Application of AhR-HEGBC-CYP1A1 regulation axis in preparation of medicine for resisting gallbladder cancer

Abstract

The invention relates to an application of an AhR-HEGBC-CYP1A1 regulation axis in preparation of a medicine for resisting gallbladder cancer. The invention provides application of shRNA of a specific interference AhR gene, shRNA of a specific interference HEGBC gene and / or shRNA of a specific interference CYP1A1 gene in preparation of drugs for targeted therapy of gallbladder carcinoma. It is found for the first time that proliferation, migration and invasion of gallbladder cancer cells can be effectively inhibited by inhibiting an AhR-HEGBC-CYP1A1 regulation axis through interference. According to the invention, sh-AhR, sh-HEGBC and sh-CYP1A1 which specifically knock down the expression levels of AhR, HEGBC and CYP1A1mRNA are designed, the expression of an AhR-HEGBC-CYP1A1 regulatory axis is interfered and inhibited by knocking down the expression levels of AhR, HEGBC and CYP1A1mRNA, and the proliferation, migration and invasion of gallbladder cancer cells are successfully inhibited, so that the AhR-HEGBC-CYP1A1 regulatory axis can be used as a therapeutic target to prepare a drug for targeted therapy of gallbladder cancer.

Description

Technical Field

[0001] The present invention relates to the application of the AhR-HEGBC-CYP1A1 regulatory axis in the preparation of anti-gallbladder cancer drugs, belonging to the field of biomedical technology. Background Art

[0002] Gallbladder cancer (GBC) is a widespread and highly invasive malignant tumor in the biliary tract system. Radical resection at an early stage is the only way to treat gallbladder cancer. Traditional radiotherapy and chemotherapy have limited therapeutic value for GBC and are difficult to further improve the prognosis of patients. To further improve the prognosis of GBC patients, it is necessary to deepen the understanding of the molecular mechanism of its occurrence and development and develop new and effective treatment methods.

[0003] More and more studies have found that the incidence of gallbladder cancer is related to chronic infection, stones, and exposure to specific chemicals in the environment. The diverse microbial communities in the gallbladder constitute a dynamic microecological balance that coordinates and restricts each other between the host and the environment. The microecological transformation caused by cholesterol stones and bacterial infection can break this microecological balance, and the proliferation of anaerobic bacteria can cause bacterial cholecystitis. Anaerobic bacteria are closely related to the occurrence and development of tumors, and their main mechanism is to participate in the metabolism of polycyclic aromatic hydrocarbons (PAHs) to produce carcinogenic effects. PAHs are organic compounds containing two or more benzene rings, including naphthalene, anthracene, pyrene, etc. The endogenous PAHs [7,8-dihydroxy-9,10-epoxybenzo(a)pyrene] produced by anaerobic metabolism have strong carcinogenicity. The aryl hydrocarbon receptor (AhR) is an intracellular receptor that can be activated by its ligand PAHs and is overexpressed in various solid tumor tissues and cell lines such as breast cancer and liver cancer. Therefore, exploring its relationship with the occurrence and development of tumors is of great significance. The endogenous PAHs produced by anaerobic bacteria may play an important role in the occurrence and development of GBC by acting on its receptor AhR.

[0004] Short hairpin RNA (shRNA) is a double-stranded RNA composed of 19-25 pairs of nucleotides and is a key component in RNA interference technology. After transfection into cells, shRNA forms a hairpin structure after transcription by the RNA Pol III promoter in the cell. In vivo, this structure is cleaved by Dicer enzyme into small interfering RNA (siRNA), and siRNA binds to the RNA-induced silencing complex (RISC), resulting in the degradation of specific mRNA, thereby inhibiting the expression of related genes. Designing shRNAs with high specificity and high interference efficiency will play an important role in the targeted therapy of gallbladder cancer. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides the application of the AhR-HEGBC-CYP1A1 regulatory axis in the preparation of anti-gallbladder cancer drugs.

[0006] The technical solution of the present invention is as follows:

[0007] The application of the AhR-HEGBC-CYP1A1 regulatory axis as a drug target in the preparation of targeted drugs for the treatment of gallbladder cancer.

[0008] Preferably according to the present invention, the AhR-HEGBC-CYP1A1 regulatory axis includes the AhR gene, the HEGBC gene, and the CYP1A1 gene.

[0009] Preferably according to the present invention, the Gene ID of the AhR gene: 196; the Gene ID of the HEGBC gene: 401585; the Gene ID of the CYP1A1 gene: 1543.

[0010] Preferably according to the present invention, the targeted drug for the treatment of gallbladder cancer takes the AhR gene, the HEGBC gene, and the CYP1A1 gene as intervention targets, and is based on interfering with AhR, HEGBC, and CYP1A1 mRNA, and can efficiently and specifically inhibit the expression levels of the AhR gene, the HEGBC gene, and the CYP1A1 gene.

