CN117180430A - New application of NLN (N-type neurolysin) in preparation of medicines for treating lung cancer - Google Patents
New application of NLN (N-type neurolysin) in preparation of medicines for treating lung cancer Download PDFInfo
- Publication number
- CN117180430A CN117180430A CN202311070241.9A CN202311070241A CN117180430A CN 117180430 A CN117180430 A CN 117180430A CN 202311070241 A CN202311070241 A CN 202311070241A CN 117180430 A CN117180430 A CN 117180430A
- Authority
- CN
- China
- Prior art keywords
- nln
- reagent
- lung cancer
- neurolysin
- lung
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102100023072 Neurolysin, mitochondrial Human genes 0.000 title claims abstract description 145
- 206010058467 Lung neoplasm malignant Diseases 0.000 title claims abstract description 125
- 201000005202 lung cancer Diseases 0.000 title claims abstract description 125
- 208000020816 lung neoplasm Diseases 0.000 title claims abstract description 125
- 239000003814 drug Substances 0.000 title claims abstract description 19
- 108090000812 Neurolysin Proteins 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims description 8
- 229940079593 drug Drugs 0.000 title claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 52
- 230000014509 gene expression Effects 0.000 claims description 35
- 239000003112 inhibitor Substances 0.000 claims description 23
- 210000004072 lung Anatomy 0.000 claims description 20
- 238000004393 prognosis Methods 0.000 claims description 16
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 claims description 14
- 238000004458 analytical method Methods 0.000 claims description 14
- 201000005249 lung adenocarcinoma Diseases 0.000 claims description 13
- 108090000623 proteins and genes Proteins 0.000 claims description 13
- 230000000694 effects Effects 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- 230000035755 proliferation Effects 0.000 claims description 9
- 206010041823 squamous cell carcinoma Diseases 0.000 claims description 9
- 108020004459 Small interfering RNA Proteins 0.000 claims description 7
- 230000002401 inhibitory effect Effects 0.000 claims description 7
- 238000012216 screening Methods 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 claims description 6
- 206010027476 Metastases Diseases 0.000 claims description 5
- 230000012292 cell migration Effects 0.000 claims description 5
- 230000009401 metastasis Effects 0.000 claims description 5
- 238000002965 ELISA Methods 0.000 claims description 4
- 230000000295 complement effect Effects 0.000 claims description 4
- 238000001943 fluorescence-activated cell sorting Methods 0.000 claims description 4
- 238000003119 immunoblot Methods 0.000 claims description 4
- 238000000760 immunoelectrophoresis Methods 0.000 claims description 4
- 238000001114 immunoprecipitation Methods 0.000 claims description 4
- 238000012744 immunostaining Methods 0.000 claims description 4
- 238000002493 microarray Methods 0.000 claims description 4
- 238000003127 radioimmunoassay Methods 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 3
- 229940122498 Gene expression inhibitor Drugs 0.000 claims description 2
- 239000004480 active ingredient Substances 0.000 claims description 2
- 239000004615 ingredient Substances 0.000 claims description 2
- 238000011282 treatment Methods 0.000 abstract description 18
- 238000003209 gene knockout Methods 0.000 abstract description 3
- 238000011269 treatment regimen Methods 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 57
- 210000001519 tissue Anatomy 0.000 description 29
- 206010028980 Neoplasm Diseases 0.000 description 24
- 241000699670 Mus sp. Species 0.000 description 22
- 238000003197 gene knockdown Methods 0.000 description 21
- 238000002474 experimental method Methods 0.000 description 10
- 241000282414 Homo sapiens Species 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 201000011510 cancer Diseases 0.000 description 6
- 150000003384 small molecules Chemical class 0.000 description 6
- 238000007920 subcutaneous administration Methods 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 238000010172 mouse model Methods 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- 101710126949 Lysin Proteins 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 108010026552 Proteome Proteins 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000002222 downregulating effect Effects 0.000 description 3
- 230000002055 immunohistochemical effect Effects 0.000 description 3
- 230000002452 interceptive effect Effects 0.000 description 3
- 230000002147 killing effect Effects 0.000 description 3
- 230000005012 migration Effects 0.000 description 3
- 238000013508 migration Methods 0.000 description 3
