Use of NLN neurolysin in the preparation of drugs for treating lung cancer

Abstract

The present invention provides a use of NLN neurolysin in preparing a drug for preventing and treating lung cancer, and the present invention belongs to the field of medical science. It is first found that the level of NLN neurolysin in lung cancer tissue of lung cancer patients is significantly higher than that in adjacent cancer tissue. The present invention verifies that knocking out the NLN neurolysin gene can effectively prevent and treat lung cancer through gene knockout, and the present invention provides a target and treatment strategy for clinical treatment of lung cancer.

Description

Technical Field

[0001] The invention belongs to the field of medical science, and specifically relates to the use of NLN neurolysin in preparing medicine for preventing and treating lung cancer. Background Art

[0002] Lung cancer is one of the most common malignant tumors in the world. Its morbidity and mortality rates are increasing year by year. Currently, its morbidity ranks first in the world, which seriously threatens human health and life. There are many treatments for lung cancer. Although new treatments such as targeted therapy and immunotherapy have achieved good results in recent years, traditional treatments including surgery, radiotherapy, and chemotherapy still occupy an important position. Drug therapy has become the main treatment for lung cancer, and further screening of targets that can be used for early treatment of lung cancer is needed.

[0003] The protein NLN (Q9BYT8-NEUL_HUMAN) neurolysin is mainly distributed in mitochondria. It contains 704 amino acids and its main function is to hydrolyze oligopeptides such as neuroserine. Current studies have found that NLN neurolysin has a related role in alcoholic cardiomyopathy.

[0004] There is no prior art that shows NLN neurolysin is associated with lung cancer. Summary of the invention

[0005] The purpose of the present invention is to provide a use of an NLN neurolysin inhibitor in preparing a drug for preventing and treating lung cancer.

[0006] The present invention provides use of an NLN neurolysin inhibitor in preparing a medicine for treating lung cancer and / or inhibiting lung cancer metastasis.

[0007] Furthermore, the NLN neurolysin inhibitor is an agent that reduces the expression of NLN neurolysin or an agent that inhibits the activity of NLN neurolysin.

[0008] Furthermore, the agent for reducing the expression of NLN neurolysin is an agent for knocking out or knocking down the NLN neurolysin gene;

[0009] Furthermore, the knockout or knockdown NLN neurolysin gene expression inhibitor is a siRNA with sequence number SEQ ID NO.1, and / or SEQ ID NO.2, and / or SEQ ID NO.3.

[0010] Furthermore, the agent for inhibiting the activity of NLN neurolysin is the following compound:

[0011]

[0012] Furthermore, the drug is a drug that inhibits the migration of lung cancer cells; the drug is a drug that reduces the proliferation rate of lung cancer cells; further, the lung cancer includes lung adenocarcinoma and lung squamous cell carcinoma.

[0013] The present invention also provides a drug for treating lung cancer and / or inhibiting lung cancer metastasis, which is a preparation prepared by using NLN neurolysin inhibitor as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients;

[0014] Furthermore, the NLN neurolysin inhibitor is a drug that knocks out the NLN neurolysin gene or reduces the activity / expression of NLN neurolysin.

[0015] The present invention also provides a use of a reagent for detecting the expression level of NLN neurolysin in preparing a lung cancer and / or lung cancer prognosis screening kit.

[0016] Furthermore, the reagents for detecting the expression level of NLN neurolysin include reagents for enzyme-linked immunosorbent assay, reagents for immunoblotting, reagents for immunoelectrophoresis, reagents for tissue immunostaining, reagents for immunoprecipitation analysis, reagents for radioimmunoassay, reagents for radioimmunodiffusion, reagents for complement fixation analysis, reagents for fluorescence-activated cell sorting, reagents for mass analysis or reagents for protein microarray.

[0017] Furthermore, the reagent for detecting NLN neurolysin is used to detect NLN neurolysin in lung tissue.

[0018] The present invention also provides a lung cancer and / or lung cancer prognosis screening kit, which comprises a reagent for detecting NLN neurolysin.

