Target CHRNA5 of pancreatic cancer and application of target CHRNA5 in diagnosis and treatment

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a target CHRNA5 of pancreatic cancer and application of the target CHRNA5 in diagnosis and treatment. In-depth experiments prove that CHRNA5 is related to dryness, metastasis and invasion of adenocarcinoma and influences the tumor microenvironment of pancreatic cancer; cHRNA5 can be used as a new therapeutic target of pancreatic cancer, and prognosis conditions of pancreatic cancer can be prompted based on expression of the gene CHRNA5. The pancreatic cancer nerve invasion, especially the Ach / CHNA5 axis, is researched, and the mechanism of the Ach / CHNA5 axis is clarified, namely the action of the Ach / CHNA5 axis in tumorigenesis and microenvironment, so that a new treatment thought is provided for pancreatic cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the pancreatic cancer target CHRNA5 and its applications in diagnosis and treatment. Background Art

[0002] Pancreatic ductal adenocarcinoma (pancreatic cancer) is one of the most destructive and lethal malignant tumors. Despite the progress in treatment methods, the survival rate of pancreatic cancer remains very low, and only about 10% of patients survive more than five years after diagnosis. The incidence of pancreatic cancer continues to rise by about 0.5%-1.0% annually, driven by risk factors such as smoking, diabetes, and chronic pancreatitis, making it a major cause of global cancer-related mortality. The clinical characteristics of pancreatic cancer are determined by its tumor microenvironment, hypoxia, immune evasion, and high levels of regulating the stem cell characteristics of tumor cells. Therefore, strengthening the understanding of the microenvironment and targeting the stemness of tumor cells is beneficial to the development of new treatment strategies for pancreatic cancer.

[0003] Perineural invasion (PNI) is a common clinicopathological feature of pancreatic cancer, defined as the presence of tumor cells near the nerve, invading at least 33% of the nerve, or infiltrating within any one of the three layers of the nerve sheath. PNI is closely related to the invasiveness of pancreatic cancer, often accompanied by severe pain, tumor metastasis, and poor prognosis. More and more studies have shown that cancer cells express neurotrophic markers, such as brain-derived neurotrophic factor and glial cell-derived neurotrophic factor, which can promote axon growth. The dynamic interaction between tumors and nerves has become a key factor in the tumor microenvironment. Understanding the potential mechanism of PNI in tumorigenesis can identify promising therapeutic targets, paving the way for more effective treatment strategies and improving the patient's condition.

[0004] In pancreatic cancer, cholinergic and sympathetic signals both play key roles in PNI, which can regulate tumor progression through different mechanisms. Recent studies have clarified that compared with norepinephrine (NE), pancreatic cancer patients with severe PNI exhibit higher acetylcholine (Ach) levels, indicating that Ach plays a central role in pancreatic cancer, promoting the progression of PNI. Ach has been shown to promote cancer cell proliferation, migration, and differentiation, as well as epithelial-mesenchymal transition (EMT), enhancing the communication between tumor cells and nerves. In addition to the direct effect on tumors, Ach also promotes immune escape and mediates inflammation. However, the role and mechanism of the key downstream muscarinic or nicotinic receptors in the tumorigenesis process of pancreatic cancer, and their regulation of immune escape are still unclear. Summary of the Invention

[0005] The present invention has demonstrated the malignant phenotype of Ach in vitro and studied its effect on pancreatic cancer. It has also verified the high expression of the Ach receptor CHRNA5 in pancreatic cancer cells and in mouse experiments. The present invention has found that downregulating CHRNA5 can significantly inhibit the malignant stemness, proliferation, colony formation, and tumor migration of pancreatic cancer, and can also affect the tumor immune escape microenvironment of pancreatic cancer.

[0006] The first aspect of the present invention discloses the use of CHRNA5 as a therapeutic target in the preparation or screening of drugs for treating pancreatic cancer, and the drugs are used to inhibit the stemness, invasion, and / or migration of pancreatic cancer.

[0007] The second aspect of the present invention discloses the use of a preparation for inhibiting CHRNA5 expression in the preparation of drugs for treating pancreatic cancer, and the drugs are used to inhibit the stemness, invasion, and / or migration of pancreatic cancer.

[0008] The third aspect of the present invention discloses the use of a preparation for inhibiting CHRNA5 expression as a targeted drug for regulating the tumor immune escape microenvironment of pancreatic cancer, and the drugs are used to inhibit the immune escape of pancreatic cancer.

[0009] The fourth aspect of the present invention discloses the use of a preparation for inhibiting CHRNA5 expression in the preparation of drugs for inhibiting the activity of myeloid-derived suppressor cells.

[0010] Preferably, the preparation includes nucleic acid inhibitors and protein inhibitors.

[0011] Preferably, the drug further includes pharmaceutically acceptable excipients; the excipients include one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, or lubricants.