- 238000010837 poor prognosis Methods 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- -1 small molecule compound Chemical class 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- SGKRLCUYIXIAHR-AKNGSSGZSA-N (4s,4ar,5s,5ar,6r,12ar)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O SGKRLCUYIXIAHR-AKNGSSGZSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 208000009956 adenocarcinoma Diseases 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000004611 cancer cell death Effects 0.000 description 2
- 230000009702 cancer cell proliferation Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 229960003722 doxycycline Drugs 0.000 description 2
- 239000003651 drinking water Substances 0.000 description 2
- 235000020188 drinking water Nutrition 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000012151 immunohistochemical method Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 201000005243 lung squamous cell carcinoma Diseases 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 238000010603 microCT Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000009826 neoplastic cell growth Effects 0.000 description 2
- 230000002685 pulmonary effect Effects 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- OOMBRKGIWITBLL-UHFFFAOYSA-N (2-fluorophenyl)methylhydrazine Chemical compound NNCC1=CC=CC=C1F OOMBRKGIWITBLL-UHFFFAOYSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- ISRGONDNXBCDBM-UHFFFAOYSA-N 2-chlorostyrene Chemical compound ClC1=CC=CC=C1C=C ISRGONDNXBCDBM-UHFFFAOYSA-N 0.000 description 1
- 201000010053 Alcoholic Cardiomyopathy Diseases 0.000 description 1
- 208000031295 Animal disease Diseases 0.000 description 1
- 206010007637 Cardiomyopathy alcoholic Diseases 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- 101710113436 GTPase KRas Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- MPAQPXALASKCOX-UHFFFAOYSA-N N=C=O.C1C(C2)CC3CC1CC2C3 Chemical compound N=C=O.C1C(C2)CC3CC1CC2C3 MPAQPXALASKCOX-UHFFFAOYSA-N 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 229920002556 Polyethylene Glycol 300 Polymers 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003929 acidic solution Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229960003767 alanine Drugs 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000000424 bronchial epithelial cell Anatomy 0.000 description 1
- 239000003560 cancer drug Substances 0.000 description 1
- 230000004709 cell invasion Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000013115 immunohistochemical detection Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 108010087904 neutravidin Proteins 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 238000000575 proteomic method Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 238000002627 tracheal intubation Methods 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a new application of NLN (N-type nlN-type neurolysin) in preparing a medicine for preventing and treating lung cancer, belonging to the field of medical science. The level of NLN neurolysin in lung cancer tissues of lung cancer patients is found for the first time to be remarkable in paracancerous tissues. According to the invention, through gene knockout, the NLN neurolysin gene can be verified to be knocked out, so that lung cancer can be effectively prevented and treated, and a novel target spot and a treatment strategy are provided for clinical treatment of lung cancer.
Description
Technical Field
The invention belongs to the field of medical science, and particularly relates to a novel application of NLN (N-type NLN-type neurolysin) in preparation of a medicine for preventing and treating lung cancer.
Background
Lung cancer is one of the most common malignant tumors in the world, the morbidity and mortality of the lung cancer are in an increasing trend year by year, and the morbidity is the first in the world at present, so that the lung cancer seriously threatens the health and life of human beings. The lung cancer treatment means are many, and although the emerging treatment means such as targeted therapy, immunotherapy and the like have good effects in recent years, the traditional treatment means including surgery, radiotherapy and chemotherapy still have important roles at present. The drug therapy becomes the most important therapeutic means for lung cancer, and further screening of targets for early treatment of lung cancer is needed.
Protein NLN (Q9 BYT 8-NEUL_HUMAN) is a neurolysin, mainly distributed in mitochondria, which contains 704 amino acids and mainly acts to hydrolyze oligopeptides such as neuroserine. The present research finds that NLN neurolysin has related effects on alcoholic cardiomyopathy.