[0019] Furthermore, the reagents for detecting the expression level of NLN neurolysin include reagents for enzyme-linked immunosorbent assay, reagents for immunoblotting, reagents for immunoelectrophoresis, reagents for tissue immunostaining, reagents for immunoprecipitation analysis, reagents for radioimmunoassay, reagents for radioimmunodiffusion, reagents for complement fixation analysis, reagents for fluorescence-activated cell sorting, reagents for mass analysis or reagents for protein microarray.

[0020] The present invention provides a use of NLN neurolysin in the preparation of a drug for treating lung cancer. The present invention finds for the first time that the level of NLN neurolysin in lung cancer tissue of lung cancer patients is significantly higher than that in adjacent tissues. The present invention verifies that knocking out the NLN neurolysin gene can effectively prevent and treat lung cancer through gene knockout, and the present invention provides a target and treatment strategy for the clinical treatment of lung cancer. In addition, the present invention can significantly reduce the proliferation and migration of lung cancer cells by inhibiting NLN expression using siRNA; using a targeted NLN-specific small molecule inhibitor, it has a killing effect on lung cancer cells, and through animal experiments, it is verified that the targeted NLN inhibitor can significantly reduce the number of lung cancer foci and can significantly treat and delay the progression of primary lung cancer in mice.

[0021] The present invention also provides a kit that can screen the risk of lung cancer and / or lung cancer prognosis in a population to be tested by detecting the level of NLN neurolysin in lung tissue: if the level of NLN neurolysin is low, the risk of lung cancer and / or lung cancer prognosis is low; if the level of NLN neurolysin is high, the risk of lung cancer and / or lung cancer prognosis is high.

[0022] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0023] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Proteomic analysis identifies differentially expressed genes in lung cancer, A: Whole proteome mass spectrometry detection process of 30 cases of lung adenocarcinoma and 30 cases of lung squamous cell carcinoma; B: Number of significantly up-regulated and down-regulated proteins in lung adenocarcinoma (squamous cell) and adjacent tissues; C: Joint analysis of proteome and transcriptome to identify differentially expressed genes (vertical axis represents proteome difference change fold, horizontal axis represents transcriptome difference change fold).

[0025] Figure 2 : NLN neurolysin is highly expressed in lung adenocarcinoma and lung squamous cell carcinoma. A: Protein expression level of NLN neurolysin gene in LUAD and LUSC; B: NLN neurolysin gene expression and prognosis.

[0026] Figure 3: Immunohistochemistry experiment of NLN neurolysin in lung cancer tissue, A: Immunohistochemistry detection of NLN neurolysin expression in human lung cancer tissue; B: Immunohistochemistry score of NLN neurolysin expression in different stages of human lung cancer; C: Expression of NLN neurolysin in tumor tissue of primary lung adenocarcinoma mice.

[0027] Figure 4 : NLN neurolysin is highly expressed in various lung cancer cells. A: mRNA levels of NLN neurolysin in different cell lines; B: protein levels of NLN neurolysin in different cell lines. Figure 5 : Interference with NLN inhibits the proliferation of lung cancer A549 cells, A: QRT-PCR detection of NLN knockdown efficiency; B: Western blot detection of NLN knockdown efficiency; C: white field pictures of control group cells (NC) and NLN knockdown group; D: proliferation results of control group cells (NC) and NLN knockdown group A549 cells; E: migration and invasion results of control group cells (NC) and NLN knockdown A549 cells.

[0028] Figure 6 : Knockdown of NLN inhibits the progression of subcutaneous tumors in nude mice. A: Doxycycline (DOX)-induced conditional knockdown of A549-Tet-shNLN cell model injected into mice with subcutaneous tumors; B: Tumor volume detection of the control group and the knockdown group; C: Tumor weight detection of the control group and the knockdown group.

