ATP6V1C1 and application of ATP6V1C1 inhibitor in preparation of medicine for inhibiting lung cancer metastasis or drug resistance

Abstract

The invention discloses application of ATP6V1C1 and an inhibitor thereof in preparing a medicine for inhibiting lung cancer or lung cancer metastasis, and belongs to the technical field of medicines. It is found for the first time that ATP6V1C1 can be used as a treatment target of lung cancer or lung cancer metastasis, and inhibition of ATP6V1C1 can effectively treat lung cancer or inhibit lung cancer metastasis. The invention provides a new important target for lung cancer treatment, and has important clinical significance and application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to ATP6V1C1 and its inhibitors in the preparation of drugs for inhibiting lung cancer or lung cancer metastasis. Background Art

[0002] Lung cancer is a malignant tumor of the bronchial mucosa or glands. Due to the exacerbation of population aging, environmental deterioration caused by the rapid development of industrialization and urbanization brought about by rapid economic growth, and the high smoking rate, the harm of lung cancer is gradually emerging.

[0003] There are significant differences in pathological characteristics, pathogenesis and treatment responses among different types of lung cancer. The efficacy of traditional chemotherapy regimens is limited, with a five-year survival rate of less than 15%. Radiotherapy has relatively large side effects, which limit the long-term effectiveness of chemotherapy. Although targeted therapy has opened the door to precision medicine, the effective rate of drugs targeting rare targets such as mutations is relatively low. Although immunotherapy has shown strong and durable efficacy in some patients with metastatic lung cancer, there is a lack of highly efficient markers to predict its effect. Therefore, it is necessary to strengthen research and exploration in order to find more effective treatment methods.

[0004] ATP6V1C1 is a subtype on the C subunit of vacuolar proton-translocating ATPase (V-ATPase). V-ATPase is composed of a hydrolytic ATP domain (V1) and a proton translocation domain (V0). The V0 domain is composed of a, c, c’, c”, d and e subunits, and six or more c subunits form a ring structure. The V1 domain is located in the cytoplasm and contains eight different subunits (A-H). There are three subtypes of the C subunit (red arrow) of the V-ATPase domain V1: ATP6V1C1, ATP6V1C2a and ATP6V1C2b. As a key component of V-ATPase, ATP6V1C1 is responsible for the assembly and function of the catalytic region. In osteoclasts, ATP6V1C1 may play an important role in regulating the formation of the sealing zone F-actin ring, which is related to the activation and function of osteoclasts. ATP6V1C1 has also been found to affect the radiation tolerance of cells. Upregulating this gene can enhance the cell proliferation ability after radiation damage, indicating that ATP6V1C1 also plays an important role in cell stress response and anti-radiation mechanism.

[0005] There is currently no relevant research on what important role ATP6V1C1 plays in lung cancer cells. Summary of the Invention

[0006] To solve the problems existing in the prior art, the present invention provides the use of ATP6V1C1 as a therapeutic target for lung cancer.

[0007] The present invention provides the use of an ATP6V1C1 inhibitor in the preparation of a drug for inhibiting lung cancer, inhibiting lung cancer metastasis or reducing the resistance of lung cancer to EGFR-TKI; the ATP6V1C1 inhibitor is a reagent for inhibiting the expression level of ATP6V1C1 or a substance for inhibiting the activity of ATP6V1C1.

[0008] Among them, the reagent for inhibiting the expression level of ATP6V1C1 is a reagent for knocking out the ATP6V1C1 gene, knocking down the ATP6V1C1 gene, or silencing the expression of the ATP6V1C1 gene.

[0009] Among them, the reagent for silencing the expression of the ATP6V1C1 gene includes siRNA for silencing the expression of the ATP6V1C1 gene. Preferably, the siRNA includes siATP6V1C1-1, siATP6V1C1-2, and siATP6V1C1-3;

[0010] The nucleotide sequence of the siATP6V1C1-1 is shown in SEQ ID NO.2 to 3;

[0011] The nucleotide sequence of the siATP6V1C1-2 is shown in SEQ ID NO.4 to 5;

[0012] The nucleotide sequence of the siATP6V1C1-3 is shown in SEQ ID NO.6 to 7.

[0013] Among them, the reagent is dronedarone hydrochloride.