No prior art exists for NLN neurolysin associated with lung cancer.
Disclosure of Invention
The invention aims to provide a new application of an NLN (N-type NLN) neurolysin inhibitor in preparation of a medicine for preventing and treating lung cancer.
The invention provides an application of NLN (N-type NLN-type neurolysin) inhibitor in preparing medicines for treating lung cancer and/or inhibiting lung cancer metastasis.
Further, the NLN neurolysin inhibitor is an agent that reduces the expression level of NLN neurolysin or an agent that inhibits the activity of NLN neurolysin.
Further, the agent for reducing the expression level of NLN neurolysin is an agent for knocking out or knocking down NLN neurolysin gene;
further, the knockdown or knockdown NLN neurolysin gene expression inhibitor is siRNA with a sequence number of SEQ ID NO.1, and/or SEQ ID NO.2, and/or SEQ ID NO. 3.
Further, the agent that inhibits NLN lysin activity is a compound of:
further, the drug is a drug that inhibits lung cancer cell migration; the medicine is used for reducing the proliferation speed of lung cancer cells; further, the lung cancer includes lung adenocarcinoma and lung squamous carcinoma.
The invention also provides a medicine for treating lung cancer and/or inhibiting lung cancer metastasis, which is a preparation prepared by taking an NLN (N-type NLN) neurolysin inhibitor as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients;
further, the NLN neurolysin inhibitor is a drug for knocking out NLN neurolysin genes or reducing NLN neurolysin activity/expression quantity.
The invention also provides application of the reagent for detecting the expression level of NLN neurolysin in preparing lung cancer and/or lung cancer prognosis screening kits.
Further, the reagent for detecting the expression level of NLN-neurolysin includes a reagent for enzyme-linked immunosorbent assay, a reagent for immunoblotting, a reagent for immunoelectrophoresis, a reagent for tissue immunostaining, a reagent for immunoprecipitation analysis, a reagent for radioimmunoassay, a reagent for complement fixation analysis, a reagent for fluorescence-activated cell sorting, a reagent for mass analysis, or a reagent for protein microarray.
Further, the reagent for detecting NLN neurolysin is used for detecting NLN neurolysin in lung tissues.
The invention also provides a lung cancer and/or lung cancer prognosis screening kit, which comprises a reagent for detecting NLN neurolysin.
Further, the reagent for detecting the expression level of NLN-neurolysin includes a reagent for enzyme-linked immunosorbent assay, a reagent for immunoblotting, a reagent for immunoelectrophoresis, a reagent for tissue immunostaining, a reagent for immunoprecipitation analysis, a reagent for radioimmunoassay, a reagent for complement fixation analysis, a reagent for fluorescence-activated cell sorting, a reagent for mass analysis, or a reagent for protein microarray.
The invention provides a new application of NLN (N-type NLN-type neurolysin) in preparing a medicine for treating lung cancer, and the invention discovers that the NLN-type neurolysin level in lung cancer tissues of a lung cancer patient is obviously higher than that of tissues beside cancer for the first time. According to the invention, through gene knockout, the NLN neurolysin gene can be verified to be knocked out, so that lung cancer can be effectively prevented and treated, and a novel target spot and a treatment strategy are provided for clinical treatment of lung cancer. After the expression of NLN is inhibited by siRNA, the proliferation and migration of lung cancer cells can be obviously reduced; the targeted NLN specific small molecule inhibitor has a killing effect on lung cancer cells, and animal experiments prove that the targeted NLN inhibitor can remarkably reduce the number of lung cancer foci and can remarkably treat and delay the progress of primary lung cancer of mice.
The invention also provides a kit which can screen the risk degree of lung cancer and/or lung cancer prognosis of the crowd to be detected by detecting the lung tissue NLN neurolysin level: if the NLN neurolysin level is low, the risk of having a lung cancer and/or lung cancer prognosis is low, and if the NLN neurolysin level is high, the risk of having a lung cancer and/or lung cancer prognosis is high.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
Fig. 1: proteomic analysis identified differentially expressed genes in lung cancer, a: mass spectrum detection flow of the whole proteome of 30 lung adenocarcinomas and 30 lung squamous carcinomas; b: lung adenocarcinoma (squamous) and paracancerous significantly up-regulates and down-regulates protein numbers; c: proteome and transcriptome combined analysis identified differential genes (vertical axis represents fold change in proteome differences and horizontal axis represents fold change in transcriptome differences).
