Application of TLQP-21 in treatment of pancreatic cancer or pancreatic cancer-depression co-disease

Abstract

The invention relates to the technical field of biology, and provides application of TLQP-21 in treatment of pancreatic cancer or pancreatic cancer-depression co-disease. The application reveals that the TLQP-21 has the effect of inhibiting pancreatic cancer progression, can improve depression-like behaviors and pain symptoms, provides a novel target for the development of pancreatic cancer and pancreatic cancer-depression co-disease medicines, also provides a new treatment strategy for the pancreatic cancer and pancreatic cancer-depression co-disease medicines, is expected to be clinically used, provides an effective treatment method, and has a wide application prospect. The life quality of pancreatic cancer patients is improved, the survival time is prolonged, and a new research direction is provided for treatment of pancreatic cancer and pancreatic cancer-depression codiseases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically, to an application of TLQP-21 in treating pancreatic cancer or pancreatic cancer-depression comorbidity. Background Art

[0002] The 5-year survival rate of pancreatic cancer is only 11%, making it one of the malignant tumors with the worst prognosis. Depression often co-occurs with cancer and is associated with poor prognosis, reduced treatment compliance, and increased cancer mortality. Pancreatic cancer is the tumor with the highest incidence of depression among digestive system tumors. Surveys show that more than 50% of pancreatic cancer patients have psychiatric symptoms, which appear 43 months earlier than physical symptoms. Although the comorbidity of pancreatic cancer and depression is significant, little is known about the mechanism of their comorbidity.

[0003] There are currently two chemotherapy treatments for pancreatic cancer, gewcitbinemab-Paclitaxel and FOLFIRINO (irinotecan.oxaliplatin.S-fluororacil and leucovorin), however, these two chemotherapy treatments are only effective in 30% of patients. Although surgical resection of the tumor is a potential cure, less than 20% of pancreatic cancer cases are resectable at diagnosis, and most patients who undergo surgery still die from local recurrence and / or metastasis. And for patients with advanced pancreatic cancer, there is still no clear cure.

[0004] Recent studies have found that nerves are an important component of the tumor microenvironment and play an important role in cancer progression. Neurotrophic factors and neuropeptides are important mediators of cancer cell-nerve interactions. The neuropeptide VGF is a secreted protein and neuropeptide precursor. Currently, there are relatively few reports on the molecular mechanism by which VGF affects tumor progression. Existing literature suggests that in lung cancer, high expression of VGF can reduce cancer cell apoptosis, while silencing VGF expression reduces tumor growth by enhancing cancer cell apoptosis. In addition, VGF can promote the migration and invasion of lung cancer lineage cells. In pancreatic endocrine tumors, VGF promotes the proliferation and metastasis of cancer cells. In glioblastoma, glioblastoma stem cells promote the growth and self-renewal of glioblastoma stem cells by autocrine VGF, while maintaining the survival of differentiated glioblastoma cells through paracrine effects and promoting tumor growth in vivo. However, the role of VGF and its derivative peptides in pancreatic cancer has not been reported.

[0005] VGF is expressed in the central and peripheral nervous systems and pancreatic islets, participates in and regulates the body's energy balance and metabolism, and has anti-depressant functions. VGF exerts different physiological functions by breaking down into a variety of polypeptides, such as TLQP-21, TLQP-62, NERP-1 and NERP-2. Among them, TLQP-21 and its receptors are enriched and expressed in the central nervous system and pancreas, and play an important role in maintaining the survival of pancreatic B cells, stimulating insulin secretion, and regulating the body's energy metabolism. Studying the effect of TLQP-21 on the progression of pancreatic cancer and its regulatory mechanism is conducive to finding a breakthrough in breaking the comorbid cycle between pancreatic cancer and depression, which is crucial to improving the quality of life and survival time of pancreatic cancer patients. Summary of the invention

[0006] The present invention aims to overcome at least one defect (shortcoming) of the above-mentioned prior art, provide an application of TLQP-21 in the treatment of pancreatic cancer or pancreatic cancer-depression comorbidity, and provide a new treatment strategy for pancreatic cancer and pancreatic cancer-depression comorbidity.

[0007] One object of the present invention is to provide a use of TLQP-21 in the preparation of a drug for treating pancreatic cancer or pancreatic cancer-depression comorbidity. In one or more embodiments of the present invention, the present invention first studied the effect of VGF-derived peptide TLQP-21 on the occurrence and development of pancreatic cancer, and found that unlike the previous characteristics of VGF promoting lung cancer and glioma, VGF-derived peptide has the effect of inhibiting the progression of pancreatic cancer and can improve its depressive-like behavior and pain symptoms. The amino acid sequence of TLQP-21 polypeptide is: TLQPPASSRRRHFHHALPPAR (SEQ ID NO.1).

[0008] Further, when TLQP-21 is used in the preparation of a drug for treating pancreatic cancer-depression comorbidity, TLQP-21 alleviates the activation of the insular cortex. In one or more embodiments of the present invention, after administering TLQP-21 to pancreatic cancer model mice, the results showed that the activation of the insular cortex brain region was alleviated, and the pain level of the mice was positively correlated with the activity of the insular cortex brain region, indicating that TLQP-21 improves pancreatic cancer-depression comorbidity by acting on pain information mediated by the insular cortex brain region.

[0009] Further, when TLQP-21 is used in the preparation of a drug for treating pancreatic cancer-depression comorbidity, TLQP-21 alleviates astrocyte-mediated neuroinflammatory reactions in brain regions, including the dorsal nucleus of the vagus nerve, the area after the back, and the nucleus of the solitary tract. In one or more embodiments of the present invention, GFAP immunofluorescence staining analysis was performed on 15 brain regions related to emotion and pain in the whole brain, and the results showed that among the 15 brain regions included in the analysis, GFAP in the area after the back and the dorsal nucleus of the vagus nerve brain regions was significantly increased in pancreatic cancer model mice; and after TLQP-21 was injected into pancreatic cancer model mice, GFAP expression in the area after the back, the dorsal nucleus of the vagus nerve brain regions, and the nucleus of the solitary tract brain regions was significantly reduced, and the inflammatory reaction of astrocytes was alleviated, thereby reducing susceptibility to depression.