[0011] Preferably according to the present invention, the targeted drug for the treatment of gallbladder cancer is siRNA, shRNA, microRNA, or a chemical inhibitor of AhR, HEGBC, and CYP1A1 mRNA.

[0012] The application of shRNA specifically interfering with the AhR gene, shRNA specifically interfering with the HEGBC gene, and / or shRNA specifically interfering with the CYP1A1 gene in the preparation of targeted drugs for the treatment of gallbladder cancer.

[0013] Preferably according to the present invention, the targeted nucleotide sequence of the shRNA specifically interfering with the AhR gene is as shown in SEQ ID NO.1-2; the targeted nucleotide sequence of the shRNA specifically interfering with the HEGBC gene is as shown in SEQ ID NO.3-4; the targeted nucleotide sequence of the shRNA specifically interfering with the CYP1A1 gene is as shown in SEQ ID NO.5-6.

[0014] Preferably according to the present invention, the molar ratio of the shRNA specifically interfering with the AhR gene, the shRNA specifically interfering with the HEGBC gene, and the shRNA specifically interfering with the CYP1A1 gene in the targeted drug for the treatment of gallbladder cancer is 1:1:1.

[0015] A targeted drug for treating gallbladder cancer, which comprises shRNA specifically interfering with the AhR gene, shRNA specifically interfering with the HEGBC gene, and / or shRNA specifically interfering with the CYP1A1 gene.

[0016] Preferably according to the present invention, the targeted nucleotide sequences of the shRNA specifically interfering with the AhR gene are as shown in SEQ ID NO.1-2; the targeted nucleotide sequences of the shRNA specifically interfering with the HEGBC gene are as shown in SEQ ID NO.3-4; the targeted nucleotide sequences of the shRNA specifically interfering with the CYP1A1 gene are as shown in SEQ ID NO.5-6.

[0017] Beneficial effects:

[0018] 1. The present invention first discovers that by interfering with and inhibiting the AhR-HEGBC-CYP1A1 regulatory axis, the proliferation, migration and invasion of gallbladder cancer cells can be effectively inhibited. The present invention designs sh-AhR, sh-HEGBC and sh-CYP1A1 that specifically knockdown the mRNA expression levels of AhR, HEGBC and CYP1A1, and interfere with and inhibit the expression of the AhR-HEGBC-CYP1A1 regulatory axis by knocking down the mRNA expression levels of AhR, HEGBC and CYP1A1, and successfully inhibits the proliferation, migration and invasion of gallbladder cancer cells. Therefore, the AhR-HEGBC-CYP1A1 regulatory axis can be used as a therapeutic target to prepare targeted drugs for treating gallbladder cancer.

[0019] 2. The present invention first discovers that targeting and interfering with the activity of the AhR-HEGBC-CYP1A1 regulatory axis can more effectively inhibit the malignant progression of gallbladder cancer compared with separately inhibiting the expression levels of the AhR gene, HEGBC gene or CYP1A1 gene. The sh-MIX designed by the present invention can simultaneously and specifically interfere with the mRNA levels of AhR, HEGBC and CYP1A1. In the case of transfecting the same dose of shRNA, cell function experiments show that compared with sh-AhR, sh-HEGBC or sh-CYP1A1, sh-MIX shows a more effective anti-proliferation and anti-metastasis effect on gallbladder cancer. Therefore, the AhR-HEGBC-CYP1A1 regulatory axis can be used as a drug target and applied to the research and preparation of targeted drugs for treating gallbladder cancer. Description of the drawings

[0020] Figure 1 It is the relevant verification result of the HEGBC gene and the PAHs-AhR signaling pathway in GBC;

[0021] In the figure, A shows the expression levels of the HEGBC gene in GBC tissues and adjacent non-tumor tissues, with GAPDH as the internal reference control; B shows the representative hematoxylin-eosin (HE) staining results in GBC tumor tissues and adjacent non-tumor tissues (magnification 40×, scale bar 50 μm); C shows the results of analyzing the correlation between high or low expression of HEGBC and cumulative survival rate in 110 GBC patients by Kaplan-Meier survival analysis. Using the median expression level of HEGBC as the cut-off value, a P<0.05 by Log-rank test indicates statistical significance; D shows the expression levels of the HEGBC gene in human non-tumor cholangiocyte cell line H69 and human GBC cell lines (GBC-SD, OCUG-1, NOZ, EH-GB2, SGC-996); E shows the KEGG functional enrichment analysis of pathways related to high expression of HEGBC; F shows a heatmap presenting the KEGG functional enrichment analysis results of differentially expressed genes (DEGs) enriched in HEGBC-related enrichment pathways between HEGBC-overexpressing GBC cells and control cells; G-H show the expression levels of the HEGBC gene and the AhR gene in GBC-SD cells and EH-GB2 cells transfected with the HEGBC overexpression plasmid. Data are expressed as mean ± standard deviation (SD); statistical significance is indicated by P<0.05, P<0.01, and P<0.001.

