatory role in the tumor microenvironment of pancreatic cancer.

[0066] To further investigate the role of FBXO9 in anti-tumor immune responses, FBXO9-overexpressing Panc-02 pancreatic cancer cells were inoculated into immunocompetent C57BL / 6 mice. Figure 9 As shown, compared with the EV control group, as Figure 10 As shown, tumor growth in the FBXO9 overexpression group was significantly inhibited, and the tumor weight was also lower than that in the control group. Flow cytometry further showed that CD8+ T cell infiltration was significantly increased in the tumor tissue of the FBXO9 overexpression group, such as... Figure 11 As shown. Furthermore, after co-culturing FBXO9-overexpressing Panc-02 cells or EV control cells with spleen cells from healthy C57BL / 6 mice for 3 days, a significantly increased apoptosis rate was observed in FBXO9-overexpressing Panc-02 cells compared to the EV control group, as shown. Figure 12 As shown in the figure, the above research results indicate that FBXO9 can promote anti-tumor immune effects.

[0067] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Clearly, those skilled in the art can make various alterations and variations to the invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of equivalents of the invention, the invention is also intended to include these modifications and variations.

Claims

1. The application of FBXO9 protein in the preparation of drugs for treating pancreatic cancer, characterized in that, The NCBI accession number for the mRNA of the FBXO9 protein is NM_012347; The drug contains the FBXO9 protein.

2. The application of the FBXO9 protein according to claim 1 in the preparation of drugs for treating pancreatic cancer, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

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