[0010] Further, when TLQP-21 is used in the preparation of a drug for treating pancreatic cancer, TLQP-21 downregulates differentially expressed genes in pancreatic tumors, and the differentially expressed genes include proinflammatory cytokine-related genes, genes related to promoting polarization of M2 macrophages, angiogenesis-related genes, and immune infiltration-related genes. Further, the proinflammatory cytokine-related genes include A2m, Ccl2, Plek, Tsc22d3, Zc3h12a, and Cxcl1; the genes related to promoting polarization of M2 macrophages include Per1, Fgl2, Adgre1, Socs3, Cc120, Rel, Herpud1, and Ccl2; the angiogenesis-related genes, such as Ccl2, E2f2, Icam1, Tlr2, and Cxcl1; the immune infiltration-related genes include Fgl2, Cdl4, Nlrp3, Syk, and Cxcl1.

[0011] The present invention analyzed the cell types in which DEGs were enriched and expressed in the pancreas of pancreatic cancer model mice, and found that the screened DEGs were expressed in various types of cells such as ductal cells, metaplastic cells, endocrine cells, endothelial cells, immune cells, pericytes and fibroblasts, and were most highly enriched in macrophages. Furthermore, the present invention used qPCR to verify the expression of some DEGs, and found that compared with the control group, the mRNA expression levels of Gpr141, Per1, Birc3, Slamf7, Icam1, Adrb2, Ccl20 and A2m in the tumor of the TLQP-21 intervention group were significantly reduced.

[0012] Another object of the present invention is to provide a use of TLQP-21 in the preparation of a drug for inhibiting the migration and growth of pancreatic cancer cells. Further, TLQP-21 inhibits the migration of pancreatic cancer cells by acting on C1QBP receptors or inhibits the growth of pancreatic cancer cells by acting on TAM cells. In one or more embodiments of the present invention, it was found through co-culture experiments of tumor cell-associated macrophages and pancreatic tumor cells that TLQP-21 can inhibit the growth of pancreatic tumor cells by regulating unknown factors secreted by TAM.

[0013] Another object of the present invention is to provide a pharmaceutical composition for treating pancreatic cancer or pancreatic cancer-related depression, comprising TLQP-21. Further, the drug is in the form of an injection.

[0014] Another object of the present invention is to provide a use of TLQP-21 in the preparation of angiogenesis-inhibiting drug, wherein the angiogenesis-inhibiting drug is a drug for inhibiting angiogenesis in pancreatic cancer lesions.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention reveals that TLQP-21 has the effect of inhibiting the progression of pancreatic cancer, and can improve its depressive-like behavior and pain symptoms, providing a new target for the development of drugs for pancreatic cancer and pancreatic cancer-depression comorbidity, and also providing a new treatment strategy for it, which is expected to be used clinically, provide an effective treatment method, improve the quality of life of pancreatic cancer patients and prolong their survival time, and also provide a new research direction for the treatment of pancreatic cancer and pancreatic cancer-depression comorbidity. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1Shown: The relationship between the change of VGF mRNA expression in pancreatic cancer tumor samples and the survival time of patients. (AD) Kaplan-Meier survival analysis of the four neurotrophic factor VGF (A), NGF (B), BDNF (C), and GDNF (D) genes in the GEPIA database and the overall survival (OS) of human pancreatic cancer (pancreative ductal adenocarcinoma, PDAC) patients (statistical analysis using the log-rank test (Log Bank Test)). (EF) Kaplan-Meier survival analysis of the neurotrophic factor VGF gene in the GEPIA database and the OS of human lung adenocarcinoma (LUAD, E) and glioblastoma multiforme (GBM, F) patients (statistical analysis using the log-rank test (Log Rank Test)). (G) Expression of the neurotrophic factor VGF gene in human PDAC patient tumor tissue (Tumor) and adjacent normal tissue (Normal) in the GEPIA database. (H) Distribution of VGF protein expression in human tissues. The data comes from the human proteomics database PreteomicsDB.

[0017] Figure 2 Shown: TLQP-21 inhibits tumor growth in orthotopically implanted pancreatic cancer model mice. (A) Schematic diagram of the experimental process of the effect of TLQP-21 on tumor progression in orthotopic pancreatic cancer model mice. (B) Quantitative analysis of tumor volume after orthotopic pancreatic cancer model mice were intraperitoneally injected with saline or TLQP-21 for 14 consecutive days. (C) Representative IVIS images of orthotopic pancreatic cancer model mice 3 days, 7 days, and 14 days after intraperitoneal injection of saline or TLQP-21. (D) Average fluorescence quantum number graph measured by IVIS on different days. (E) Total fluorescence quantum number graph measured by IVIS on different days All data are expressed as mean ± SEM. and analyzed by Student's t tests. *p<0.05.

[0018] Figure 3Shown: TLQP-21 alleviates pain and depressive behavior in pancreatic cancer model mice. (A) Schematic diagram of the experimental process of TLQP-21 affecting emotional behavior and pain in orthotopically implanted pancreatic cancer model mice; Sham: mice that underwent sham surgery but did not have tumors, and were injected with saline every day; KPC+Saline: mice with tumors, and were injected with saline every day; KPC+TLQP-21: mice with tumors, and were injected with TLQP-21 (5ug / g / d) every day; OF: Open field test; SIT: social interaction test; VFT: von frey test; TST: tail suspension test. (B) Body weight changes of mice every three days after tumor implantation. (C) Tumor volume of animals in the KPC+Saline and KPC+TLQP-21 groups 20 days after tumor implantation, analyzed by Student's t tests, n=9-12. (D) Representative images of the trajectories of the three groups of mice in OF. (E) Total moving distance of the three groups of mice in OF, analyzed by One-way Anova, n=10-12. (F) Changes in pain threshold of VFT of the three groups of mice before and after modeling, analyzed by One-way Anova, multiple comparison correction by Tukey test, a=5-6. (G) Immobility time of the three groups of mice in TST, analyzed by One-way Anova, multiple comparison correction by Kruskal-Wallis test w / uncorrected Dunn's test, n=10-12. (H) Trajectory representative heat map of the three groups of mice in SIT. (I) Total exploration time of the three groups of mice in SIT and social interaction index (DI%) of mice, analyzed by One-way Anova, multiple comparison by Fisher's LED test, n=10-12. All data are expressed as mean=SEM.