[0012] The fifth aspect of the present invention discloses the use of a reagent for detecting the expression level of CHRNA5 or a product including the reagent in the preparation of a diagnostic agent for pancreatic ductal adenocarcinoma.

[0013] Wherein, the product includes a chip, a kit, or a nucleic acid membrane strip.

[0014] Wherein, the reagent is selected from a probe that specifically recognizes the CHRNA5 gene, a primer that specifically amplifies the CHRNA5 gene, or a protein that specifically binds to the CHRNA5 gene; the drug is used to inhibit the proliferation, invasion, and / or migration of pancreatic cancer.

[0015] The sixth aspect of the present invention discloses the use of a preparation for inhibiting CHRNA5 expression and a preparation for inhibiting Ach expression in the combined preparation of drugs for treating pancreatic cancer.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] Through in-depth experiments, the present invention proves that CHRNA5 is related to adenocarcinoma stemness, metastasis, and invasion, and affects the tumor immune escape microenvironment of pancreatic cancer. Specifically, it regulates CCL20 through the CAMK2 / AKT pathway, thereby affecting the activity of MDSC, the core immune cells of pancreatic cancer; CHRNA5 can be used as a new therapeutic target for regulating tumor stemness in pancreatic cancer, and CHRNA5 can become a new therapeutic target for immune escape in pancreatic cancer. Based on the expression of the gene CHRNA5, the prognosis of pancreatic cancer can be predicted.

[0018] The present invention's protocol studies pancreatic cancer neural invasion, especially the Ach / CHNA5 axis, and clarifies its mechanism: the role of the Ach / CHRNA5 axis in tumorigenesis and the immune escape microenvironment, providing new treatment ideas for the treatment of pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Showing that acetylcholine and cholinergic signaling are overactivated in pancreatic ductal adenocarcinoma (PDAC).

[0020] Figure 2 Showing that acetylcholine promotes the malignant phenotype of pancreatic ductal adenocarcinoma (PDAC) in vitro.

[0021] Figure 3 Showing that CHRNA5 exacerbates tumor progression in vitro and in vivo.

[0022] Figure 4 Showing that CHRNA5 remodels the tumor immune escape microenvironment. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0024] Example 1: Ach and cholinergic signaling are overactivated in pancreatic cancer

[0025] Given that PNI is a common feature of pancreatic cancer, this study re-analyzed the RNA sequencing data of pancreatic cancer from pancreatic cancer patients and normal human tissues in the TCGA combined with GTEx datasets. KEGG analysis showed that the neuroactive ligand-receptor interaction pathway was enriched in pancreatic cancer (Figure 1A). Similarly, GO analysis demonstrated the enrichment of neuro-related enriched pathways during the tumorigenesis of pancreatic cancer ( Figure 1B). GSEA analysis also confirmed the enrichment of nerve-related pathways in pancreatic cancer patients, indicating a strong link between PNI and pancreatic cancer. Figure 1 C). It has been reported that the concentration of Ach increases in the tumor tissues of pancreatic cancer patients with severe PNI. Consistent with the previous results, in the reanalysis of the present invention, it was found that the expressions of CHAT and SLC18A3, which can produce Ach, were significantly upregulated in the tissues of pancreatic cancer patients, which may explain the increase in Ach levels. Figure 1 D). In addition, CHAT and SLC18A3 have high accuracy in the diagnosis and prediction of pancreatic cancer, with AUC values of 0.682 and 0.753 respectively. Figure 1 E). In addition, the protein levels of CHAT and VACHT both increased in the mouse orthotopic tumor model of the present invention.

[0026] Example 2. Ach promotes the malignant phenotypes of pancreatic cancer in vitro

[0027] To study the role of Ach in vitro, the present invention explored its effects on PANC-01 and PANC-02 cell lines at different concentrations. In PANC-01 cells, CCK8 and Transwell assays showed that 100 μM Ach significantly promoted cell proliferation, invasion, and migration. Figure 2 A - B). Similarly, Ach enhanced these malignant phenotypes of PANC-02 cells in a dose-dependent manner. Figure 2 C - D). Based on these findings, 100 μM Ach was selected for subsequent experiments. In both cell lines, Ach treatment significantly increased colony formation. Figure 2 E - F). In addition, exogenous Ach promoted the stemness of tumor cells, such as increasing the formation of tumor cell stem spheres and upregulating cancer stem cell (CSC) marker genes. Figure 2 G - J). These results indicate that Ach contributes to the proliferation, invasion, migration, and stemness phenotypes of pancreatic cancer cells.