[0022] Figure 2 It is the relevant verification results for the activation of the PAHs-AhR signaling pathway in GBC cells;

[0023] In the figure, A shows the expression levels of the AhR gene in human gallbladder epithelial cells (H69) and GBC cell lines (GBC-SD and EH-GB2); B shows the expression levels of the AhR gene in GBC tissues and adjacent non-tumor tissues; C shows the verification of the correlation between the AhR gene and the HEGBC gene by Pearson correlation analysis (n = 110 GBC cases); D shows the detection of the expression of AhR protein in GBC tissues and adjacent non-tumor tissues by immunohistochemistry (IHC) (magnification 10×, scale bar 200 μm); E shows the detection of the activation of AhR in GBC cell lines (GBC-SD and EH-GB2) using an AhR-dependent luciferase reporter gene. Statistical significance is indicated by P<0.05, P<0.01, and P<0.001.

[0024] Figure 3 It is the relevant verification results for the participation of the CYP1A1 gene in the in vitro proliferation, migration, and invasion of GBC cells mediated by PAHs-AhR / HEGBC;

[0025] In the figure, A shows the protein expression levels of CYP1A1, CYP1B1, and CYP2S1 in GBC-SD cells and EH-GB2 cells after treatment with 5 μmol / L PAHs or vehicle (0.1% DMSO) respectively; B shows the protein expression level of CYP1A1 in GBC-SD cells and EH-GB2 cells after transfection with sh-HEGBC or sh-AhR and treatment with PAHs; C shows the proliferation rate of transfected GBC-SD cells and EH-GB2 cells quantitatively detected by CCK-8 assay; D-E show the migration and invasion abilities of transfected GBC-SD and EH-GB2 cells evaluated by Transwell assay (scale bar = 50 μm), and the data are expressed as mean ± standard deviation (SD); *P < 0.05, **P < 0.01, ***P < 0.001.

[0026] Figure 4 It is the relevant verification result that the CYP1A1 gene is involved in the in vivo tumor growth and metastasis of GBC mediated by PAHs-AhR / HEGBC.

[0027] In the figure, A shows the representative images of tumors in mice, scale bar = 1 cm; B shows the results of tumor volume detected once a week after subcutaneous injection of transfected GBC cells into nude mice; C shows the weight of subcutaneous tumors detected in mice on the 28th day after injection; D shows the protein levels of AhR and CYP1A1 in mouse tumor tissues; E shows the results of analyzing the metastatic nodules in mice of xenograft tumor models by bioluminescence imaging (5 mice in each group); F shows the expression levels of AhR and CYP1A1 proteins in mouse tumor tissues detected by immunohistochemistry (IHC) (magnification 400×, scale bar = 20 μm), and the data are expressed as mean ± standard deviation (SD); *P < 0.05, **P < 0.01, ***P < 0.001. Specific embodiments

[0028] The technical solutions of the present invention will be further described below in combination with specific experimental examples, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the examples are all ordinary commercially available products without special instructions.

[0029] Human GBC cell lines GBC-SD, OCUG-1, NOZ, EH-GB2, SGC-996, and human biliary epithelial cells H69 were all purchased from Procell (Wuhan, China).

[0030] Hairless BALB / c mice (male, six weeks old, weighing 22-25 grams) were purchased from Charles River.

[0031] The overexpression plasmid pcDNA3.1-HEGBC was purchased from GenePharma, Shanghai, China.

[0032] The sh-AhR targeting sequence is double-stranded, and the specific sequence information is as follows:

[0033] sh-AhR#1: 5′-CCCAGACAGUAGUCUGUUAUA-3' (SEQ ID NO.1),

[0034] sh-AhR#2: 5′-UAACAGACUACUGUCUGGGGG-3' (SEQ ID NO.2).

[0035] The sh-HEGBC targeting sequence is double-stranded, and the specific sequence information is as follows:

[0036] sh-HEGBC#1: 5′-GGAGCTTCCAGAAGTGGTTTC-3' (SEQ ID NO.3),

[0037] sh-HEGBC#2: 5′-GCTG ATGAGAGACATGTTTGT-3' (SEQ ID NO.4).

[0038] The sh-CYP1A1 targeting sequence is double-stranded, and the specific sequence information is as follows:

[0039] sh-CYP1A1#1: 5′-GAGAAGUUCUACAGCUUCAUG-3' (SEQ ID NO.5),

[0040] sh-CYP1A1#2: 5′-UGAAGCUGUAGAACUUCUCAU-3' (SEQ ID NO.6).

[0041] The above shRNAs can all be obtained by artificial synthesis according to the sequence information.

