Application of fosl1 as a target in preparation of drugs for preventing or treating mucinous lung adenocarcinoma

Abstract

The application belongs to the field of biological medicine, and specifically discloses application of FOSL1 as a target in preparation of a mucinous lung adenocarcinoma drug, wherein FOSL1 is taken as a target, expression of FOSL1 is inhibited, and formation of mucinous lung adenocarcinoma cells is significantly reduced, and proliferation of the mucinous lung adenocarcinoma cells is effectively inhibited. The research result of the application makes FOSL1 expected to become a potential treatment target of mucinous lung adenocarcinoma, provides a new strategy for mucinous lung adenocarcinoma treatment, and has important application value in drug research and development and vaccine development.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to the application of FOSL1 as a target in the preparation of drugs for the prevention or treatment of mucinous lung adenocarcinoma. Background Technology

[0002] Lung adenocarcinoma (LUAD) is the most common histological subtype of lung adenocarcinoma, accounting for approximately 45.3% of all lung adenocarcinoma cases. It is one of the leading causes of cancer-related deaths. Based on pathological features, lung adenocarcinoma can be divided into mucinous lung adenocarcinoma and non-mucinous lung adenocarcinoma, which differ significantly in molecular characteristics, treatment response, and prognosis.

[0003] Currently, the main treatments for lung adenocarcinoma include surgical resection, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. However, the development of chemotherapy resistance is a major cause of treatment failure and poor patient prognosis. Studies have shown that the transcription factor FOSL1 (FOS-like antigen 1, AP-1 transcription factor subunit) is abnormally expressed in various tumors and participates in regulating the proliferation, invasion, metastasis, and drug resistance of tumor cells. As an important member of the AP-1 transcription factor family, FOSL1 is a major effector molecule of the RAS-ERK1 / 2 signaling pathway and plays a key role in the occurrence and development of lung adenocarcinoma.

[0004] Although the potential value of FOSL1 in cancer treatment has been recognized, its specific mechanism of action in lung adenocarcinoma, particularly in differentiating between mucinous and non-mucinous subtypes, and its systemic application as a therapeutic target have not been reported. Therefore, exploring the application of FOSL1 as a therapeutic target for lung adenocarcinoma has significant clinical implications. Summary of the Invention

[0005] To address the aforementioned problems, the primary objective of this invention is to provide a universal therapeutic strategy targeting FOSL1 and its signaling pathway for patients with mucinous lung adenocarcinoma, namely, the application of FOSL1 as a target in the preparation of drugs for the prevention or treatment of mucinous lung adenocarcinoma.

[0006] The specific technical solution of the present invention includes: This invention provides the application of FOSL1 as a target in the preparation of drugs for the prevention or treatment of mucinous lung adenocarcinoma.

[0007] As a further optimization of the present invention, the replication and proliferation of mucinous lung adenocarcinoma cells are inhibited by drug inhibition of FOSL1 expression.

[0008] As a further optimization of the present invention, the method of inhibiting FOSL1 by drugs is to use FOSL1 inhibitors for inhibition.

[0009] As a further optimization of the present invention, the FOSL1 inhibitor is irinotecan or SN-38.

[0010] As a further optimization of the present invention, the drug for preventing or treating mucinous lung adenocarcinoma refers to any one of the following (1)-(5): (1) Drugs used to prevent or treat infection with mucinous lung adenocarcinoma cells; (2) Drugs used for the prevention or treatment of mucinous lung adenocarcinoma; (3) Drugs used to inhibit the replication and proliferation of mucinous lung adenocarcinoma cells; (4) Drugs used to inhibit the lesioning effect of mucinous lung adenocarcinoma cells; (5) Inhibitor of mucinous lung adenocarcinoma cells.

[0011] The present invention also provides a drug for the prevention or treatment of mucinous lung adenocarcinoma, said drug being a FOSL1 inhibitor.

[0012] As a further optimization of the present invention, the PLK inhibitor is irinotecan or SN-38.