[0019] Figure 4Shown: TLQP-21 down-regulates the expression level of c-FOS, an active marker, in specific brain regions of pancreatic cancer model mice. (A) Mouse brain map related to the 15 brain regions included in the whole brain c-FOS analysis. The red color in the figure represents the brain regions where the c-FOS expression level in the KPC+Saline group increased compared with the Sham group, and the c-FOS expression level decreased after TLQP-21 administration (KPC+TLQP-21 group). Gray represents the brain regions with no significant changes. (B) c-FOS-positive cell density (number of c-FOS-positive cells / area of ​​target region), fold change of the means of the three groups (all data are expressed as mean ± SEML. and analyzed by Student's ttests. IL: n = 9, 10, 12; PL: n = 8, 10, 12; IC: n = 9, 10, 12; LS: n = 9, 9, 12; BNST: n = 9, 9, 12; MPO: n = 9, 9, 11; RE: n = 9, 10, 12; PVN: n = 9, 10, 12; MH: n = 9, 10, 12; LH: n = 9, 10, 12; dHC: n = 9, 10, 12; ARC: n = 9, 10, 12; AP: n = 9, 10, 12; DMV: n = 9, 10, 12: NTS: n = 9, 9, 12). (C) Representative immunofluorescence staining of c-FOS expression in the IC brain region of three groups of mice, scale bar = 300 μm. (D) Correlation analysis between the change of pain threshold of mice in VFT experiment and the density of c-FOS positive neurons in the IC brain region (linear regression and Person correlation analysis), n = 15. *p < 0.05 ***p < 0.0001.

[0020] Figure 5Shown: TLQP-21 down-regulates GFAP expression in astrocytes in specific brain regions of pancreatic cancer model mice. (A) Mouse brain atlas related to 15 brain regions included in the whole-brain GFAP analysis. The red color in the figure represents the brain regions where GFAP expression levels increased in the KPC+Saline group compared with the Sham group, and where GFAP expression levels decreased after TLQP-21 administration (KPC+TLQP-21 group). Gray represents the brain regions with no significant changes. (B) The ratio of GFAP fluorescence intensity to the target brain area (GFAP fluorescence intensity / target area), and the fold change of the means of the three groups (all data are expressed as mean ± SEML. and analyzed by Student's t tests. IL: n = 9, 10, 12; PL: n = 9, 9, 12; IC: n = 10, 9, 11; LS: n = 10, 9, 12; BNST: n = 10, 9, 12; MPO: n = 10, 9, 12; RE: n = 10, 8, 10; PVN: n = 10, 10, 12; MH: n = 10, 8, 11; LH: n = 10, 10, 12; dHC: n = 10, 10, 12; ARC: n = 10, 10, 12; AP: n = 10, 10, 12; DMV: n = 9, 10, 12; NTS: n = 10, 10, 12). (C) Percentage of GFAP fluorescence coverage area (GFAP fluorescence coverage area / target area*100), fold change of the mean of the three groups (all data are expressed as mean ± SEM and expressed by Student's (D) Representative immunofluorescence staining of GFAP expression in the AP, DMV and NTS brain regions of the three groups of mice. Scale bar = 300 μm. *p<0.05**p<0.001***p<0.0001.

[0021] Figure 6Shown: TLQP-21 inhibits the migration of pancreatic cancer cells. (A) Schematic diagram of the experiment to detect the proliferation (CCK8 assay) and migration (Transwell) of tumor cells after TLQP-21 treatment. (B) CCK8 assay to detect the effect of 48h treatment of TLQP-21 on the proliferation ability of KPC cells. (C) CCK8 assay to detect the effect of 48h treatment of TLQP-21 on the proliferation ability of Pan02 cells. (D) Representative image of the effect of 18h treatment of TLQP-21 on the migration ability of KPC cells by Transwell migration assay, scale bar = 300μm. (E) Representative image of the effect of 18h treatment of TLQP-21 on the migration ability of KPC cells by Transwell migration assay. (F) Representative image of the effect of 18h treatment of TLQP-21 on the migration ability of Pan02 cells by Transwell migration assay, scale bar = 300μm. (G) Representative image of the effect of 18h treatment of TLQP-21 on the migration ability of Pan02 cells by Transwell migration assay. All data are expressed as mean ± SEML and analyzed by One-way ANOVA.