[0042] Example 1

[0043] 1. Tumors and adjacent non-tumor tissues of 110 groups of GBC patients diagnosed in Quzhou People's Hospital from 2018 to 2023 were collected. In addition, gallbladder tissue samples of 15 GBC patients and 15 gallstone patients in the same hospital during the same period were collected. None of the patients received chemotherapy or radiotherapy before surgery and signed the informed consent form. All samples were immediately frozen in liquid nitrogen and stored at -80°C after collection. All patients were informed and consented, and the study was approved by the Human Biomedical Research Ethics Committee of Quzhou People's Hospital (approval number: 2014-015).

[0044] 2. Human GBC cell lines (GBC-SD, OCUG-1, NOZ, EH-GB2, SGC-996) and human biliary epithelial cells (H69) were respectively inoculated in Dulbecco's modified Eagle's medium (Gibco BRL, Grand Island, New York, USA) containing 10% fetal bovine serum and cultured under the conditions of 37 °C and 5% carbon dioxide until the logarithmic growth phase of the cells was reached.

[0045] Drug treatment of GBC cells: The human GBC cell lines (GBC-SD, OCUG-1, NOZ, EH-GB2, SGC-996) and human biliary epithelial cells (H69) cultured to the logarithmic growth phase of the cells were continuously cultured. Then, the time interval for changing the culture medium was set to once every 48 hours. Each time the culture medium was changed, the cells were exposed to 0 μmol / L, 2.5 μmol / L, 5 μmol / L or 10 μmol / L of 7,8-dihydrodihydroxy-9,10-epoxybenzo[a]pyrene (PAHs, purchased from SIGMA) for 16 hours to obtain cells treated with polycyclic aromatic hydrocarbons (PAHs). At the same time, in the same way, DMSO with the same concentration was used as the control group of cells.

[0046] 3. The overexpression plasmid pcDNA3.1-HEGBC was transfected into PAH-treated GBC-SD cells and EH-GB2 cells respectively using Lipofectamine 3000 (Catalog number: L3000015, Life Technologies, Carlsbad, CA) reagent. At the same time, cells transfected with the empty plasmid were used as the blank control to obtain OE-HEGBC cells and OE-NC cells.

[0047] Among them, OE-HEGBC cells are GBC-SD cells and EH-GB2 cells transfected with the overexpression plasmid pcDNA3.1-HEGBC; OE-NC cells are GBC-SD cells and EH-GB2 cells transfected with the empty plasmid.

[0048] 4. The tumor tissue (GBC) and adjacent non-tumor tissue (Control) obtained in the first point of this example were first subjected to qRT-PCR analysis to determine the difference in the expression levels of the HEGBC gene (lncRNA HEGBC) in the tumor tissue (GBC) and adjacent non-tumor tissue (control) (a total of 3 groups of parallel determinations were performed). Then, they were fixed with formalin and embedded in paraffin, and stained with hematoxylin and eosin (HE). The staining was evaluated by scanning the entire tissue specimen under a low-power magnifying glass (×10) and confirmed under a high-power magnifying glass (×40). Finally, Kaplan-Meier survival analysis was performed, and the results are shown as Figure 1 shown in A - C.

[0049] 5. The human GBC cell lines (GBC-SD, OCUG-1, NOZ, EH-GB2, SGC-996) obtained in the second point of this example after polycyclic aromatic hydrocarbon treatment were subjected to qRT-PCR analysis to determine the differential expression levels of the HEGBC gene (lncRNA HEGBC) in each cell. The results are as Figure 1 shown in

[0050] Figure D. Figure 1 6. The OE-HEGBC cells and OE-NC cells obtained in the third point of this example were first subjected to RNA sequencing (RNA-seq), and then KEGG functional enrichment analysis was performed based on the RNA sequencing result data. Finally, the differential expression levels of the HEGBC gene and the AhR gene in the OE-HEGBC cells and OE-NC cells were determined by qRT-PCR. The results are as

[0051] shown in Figure 1 Figures E - H. Figure 1 Figure 1 As shown in Figure 1 Figures A - C, compared with adjacent non-tumor tissues (Control), the expression level of the HEGBC gene in tumor tissues (GBC) was significantly higher (P < 0.001, Figure A). HE staining showed obvious differences between tumor tissues (GBC) and adjacent non-tumor tissues (Control)

[0052] Figure B), and the expression level of the HEGBC gene in GBC tissue tumors increased significantly. Kaplan-Meier survival analysis showed that the survival rate of GBC patients with high expression of the HEGBC gene was poor <00,00122>Figure C).