[0029] Figure 7 :Anti-tumor detection of NLN-specific small molecule inhibitor NR2, A: Synthesis route and crystal structure of NLN small molecule compound; B: IC of NLN small molecule compound in various lung cancer cell lines 50 Detection

[0030] Figure 8 :Targeted NLN small molecule inhibitor treatment delays the progression of primary cancer in mice. A: Surface observation of lung tissue samples from normal C57 mice, primary adenocarcinoma solvent group, and primary lung cancer treatment and administration group;

[0031] B: HE staining was used to observe the number of lung nodules in mice in different groups; C: Micro-CT imaging of the lungs after NR2 treatment, WT, wild-type C57 adult mice; KRASG12D / P53- / -, primary lung cancer model mice; NR2-Treatment, primary lung cancer model mice were treated with NR2; 3D, three-dimensional lung reconstruction; Coronal, coronal plane; Axial, cross-section; Sagittal, sagittal plane DETAILED DESCRIPTION

[0032] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0033] The CAS number of NR2 is: 605632-58-4, and the structural formula is:

[0034]

[0035] Example 1: Relationship between NLN neurolysin in lung cancer tissue and lung cancer

[0036] 1. Experimental Methods

[0037] (1) NLN neurolysin is expressed at an elevated level in lung cancer tissues and is associated with poor prognosis in patients

[0038] A total of 60 pairs of samples were collected, including 30 pairs of adenocarcinoma tissue samples and their adjacent tissue samples, and 30 pairs of squamous cell carcinoma tissue samples and their adjacent tissue samples, for protein absolute quantitative paired analysis of NLN neurolysin expression levels. (2) Immunohistochemistry

[0039] Immunohistochemical staining was performed on tumor tissues and distal control tissues from 11 patients with lung adenocarcinoma and 15 patients with lung squamous cell carcinoma, and professional pathologists were asked to score the NLN expression intensity.

[0040] (3) NLN expression in various cell lines

[0041] qRT-PCR (reverse transcription quantitative PCR) was used to detect the high expression of NLN neurolysin in different cell lines. The cell lines include BESA-2B cells (human bronchial epithelial cells), A549 cells (lung cancer human alveolar basal epithelial cells), H1299 cells (human lung cancer cells), PC-9 cells (human lung cancer cells), H1975 cells (human lung adenocarcinoma cells), SK-MES-1 cells (lung squamous cell carcinoma), H266 cells (lung squamous cell carcinoma), Calu-1 cells (lung adenocarcinoma cells).

[0042] (4) Interfering with NLN neurolysin to induce lung cancer cell death and inhibit its proliferation

[0043] The present invention designs and synthesizes siRNAs that specifically knock down NLN, namely si-NLN-1, si-NLN-2, and si-NLN-3, and uses a liposome transfection method to perform siRNA interference on A549 cells. After incubation for 72 hours, the NLN knockdown effect is detected by fluorescent quantitative PCR (QRT-PCR) and protein blotting (Western blot) methods, and the effects of NLN knockdown on A549 cell proliferation and migration are also detected.

[0044] Among them, si-NLN-1, si-NLN-2, and si-NLN-3 represent three interfering RNAs designed for NLN. The sequences of si-NLN-1, si-NLN-2, and si-NLN-3 are as follows:

[0045] si-NLN-1(SEQ ID NO.1):CCAGCUACCUUUGGACAUU

[0046] si-NLN-2(SEQ ID NO.2):GGUGAGGACUUACUUUCAU

[0047] si-NLN-3(SEQ ID NO.3):GCUGACUUCGUCCUUGAAA

[0048] (5) Effect of downregulating NLN neurolysin on subcutaneous tumor formation in mice

[0049] A doxycycline (DOX)-induced NLN neurolysin conditional knockdown cell model was constructed and subcutaneous tumor experiments were performed.

[0050] ① The Tet-on tetracycline (Tet) inducible expression system was used to construct a cell model for conditional knockdown of NLN expression. The specific steps are as follows:

[0051] 1) Construction of NLN sh1 Tet-pLKO-puro plasmid with Tet-on lentiviral backbone;

[0052] 2) Packaging lentivirus;

[0053] 3) The virus was used to infect A549 cells, and puromycin selection (puro selection) was performed 72 hours later to successfully obtain the NLN conditional knockdown A549 cell model;

[0054] 4) At the in vitro cell level, NLN knockdown can be induced by simply adding 1ug / ml DOX to the culture medium for 48h, and an A549-Tet-shNLN cell model with conditional NLN knockdown was constructed.