[0022] Figure 7Show: TLQP-21 inhibits pancreatic cancer cell migration by binding to C1QBP receptor. (A) Expression of neurotrophic factor receptor CIQBP gene in human PDAC patient tumor tissue (Tumor) and adjacent normal tissue (Normal) in the GEPLA database. (B) Expression of neurotrophic factor receptor C3AR1 gene in human PDAC patient tumor tissue (Tumor) and adjacent normal tissue (Normal) in the GEPIA database. (C) Correlation analysis diagram between the expression of neurotrophic factor VGF and its receptor C1QBP gene in the GEPIA database (linear regression and Person correlation analysis). (D) qPCR verified that TLQP-21 receptor C1qbp was expressed on tumor cells, and BV2 microglia were used as positive controls. (E) UMAP diagram shows the main cell types in the pancreas of PtfIa-CreER, LSL-Kras-GI2D, LSL-tdTomato (PRT) mice injected with tamoxifen for 17 days, 6 weeks, 3 months, 5 months, 9 months and 15 months. The data are from an article published by Yehuda Schlesinger et al. in 2020 on single-cell transcriptome analysis of pancreatic preinvasive lesions and cancer in mice (Schlesinger Y, Yosefov-Levi O, Kolodkin-Gal D, et al. Single-cell transcriptomes of pancreatic preinvasive lesions and Nat Commun, 2020, 11(1): 4516). (F) Distribution of C1qbpmRNA in mouse tissues. (G) Distribution of C3ar1 mRNA in mouse tissues. (H) Representative image of the effect of competitive inhibition of 0.1uM C1qbp mAb on KPC cell migration after 0.luM TLQP-21 in Transwell migration assay, scale bar = 300μm. (I) Effect of competitive inhibition of 0.1uM C1qbp mAb on KPC cell migration ability after 0.1uM TLQP-21 in Transwell migration assay - gray columns are Ctrl group. (J) Representative figure of the effect of competitive inhibition of TLQP-21 by C1qbp mAb on the migration of Pan02 cells in Transwell migration assay, scale bar = 300um. (K) Effect of competitive inhibition of TLQP-21 by C1qbp mAb on the migration ability of Pan02 cells in Transwell migration assay - gray columns are Ctrl group. All data are expressed as mean + SEML. and analyzed by One-way ANOVA. *p < 0.05.

[0023] Figure 8 Shown: TLQP-21 regulates gene expression profiles and functional pathways in pancreatic tumor tissue. (A) Schematic diagram of the experimental and analytical process. An orthotopic pancreatic cancer mouse model was established. Starting from the second day after tumor implantation, mice were continuously injected with TLQP-21 IP (control group was injected with saline). Saline: mice with tumor implantation were injected with saline IP every day; TLQP-21: mice with tumor implantation were injected with TLQP-21 (5ug / g / d) every day. Mice were subjected to IVIS on days 7 and 14 after tumor implantation to observe the growth of in vivo tumors. After confirming that TLQP-21 slowed the growth of pancreatic cancer tumors, tumors were freshly sampled on day 15 after tumor implantation, and RNA was extracted from tumors. (B) Cluster heat map of differentially expressed genes (DGEs) (DGE screening condition: p value < 0.01). Scale (right): log2 (CPM). (C) DGE volcano plot: fold change and p value of the TLQP-21 group compared with the Saline group. Red: upregulated expression, blue: downregulated expression. (D) UMAP diagram showing the main cell types in the pancreas of Ptf1a-CreER, LSL-Kras-G12D, LSL-tdTomato (PRT) mice 17 days, 6 weeks, 3 months, 5 months, 9 months and 15 months after injection of tamoxifen. The data are from an article published in 2020 by Yehuda Schlesinger et al. on single-cell transcriptome analysis of pre-invasive lesions and cancers in the mouse pancreas. (E) Distribution diagram of mRNA of all DGEs in mouse tissues. (F) qPCR verification of the expression of selected DGEs compared with the mean of the Saline group (all data are expressed as mean ± SEM. and analyzed by Student's t tests). (G) Gene Set Enrichment Analysis (GSEA), enrichment screening conditions: enrichment score (NES) absolute value> 0; false discovery rate (FDR) < 0.25. GO-BP enrichment analysis, GO-CC enrichment analysis, GO-MF enrichment analysis, KEGG enrichment analysis. Red: items enriched in the Saline group, blue, items enriched in the TLQP-21 group. Scale: Dots represent: -log 10 FDRq value. Color represents: NES.

[0024] Fig. 9Shows: TLQP-21 indirectly inhibits pancreatic cancer cell growth by acting on TAM. (A) Flowchart of TAM cell sorting. TAM cells were sorted by FACS, and the sorted TAM cells were co-cultured with KPC cells using a non-contact Transwell system. (B) TAM cell sorting strategy (CD45 + CD11b + F4 / 80 + ), representative graph of flow cytometry. (C) Schematic diagram of TAM cells co-cultured with KPC for 48 h using a non-contact Transwell system (0.4 mm pore size). (D) Representative graph of KPC cell growth in the lower chamber after TAM cells were co-cultured with KPC for 48 h, scale bar = 100 μm. (E) Statistical graph of the number of KPC cells in the lower chamber after TAM cells were co-cultured with KPC for 48 h. (F) Schematic diagram of KPC co-cultured with KPC (control experiment) for 48 h using a non-contact Transwell system (0.4 μm pore size). (G) Representative graph of KPC cell growth in the lower chamber after KPC co-cultured with KPC for 48 h, scale bar = 100 μm. (H) Statistical graph of the number of KPC cells in the lower chamber after KPC co-cultured with KPC for 48 h. All data are expressed as mean ± SEM. and analyzed by Student's t test. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0026] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0027] The present invention is now further described in conjunction with specific examples. The following examples are only for explaining the present invention, but do not constitute a limitation of the present invention. The test samples and test processes used in the following examples include the following (if the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, if not otherwise specified, can all be obtained from commercial channels). Example 1

[0028] 1. Relationship between the expression levels of different neurotrophic factors and the survival time of tumor patients