Fig. 2: NLN neurolysin is highly expressed in lung adenocarcinoma and lung squamous carcinoma, a: protein expression levels of NLN lysin gene at LUAD and luc; b: NLN neurolysin gene expression and prognosis.
Fig. 3: NLN neurolysin was tested in lung cancer tissue immunohistochemistry, A: immunohistochemical detection of NLN neurolysin expression in human lung cancer tissue; b: immunohistochemical scoring of expression of NLN lysin in different stages of human lung cancer; c: expression of NLN neurolysin in tumor tissue of primary lung adenocarcinoma mice.
Fig. 4: NLN neurolysin is highly expressed in various lung cancer cells, a: levels of mRNA of NLN neurolysin in different cell lines; b: protein levels of NLN neurolysin in different cell lines. Fig. 5: interference NLN inhibits proliferation of lung cancer A549 cells, wherein the A is QRT-PCR detection NLN knockdown efficiency; western blot detection NLN knockdown efficiency; c: control group cells (NC) and NLN knockdown group whitefield pictures; proliferation results of control group cells (NC) and NLN knockout group A549 cells; e: control group cells (NC) and NLN knockdown a549 cell migration, invasion results.
Fig. 6: knocking down NLN inhibits progression of nude mice subcutaneous tumor, a: doxycycline DOX induced conditional knockdown A549-Tet-shNLN cell model injection mice subcutaneous tumor experiment; b: tumor volume detection of a control group and a knockdown group; and C, detecting the tumor weight of the control group and the knockdown group.
Fig. 7: anti-tumor detection of a target NLN specific small molecule inhibitor NR2, wherein the synthetic route and crystal structure of the NLN small molecule compound are as follows; NLN small molecule compound in various lung cancer cell strains IC 50 Detection of
Fig. 8: targeting NLN small molecule inhibitor treatment, delay of primary cancer progression in mice, a: normal C57 mice, primary adenocarcinoma solvent group, primary lung cancer treatment dosing group mice lung tissue surface observation;
b: HE staining observed the number of lung tissue nodules in different groups of mice; c, carrying out lung Micro-CT imaging, WT and wild C57 adult mice after NR2 treatment; KRASG12D/P53-/-, primary lung cancer model mice; NR2 Treatment is carried out on NR2-Treatment on primary lung cancer model mice; 3D, three-dimensional pulmonary reconstruction; coronal, coronal plane; axial, cross section; sagittal, sagittal plane
Detailed Description
The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.
The CAS for NR2 is: 605632-58-4, the structural formula is:
EXAMPLE 1 relationship between NLN-neurolysin and lung cancer in lung cancer tissue
1. Experimental method
(1) NLN-neurolysin is expressed in lung cancer tissue and is associated with poor prognosis in patients
60 pairs of samples including 30 pairs of adenocarcinoma tissue samples and other cancer tissue samples and 30 pairs of squamous carcinoma tissue samples and other cancer tissue samples are collected, and protein absolute quantitative pairing analysis is carried out on the expression level of NLN neurolysin. (2) Immunohistochemical experiments
Immunohistochemical methods tumor tissues and distal control tissues of 11 lung adenocarcinoma and 15 lung squamous carcinoma patients were stained, and a doctor of a specialized doctor was asked to score the intensity of NLN expression.
(3) NLN expression in various cell lines
qRT-PCR (reverse transcription quantitative PCR) was used to detect high expression of NLN-neurolysin in different cell lines. 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).