[0014] The present invention also provides a drug for inhibiting lung cancer, inhibiting lung cancer metastasis or reducing the resistance of lung cancer to EGFR-TKI. It is a pharmaceutical preparation prepared with an ATP6V1C1 inhibitor as an active ingredient and pharmaceutically acceptable excipients or auxiliary components; the ATP6V1C1 inhibitor is a reagent for inhibiting the expression level of ATP6V1C1 or a substance for inhibiting the activity of ATP6V1C1.

[0015] Among them, the reagent for inhibiting the expression level of ATP6V1C1 is a reagent for knocking out the ATP6V1C1 gene, knocking down the ATP6V1C1 gene, or silencing the expression of the ATP6V1C1 gene.

[0016] Among them, the reagent for silencing the expression of the ATP6V1C1 gene includes siRNA for silencing the expression of the ATP6V1C1 gene. Preferably, the siRNA includes siATP6V1C1-1, siATP6V1C1-2, and siATP6V1C1-3;

[0017] The nucleotide sequence of the siATP6V1C1-1 is shown as SEQ ID NO.2-3;

[0018] The nucleotide sequence of the siATP6V1C1-2 is shown as SEQ ID NO.4-5;

[0019] The nucleotide sequence of the siATP6V1C1-3 is shown as SEQ ID NO.6-7.

[0020] Wherein, the substance is dronedarone hydrochloride.

[0021] The present invention finally provides the use of dronedarone hydrochloride combined with siATP6V1C1 in the preparation of a drug for inhibiting lung cancer, inhibiting lung cancer metastasis or reducing the drug resistance of lung cancer to EGFR-TKI, and the siRNA includes siATP6V1C1-1 and siATP6V1C1-2, as well as siATP6V1C1-3;

[0022] The nucleotide sequence of the siATP6V1C1-1 is shown as SEQ ID NO.2-3;

[0023] The nucleotide sequence of the siATP6V1C1-2 is shown as SEQ ID NO.4-5;

[0024] The nucleotide sequence of the siATP6V1C1-3 is shown as SEQ ID NO.6-7.

[0025] In the present invention, the ATP6V1C1 inhibitor can be a gene drug, a chemical drug or a biological drug. This drug can target ATP6V1C1 at the gene level or protein level, and can inhibit the expression of ATP6V1C1 protein by silencing the gene or blocking the transcription and translation of ATP6V1C1, or block its downstream signal transduction by inhibiting the active site of the ATP6V1C1 protein, and finally achieve the purpose of inhibiting lung cancer, inhibiting lung cancer metastasis and reducing the drug resistance of lung cancer to EGFR-TKI.

[0026] Obviously, according to the above content of the present invention, according to the common general technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, other various forms of modifications, substitutions or changes can be made.

[0027] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 ATP6V1C1 promotes tumor cell migration. A. A549 cells were transfected with multiple siRNAs, and the protein level of ATP6V1C1 was detected by Western blot 48 h later; B. The mRNA level of ATP6V1C1 was detected by qRT-PCR 24 h later; C. Fluorescence microscopy showed the lentivirus transfection efficiency (4×); D. The protein levels of ATP6V1C1 in stably transfected A549 cells with knockdown and overexpression were detected by Western blot; E-F. The mRNA levels of ATP6V1C1 in stably transfected A549 cells with knockdown and overexpression were detected by qRT-PCR; G. The change in cell migration ability after knocking down ATP6V1C1 in A549 / PC9 / H1975 cells was detected by Transwell assay (10×); H. The bar graph showed the change in cell migration ability after knocking down ATP6V1C1 in A549 / PC9 / H1975 cells; I. The change in migration ability of A549 cells with stable knockdown / overexpression of ATP6V1C1 was detected by Transwell assay (10×); J. The bar graph showed the change in migration ability of A549 cells with stable knockdown / overexpression of ATP6V1C1 (**, P < 0.01; unpaired t-test).

[0029] Figure 2 ATP6V1C1 promotes tumor cell metastasis in vivo; A. In vivo live imaging showed the tumor growth and metastasis in mice; B. Day 40 showed the magnetic resonance imaging (T2) of mouse #1 in the OV.ATP6V1C1 group; CD. Gross morphology and HE staining of primary lung adenocarcinoma PDX tissues. E. Immunohistochemistry showed the expression of ATP6V1C1 in tumor tissues of different groups; F. The survival curve showed the survival time of mice in different groups (P > 0.05).