[0013] In summary, the beneficial effects of the present invention are as follows: This invention experimentally verified that FOSL1 is a key hub gene for the prognosis of lung adenocarcinoma, and its high expression is significantly associated with poor patient prognosis. FOSL1 regulates the sensitivity of lung adenocarcinoma cells to SN-38 and cisplatin. In non-mucinous lung adenocarcinoma, FOSL1 overexpression enhances chemosensitivity. In mucinous lung adenocarcinoma, FOSL1 knockdown alters chemosensitivity. FOSL1 exerts its anti-tumor effect by influencing cell cycle and apoptosis pathways. This provides experimental evidence for the development of FOSL1-targeted therapies for lung adenocarcinoma. This invention reveals the application value of FOSL1 as a therapeutic target for lung adenocarcinoma and provides a new strategy for the precision treatment of lung adenocarcinoma. Attached Figure Description

[0014] Figure 1 Volcano plot showing differentially expressed genes between mucinous and non-mucinous lung adenocarcinoma (circles represent expressed genes in the two groups, red indicates gene upregulation, and blue indicates gene downregulation); Figure 2 Kaplan-Meier survival curves for the FOSL1 gene and overall survival (OS); Figure 3 The expression differences of nine hub genes in the GDSC database for non-mucinous lung adenocarcinoma and mucinous lung adenocarcinoma (Table 2) and transcriptome sequencing data of PDO samples of non-mucinous lung adenocarcinoma and mucinous lung adenocarcinoma (Table 3); Figure 4The results of qPCR experiments for nine candidate genes (MMP1, SERPINE1, ITGB4, ITGA6, LAMC2, LAMB3, TLR2, FOSL1, ITGA5) in four lung adenocarcinoma cell lines (mucinous: NCI-H292; non-mucinous: H1975, A549, PC9); Figure 5 WB band grayscale analysis of nine candidate genes (MMP1, SERPINE1, ITGB4, ITGA6, LAMC2, LAMB3, TLR2, FOSL1, ITGA5) in four lung adenocarcinoma cell lines (mucinous: NCI-H292; non-mucinous: H1975, A549, PC9); Figure 6 qPCR and WB validation images for FOSL1 gene overexpression and knockdown experiments in NCI-H292 cells; Figure 7 Figures showing qPCR and WB validation of FOSL1 gene overexpression and knockdown experiments in PC-9 cells; Figure 8 Cell proliferation assay in NCI-H292 cells; Figure 9 Cell proliferation assay in PC-9 cells; Figure 10 Flow cytometry analysis of apoptosis in NCI-H292 cells; Figure 11 Flow cytometry analysis of apoptosis in PC-9 cells; Figure 12 Flow cytometry analysis of cell cycle in NCI-H292 cells; Figure 13 This is a flow cytometry diagram of the cell cycle in PC-9 cells. Detailed Implementation

[0015] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0016] I. Materials Unless otherwise specified, all methods used in this application are conventional methods known to those skilled in the art, and all reagents and materials used are commercially available products unless otherwise specified.

[0017] II. Methods 2.1 Bioinformatics screening of FOSL1 as a prognostic hub gene for lung adenocarcinoma (1) Data sources and difference analysis Transcriptome and clinical survival (OS) data for lung adenocarcinoma were downloaded from the TCGA database (Xena website), totaling 525 tumor samples. Transcriptome sequencing was also performed on organoids (PDO) from 3 patients with non-mucinous lung adenocarcinoma and 3 patients with mucinous lung adenocarcinoma.

[0018] Differentially identified genes (DEGs) were screened using the limma software package, with the screening criteria being |logFC|>1 and p<0.05. A total of 700 differentially identified genes (DEGs) were obtained. Figure 1 As shown, this includes 614 downregulated genes and 86 upregulated genes.

[0019] (2) Survival analysis and hub gene identification Kaplan-Meier survival analysis was performed on the differentially expressed genes using the R package "survival". The screening criterion was KM Pvalue < 0.05, and 88 differentially expressed genes associated with prognosis were obtained.