[0029] In order to explore the potential effects of different neurotrophic factors on pancreatic cancer survival, this example first used the GEPIA database to analyze whether there is a correlation between the expression of four neurotrophic factors, including VGF, NGF, BDNF and GDNF in pancreatic cancer tumor samples and the overall survival time of human pancreatic cancer patients ( Figure 1 A-1D), the results showed that the overall survival time of patients with high expression of VGF mRNA in tumor samples was significantly longer than that of patients with low expression of VGF mRNA ( Figure 1 A), while the mRNA expression of NGF, BDNF and GDNF had no significant correlation with the overall survival time of human pancreatic cancer patients ( Figure 1 B-1D). Previous studies have reported that VGF plays a role in promoting the occurrence and development of tumors in the progression of lung cancer (Hwang W, Chiu YF, Kuo MH, et al. Expression of Neuroendocrine Factor VGF in Lung Cancer Cells Confers Resistance to ECFR Kinase Inhibitors and Triggers Epithelial-to Mesenchymal Transition [J]. Cancer Res, 2017, 77 (11): 3013-26) and glioma (Wang X, Prager BC, Wu O, et al. Reciprocal Signaling between Glioblastoma stem Cells and Differentiated Tumor Cells Promotes Malignant Progression [J]. Cell Stem Cell, 2018 22 (4): 514-28e5). In order to verify the reliability of GEPIA data, the inventors also inquired whether there is a correlation between the expression level of neurotrophic factor VGF mRNA and the overall survival time of human lung adenocarcinoma and glioma patients ( Figure 1 E-1F). The results showed that high expression of VGF mRNA in tumor samples significantly reduced the expression of VGF in human lung adenocarcinoma ( Figure 1 E) and glioma ( Figure 1 F) Overall survival time of patients. It is worth noting that there is no significant difference in the expression of VGF mRNA in tumor tissues and adjacent normal tissues of human pancreatic cancer patients ( Figure 1G), this example further uses the human proteomics database ProteowicsDB to analyze the distribution of VGF protein in human tissues. The results show that under normal physiological conditions, the expression level of VGF protein is highest in pancreatic islets ( Figure 1 H). This suggests that under normal physiological conditions, VGF may play an important physiological role in the pancreas.

[0030] In general, the research data of this example show that in human lung adenocarcinoma tumor samples and glioma samples, high expression of VGF significantly reduced the overall survival time of patients, while in human pancreatic cancer patient tumor samples, high expression levels of VGF significantly increased the survival time of patients.

[0031] TLQP-21 inhibits tumor progression in pancreatic cancer model mice

[0032] In order to study the effect of TLQP-21 on tumor growth, this example established an orthotopic pancreatic cancer mouse model. Starting from the second day after tumor inoculation, mice were continuously intraperitoneally injected with TLQP-21 (the control group was injected with normal saline). On the 3rd, 7th and 14th days after tumor inoculation, the mice were subjected to in vivo imaging (IVIS) to observe the growth of the tumor in vivo. On the 15th day, the mice were sacrificed to measure the tumor volume ( Figure 2 A). The results showed that compared with the control solvent injection group, the administration of TLQP-21 to pancreatic cancer model mice significantly reduced the tumor volume ( Figure 2 B). The results of IVIS showed that compared with the control group, the tumor size of the mice in the TLQP-21 intervention group did not change significantly on the 3rd and 7th days after tumor inoculation, but on the 14th day after tumor inoculation, the average fluorescence quantum number of the tumor of the mice in the TLQP-21 intervention group ( Figure 2 D) and the total fluorescence quantum number ( Figure 2 E) significantly decreased.

[0033] Overall, the above data results suggest that VGF-derived peptide TLQP-21 can inhibit the growth of pancreatic cancer.

[0034] TLQP-21 can alleviate depressive-like behavior and pain in pancreatic cancer model mice

[0035] In order to explore the effect of VGF-derived peptide TLQP-21 on the emotional behavior and pain of pancreatic cancer model mice, this example used an orthotopic pancreatic cancer mouse model and divided the animals into 3 groups:

[0036] (1) Animals in the sham group received sham surgery but no tumor was implanted, and received intraperitoneal injections (IP) of normal saline once a day starting from the second day after surgery;

[0037] (2) Animals in the tumor control (KPC+Saline) group received an injection of normal saline once a day starting from the second day after tumor implantation;

[0038] (3) The animals in the experimental (KPC+TLQP-21) group received an injection of TLQP-21 (5ug / g / d) once a day starting from the second day after tumor implantation. The sequence of TLQP-21 is:

[0039] Starting from the 14th day after surgery, the mice were subjected to a series of behavioral tests to evaluate their depression-like and pain behaviors. The mice were perfused and sampled 72 hours after the completion of the behavioral tests ( Figure 3 A). The results showed that the body weight of the three groups of mice increased steadily after tumor implantation, and there was no significant difference between the groups ( Figure 3 B). Compared with the KPC+Saline group, administration of TLQP-21 to pancreatic cancer model mice significantly reduced tumor volume ( Figure 3 C) In the OF test, there was no significant difference in the total movement distance of the three groups of mice, indicating that the mice's movement ability was not affected. Figure 3 D and Figure 3 E). In the VFT test, the mechanical threshold of the animals in the KPC-Saline group was significantly decreased compared with the Sham group, indicating that the pancreatic cancer model mice had pain sensitization; and the administration of TLQP-21 to pancreatic cancer model mice significantly increased the mechanical pain threshold of the mice and improved the pain sensitization of pancreatic pain model mice ( Figure 3 F). In the tail suspension test (TST), the immobility time of animals in the KPC+Saline group was significantly increased compared with that in the Sham group, while administration of TLQP-21 significantly reduced the immobility time of pancreatic cancer model mice ( Figure 3 G). In the social interaction test (SIT), the exploration time of the KPC-Saline group animals towards the unfamiliar mice was significantly reduced compared with the Sham group, while the administration of TLQP-21 significantly increased the exploration time of the pancreatic pain model mice towards the unfamiliar mice ( Figure 3 H and Figure 3 I). There was no significant difference in the total exploration time of the three groups of mice in the SIT test, indicating that the movement and exploration abilities of the mice were not changed ( Figure 3 H and Figure 3 I).

[0040] Overall, the above experimental results show that pancreatic cancer model mice exhibit depressive-like behaviors such as despair and social disorders, and are sensitive to pain, and intraperitoneal injection of TLQP-21 can significantly alleviate the depressive-like behaviors and pain of pancreatic cancer model mice.

[0041] 4. The effect of TLQP-21 on the expression level of c-FOS, a functional activity marker in the brain area of ​​pancreatic cancer model mice.