(4) Interfering NLN neurolysin to induce lung cancer cell death and inhibit proliferation thereof
The invention designs and synthesizes siRNA for specifically knocking down NLN, namely si-NLN-1, si-NLN-2 and si-NLN-3, adopts a liposome transfection method to perform siRNA interference on A549 cells, detects NLN knocking down effect through a fluorescence quantitative PCR (QRT-PCR) method and a Western blot method after incubation for 72 hours, and detects the influence of proliferation and migration of the A549 cells after NLN knocking down.
Wherein si-NLN-1, si-NLN-2, si-NLN-3 represent three interfering RNAs designed for NLN, respectively. The sequences of si-NLN-1, si-NLN-2 and si-NLN-3 are as follows:
si-NLN-1(SEQ ID NO.1):CCAGCUACCUUUGGACAUU
si-NLN-2(SEQ ID NO.2):GGUGAGGACUUACUUUCAU
si-NLN-3(SEQ ID NO.3):GCUGACUUCGUCCUUGAAA
(5) Down-regulating influence of NLN neurolysin on mouse subcutaneous neoplasia
And constructing a doxycycline DOX-induced NLN neurolysin conditional knockdown cell model, and performing a subcutaneous tumor experiment.
(1) A Tet-on tetracycline (Tet) induction regulation expression system is adopted to construct a cell model for conditionally knocking down NLN expression, and the specific steps are as follows:
1) Constructing NLN sh1 Tet-pLKO-puro plasmid of a Tet-on lentiviral skeleton;
2) Packaging the lentivirus;
3) Carrying out puromycin screening (puro screening) after virus infection of A549 cells for 72 hours, and successfully obtaining NLN conditional knockdown A549 cell model;
4) In vitro cell level can induce NLN to knock down only by containing 1ug/ml DOX in a culture medium for 48 hours, and an A549-Tet-shNLN cell model with NLN conditional knockdown is constructed.
(2) Node mice were vaccinated with conditional knockdown of a549-Tet-shNLN cells, 200 ten thousand/mouse, monitored for neoplastic progression, and given two groups of 6 mice each, with DOX (2 mg/ml) in drinking water, i.e., NLN knockdown group (sh-NLN), and normal drinking water in normal group (sh-Ctrl).
(3) Tumor progression was monitored by euthanizing mice at day 28, measuring tumor size and weight, and photographing.
(7) Synthesis of targeted NLN specific inhibitor NR2 kills multiple lung cancer cell lines
The method is as follows, synthesis of inhibitors specifically targeting NLN anddesignated NR2, IC for in vitro multiple lung cancer cell lines 50 And (5) detecting.
And NR2 synthesis, namely, 2-chlorostyrene and 2-fluorobenzylhydrazine are used as starting materials, a disubstituted pyrazole compound a is constructed in one step by condensing with paraformaldehyde under the condition of mixed acid, the compound a and L-alanine protected by Boc are condensed to obtain a three-dimensional mixture b, the three-dimensional mixture b is resolved by column chromatography to smoothly obtain a compound c with an R configuration, then the compound c is subjected to Boc protecting group removal in an acidic solution to obtain a compound d, finally, the compound d and adamantane isocyanate are condensed to obtain a target product, namely NR2, and molecular crystals of the target product are obtained through post-treatment purification by a recrystallization method.
(8) Evaluation of NR2 Effect on treatment of Primary Lung cancer in mice
The most widely used international lung cancer animal model is a KRASG 12D/P53-/-mouse model, and the activation of Kras mutant and the inactivation of P53 can be realized by nasal instillation or intubation inhalation of adenovirus carrying Cre expression genes or hybridization with Cre transgenic mice specific to lung epithelial cells, so that lung cancer is caused, and the work of Michel DuPage et al published in 2009 can be referred to, and the model construction process is as follows:
1) The mice are selected to carry K-ras LSL-G12D/+' over 6 weeks old; mice of the p53fl/fl (KP) genotype;
2) The cre virus is dripped into the trachea of a mouse by adopting a cannula inhalation method, and the virus quantity is 1 multiplied by 10 8 Tu/ml/each was made up to a volume of 100 μl with PBS;
3) The model has the characteristics of obvious tumor characteristics, large cubic cells, deep cell nucleus staining, increased nuclear mitosis, obvious nucleolus and unsmooth nuclear membrane of tumor cells. This animal disease modeling approach is illustrated to have utility (see fig. 8B).