[0053] As shown in Figure 1 Figure D, after polycyclic aromatic hydrocarbon treatment, the HEGBC gene showed a higher expression level in human GBC cell lines (GBC-SD, OCUG-1, NOZ, EH-GB2, SGC-996). Figure 1 Figure 1 As shown in Figure 1 Figures E - H, RNA-seq identified a total of 747 downregulated genes and 709 upregulated genes (|log FC| ≥ 1). KEGG functional enrichment analysis showed the top 12 pathways related to high expression of HEGBC Figure E). The heatmap showed that the differentially expressed genes (DEGs) including AhR in KEGG functional enrichment analysis were enriched in chemical carcinogenesis - DNA adducts, chemical carcinogenesis - receptor activation, bile secretion, cytochrome P450 metabolism of xenobiotics, and AhR signaling pathway Figure F). Finally, it was also observed that HEGBC and AhR mRNA were significantly overexpressed in OE-HEGBC cells, and the expression level was about twice that of OE-NC cells (P < 0.001, Figures G, H).

[0054] Based on the above results, the inventors of the present application speculated that the AhR gene is the upstream regulatory gene of the HEGBC gene, and the AhR-HEGBC signaling pathway is involved in the development process of gallbladder cancer mediated by HEGBC.

[0055] Example 2

[0056] 1. Perform qRT-PCR analysis on the polycyclic aromatic hydrocarbon-treated GBC-SD cells, EH-GB2 cells, and H69 cells obtained in point 2 of Example 1 to measure the differences in the expression levels of the AhR gene in each cell. The results are as Figure 2 shown in

[0057] A. Figure 2 2. Perform qRT-PCR analysis on the tumor tissue (GBC) and adjacent non-tumor tissue (Control) obtained in point 1 of this example to measure the differences in the expression levels of the AhR gene in the tumor tissue (GBC) and adjacent non-tumor tissue (control). The results are as shown in

[0058] B. Figure 2 3. Use Pearson correlation analysis to analyze the correlation between the expression level of AhR and the expression of the HEGBC gene (lnc HEGBC) in the tumor tissue (GBC). The results are as shown in

[0059] C. Figure 2 4. Fix the tumor tissue (GBC) and adjacent non-tumor tissue (Control) obtained in point 1 of this example with formalin, embed them in paraffin, and perform IHC staining. Evaluate the staining by scanning the entire tissue specimen under a low-power magnifying glass (×10) and confirm it under a high-power magnifying glass (×40). The results are as shown in

[0060] D. Figure 2 5. Using the AhR-dependent luciferase reporter gene (pXRE4-SV40-Luc) as a marker, use the TransIT keratinocyte transfection reagent (E7-0084; Geneflow) to transfect the Renilla luciferase control vector (pRLTK, Promega) into H69 cells, GBC-SD cells, and EH-GB2 cells. After ligand treatment for 48 hours, using the normalized firefly value as an internal reference, analyze the cells using the dual luciferase reporter system. The results are as Figure 2 shown in

[0061] As Figure 2 shown in

[0062] A, the expression levels of the AhR gene in GBC-SD cells and EH-GB2 cells are higher than those in H69 cells, which is consistent with the high-expression trend of the HEGBC gene in GBC (P<0.01, P<0.001). <000015i>

[0062] AsFigure 2 As shown in B, the AhR gene level in GBC tissues was significantly higher than that in adjacent non-tumor tissues (P < 0.001).

[0063] As shown in Figure 2 C, correlation analysis showed a positive correlation between the expression levels of AhR mRNA and HEGBC gene in GBC tissues (r = 0.2732, P = 0.0039).

[0064] As shown in Figure 2 D, the expression level of AhR gene in GBC tumor tissues increased significantly.

[0065] As shown in Figure 2 E, the activation of AhR gene was prevalent in GBC-SD cells and EH-GB2 cells (P < 0.01).

[0066] Based on the above results, the inventors of this application further confirmed that the AhR gene is an upstream regulatory gene of the HEGBC gene, and the AhR-HEGBC signaling pathway is involved in the development of gallbladder cancer mediated by HEGBC.

[0067] Example 3

[0068] 1. Western blot detection was performed on GBC-SD cells, EH-GB2 cells and control cells (DMSO) treated with polycyclic aromatic hydrocarbons (PAH) obtained in the second point of Example 1 to determine the differences in the expression levels of CYP1A1, CYP1B1 and CYP2S1 proteins in GBC-SD cells, EH-GB2 cells and control cells (DMSO) after treatment with polycyclic aromatic hydrocarbons. The results are as Figure 3 shown in A.