[0030] Figure 3ATP6V1C1 expression level and Dro-Hcl treatment sensitivity; A. Immunohistochemistry shows the ATP6V1C1 expression levels in EGFR-L858R PDX of different treatment groups (10×); B. Column chart shows the immunohistochemical ATP6V1C1 expression levels in tumor tissues of each group (*, P < 0.05; **, P < 0.01; unpaired t-test); C. Line chart shows the inhibitory effect of Dro-Hcl on the proliferation of subcutaneous lung adenocarcinoma in mice (n = 5, 5 mg / kg, dronedarone hydrochloride intraperitoneal injection, once every 2 days); D. In vivo live imaging shows the growth and metastasis of lung adenocarcinoma overexpressing ATP6V1C1 in the lungs of mice (day 10 / 40, dronedarone hydrochloride 15 mg / kg, intraperitoneal injection, once every other day); -siATP6V1C1 / siATP6V1C1-1: treated with siATP6V1C1; siNC / NC / Control: corresponding blank control group; Dro-Hcl / Dronedarone hydrochloride: treated with dronedarone hydrochloride.

[0031] Figure 4 Treatment of lung adenocarcinoma PDX with Dro-Hcl combined with siATP6V1C1; A-B. Line charts show the changes in relative tumor volume (RTV) of EGFR-L858R PDX NOD-SCID mice treated with Dro-Hcl (5 mg / kg), gefitinib (5 mg / kg), siNC (1 OD), and siATP6V1C1 (1 OD) carriers (mean ± standard deviation, unpaired t-test); C. Line chart shows the changes in relative tumor volume (RTV) of EGFR-790M PDX NOD-SCID mice treated with Dro-Hcl (5 mg / kg), siNC (1 OD), and siATP6V1C1 (1 OD) carriers (mean ± standard deviation, unpaired t-test); D. Molecular docking of ATP6V1C1 and Dro-Hcl was simulated using AutoDock software. Detailed implementation manners

[0032] The raw materials and equipment used in the specific implementation manners of the present invention are all known products and are obtained by purchasing commercially available products.

[0033] Among them, the nucleotide sequence of the target gene ATP6V1C1 specifically involved is shown as NCBI Gene: 528, Ensembl: ENSG00000155097; the siRNA used to silence the target gene ATP6V1C1 targets a specific nucleotide sequence SEQ ID NO.1 in the target gene: GGACTTGGTTACTTATATA;

[0034] Nucleotide sequence of siATP6V1C1-1:

[0035] Sense GUUCGUGACUUCCAGUAUATT(SEQ ID NO.2)

[0036] antisense UAUACUGGAAGUCACGAACTT(SEQ ID NO.3);

[0037] Nucleotide sequence of siATP6V1C1-2:

[0038] Sense CUACUUCAGCCCAAUAAGATT(SEQ ID NO.4):

[0039] antisense UCUUAUUGGGCUGAAGUAGTT(SEQ ID NO.5):

[0040] Nucleotide sequence of siATP6V1C1-3:

[0041] Sense GUUCGUGACUUCCAGUAUATT(SEQ ID NO.6):

[0042] antisense UAUACUGGAAGUCACGAACTT(SEQ ID NO.7)

[0043] Nucleotide sequence of siNC blank control:

[0044] Sense UUCUCCGAACGUGUCACGUTT(SEQ ID NO.8)

[0045] antisense ACGUGACACGUUCGGAGAATT(SEQ ID NO.9)

[0046] Chemically synthesized by Tsingke (China) Co., Ltd.

[0047] In previous experiments, lung cancer cell lines: A549, PC-9, H1975 and primary tumor cells LC-HX01 from lung cancer patients were used as in vitro models. Six designed siRNAs were used to silence ATP6V1C1 in lung cancer cells. After 24 hours, western blot experiments ( Figure 1 A) and RT-PCR experiments ( Figure 1B), experiments showed that the silencing efficiency of siRNA-2, siRNA-4, and siRNA-5 on ATP6V1C1 was higher than 80%, and they were named siATP6V1C1-1 and siATP6V1C1-2 respectively. Another siATP6V1C1-3 was used to design shRNA lentivirus, and at the same time, an overexpressed ATP6V1C1-EGFP lentivirus was designed for verifying the correlation between ATP6V1C1 and lung cancer progression.