[0055] ②Node mice were inoculated with conditional knockdown A549-Tet-shNLN cells, 2 million per mouse, and the tumor progression was monitored. On the 7th day, they were divided into two groups, with 6 mice in each group. The mice in each group were given drinking water containing DOX (2 mg / ml), namely the NLN knockdown group (sh-NLN), and the mice in the normal group (sh-Ctrl) were given normal drinking water.

[0056] ③Monitor tumor progression: Euthanize mice on day 28, measure tumor size and weight, and take photos.

[0057] (7) Synthetic targeting of NLN-specific inhibitor NR2 to kill multiple lung cancer cell lines

[0058] An inhibitor specifically targeting NLN was synthesized and named NR2, and IC of various lung cancer cell lines was performed in vitro as follows. 50 Detection.

[0059] NR2 synthesis steps: 2-chlorostyrene and 2-fluorophenylhydrazine are used as starting materials, and a disubstituted pyrazole compound a is constructed in one step by condensation with polyformaldehyde under mixed acid conditions. Compound a is condensed with Boc-protected L-alanine to obtain a stereo mixture b. The stereo mixture b is separated by column chromatography to smoothly obtain an R-configured compound c, and then compound c is deprotected from the Boc protecting group in an acidic solution to obtain compound d. Finally, compound d is condensed with adamantane isocyanate to obtain the target product, namely NR2, and the molecular crystal of the target product is obtained by post-treatment and purification by a recrystallization method.

[0060] (8) Evaluation of the effect of NR2 in treating primary lung cancer in mice

[0061] At present, the most widely used animal model of lung cancer in the world is the KRASG12D / P53- / - mouse model. The activation of Kras mutants and the inactivation of p53 can be achieved by nasal instillation or inhalation of adenovirus carrying the Cre expression gene or hybridization with lung epithelial cell-specific Cre transgenic mice, thereby leading to the occurrence of lung cancer. For details, please refer to the work published in Nature protocol by Michel Du Page et al. in 2009. The model construction process is as follows:

[0062] 1) Mice were selected that were over 6 weeks old and carried the K-rasLSL-G12D / +; p53fl / fl (KP) genotype;

[0063] 2) Using the intubation inhalation method, the Cre virus was instilled into the mouse trachea with a virus dose of 1 × 10 8 Tu / ml / mouse, each was made up to 100 μl volume with PBS;

[0064] 3) The model showed proliferative lesions on the 10th day of modeling, and significant nodules appeared on the surface of the mouse lungs on the 30th day. After sampling, HE staining was performed to determine that primary adenocarcinoma had formed in the lungs, which was characterized by obvious tumor characteristics, large cuboidal cells, darkly stained nuclei, increased nuclear mitosis, obvious nucleoli, and rough nuclear membranes of tumor cells. This shows that this animal disease modeling method is practical (see Figure 8 B).

[0065] After the Cre virus induced primary lung cancer model mice successfully, the primary lung cancer model mice were divided into two groups, with 6 mice in each group:

[0066] One group was the NR2 treatment group, which was given NR2 (10% (v / v) DMSO + 10% (v / v) PEG300 + 0.9% (w / v) NaCl) starting on the 6th day, 100 mg / kg / mouse, intraperitoneal injection, once every two days, for a total of 6 doses. On the 28th day, the mice were euthanized for sampling.

[0067] One group was the control group, which was operated in the same manner as the drug-treated group, except that only the same dose of NR2 solvent was injected as in the drug-treated group. WT mice were used as the natural control group.