[0069] 2. sh-HEGBC and sh-AhR were transfected into GBC-SD cells treated with polycyclic aromatic hydrocarbons (PAH) obtained in the second point of Example 1 using Lipofectamine 3000 (Catalog No. L3000015, Life Technologies, Carlsbad, CA) reagent. At the same time, GBC-SD cells transfected with sh-NC and treated with polycyclic aromatic hydrocarbons (PAH) and control cells (DMSO) were used as controls to obtain DMSO cells (GBC-SD), PAH cells (GBC-SD), PAH-sh-HEGBC cells (GBC-SD) and PAH-sh-AhR cells (GBC-SD); in the same way, DMSO cells (EH-GB2), PAH cells (EH-GB2), PAH-sh-HEGBC cells (EH-GB2) and PAH-sh-AhR cells (EH-GB2) were obtained;

[0070] Then, the above groups of cells were subjected to Western blot detection to determine the differences in the expression levels of CYP1A1 protein in the above groups of cells. The results were as Figure 3 shown in

[0071] 3. Using Lipofectamine 3000 (Catalog No. L3000015, Life Technologies, Carlsbad, CA) reagent, sh-HEGBC, sh-AhR, and sh-CYP1A1 were transfected into the polycyclic aromatic hydrocarbon (PAH)-treated GBC-SD cells obtained in the second point of Example 1 respectively. At the same time, the PAH-treated GBC-SD cells transfected with sh-NC and the control group cells (DMSO) were used as controls to obtain DMSO cells (GBC-SD), PAH cells (GBC-SD), PAH-sh-HEGBC cells (GBC-SD), PAH-sh-AhR cells (GBC-SD), and PAH-sh-CYP1A1 cells (GBC-SD); according to the same method, DMSO cells (EH-GB2), PAH cells (EH-GB2), PAH-sh-HEGBC cells (EH-GB2), PAH-sh-AhR cells (EH-GB2), and PAH-sh-CYP1A1 cells (EH-GB2) were obtained;

[0072] After 48 hours, the above groups of cells were collected and the cells of each group were re-cultured in 96-well plates (2,000 cells / well), and the CCK-8 assay was used to detect the changes in the viability of GBC-SD cells. The results were as Figure 3 shown in

[0073] 4. The cells obtained in the third point of this example were used in the Transwell assay to detect the cell migration and invasion abilities. The results were as Figure 3 shown in

[0074] As Figure 3 shown in

[0075] As Figure 3As shown in Figure B, sh-AhR and sh-HEGBC significantly inhibited the increase in the expression level of CYP1A1 protein in GBC-SD cells and EH-GB2 cells after polycyclic aromatic hydrocarbon (PAH) treatment (P<0.01, P<0.001). These data indicate that PAH-induced CYP1A1 expression depends on the AhR-HEGBC signaling pathway.

[0076] As shown in Figure 3 Figure C, sh-CYP1A1, sh-AhR and sh-HEGBC significantly inhibited the proliferation of GBC-SD cells and EH-GB2 cells after polycyclic aromatic hydrocarbon (PAH) treatment (P<0.001).

[0077] As shown in Figure 3 Figures D-E, sh-CYP1A1, sh-AhR and sh-HEGBC significantly inhibited the migration and invasion of GBC-SD cells and EH-GB2 cells after polycyclic aromatic hydrocarbon (PAH) treatment (P<0.001).

[0078] Based on the above results, the inventors of this application speculated that the CYP1A1 gene is a downstream effector gene of the HEGBC gene, and the HEGBC-CYP1A1 signaling pathway is involved in the HEGBC-mediated development process of gallbladder cancer.

[0079] Example 4

[0080] 1. Expose GBC cells to 5 μM of 7,8-dihydroxy-9,10-epoxybenzo[a]pyrene for 16 h to obtain GBC-SD cells after polycyclic aromatic hydrocarbon (PAH) treatment. At the same time, according to the same method, use DMSO at the same concentration as the control group of GBC-SD cells.

[0081] Then, transfect sh-HEGBC, sh-AhR, and sh-CYP1A1 into GBC-SD cells after polycyclic aromatic hydrocarbon (PAH) treatment, respectively. At the same time, use GBC-SD cells transfected with sh-NC after polycyclic aromatic hydrocarbon (PAH) treatment and control group cells (DMSO) as controls to obtain DMSO cells (GBC-SD), PAH cells (GBC-SD), PAH-sh-HEGBC cells (GBC-SD), PAH-sh-AhR cells (GBC-SD), and PAH-sh-CYP1A1 cells (GBC-SD);

[0082] Next, mice of the BALB / c strain were separately injected into the spleen with DMSO cells (GBC-SD), PAH cells (GBC-SD), PAH-sh-HEGBC cells (GBC-SD), PAH-sh-AhR cells (GBC-SD), and PAH-sh-CYP1A1 cells (GBC-SD) in amounts twice that of the normal subcutaneous injection volume, to obtain corresponding subcutaneous xenograft tumor model mice;

[0083] Finally, the mice in each group were cultured for four weeks, measured once daily with calipers within the four weeks, and linear regression analysis was performed to calculate the tumor volume. The calculation formula was: a × b 2 × 0.5 (where a is the longest diameter and b is the shortest diameter). After culturing for four weeks, the mice were euthanized, the gallbladder cancer tumors of the mice were removed, the tumor size was evaluated, and the tumor weight was measured. The results are as Figure 4 shown in A and C.