[0048] Example 1 Verification of the correlation between ATP6V1C1 and lung cancer progression

[0049] I. In vitro experiments

[0050] 1. Method

[0051] 1.1 Use lentivirus to transfect adherent cells to construct OV.NC / ATP6V1C1 and sh.NC / ATP6V1C1 cells. Among them, OV.NC cells were transfected with a blank control lentivirus without GFP protein and without integrated siATP6V1C1; OV.ATP6V1C1 cells were transfected with an overexpressed ATP6V1C1-EGFP lentivirus; sh.NC cells were transfected with a blank control lentivirus with GFP label and without integrated siATP6V1C1; sh.ATP6V1C1 cells were transfected with a GFP-labeled ATP6V1C1-silencing lentivirus (i.e., the shRNA lentivirus designed by siATP6V1C1-3). Taking A549 cells as an example for the specific transfection method:

[0052] ① Inoculate cells with good growth status into a 24-well plate at a density of 3 plates, with a density of 5 / ml, and place it in a 37°C incubator with 5% CO2 to ensure that the cell confluence rate reaches 30% - 50% when infected with the virus the next day.

[0053] ② Use an appropriate amount of virus for infection (the amount of virus per well (μl) = MOI × number of cells / virus titer (TU / ml) × 1000), and at the same time add the transfection reagent polybrene (1 μg / ml).

[0054] ③ 24 hours after virus transfection, aspirate the culture medium containing the virus and replace it with fresh complete culture medium, and place it in a 37°C incubator with 5% CO2.

[0055] ④ After infection, wait until the cell state is stable and the cell confluence is about 80% - 90%, and replace it with fresh complete culture medium containing 8 μg / ml Puromycin to screen for stably transfected cell lines. For lentiviruses carrying the GFP gene, fluorescence can be observed under a fluorescence microscope to calculate the transfection efficiency.

[0056] 1.2 Detection of mRNA levels of ATP6V1C1 in A549 / HCC827 / PC9 cells transfected with siRNA by qRT-PCR

[0057] Total RNA was extracted from cells 24 h after siRNA transfection using TRIzol reagent (ThermoFisher) according to the manufacturer's instructions. cDNA was reverse-transcribed using a reverse transcription kit (Vazyme). Using 100 ng of cDNA as a template, qRT-PCR reactions were performed using a 2×SYBR Green PCR kit (Vazyme) and a Bio-Rad CFX real-time PCR system. The data were normalized using the expression of GAPDH and ACTIN as internal references.

[0058] 1.3 Detection of protein expression levels of ATP6V1C1 in A549 / HCC827 / PC9 cells transfected with siRNA by Western blot

[0059] Cells 48 h after siRNA transfection were lysed in RIPA buffer containing protease inhibitors. The total protein concentration was determined using the BCA method. Equal amounts of protein were separated using a 4-20% SDS-PAGE gel. After electrophoresis, the proteins were transferred to a PVDF membrane, blocked with 5% bovine serum albumin, and then incubated overnight at 4°C with a primary antibody against ATP6V1C1 (Abclonal). After washing 3 times with TBST, the membrane was incubated with an HRP-conjugated goat anti-rabbit or anti-mouse secondary antibody (Abclonal) for 2 h at room temperature. The gray value of the protein bands was observed using enhanced chemiluminescence.

[0060] 2. Results

[0061] Based on lentiviruses for silencing and overexpression, stable cell models for silencing and overexpressing ATP6V1C1 were constructed respectively. Under a fluorescence microscope, it could be observed that both the silencing control group (sh.NC) and the silencing target gene group (sh.ATP6V1C1) cells could stably express the viral vector with green fluorescent protein; the overexpressing target gene group (OV.ATP6V1C1) cells could stably express ATP6V1C1-EGFP green fluorescent protein, and the control group (OV.NC) cells did not express ATP6V1C1-EGFP( Figure 1 C).