[0028] Example 3. CHRNA5 exacerbates tumor progression in vitro and in vivo

[0029] Since the role of CHRNA5 in pancreatic cancer has not been explored to a great extent, the present invention designed siRNA to knock out CHRNA5 in PANC-02 cells to study its role. qRT-PCR and Western blot analysis confirmed the knockout efficiency of CHRNA5. Figure 3 A - B). Among these siRNAs, Si-1 achieved the highest inhibitory efficiency, leading to the selection of its sequence for the synthesis of Sh-CHRNA5 plasmid. Transfection with Sh-CHRNA5 significantly reduced the proliferation of PANC-02 cells. Figure 3C). Transwell assays further showed a significant decrease in invasion and migration after CHRNA5 silencing ( Figure 3 D). In addition, colony formation was significantly inhibited in cells treated with Sh-CHRNA5 ( Figure 3 E). More importantly, inhibition of CHRNA5 reduced sphere formation and downregulated CSC marker genes, indicating a decrease in tumor stemness ( Figure 3 F-G). To investigate the role of CHRNA5 in vivo, PANC-02 cells were infected with lentiviruses carrying Sh-NC or Sh-CHRNA5 to establish stable knockout cell lines. An orthotopic tumor model was established in C57BL / 6J mice. Compared with the Sh-NC group, tumors derived from Sh-CHRNA5 cells showed a significant reduction in tumor-forming ability ( Figure 3 H). The knockout efficiency was verified by qRT-PCR and Western blotting ( Figure 3 I-J). In addition, tumors in mice in the Sh-CHRNA5 group were significantly smaller, as reflected by reduced tumor volume and weight ( Figure 3 K-L). Immunohistochemical staining confirmed a decrease in Ki-67 expression, indicating a reduction in tumor stemness in the Sh-CHRNA5 group ( Figure 3 M).

[0030] Example 4. CHRNA5 remodels the tumor immune escape microenvironment

[0031] Given the crucial role of the tumor immune escape microenvironment, the present invention then attempted to investigate whether CHRNA5 could regulate the tumor-associated immune escape microenvironment. To explore this, orthotopic pancreatic cancer models were constructed in nude mice and C57BL / 6 mice. In vivo experiments demonstrated that knockout of CHRNA5 significantly reduced tumor formation and ki-67 levels in nude mice, and this inhibitory effect was more obvious in C57BL / 6 mice ( Figure 4 A-E), indicating that CHRNA5 plays an important role in the immune microenvironment. GO analysis revealed the enrichment of immune-related pathways, such as leukocyte migration, further supporting the involvement of CHRNA5 in immune regulation ( Figure 4 F). Heatmap analysis of leukocyte migration-related genes showed that CHRNA5 might affect chemokines such as CCL20, CXCL9, and CTSG, which might mediate the chemotaxis and infiltration of myeloid cells ( Figure 4 G). qRT-PCR in PANC-02 and mice verified the downregulation of CCL20 after CHRNA5 inhibition ( Figure 4H, I). CCL20 is a key mediator of immune cell recruitment, particularly myeloid-derived suppressor cells (MDSCs) and CD4+ T cells. Flow cytometry confirmed that CHRNA5 knockout led to a significant reduction in MDSCs, especially polymorphonuclear MDSCs (PMN-MDSCs), and an increase in the proportion of CD8+ T cells ( Figure 4 J-L). These results highlight the possible role of CHRNA5 in regulating MDSC recruitment by modulating CCL20, playing an important role in the tumor immune escape microenvironment of pancreatic cancer.

[0032] As described above, only the preferred specific embodiments of the present invention are provided, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, shall be covered by the protection scope of the present invention.

Claims

1. Use of CHRNA5 as a therapeutic target in the preparation or screening of drugs for treating pancreatic cancer, wherein the drugs are used to inhibit the stemness, invasion and / or migration of pancreatic cancer.

2. Use of a preparation for inhibiting CHRNA5 expression in the preparation of a drug for treating pancreatic cancer, wherein the drug is used to inhibit the stemness, invasion and / or migration of pancreatic cancer.

3. Use of a preparation that inhibits CHRNA5 expression as a targeted drug for regulating the pancreatic cancer immune escape microenvironment, wherein the drug is used to inhibit the immune escape of pancreatic cancer.

4. Use of preparations that inhibit CHRNA5 expression in the preparation of drugs that inhibit the activity of myeloid-derived suppressor cells.

5. The use according to any one of claims 2 to 4, characterized in that: The preparation includes nucleic acid inhibitors and protein inhibitors.

6. The use according to any one of claims 2 to 4, characterized in that: The medicine also includes pharmaceutically addable excipients; the excipients include one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers or lubricants.

7. Use of a reagent for detecting CHRNA5 expression level or a product comprising the reagent in the preparation of a method for diagnosing pancreatic ductal adenocarcinoma.

8. The use according to claim 7, characterized in that: The product includes a chip, a test kit or a nucleic acid membrane strip.

9. The use according to claim 7, characterized in that: The reagent is selected from a probe that specifically recognizes the CHRNA5 gene, a primer that specifically amplifies the CHRNA5 gene, or a protein that specifically binds to the CHRNA5 gene; and the drug is used to inhibit the proliferation, invasion and / or migration of pancreatic cancer.

10. Use of a preparation for inhibiting CHRNA5 expression and a preparation for inhibiting Ach expression in combination for preparing a drug for treating pancreatic cancer.

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