[0020] A PPI network was constructed using the STRING database. The CytoHubba plugin of Cytoscape 3.9 software was used to identify hub genes using five algorithms: EPC, MCC, DMNC, MNC, and Stress. The intersection of the top 15 gene sets of the five algorithms was used to obtain 9 hub genes (MMP1, ITGB4, ITGA6, LAMC2, LAMB3, SERPINE1, TLR2, FOSL1, and ITGA5), which are considered as key differentially expressed genes for the prognosis of non-mucinous lung adenocarcinoma and mucinous lung adenocarcinoma.

[0021] In addition, such as Figure 2 As shown, TCGA survival analysis confirmed that high expression of all nine hub genes was significantly associated with poor prognosis in patients with lung adenocarcinoma (P<0.05).

[0022] (3) Verification of drug resistance genes Download drug ICs from the GDSC database (https: / / www.cancerrxgene.org / ). 50 Data and transcriptome data were used to screen for the corresponding cell lines and IC50 values ​​for irinotecan. 50 Data matrix, based on the IC50 of irinotecan in all cell lines 50 The median divided the cell lines into sensitive and resistant groups, and we performed differential gene expression analysis, focusing on the differential expression of the hub gene in the GDSC database. Figure 3Table 2 shows that MMP1 and SERPINE1 were downregulated in the GDSC database, and their expression was also downregulated in transcriptome sequencing data of PDO samples from both non-mucinous lung adenocarcinoma and mucinous lung adenocarcinoma. Figure 3 (See Table 3 in the table). The expression differences between MMP1 and SERPINE1 were validated in a tumor drug sensitivity multi-omics database, suggesting that MMP1 and SERPINE1 may be key genes for irinotecan resistance in mucinous lung adenocarcinoma.

[0023] 2.2 Cell line RT-PCR validation bioinformatics analysis Based on previous treatment of primary lung adenocarcinoma organoids with SN-38 (the active metabolite of irinotecan) and subsequent transcriptome sequencing analysis, nine candidate genes (MMP1, ITGB4, ITGA6, LAMC2, LAMB3, SERPINE1, TLR2, FOSL1, and ITGA5) were screened. To further investigate the expression changes of these genes in lung adenocarcinoma cell lines, clarify the molecular mechanisms and biological functions of key candidate genes in the SN-38 drug response, and provide a basis for studying the mechanism of lung adenocarcinoma sensitivity to SN-38 through in vitro and in vivo experiments, the specific experimental methods are as follows: (1) Cell culture and drug treatment Lung adenocarcinoma cell lines (mucinous: NCI-H292; non-mucinous: H1975, A549, PC9) were selected and routinely cultured in RPMI-1640 medium with 10% FBS (fetal bovine serum) and maintained stable growth in an incubator at 37℃ and 5% CO2.

[0024] The cells were divided into 3×4 groups, totaling 12 groups: control group, SN-38 group, and Cisplatin group; Cell samples were collected after processing for subsequent RNA and protein extraction and functional analysis.

[0025] (2) qPCR verification of candidate gene expression Total RNA was extracted using the Trizol method and reverse transcribed into cDNA. The relative expression levels of nine candidate genes (MMP1, ITGB4, ITGA6, LAMC2, LAMB3, SERPINE1, TLR2, FOSL1, and ITGA5) were detected by qPCR. Each experiment was performed in triplicate, and data were presented as follows: -ΔΔCt We calculated the fold change in expression and analyzed the regulatory trends and consistency of SN-38 and Cisplatin on gene expression.

[0026] The experiment was divided into 3 × 4 groups, for a total of 12 groups: control group, SN-38 group, and Cisplatin group. like Figure 4 As shown in the figure, the q-PCR results identified three genes: ITGB4, TLR2, and FOSL1.

[0027] 2.3. Verification of q-PCR results by wb analysis of cell lines Based on the previous q-PCR results, three genes, ITGB4, TLR2, and FOSL1, were screened. The expression of these three indicators was verified by Western blotting. The specific experimental method is as follows: (1) Cell culture and drug treatment Lung adenocarcinoma cell lines (mucinous: NCI-H292; non-mucinous: H1975, A549, PC9) were selected and routinely cultured in RPMI-1640 medium containing 10% FBS. Stable growth was maintained at 37℃ in a 5% CO2 incubator. The cells were divided into 3×4 groups, totaling 12 groups: control group, SN-38 group, and Cisplatin group; GI of drugs 50 Cell samples were collected after 24 hours of concentration treatment for protein extraction and Western blot (WB) experiments.