[0062] The expression of ATP6V1C1 was qualitatively and quantitatively analyzed by western blot and RT-PCR experiments, Figure 1 D, Figure 1 E, F demonstrated that the stable transfected strains had good silencing and overexpression effects.

[0063] On this basis, in A549, PC-9, and H1975 cell lines respectively, siATP6V1C1-1 and siATP6V1C1-2 were used to silence the expression of ATP6V1C1, and the migration changes of lung cancer cells before and after silencing were studied through transwell experiments. Crystal violet staining observation showed that compared with the blank control group (siNC), both siATP6V1C1-1 and siATP6V1C1-2 could reduce the migration of lung cancer cells ( Figure 1 G), and there were significant statistical differences ( Figure 1 H). Using the A549 stable transfected cell line, it was observed that compared with the control group, the number of migrated cells in the ATP6V1C1-silenced stable transfected cell line was significantly reduced, and the number of migrated cells in the ATP6V1C1-overexpressing stable transfected cell line was significantly increased, and there were significant statistical differences ( Figure 1 I). Further, stable transfected cell lines with silenced and overexpressed ATP6V1C1 were constructed using the primary tumor cells LC-HX01 of lung cancer patients, and the results were consistent with those of the A549 stable transfected cell line ( Figure 1 J).

[0064] It can be seen from the above in vitro experiments that siATP6V1C1-1, siATP6V1C1-2, and siATP6V1C1-3 have good silencing effects on the ATP6V1C1 gene in lung cancer cell lines and can be used as ATP6V1C1 inhibitors; transfecting lung cancer cell lines with siATP6V1C1-1 and siATP6V1C1-2 can reduce the migration of lung cancer cells, thereby playing a role in inhibiting the metastasis of lung cancer, indicating that ATP6V1C1 inhibitors have the effect of inhibiting the metastasis of lung cancer.

[0065] II. In vivo experiments

[0066] 1. Methods

[0067] 1.1 Construction of primary tumor cells

[0068] ① For primary lung adenocarcinoma, the tumor tissue was mechanically chopped and digested in DMEM / F12 (Gibco, 10565018) supplemented with 2.5% heat-inactivated FBS with 1 mg / ml collagenase I (Gibco, 17101015) and 0.5 mg / ml collagenase IV (Gibco, 171041019) on a shaker at 37°C for 30 minutes, terminated with sterile PBS, and filtered through a 100 μm sieve.

[0069] ② The obtained cell suspension was centrifuged at 4°C and 1200 g for 5 min to remove the supernatant. The precipitated cells were washed once with PBS, and a single-cell suspension was prepared by adding complete medium.

[0070] ③For primary tumor cells, the cells were cultured in DMEM (Gibco, 10313021) with 10% FBS and 1% PSN medium.

[0071] ④Primary adherent lung adenocarcinoma cells were transfected with lentivirus. The specific operation was the same as the steps of constructing OV.NC / ATP6V1C1 and sh.NC / ATP6V1C1 cells in item 1.1 of "I. In vitro experiments".

[0072] 1.2 Construction of orthotopic lung adenocarcinoma PDX model

[0073] ①Primary PDC-LUAD was transfected with HBLV-luciferase-PURO (HANBIO) and purified with Puromycin to construct PDC-LUAD-LUC cells. Then, the cells were transfected with sh.NC / sh.ATP6V1C1 / OV.NC / OV.ATP6V1C1 lentivirus to construct PDC-LUAD-LUC-sh.NC / sh.ATP6V1C1 / OV.NC / OV.ATP6V1C1 cell lines.

[0074] ②D-luciferin was dissolved in complete medium to prepare a working solution of 150 μg / ml. Cells were seeded in gradient in a 96-well cell culture plate, 1× luciferin working solution was added, and then the luciferase activity of the cells was detected by a multifunctional microplate reader in LUC mode for image analysis. The image analysis showed that the cells showed gradient bioluminescence signals for subsequent experiments.