[0068] 2. Experimental Results

[0069] (1) NLN expression is elevated in lung cancer tissues and is associated with poor prognosis in patients

[0070] Protein absolute quantitative analysis showed that the expression of NLN neurolysin was significantly increased in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Figure 2 A), indicating that NLN neurolysin is significantly associated with lung cancer. Figure 2 As can be seen in B, 50 months after detection, the survival rate of patients with high NLN neurolysin expression was about 50%, which was much lower than the survival rate of patients with low NLN neurolysin expression, which was about 85%. The high (n = 35), low = (68) groups were all lung cancer patients. Among them, the NLN neurolysin level in the high group was higher than the iBAQ value of 10520257.12, and the NLN neurolysin level in the low group was lower than the iBAQ value of 10520257.12, indicating that NLN neurolysin was significantly associated with the poor prognosis of lung cancer patients ( Figure 2 B).

[0071] (2) Immunohistochemistry results confirmed that NLN neurolysin is highly expressed in lung cancer tissues

[0072] Immunohistochemical staining of tumor tissues and distal control tissues from 11 patients with lung adenocarcinoma and 15 patients with lung squamous cell carcinoma showed that the expression of NLN neurolysin in tumor tissues was significantly increased ( Figure 3 A, 3B). The same results were obtained in the tumor tissue of the mouse primary lung cancer model constructed in Example 1 (8) ( Figure 3 C). ADC stands for lung adenocarcinoma, SCC stands for lung squamous cell carcinoma, and stage I, stage II, and stage III refer to stage 1, stage 2, and stage 3 of the clinical tumor TNM staging, respectively.

[0073] (3) NLN neurolysin is highly expressed in a variety of cell lines

[0074] qRT-PCR assay confirmed that NLN was highly expressed in BESA-2B, A549, H1299, PC-9, cell lines ( Figure 4A), Western blot analysis confirmed that NLN was highly expressed in a variety of lung cancer cell lines (BESA-2B, A549, H1299, PC-9, H1975, SK-MES-1, H266, Calu-1) ( Figure 4 B).

[0075] The experimental results showed that NLN neurolysin was abnormally upregulated in lung cancer, and the downregulation of NLN neurolysin was a prognostic factor for lung cancer patients. Lung cancer patients with high NLN neurolysin expression had a poor prognosis, while lung cancer patients with low NLN neurolysin expression had a good prognosis. NLN neurolysin can be used for auxiliary diagnosis of clinical lung cancer and / or lung cancer prognosis, and can be used to evaluate the therapeutic effect of lung cancer drugs.

[0076] (4) Interfering with NLN neurolysin to induce lung cancer cell death and inhibit its proliferation

[0077] A549 cells were subjected to siRNA interference, and the knockdown effect of NLN was detected by fluorescence quantitative PCR (QRT-PCR) and protein blotting (Western blot) detection methods. The results showed that NLN neurolysin was significantly inhibited ( Figure 5 A, 5B). Microscopic observation revealed that after NLN knockdown, cells showed a significant death phenotype ( Figure 5 C). CCK8 experiments showed that the use of si-NLN-1, si-NLN-2, and si-NLN-3 to inhibit NLN expression significantly inhibited the proliferation of A549 cells ( Figure 5 D). Transwell assay results showed that downregulation of NLN neurolysin inhibited A549 cell migration and invasion ( Figure 5 E).

[0078] The experimental results show that interfering with NLN neurolysin can inhibit the proliferation of lung cancer cells, promote the occurrence of lung cancer cell apoptosis, and inhibit the migration of lung cancer cells. Reagents that interfere with or knock down the expression level of NLN neurolysin can treat lung cancer or inhibit lung cancer metastasis. The experimental results also further verified that NLN neurolysin is closely related to lung cancer and its prognosis, and the two have a significant correlation.

[0079] (5) Effect of downregulating NLN on subcutaneous tumor formation in mice

[0080] After obtaining the NLN conditional knockdown A549 cell model, 1ug / ml DOX was added to the culture medium. After 48h of induction, it was found that the expression of NLN was significantly inhibited at the mRNA and protein levels, indicating that the NLN conditional knockdown A549-Tet-shNLN cell model was successfully constructed. Subcutaneous tumor experiments were performed. The experimental results showed that after NLN was knocked down, the development of A549 subcutaneous tumors was significantly inhibited ( Figure 6A). As the tumor progressed, the size of the tumor was measured, and the results showed that downregulation of NLN neurolysin significantly inhibited tumor growth ( Figure 6 B), Tumor weight statistics showed the same trend ( Figure 6 C).