[0084] 2. Western blot detection was performed on the gallbladder cancer tumor tissues of the mice obtained in point 1 of this example. The differences in the protein expression levels of AhR and CYP1A1 in the gallbladder cancer tumor tissues of the mice in each group were measured. The results are as Figure 4 shown in D.

[0085] 3. To detect the metastasis of GBC in mice, subcutaneous xenograft tumor model mice were constructed according to the method described in point 1 of this example. GBC cells stably expressing luciferase (1 × 10) were suspended in 200 μL of PBS buffer and injected into the lateral part of the tail vein of the mice. After four weeks, the mice were anesthetized with isoflurane and intraperitoneally injected with D-luciferin sodium salt (150 mg / kg). The GBC cells were detected using the in vivo imaging system Xenogen IVIS (PerkinElmer, Massachusetts, USA). The results are as Figure 4 shown in E.

[0086] 4. The mice obtained in point 6 of this example were euthanized, then the gallbladder cancer tumors of the mice were removed, the tumor conditions were evaluated, and the tumor tissues of the mice were collected for immunohistochemistry (IHC) detection. The results are as Figure 4 shown in F.

[0087] From Figure 4 A - C, it can be seen that after treatment with polycyclic aromatic hydrocarbons (PAH), the GBC-SD cells significantly increased the tumor growth rate, while shCYP1A1, sh-AhR, and sh-HEGBC inhibited the increase in tumor volume and weight induced by PAH (P < 0.001).

[0088] From Figure 4It was found that after treatment with polycyclic aromatic hydrocarbons (PAHs), the expression of AhR protein and CYP1A1 protein in GBC-SD cells promoted the growth of tumors in mice, while sh-AhR inhibited the increase in AhR protein levels induced by PAHs. shCYP1A1, sh-AhR, and sh-HEGBC reversed the upregulation of CYP1A1 protein levels induced by PAHs (P<0.05, P<0.01, P<0.001).

[0089] As Figure 4 shown in Fig. E, 4 weeks after tail vein injection, GBC-SD cells treated with polycyclic aromatic hydrocarbons (PAHs) significantly promoted cell metastasis, while shCYP1A1, sh-AhR, and sh-HEGBC inhibited the increase in tumor metastasis induced by PAHs.

[0090] As Figure 4 shown in Fig. F, sh-HEGBC and sh-AhR inhibited the increase in AhR protein levels in tumor tissues of mice induced by PAHs, while shCYP1A1, sh-AhR, and sh-HEGBC reversed the upregulation of CYP1A1 protein levels induced by PAHs.

[0091] Based on the above results, the inventors of the present application further confirmed that the CYP1A1 gene is a downstream effector gene of the HEGBC gene, and the HEGBC-CYP1A1 signaling pathway is involved in the development of gallbladder cancer mediated by HEGBC.

[0092] Example 5

[0093] Based on the results of Examples 1 to 4 above, the expression level of HEGBC in gallbladder cancer was abnormally upregulated, and was significantly negatively correlated with the poor prognosis of patients. At the same time, the inventors of the present application found that the HEGBC gene upregulated the CYP1A1 mRNA level by binding to AhR, thereby promoting the development of gallbladder cancer. Accordingly, the inventors deduced that simultaneously interfering with the expression levels of AhR, HEGBC, and CYP1A1 mRNA would play a significant anti-cancer role in gallbladder cancer. That is, by interfering with and inhibiting the AhR-HEGBC-CYP1A1 regulatory axis, the proliferation, migration, and invasion of gallbladder cancer cells can be effectively inhibited. Moreover, compared with interfering with a single mRNA, simultaneously interfering with the expression levels of AhR, HEGBC, and CYP1A1 mRNA would play a more significant anti-cancer role in gallbladder cancer.

[0094] Example 6

[0095] Mix sh-AhR, sh-HEGBC, and sh-CYP1A1 at a molar ratio of 1:1:1 to obtain sh-MIX. Then, use Lipofectamine 3000 (Catalog No. L3000015, Life Technologies, Carlsbad, CA) reagent to transfect GBC-SD cells with the same dose (volume 3 μL, concentration 10 μM) of sh-NC (sh-RNAs negative control, a conventional existing sequence) and sh-MIX respectively. After 48 hours of transfection, collect the cells and extract RNA, and perform reverse transcription-real-time fluorescence quantitative PCR. Using the ACTB mRNA level as an internal reference, calculate the relative expression levels of AhR, HEGBC, and CYP1A1 mRNA according to the formula 2 -ΔΔCT to detect the relative expression levels of AhR, HEGBC, and CYP1A1 mRNA.

[0096] According to the detection by reverse transcription-real-time fluorescence quantitative PCR, compared with the transfection of sh-NC, the transfection of sh-MIX can significantly down-regulate the expression levels of AhR, HEGBC, and CYP1A1 mRNA in GBC-SD cells.