[0075] ③6- to 8-week-old Balb / c nod / scid mice were randomly divided into a control group, an ATP6V1C1 overexpression group, a group with silenced ATP6V1C1, and a silenced control group. They were anesthetized with isoflurane gas, the hair on the left chest wall of the mice was removed, the skin was incised with scissors, the subcutaneous tissue was separated to the parietal pleura, and about 5×10 6 cells

[0076] PDC-LUAD-LUC-OV.NC / ATP6V1C1 / PDC-LUAD-LUC-sh.NC / sh.ATP6V1C1 cells were inoculated into the left lung of the mice. Among them, the control group was inoculated with PDC-LUAD-LUC-OV.NC, the ATP6V1C1 overexpression group was inoculated with PDC-LUAD-LUC-OV.ATP6V1C1, the group with silenced ATP6V1C1 was inoculated with PDC-LUAD-LUC-sh.ATP6V1C1 cells, and the silenced control group was inoculated with

[0077] PDC-LUAD-LUC-sh.NC, and then the incision was sutured.

[0078] ④ From the second week after the operation, the mice were examined weekly using a small animal in vivo imager. The mice were anesthetized with isoflurane gas. A fluorescein working solution at a dose of 150 mg / kg was administered at a volume of 10 μL / g. (For example, a 10 g mouse was injected with 100 μL of the working solution and given 1.5 mg of fluorescein.) Imaging analysis was performed 10 - 15 minutes after intraperitoneal injection of fluorescein.

[0079] ⑤ Record the tumor location and the luciferase signal intensity. Use LivingImage software (PerkinElmer) to obtain images. Select the lucifiearse mode for shooting, set the exposure time to automatic, and normalize all images using the same scale.

[0080] 1.3 Treatment experiments on the PDX model of lung adenocarcinoma

[0081] (1) Two LUAD PDX models from the PDX model biobank established in previous studies were used: ① PDX-L858R was derived from a patient with EGFR exon 21 L858R who had not received drug treatment. Its characteristic is sensitivity to the first-generation TKI gefitinib and the chemotherapy drug cisplatin. ② PDX-790M was derived from a patient with EGFR exon 20 T790M. Its characteristic is resistance to the first-generation TKI gefitinib and sensitivity to the chemotherapy drug cisplatin.

[0082] (2) Use vernier calipers to measure the major axis and minor axis of the subcutaneous tumor tissue of the mice in the LUAD PDX model. The tumor volume of the mice was calculated as tumor volume d (mm3) = major axis l (mm) × minor axis w2 (mm) / 2. When the tumor volume was approximately 150 mm3, drug intervention was performed on the mice.

[0083] (3) Randomly divide the mice into 6 groups (n > 5): control group (Control), gefitinib treatment group (Gefitinib), dronedarone hydrochloride treatment group (Dro-Hcl), gefitinib + dronedarone hydrochloride group (Gefitinib+Dro-Hcl), dronedarone hydrochloride + siNC treatment group (siNC+Dro-Hcl), dronedarone hydrochloride + siATP6V1C1 treatment group (siATP6V1C1-1+Dro-Hcl). The drugs in each of the above groups were intraperitoneally injected into the mice once every 2 days. The injection dose of dronedarone hydrochloride was 5 mg / kg, and the injection dose of gefitinib hydrochloride was 2.5 mg / kg. siNC and siATP6V1C1 were infiltrated into the subcutaneous tumor tissue of the mice once every 2 days at a dose of 1 OD + 25 μL of Entranste.

[0084] (4) Tumor progression was calculated using the relative tumor volume (RTV), and the calculation formula was: RTV = Vt / V0. Where V0 was the tumor volume at the time of drug administration (i.e., d0), and Vt was the tumor volume at each drug administration (i.e., dn).