[0042] In order to further explore the neural mechanism of VGF-derived peptide TLQP-21 in improving pancreatic cancer-depression comorbidity, in this example, 72 hours after the end of behavioral studies, the three groups of mice were perfused and samples were collected. The mouse brain tissue was frozen and sectioned, and c-FOS immunofluorescence staining analysis was performed on 15 brain regions related to emotion and pain to screen for brain regions with changed activity ( Figure 4 A). c-FOS is a protein encoded by an immediate-early gene and is often used as an indicator of neuronal activity. Increased expression of c-FOS represents increased activity in the brain region (Guzowski JF, Timlin J A. Roysam B, et al. Mapping behaviorally relevant neural circuits with immediate-early gene expression [J]. Curr Opin Neurobiol, 2005, 15 (5): 599-606). The results showed that among the 15 brain regions included in the analysis, compared with the mice without cancer (Sham), the density of c-FOS-positive cells in the PL and LS brain regions of the mice in the group with KPC cells implanted in the pancreas and injected with saline (KPC+Saline) increased significantly (). Compared with the sham group, the density of c-FOS-positive cells in the PL, LS, PVN, LH and dHC brain regions of the pancreatic cancer model mice injected with TLQP-21 (KPC-TLQP21) increased significantly ( Figure 4 B). It is worth noting that compared with the Sham group, the density of c-FOS-positive cells in the insular cortex (IC) of mice in the KPC+Saline group increased significantly, and this regulation was significantly alleviated by TLQP-21 intervention ( Figure 4 B and Figure 4 C). According to literature reports, IC has the function of regulating emotions, pain, motivation and cognition, and is also the main cortex for receiving and processing visceral sensory information (Gogolla N. The insular cortex D. Curr Biol, 2017, 27 (12): R580-R6). Further correlation analysis showed that the change in pain threshold in VFT of pancreatic cancer model mice was significantly negatively correlated with the density of c-FOS positive cells in the IC brain region, indicating that the higher the pain sensitivity of pancreatic cancer model mice, the stronger the activity of the IC brain region ( Figure 4 D).

[0043] Overall, the above results showed that the IC brain region was activated in pancreatic cancer model mice, and the activation of the IC brain region was alleviated after administering TLQP-21 to pancreatic cancer model mice. In addition, the pain level of the mice was positively correlated with the activity of the IC brain region, suggesting that pain information mediated by the IC brain region may be one of the neural mechanisms by which TLQP21 improves pancreatic cancer-depression comorbidity.

[0044] 5. The effect of TLQP-21 on the expression level of inflammatory marker GFAP in astrocytes in the brain region of pancreatic cancer model mice.

[0045] In order to further explore the neural mechanism of VGF-derived peptide TLQP-21 in improving pancreatic cancer-depression comorbidity, this example performed GFAP immunofluorescence staining analysis on 15 brain regions related to emotion and pain ( Figure 5 A). GFAP is a biomarker of inflammatory astrocytes, and its increased expression often indicates astrocyte-mediated neuroinflammation (Sofironiew M V. Molecular dissection of reactive astrogliosis and glial scarformation [J] Trends Neurosci. 2009. 32 (12): 638-47). The results showed that among the 15 brain regions included in the analysis, only the GFAP (including fluorescence intensity and percentage of fluorescence coverage area) in the area postrema (AP) and DMV of the brainstem increased significantly in pancreatic cancer model mice (KPC + Saline group) compared with the Sham group; administration of TLQP-21 to pancreatic cancer model mice significantly inhibited the expression of GFAP in the AP and DMV regions ( Figure 5 B-5D). Compared with the sham group, the expression level of GFAP in the nucleus of the solitary tract (NTS) of pancreatic cancer model mice did not change significantly, but the administration of TLQP-21 to pancreatic cancer model mice could also significantly reduce the expression of GFAP in this brain region ( Figure 5 B-5D).

[0046] Overall, the above results indicate that astrocytes in the DVC (DMV, AP and NTS are collectively referred to as the DVC brain region) of pancreatic cancer model mice showed an inflammatory response, and that administration of TLQP-21 to pancreatic cancer model mice could alleviate the inflammatory response of DVC astrocytes, suggesting that the inflammatory activation of astrocytes in the DVC brain region may also be one of the neural mechanisms by which TLQP-21 improves pancreatic cancer-depression comorbidity.

[0047] 6. Direct effects of TLQP-21 on pancreatic tumor cells

[0048] In order to explore the direct effect of TLQP-21 on pancreatic cancer cells, this example used different concentrations of TLQP-21 to treat KPC cells, and the effect of TLQP-21 treatment on the proliferation ability of pancreatic ulcer cells was detected by CCK8 experiment; the effect of TLQP-21 treatment on the migration ability of pancreatic cancer cells was detected by Transwell experiment ( Figure 6 A). The results of CCK8 cell proliferation assay showed that 0.1-1uM TLQP-21 treatment did not affect the proliferation ability of KPC ( Figure 6 B). To further verify the above results, this example also detected the effect of TLQP-21 on the proliferation of another pancreatic cancer cell line Pan02. Figure 6 As shown in C, 0.1-1 μM TLQP-21 treatment did not affect the proliferation ability of Pan02. The results of Transwell migration assay showed that KPC ( Figure 6 D and 6E) or Pan02( Figure 6 F and 6G) ​​The number of cells passing through the transwell was significantly reduced compared with the control group.

[0049] Overall, the above experimental results show that TLQP-21 does not directly inhibit the growth of cells, but can inhibit the migration of pancreatic cancer cells.