[0028] (2) Verify changes in protein expression Total protein was extracted using RIPA lysis buffer, quantified using the BCA method, separated by electrophoresis, and transferred to a membrane. The protein was incubated with a specific primary antibody (candidate antibody), and the grayscale changes were analyzed using ImageJ after color development.

[0029] The experiment was divided into 3×4 groups, for a total of 12 groups: control group, SN-38 group, and Cisplatin group.

[0030] like Figure 5 As shown, the FOSL1 gene was screened out, and the following further validated its function through the construction and functional verification of stable FOSL1 gene transgenic strains.

[0031] 2.4 Construction and validation of FOSL1 overexpression and knockdown stable transgenes (1) Construction of stable overexpression and knockdown cell lines Based on the preliminary screening results, the target gene FOSL1 was selected, and lentiviruses were designed to infect target cells (mucinous lung adenocarcinoma cells NCI-H292 and non-mucinous lung adenocarcinoma cells PC-9). Stable knockdown and overexpression cell lines were obtained by screening with puromycin, and the construction effect was verified by qPCR and WB.

[0032] The experiment consisted of 7×2×2 groups, totaling 28 groups: WT, OE NC, OE FOSL1, sh NC, shFOSL1-1, shFOSL1-2, and shFOSL1-3 in mucinous lung adenocarcinoma NCI-H292 and non-mucinous lung adenocarcinoma PC-9, respectively.

[0033] Experimental results are as follows Figure 6-7 As shown, in NCI-H292 cells, qPCR results showed that compared with the NC group, FOSL1 mRNA expression was significantly increased in the NCI-H292-FOSL1 overexpression stable transgenic strain, while FOSL1 mRNA expression was significantly decreased in the shFOSL1-2 and shFOSL1-3 groups of the three NCI-H292-FOSL1 knockdown stable transgenic strains. Western blot results showed that compared with the NC group, FOSL1 protein expression was significantly increased in the NCI-H292-FOSL1 overexpression stable transgenic strain, while FOSL1 protein expression was significantly decreased in all three NCI-H292-FOSL1 knockdown stable transgenic strains, with the shFOSL1-3 group showing the most significant decrease. In PC-9 cells, qPCR results showed that, compared with the NC group, FOSL1 mRNA expression was significantly increased in PC-9-FOSL1 overexpression stable transgenic cells, while FOSL1 mRNA expression was significantly decreased in all three PC-9-FOSL1 knockdown stable transgenic cells, with the shFOSL1-2 group showing the most significant decrease. Western blot results showed that, compared with the NC group, FOSL1 protein expression was significantly increased in PC-9-FOSL1 overexpression stable transgenic cells, while FOSL1 protein expression was significantly decreased in all three PC-9-FOSL1 knockdown stable transgenic cells, with the shFOSL1-2 group showing the most significant decrease.

[0034] (2) Cell proliferation Stable overexpression and knockdown cell lines obtained from different treatment groups in experiment (1) were seeded into 96-well plates. Cell proliferation rates were continuously detected at 0, 24, 48, and 72 h using the CTG method. The concentration of each drug treatment was its corresponding GI. 50 concentration.

[0035] Experimental results are as follows Figure 8-9 As shown, in NCI-H292 cells, compared to the control groups, the effect of Cisplatin GI... 50 (cisplatin GI) 50 After treatment with SN-38 GI, the cell proliferation rate of each cell group showed a significant decreasing trend with the extension of drug action time. Compared with the control groups, the cell proliferation rate of each cell group decreased significantly after treatment with SN-38 GI. 50 After concentration treatment, the cell proliferation rate of each experimental group showed a significant decreasing trend with the extension of drug treatment time; In PC-9 cells, compared to the control groups, the effect of Cisplatin GI... 50 After concentration treatment, the cell proliferation rate of the PC-9 WT cell group showed a significant decreasing trend with prolonged drug treatment time, while no significant time-dependent proliferation inhibition was observed in other groups; compared with the control groups, the cell proliferation rate of the SN-38 GI group was significantly lower. 50 After concentration treatment, the cell proliferation rate of each experimental group showed a significant decreasing trend with the extension of drug action time.