[0085] 2. Results

[0086] After transfecting the stable cell line constructed from the patient's primary cells with luciferase lentivirus, it was surgically injected into the lungs of NOD·SCID mice to construct an orthotopic transplantation model of patient-derived lung cancer cells, with 8 mice in each group. The progression of lung cancer in mice was observed by in vivo imaging every 10 days. At the 40th day, 3 mice in the group overexpressing ATP6V1C1 showed distant metastases, while no metastases occurred in the control group ( Figure 2 A). Multiple distant metastases in the group overexpressing ATP6V1C1 were further confirmed by MRI magnetic resonance imaging ( Figure 2 B). Anatomical observation of the lung tissues of mice in each group showed that compared with the control group, a large number of nodules appeared in both lungs in the group overexpressing ATP6V1C1 ( Figure 2 C), and HE staining showed that the number of lung metastases in the group overexpressing ATP6V1C1 increased significantly ( Figure 2 D), indicating that overexpression of ATP6V1C1 could promote lung cancer metastasis. Compared with the silencing control group, about 2 nodules were visible in the lungs of 3 mice, and only in-situ lesions were seen in each mouse in the group silencing ATP6V1C1, indicating that silencing of ATP6V1C1 could inhibit lung cancer metastasis ( Figure 2 C, D). The expression level of ATP6V1C1 in the lung cancer tissues of each group was detected by immunohistochemical staining, further verifying that good silencing and overexpression effects were maintained in vivo for lung cancer ( Figure 2 E). After the mice in each group died naturally, survival rates were statistically analyzed. The results showed that silencing of ATP6V1C1 could significantly increase the overall survival rate of mice, while overexpression had the opposite effect ( Figure 2 F).

[0087] Further drug treatment of the constructed orthotopic lung adenocarcinoma PDX model found that: Dronedarone hydrochloride could significantly inhibit the expression of ATP6V1C1 in the tumor tissues of the lung cancer PDX model ( Figure 3 A), and its in vivo inhibition efficiency was close to that of the positive control group siATP6V1C1 ( Figure 3 B). The tumor tissues of lung cancer patients were inoculated subcutaneously into NOD·SCID mice to construct a lung cancer PDX model, and a pharmacodynamic experiment was carried out by intraperitoneal injection of Dronedarone hydrochloride. The results showed that Dronedarone hydrochloride could significantly inhibit the growth of lung cancer ( Figure 3C). After further orthotopically inoculating primary cells of patients in the control group (OV.NC) and the ATP6V1C1 overexpression group (OV.ATP6V1C1) into NOD·SCID mice and then intraperitoneally injecting dronedarone hydrochloride, it was observed by in vivo imaging 40 days later that the orthotopic lung cancer in the control group mice significantly shrank or progressed slowly, while half of the mice in the ATP6V1C1 overexpression group died and there was no obvious therapeutic effect. Figure 3 D), indicating that overexpression of ATP6V1C1 eliminated the therapeutic effect of dronedarone hydrochloride and conversely proving that it might be an inhibitory target of dronedarone hydrochloride.

[0088] In vivo verification was carried out using lung adenocarcinoma PDX mice with EGFR-L858R, and the results showed Figure 4 A) After using siATP6V1C1-1 to inhibit the expression of ATP6V1C1, the inhibitory effect of dronedarone hydrochloride on lung adenocarcinoma could be increased. Further, it was also found that Figure 4 B), combining dronedarone hydrochloride with gefitinib had a better inhibitory effect on tumor growth. In addition, verification using EGFR-T790M mutant lung adenocarcinoma PDX mice resistant to first-generation TKIs found that Figure 4 C) In EGFR-resistant lung cancer, by inhibiting the expression of ATP6V1C1, the therapeutic sensitivity of dronedarone hydrochloride to tumors could be improved.

[0089] In view of the effect of the ATP6V1C1 inhibitor in the treatment of lung cancer, further research and analysis were carried out on its mechanism of action with the target. Specifically, through the X-ray crystal structure of ATP6V1C1 (PDB: 6WM2) in the protein database, the protonation state of the small molecule was set to pH = 7.4, and the compound was expanded to a 3D structure using Open Babel. A series of preparations were carried out on the receptor protein and the ligand using the AutoDock tool (ADT3). The docking box was generated by the AutoGrid program, and molecular docking was carried out using AutodockVina (1.2.0). The optimal binding conformation was selected to analyze the interactions. Figure 4 D). The discovered interaction forces were analyzed and classified according to their interactions. The receptor protein and the small molecule directly formed multiple groups of interaction forces. For example, LYS298 of ATP6V1C1 formed a hydrogen bond with the dronedarone hydrochloride ligand. Under the action of these interaction forces, the binding energy of the protein-small molecule complex was -6.0 kcal / mol, showing good overall performance, thus playing a significant role in inhibiting the expression of ATP6V1C1.