[0097] Example 7

[0098] Use Lipofectamine 3000 (Catalog No. L3000015, Life Technologies, Carlsbad, CA) reagent to transfect GBC-SD cells with the same dose (volume 3 μL, concentration 10 μM) of sh-NC, sh-MIX, sh-AhR, sh-HEGBC, and sh-CYP1A1 respectively. After 48 hours of transfection, collect the transfected cells and re-culture the different transfected cells in 96-well plates (2,000 cells / well), divided into 5 groups in total, and use the CCK-8 assay to detect the changes in the viability of GBC-SD cells.

[0099] According to the detection by the CCK-8 assay, the cell proliferation ability of GBC-SD cells transfected with sh-MIX, sh-AhR, sh-HEGBC, and sh-CYP1A1 is significantly weaker than that of the control group cells. However, sh-MIX shows a more significant inhibitory effect on cell proliferation compared with the same dose of sh-AhR, sh-HEGBC, or sh-CYP1A1.

[0100] Example 8

[0101] The GBC-SD cells were transfected with the same dose (3 μL in volume, 10 μM in concentration) of sh-NC, sh-MIX, sh-AhR, sh-HEGBC, and sh-CYP1A1 using Lipofectamine 3000 (Catalog No. L3000015, Life Technologies, Carlsbad, CA). The transfected cells were collected 48 hours later and re-cultured in 96-well plates (2,000 cells / well). There were a total of 5 groups, and the Transwell assay was used to detect the cell migration and invasion abilities. [[ID=[]]] [[ID=[]]]

[0102] According to the results of the Transwell assay, under the condition of transfection with the same dose of sh-RNAs, compared with the transfection of sh-AhR, sh-HEGBC, or sh-CYP1A1, the GBC-SD cells transfected with sh-MIX showed weaker cell migration and invasion abilities. This indicates that simultaneously interfering with the expression levels of AhR, HEGBC, and CYP1A1 genes has a more significant anti-cancer effect than knocking down the expression of any one of these genes alone.

Claims

1. Application of the AhR-HEGBC-CYP1A1 regulatory axis as a drug target in the preparation of targeted drugs for treating gallbladder cancer.

2. The application according to claim 1, wherein The AhR-HEGBC-CYP1A1 regulatory axis includes the AhR gene, the HEGBC gene, and the CYP1A1 gene; the Gene ID of the AhR gene: 196; the Gene ID of the HEGBC gene: 401585; the Gene ID of the CYP1A1 gene: 1543.

3. The application according to claim 1, wherein The targeted drug for treating gallbladder cancer takes the AhR gene, the HEGBC gene, and the CYP1A1 gene as intervention targets, and based on interfering with AhR, HEGBC, and CYP1A1 mRNA, can efficiently and specifically inhibit the expression levels of the AhR gene, the HEGBC gene, and the CYP1A1 gene.

4. The application according to claim 1, characterized in that, The targeted drug for treating gallbladder cancer is siRNA, shRNA, microRNA, or a chemical inhibitor of AhR, HEGBC, and CYP1A1 mRNA.

5. Application of shRNA specifically interfering with the AhR gene, shRNA specifically interfering with the HEGBC gene, and / or shRNA specifically interfering with the CYP1A1 gene in the preparation of drugs for targeted treatment of gallbladder cancer.

6. The application according to claim 5, characterized in that, The targeted nucleotide sequence of the shRNA specifically interfering with the AhR gene is shown as SEQ ID NO.1-2; the targeted nucleotide sequence of the shRNA specifically interfering with the HEGBC gene is shown as SEQ ID NO.3-4; the targeted nucleotide sequence of the shRNA specifically interfering with the CYP1A1 gene is shown as SEQ ID NO.5-6.

7. The application according to claim 5, characterized in that, In the targeted drug for treating gallbladder cancer, the molar ratio of shRNA specifically interfering with the AhR gene, shRNA specifically interfering with the HEGBC gene, and shRNA specifically interfering with the CYP1A1 gene is 1:1:

1.

8. A targeted drug for treating gallbladder cancer, characterized in that, The targeted drug for treating gallbladder cancer contains shRNA specifically interfering with the AhR gene, shRNA specifically interfering with the HEGBC gene, and / or shRNA specifically interfering with the CYP1A1 gene.

9. The targeted drug for treating gallbladder cancer according to claim 8, wherein, The targeted nucleotide sequence of the shRNA specifically interfering with the AhR gene is shown as SEQ ID NO.1-2; the targeted nucleotide sequence of the shRNA specifically interfering with the HEGBC gene is shown as SEQ ID NO.3-4; the targeted nucleotide sequence of the shRNA specifically interfering with the CYP1A1 gene is shown as SEQ ID NO.5-6.

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