[0036] (3) Apoptosis After treating cells in each group with different drugs for 72 hours, the proportions of early and late apoptosis were detected by Annexin V-APC / 7-AAD apoptosis kit double staining flow cytometry; the concentration of each drug was corresponding to its GI. 50 concentration.

[0037] Experimental results are as follows Figure 10-11 As shown, in NCI-H292 cells, compared to the NCI-H292 WT group, the NCI-H292WT Cisplatin GI 50 NCI-H292 WT SN-38 GI 50 Early and mid-to-late apoptosis levels were significantly increased in the NCI-H292OENC group; compared to the NCI-H292OENC group, the NCI-H292OENC Cisplatin GI 50 NCI-H292 OENC SN-38 GI 50 Early and mid-to-late apoptosis levels were significantly increased in the group; compared to the NCI-H292 OE-FOSL1 group, the NCI-H292 OE-FOSL1 group had significantly higher levels of Cisplatin GI. 50 NCI-H292 OE-FOSL1 SN-38 GI 50 Early and mid-to-late apoptosis levels were significantly increased in the NCI-H292 KDNC group; compared to the NCI-H292 KDNC group, the NCI-H292 KDNC Cisplatin GI 50 NCI-H292 KDNC SN-38 GI 50 Early and mid-to-late apoptosis levels were significantly increased in the group; compared with the NCI-H292 KD-FOSL1 group, the NCI-H292 KD-FOSL1 Cisplatin GI 50 NCI-H292 KD-FOSL1 SN-38 GI 50 Early apoptosis levels were significantly increased in the group; In PC-9 cells, compared to the PC-9 WT group, PC-9 WT Cisplatin GI 50PC-9 WT SN-38 GI 50 Early and mid-to-late apoptosis levels were significantly increased in the PC-9 OENC group; compared to the PC-9 OENC group, the PC-9 OENC Cisplatin GI 50 PC-9 OENC SN-38 GI 50 Early and mid-to-late apoptosis levels were significantly increased in the PC-9 OE-FOSL1 group; compared to the PC-9 OE-FOSL1 group, the PC-9 OE-FOSL1 Cisplatin GI 50 PC-9 OE-FOSL1 SN-38 GI 50 Early and mid-to-late apoptosis levels were significantly increased in the PC-9 KDNC group; compared to the PC-9 KDNC group, the PC-9 KDNC Cisplatin GI 50 PC-9 KDNC SN-38 GI 50 Early and mid-to-late apoptosis levels were significantly increased in the PC-9 KD-FOSL1 group; compared to the PC-9 KD-FOSL1 group, the PC-9 KD-FOSL1 CisplatinGI group showed significantly higher levels of apoptosis. 50 PC-9 KD-FOSL1 SN-38 GI 50 Early apoptosis levels were significantly increased in the group. (4) Cell cycle After treating cells in each group with different drugs for 72 hours, the cells were fixed with 70% ethanol, stained with PI, and analyzed by flow cytometry. The changes in the proportion of cells in each phase (G0 / G1, S, G2 / M) were calculated to determine the cell cycle regulation effect of the target gene under the action of SN-38. The concentration of each drug treatment was its corresponding GI. 50 concentration.