[0050] 7. TLQP-21 inhibits pancreatic cancer cell migration by relying on its receptor C1QBP

[0051] TLQP21 mainly has two receptors, C1QBP and C3AR1 (Sahu BS, Nguyen ME, Rodriguez P, et al. The molecular identity of the TLQP-21 peptide receptor [J] Cell Mol Life Sci. 2021, 78 (23): 7133-44). In this example, the GEPIA database was first used to analyze the C1QBP ( Figure 7 A) and C3AR1( Figure 7 B) Changes in mRNA expression. We found that the mRNA expression levels of these two receptors in human pancreatic tumor tissues were significantly higher than those in normal tissues; and the expression level of C1QBP was about twice that of C3AR1. We performed a correlation analysis on the expression levels of VGF and the above two receptors and found that the expression levels of C1QBP and VGF were significantly positively correlated ( Figure 7C), while there was no significant correlation between the expression levels of C3AR1 and VGF (not shown in the figure). Further, this example analyzes the expression of the above two receptors in mouse pancreatic cancer cells and animal models. The inventors used a single-cell transcriptome data of mouse pancreatic precancerous lesions and pancreatic cancer progression published by Schlesiger et al. in 2020 (Schlesinger Y, Yosefov-Levi O, Kolodkin-Gal D, et al. Single-cell transcriptomes pancreatic preinvasive lesions and cancer reveal acinarmetaplastic cells'heterogeneity D Nat Commun, 2020, 11 (1): 4516) to analyze the cell types expressing C1qbp and C3ar1 mRNA in the pancreas of pancreatic cancer model mice ( Figure 7 E-7G). UMAP clustering diagram showed that there were ductal cells, metaplastic cells, acinar cells, endocrine cells, endothelial cells, immune cells, pericytes and fibroblasts in mouse pancreatic cancer tissues. C3ar1 mRNA was mainly expressed in macrophages, while C1qbp mRNA was expressed in a variety of cell types including metaplastic cells, suggesting that the target of TLQP-21 on pancreatic cancer cells might be C1QBP ( Figure 7 E-7G). To verify the above hypothesis, we first used qPCR to detect whether C1qbp mRNA was expressed in KPC and Pan02 cells, and BV2 microglia were used as positive control. The results showed that C1qbp mRNA was expressed in both KPC and Pan02 cells ( Figure 7 D). Then, this example uses a monoclonal neutralizing antibody of the C1QBP receptor to block the C1QBP signal to evaluate the role of C1QBP in TLQP-21 inhibiting pancreatic cancer cell migration. The results of the Transwell migration assay showed that TLQP21 significantly reduced the migration ability of KPC and Pan02 cells, while blocking C1QBP restored the number of cells passing through the Transwell to the level of the control group.

[0052] Overall, the above data indicate that TLQP-21 inhibits cancer cell migration by acting on the C1QBP receptor on the membrane of pancreatic cancer cells ( Figure 7 H-7K).

[0053] 8. Effect of TLQP-21 on gene expression profile of pancreatic tumors in mice

[0054] In order to explore the molecular mechanism by which TLQP-21 slows down the progression of pancreatic cancer, this example performed whole transcriptome sequencing (RNA sequencing, RNA-Seq) analysis on pancreatic cancer tumors in the TLQP-21 intervention group and the control group. This example established an orthotopic pancreatic cancer model mouse. Starting from the second day after tumor inoculation, the mice were continuously intraperitoneally injected with TLQP-21 (5ug / g / d), and the control group was injected with normal saline. Samples were collected on the 15th day after tumor inoculation, and pancreatic tumor RNA was extracted. After RNA quality inspection (RNA integrity number, RIN value>7), 3-4 independent samples in each group (1-2 animals mixed into one independent sample) were library constructed and RNA-Seq was performed, and the results of RNA-Seq were subjected to differential gene expression analysis and gene enrichment analysis (Gene Set Enrichment Analysis, GSEA) ( Figure 8 A). In this example, the RNA-Seq results were analyzed using the DESeq2 package of the R language. The screening condition was p value < 0.01, and 194 differentially expressed genes (DEGs) were obtained, of which 47 genes were up-regulated and 147 were down-regulated. The inventors used the pheatmap package, ggplot2, and ggpubr packages of the R language to visualize the DEG analysis results as a cluster heat map ( Figure 8 B) and volcano plot ( Figure 8C). Notably, the significantly downregulated DEGs included: pro-inflammatory cytokine-related genes, such as A2m, Ccl2, Plek, Tsc22d3, Zc3h12a, and Cxcl1; genes promoting M2 macrophage polarization, such as Per1, Fgl2, Adgre1, Socs3, Cc120, Rel, Herpud1, and Ccl2; angiogenesis-related genes, such as Ccl2, E2f2, Icam1, Tlr2, and Cxcl1; and genes related to immune infiltration, such as Fgl2, Cdl4, Nlrp3, Syk, and Cxcl1. The inventors used the single-cell transcriptome data of pancreatic preinvasive lesions and pancreatic cancer progression in mice published by Schlesinger et al. in 2020 (Schlesinger Y, Yosefov-Levi O, Kolodkin-Gal D, et al. Single-cell transcriptomes pancreatic preinvasive lesions and cancer reveal acinar metaplastic cells'heterogeneity[J]Nat Commun, 2020, 11(1):4516) to analyze the cell types in which DEGs were enriched in the pancreas of pancreatic cancer model mice ( Figure 8 D). The results showed that the screened DEGs were expressed in various cell types, including ductal cells, metaplastic cells, endocrine cells, endothelial cells, immune cells, pericytes, and fibroblasts, but were most highly enriched in macrophages ( Figure 8 D and 8E).

[0055] In this example, qPCR was further used to verify the expression of some DEGs, and it was found that compared with the control group, the mRNA expression levels of Gpr141, Per1, Birc3, Slamf7, Icam1, Adr2, Ce120 and 42m in the tumors of the TLQP-21 intervention group were significantly decreased ( Figure 8 F).

[0056] In this example, GSEA was used to perform gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on the gene expression profiles of the TLQP-21 intervention group and the control group. The GO database standardized the description of gene products from the perspectives of molecular function (Gene Ontology Molecular Function, GO-MF), biological pathways (Gene Ontology Biological Process, GO-BP) and location in cells (Gene Ontology Cellular Component, GO-CC) (Reimand], Isserlin R, Voisin V, et al. Pathway enrichment analysis and visualization of omics data using g: Profiler, GSEA, Cytoscape and Enrichment Map. Nat Protoc, 2019 14 (2): 482-517.). KEGG is a database that systematically analyzes the signaling pathways of gene products in cells (Reimand J, Isserlin R, Voisin V, et al. Pathway enrichment analysis and visualization of omics data using g: Profiler, GSEA, Cytoscape and Enrichment Map [J]. Nat Protoc, 2019 14 (2) 482-517.). The results showed that after pancreatic cancer model mice were given TLQP-21, the items enriched in the genes expressed by pancreatic tumors were mainly related to gene expression regulation processes such as ribosome structure composition, RNA polymerase and RNA methyltransferase activation, and RNA metabolic processes; while the items with down-regulated enrichment in pancreatic cancer model mice were mainly related to immune processes such as neutrophil migration, myeloid leukocyte activation, cell response to biological stimuli, interactions between cytokines and their receptors, hematopoietic cell lineage, and cell adhesion.