[0081] The experimental results showed that knocking down the NLN neurolysin gene could significantly inhibit lung cancer tumor growth.

[0082] (6) Targeting NLN-specific small molecule inhibitor NR2 kills multiple lung cancer cell lines

[0083] Based on existing research, a specific small molecule inhibitor targeting NLN, NR2, was synthesized. Its synthesis route is as follows: Figure 7 A) and IC of different lung cancer cell lines were performed 50 The results showed that the inhibitor had a killing effect on a variety of lung cancer cell lines ( Figure 7 B), which include A549 cells, H1299 cells, H157 cells and PC-9 cells.

[0084] (7) NR2 treatment delays the progression of primary lung cancer in mice

[0085] The present invention established a KRASG12D / P53- / - mouse primary lung cancer model and divided it into two groups for inhibitor treatment experiments. The natural mice were used as WT mice, and the lung surface was observed after sampling. The results showed that the number of lung surface cancer foci in the drug-treated group was significantly reduced ( Figure 8 A). HE staining of lung tissue sections obtained the same results ( Figure 8 B). Micro-CT imaging was performed simultaneously. 320 views were obtained for each section. MicroView software was used to perform 3D reconstruction of the lungs. The volume tools and measurement tools in MicroView were used for rendering and quantification. It can be seen that the number of lung cancer lesions in the drug-treated group was significantly reduced ( Figure 8 C) Experiments showed that NR2 can significantly delay the progression of primary lung cancer in mice.

[0086] The experimental results show that the NLN neurolysin inhibitor NR2 can inhibit the proliferation of lung cancer cells and kill multiple lung cancer cell lines. The NLN neurolysin inhibitor NR2 can treat lung cancer or delay the progression of primary lung cancer in mice.

[0087] In summary, the present invention provides a use of NLN neurolysin in the preparation of a drug for treating lung cancer. The present invention first discovered that the level of NLN neurolysin in lung cancer tissue of lung cancer patients is significantly higher than that in adjacent tissues. The present invention verifies that knocking out the NLN neurolysin gene can effectively prevent and treat lung cancer through gene knockout, and the present invention provides a target and treatment strategy for the clinical treatment of lung cancer. In addition, the present invention can significantly reduce the proliferation and migration of lung cancer cells by inhibiting NLN expression using siRNA; using a targeted NLN-specific small molecule inhibitor, it has a killing effect on lung cancer cells, and through animal experiments, it has been verified that the targeted NLN inhibitor can significantly reduce the number of lung cancer foci and can significantly treat and delay the progression of primary lung cancer in mice.

[0088] The present invention also provides a kit that can screen the risk of lung cancer and / or lung cancer prognosis in a population to be tested by detecting the level of NLN neurolysin in lung tissue: if the level of NLN neurolysin is low, the risk of lung cancer and / or lung cancer prognosis is low; if the level of NLN neurolysin is high, the risk of lung cancer and / or lung cancer prognosis is high.

Claims

1. Use of an NLN neurolysin inhibitor in the preparation of a drug for treating lung cancer and / or inhibiting lung cancer metastasis; the NLN neurolysin inhibitor is an agent that reduces the expression of NLN neurolysin or an agent that inhibits the activity of NLN neurolysin; the agent that reduces the expression of NLN neurolysin is an agent that knocks out or knocks down the NLN neurolysin gene; the agent that inhibits the activity of NLN neurolysin is NR2, and the CAS number of NR2 is: 605632-58-4; The sequence number of the reagent for knocking out or knocking down the NLN neurolysin gene is siRNA of SEQ ID NO.1, and / or SEQ ID NO.2, and / or SEQ ID NO.

3.

2. The use according to claim 1, characterized in that The drug is a drug that inhibits the migration of lung cancer cells; and / or, the drug is a drug that reduces the proliferation rate of lung cancer cells.

3. The use according to claim 2, characterized in that: The lung cancer includes lung adenocarcinoma and lung squamous cell carcinoma.

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