[0038] Experimental results are as follows Figure 12-13 As shown, in NCI-H292 cells, compared to the NCI-H292 WT group, the NCI-H292WT Cisplatin GI 50 NCI-H292 WT SN-38 GI 50 The group was stationary in phase G2; compared to the NCI-H292 OENC group, the NCI-H292 OENC Cisplatin GI 50 Stagnant in G0+G1 phase, NCI-H292 OENC SN-38 GI 50 Stagnation in S phase; NCI-H292 OE-FOSL1 Cisplatin GI compared to NCI-H292 OE-FOSL1 group 50 NCI-H292 OE-FOSL1SN-38 GI 50The group stagnated to phase S; compared to the NCI-H292 KDNC group, the NCI-H292 KDNC Cisplatin GI 50 The group was stationary in phase S; compared with the NCI-H292 KD-FOSL1 group, the NCI-H292 KD-FOSL1 Cisplatin GI 50 NCI-H292 KD-FOSL1 SN-38 GI 50 The group stalled in the G0+G1 period; In PC-9 cells, compared to the PC-9 WT group, PC-9 WT Cisplatin GI 50 PC-9 WT SN-38 GI 50 The group stalled in phase G2; compared to the PC-9 OENC group, the PC-9 OENC Cisplatin GI 50 Stagnation in G2 phase; PC-9 OE-FOSL1 Cisplatin GI relative to PC-9OE-FOSL1 group 50 PC-9 OE-FOSL1 SN-38 GI 50 The group stalled to phase G2; compared to the PC-9 KDNC group, the PC-9 KDNC Cisplatin GI 50 PC-9 KDNC SN-38 GI 50 The group stalled in phase G2; compared to the PC-9 KD-FOSL1 group, the PC-9 KD-FOSL1 Cisplatin GI 50 PC-9 KD-FOSL1 SN-38 GI 50 The group stalled in G2.

[0039] In summary, this invention experimentally verified that FOSL1 is a key hub gene for the prognosis of lung adenocarcinoma, and its high expression is significantly associated with poor patient prognosis. FOSL1 regulates the sensitivity of lung adenocarcinoma cells to SN-38 and cisplatin. In non-mucinous lung adenocarcinoma (PC-9), FOSL1 overexpression enhances chemosensitivity. In mucinous lung adenocarcinoma (NCI-H292), FOSL1 knockdown alters chemosensitivity. FOSL1 exerts its anti-tumor effect by influencing cell cycle and apoptosis pathways. This provides experimental evidence for the development of FOSL1-targeted therapies for lung adenocarcinoma. This invention reveals the application value of FOSL1 as a therapeutic target for lung adenocarcinoma and provides a new strategy for the precision treatment of lung adenocarcinoma.

[0040] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various improvements without departing from the concept of the present invention, and these improvements all fall within the scope of protection of the present invention.

Claims

1. Application of FOSL1 as a target in the preparation of drugs for the prevention or treatment of mucinous lung adenocarcinoma.

2. The application of FOSL1 as a target in the preparation of drugs for the prevention or treatment of mucinous lung adenocarcinoma according to claim 1, characterized in that, By inhibiting FOSL1 expression with drugs, the replication and proliferation of mucinous lung adenocarcinoma cells can be suppressed.

3. The application of FOSL1 as a target in the preparation of drugs for the prevention or treatment of mucinous lung adenocarcinoma according to claim 2, characterized in that, The method of inhibiting FOSL1 through drugs involves using FOSL1 inhibitors for inhibition.

4. The application of FOSL1 as a target in the preparation of drugs for the prevention or treatment of mucinous lung adenocarcinoma according to claim 3, characterized in that, The FOSL1 inhibitor is irinotecan or SN-38.

5. The application of FOSL1 as a target in the preparation of drugs for the prevention or treatment of mucinous lung adenocarcinoma according to claim 1, characterized in that, The drugs mentioned for the prevention or treatment of mucinous lung adenocarcinoma refer to any one of the following (1)-(5): (1) Drugs used to prevent or treat infection with mucinous lung adenocarcinoma cells; (2) Drugs used for the prevention or treatment of mucinous lung adenocarcinoma; (3) Drugs used to inhibit the replication and proliferation of mucinous lung adenocarcinoma cells; (4) Drugs used to inhibit the lesioning effect of mucinous lung adenocarcinoma cells; (5) Inhibitor of mucinous lung adenocarcinoma cells.

6. A drug for the prevention or treatment of mucinous lung adenocarcinoma, characterized in that, The drug in question is a FOSL1 inhibitor.

7. A drug for the prevention or treatment of mucinous lung adenocarcinoma according to claim 6, characterized in that, The PLK inhibitor is irinotecan or SN-38.

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