[0090] From the above in-vivo experiments, it can be seen that silencing ATP6V1C1 can inhibit lung cancer metastasis and significantly increase the overall survival rate of lung cancer mice; dronedarone hydrochloride can inhibit the expression of ATP6V1C1 in lung cancer tissues, and it is an ATP6V1C1 inhibitor, which can significantly inhibit the growth of lung cancer by inhibiting the expression of ATP6V1C1; the combined application of dronedarone hydrochloride and siATP6V1C1 can reduce the EGFR-TKI drug resistance of lung cancer and improve the therapeutic effect of lung cancer.

[0091] In summary, this invention has demonstrated through in-vitro and in-vivo experiments that silencing ATP6V1C1 or inhibiting the activity / expression of ATP6V1C1 can effectively inhibit lung cancer, inhibit lung cancer metastasis, and reduce the EGFR-TKI drug resistance of lung cancer. This invention provides an important target for clinical treatment and has important clinical significance and application prospects.

Claims

1. Use of an ATP6V1C1 inhibitor in the preparation of a drug for inhibiting lung cancer, inhibiting lung cancer metastasis, or reducing EGFR-TKI resistance in lung cancer, characterized in that: The ATP6V1C1 inhibitor is an agent that inhibits the expression level of ATP6V1C1 or a substance that inhibits the activity of ATP6V1C1.

2. The use according to claim 1, characterized in that: The reagent for inhibiting the expression level of ATP6V1C1 is a reagent for knocking out the ATP6V1C1 gene, knocking down the ATP6V1C1 gene, or silencing the expression of the ATP6V1C1 gene.

3. The use according to claim 2, characterized in that: The reagent for silencing the expression of the ATP6V1C1 gene includes siRNA for silencing the expression of the ATP6V1C1 gene.

4. The use according to claim 3, characterized in that: The siRNA includes siATP6V1C1-1, siATP6V1C1-2, and siATP6V1C1-3; The nucleotide sequence of the siATP6V1C1-1 is shown in SEQ ID NOs. 2 to 3; The nucleotide sequence of the siATP6V1C1-2 is shown in SEQ ID NOs. 4 to 5; The nucleotide sequence of the siATP6V1C1-3 is shown in SEQ ID NOs. 6 to 7.

5. The use according to claim 1, characterized in that: The reagent is dronedarone hydrochloride.

6. A drug for inhibiting lung cancer, inhibiting lung cancer metastasis, or reducing EGFR-TKI resistance in lung cancer, characterized in that: The invention relates to a pharmaceutical preparation which is prepared by taking an ATP6V1C1 inhibitor as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients; the ATP6V1C1 inhibitor is a reagent for inhibiting the expression level of ATP6V1C1 or a substance for inhibiting the activity of ATP6V1C1.

7. The use according to claim 6, characterized in that: The reagent for inhibiting the expression level of ATP6V1C1 is a reagent for knocking out the ATP6V1C1 gene, knocking down the ATP6V1C1 gene, or silencing the expression of the ATP6V1C1 gene; The reagent for silencing the expression of the ATP6V1C1 gene includes siRNA for silencing the expression of the ATP6V1C1 gene.

8. The use according to claim 7, characterized in that: The siRNA includes siATP6V1C1-1, siATP6V1C1-2, and siATP6V1C1-3; The nucleotide sequence of the siATP6V1C1-1 is shown in SEQ ID NOs. 2 to 3; The nucleotide sequence of the siATP6V1C1-2 is shown in SEQ ID NOs. 4 to 5; The nucleotide sequence of the siATP6V1C1-3 is shown in SEQ ID NOs. 6 to 7.

9. The use according to claim 7, characterized in that: The substance is dronedarone hydrochloride.

10. Use of dronedarone hydrochloride in combination with siATP6V1C1 in the preparation of a drug for inhibiting lung cancer, inhibiting lung cancer metastasis, or reducing EGFR-TKI resistance in lung cancer, characterized in that: The siRNA includes siATP6V1C1-1, siATP6V1C1-2, and siATP6V1C1-3; The nucleotide sequence of the siATP6V1C1-1 is shown in SEQ ID NOs. 2 to 3; The nucleotide sequence of the siATP6V1C1-2 is shown in SEQ ID NOs. 4 to 5; The nucleotide sequence of the siATP6V1C1-3 is shown in SEQ ID NOs. 6 to 7.

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