[0057] 9. TLQP-21 indirectly inhibits the growth of pancreatic cancer cells by acting on TAM

[0058] In order to further explore the molecular mechanism of TLQP-21 in slowing down the progression of pancreatic cancer, this example used fluorescence-activated cell sorting (FACS) to sort TAM cells (CD45 + CD11b + F4 / 80 + )( Fig. 9 A and 9B), the sorted TAM cells were co-cultured with KPC cells. In this example, TAM cells were co-cultured with pancreatic cancer cells KPC using a non-contact Transwell system for 48 hours, 1uM TLQP-21 or control solution was added to the upper and lower chambers, and the number of KPC cells after co-culture was detected by a cell counter ( Fig. 9 C). The results showed that compared with the control solution treatment group, the number of KPC cells decreased significantly after TLQP-21 treatment of the TAM-KPC co-culture system for 48 hours ( Fig. 9 D and 9E), suggesting that TLQP-21 can inhibit the growth of KPC cells by affecting the factors secreted by TAM cells. This example also used the Transwell system to observe the direct effect of TLQP-21 on pancreatic cancer cells. KPC cells were cultured in both the upper and lower chambers, and 1uM TLQP-21 or control solution was added. After 48h of culture, the number of KPC cells in the lower chamber was detected ( Fig. 9 F). The results showed that there was no significant difference in the number of KPC cells after TLQP-21 treatment of the KPC-KPC co-culture system for 48 hours compared with the control solution treatment group ( Fig. 9 G and 9H). This result is consistent with Figure 6 The results of the CCK8 experiment were consistent with those of the control group, indicating that TLQP-21 did not directly inhibit the growth of KPC cells.

[0059] Overall, the above data results indicate that TLQP-21 indirectly inhibits the growth of pancreatic cancer cells by acting on TAM.

[0060] Overall, through experimental data analysis, we found that TLQP-21 inhibits pancreatic cancer tumor growth and can also improve pain symptoms and depression-like symptoms in pancreatic cancer model mice. It also revealed that TLQP-21 inhibits cancer cell migration by acting on the C1QBP receptor on the pancreatic cancer cell membrane, and inhibits the growth of pancreatic tumor cells by acting on TAM cells. And TLQP-21 improves pancreatic cancer-depression comorbidity by relieving the activation of the IC brain region and relieving the inflammatory response of astrocytes in the DVC brain region. At the same time, it was disclosed that TLQP-21 can downregulate some differentially expressed genes. This provides a new target for the development of pancreatic cancer and pancreatic cancer-depression comorbidity drugs, and also provides a new treatment strategy for it. It is expected to be used clinically, provide effective treatment methods, improve the quality of life of pancreatic cancer patients and prolong the survival time of patients, and also provide a new research direction for the treatment of pancreatic cancer and pancreatic cancer-depression comorbidity.

[0061] The sequence of TLQP-21 used in the above experiments is: TLQPPASSRRRHFHHALPPAR (SEQ ID NO. 1).

[0062] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Use of TLQP-21 in the preparation of drugs for treating pancreatic cancer or pancreatic cancer-depression comorbidity.

2. The use according to claim 1, characterized in that: The amino acid sequence of TLQP-21 is: TLQPPASSRRRRHFHHALPPAR (SEQ ID NO. 1).

3. The use according to claim 1, characterized in that: When TLQP-21 is used in the preparation of a drug for treating pancreatic cancer-depression comorbidity, TLQP-21 alleviates the activation of the insular cortex; when TLQP-21 is used in the preparation of a drug for treating pancreatic cancer-depression comorbidity, TLQP-21 alleviates astrocyte-mediated neuroinflammatory responses in brain regions including the dorsal nucleus of the vagus nerve, the area postrema, and the nucleus tractus solitarius.

4. The use according to claim 1, characterized in that: When TLQP-21 is used in the preparation of a drug for treating pancreatic cancer, TLQP-21 downregulates differentially expressed genes in pancreatic tumors, including pro-inflammatory cytokine-related genes, genes related to promoting M2 macrophage polarization, angiogenesis-related genes, and expressions of genes related to immune infiltration.

5. The use according to claim 4, characterized in that: The pro-inflammatory cytokine-related genes include A2m, Ccl2, Plek, Tsc22d3, Zc3h12a and Cxcl1; the related genes promoting M2 macrophage polarization include Per1, Fgl2, Adgre1, Socs3, Cc120, Rel, Herpud1 and Ccl2; the angiogenesis-related genes, such as Ccl2, E2f2, Icam1, Tlr2 and Cxcl1; the immune infiltration-related genes include Fgl2, Cdl4, Nlrp3, Syk and Cxcl1.

6. Application of TLQP-21 in the preparation of drugs for inhibiting the migration and growth of pancreatic cancer cells.

7. The use according to claim 6, characterized in that: TLQP-21 inhibits pancreatic cancer cell migration by acting on C1QBP receptor or inhibits pancreatic cancer cell growth by acting on TAM cells.

8. A pharmaceutical composition for treating pancreatic cancer or pancreatic cancer-related depression, characterized in that: Including TLQP-21.

9. The pharmaceutical composition according to claim 8, characterized in that The dosage form of the drug is injection.

10. Use of TLQP-21 in the preparation of angiogenesis-inhibiting drugs, wherein the angiogenesis-inhibiting drugs are drugs for inhibiting angiogenesis in pancreatic cancer lesions.

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