After Cre virus induced primary lung cancer model mice were successful, the primary lung cancer model mice were divided into two groups of 6:
one group was the administration group (i.e., NR2 treatment), and NR2 (10% (v/v) DMSO+10% (v/v) PEG300+0.9% (w/v) NaCl), 100 mg/kg/person, was administered by intraperitoneal injection, once every two days, for a total of 6 administrations, was started on day 6. Mice were euthanized on day 28.
One group is a control group, and is operated in the same manner as the administration group except that only the solvent of NR2 at the same dose as the administration group is injected. WT mice were used as a natural control group.
2. Experimental results
(1) NLN is expressed in lung cancer tissue and is associated with poor patient prognosis
Absolute quantitative analysis of the protein showed a significant increase in expression of NLN lysin in lung adenocarcinoma (LUAD) and lung squamous carcinoma (luc) (fig. 2A), indicating that NLN lysin is significantly associated with lung cancer. As can be seen from FIG. 2B, the survival rate of NLN-neurolysin-high-expression patients is about 50% and is about 85% far lower than that of NLN-neurolysin-low-expression patients when the detection is carried out for 50 months. The high (n=35), low= (68) groups are all lung cancer patients, wherein the high group NLN neurolysin was higher than the iBAQ value 10520257.12, and the low group NLN neurolysin was lower than the iBAQ value 10520257.12, indicating that NLN neurolysin was significantly correlated with poor prognosis in lung cancer patients (fig. 2B).
(2) Immunohistochemical results confirm that NLN (N-type neurolysin) is highly expressed in lung cancer tissues
Immunohistochemical methods stained tumor tissues and distal control tissues of 11 lung adenocarcinoma and 15 lung squamous carcinoma patients, and the results showed that expression of NLN neurolysin in tumor tissues was significantly elevated (fig. 3a,3 b). The same results were obtained in the tumor tissue of the mouse primary lung cancer model constructed in example 1 item (8) (FIG. 3C). ADC is lung adenocarcinoma, SCC is lung squamous carcinoma, stageI, stageII, stageIII refers to stage 1, stage 2 and stage 3 of TNM stage of clinical tumor respectively.
(3) NLN-neurolysin is highly expressed in a variety of cell lines
qRT-PCR assays demonstrated high expression of NLN in BESA-2B, A549, H1299, PC-9, cell lines (FIG. 4A), and Western blot assays demonstrated high expression of NLN in various lung cancer cell lines (BESA-2B, A549, H1299, PC-9, H1975, SK-MES-1, H266, calu-1) (FIG. 4B).
Experimental results show that NLN neurolysin is abnormally up-regulated in lung cancer, NLN neurolysin is down-regulated as a prognosis predictor of lung cancer patients, lung cancer patients with high NLN neurolysin expression have poor prognosis, and lung cancer patients with low NLN neurolysin expression have good prognosis. NLN neurolysin can be used for clinical lung cancer and/or lung cancer prognosis auxiliary diagnosis, and can be used for evaluating the treatment effect of lung cancer drugs.
(4) Interfering NLN neurolysin to induce lung cancer cell death and inhibit proliferation thereof
siRNA interference is carried out on A549 cells, NLN knockdown effect is detected through fluorescent quantitative PCR (QRT-PCR) and Western blot (Western blot) detection methods, and the result shows that NLN neurolysin is significantly inhibited (FIGS. 5A and 5B). After microscopic observation, NLN knockdown was found, the cells developed a significant death phenotype (fig. 5C). CCK8 experiments showed significant inhibition of A549 cell proliferation following inhibition of NLN expression using si-NLN-1, si-NLN-2, si-NLN-3 (FIG. 5D). Transwell experiments showed that after down-regulating NLN neurolysin, A549 cell migration and invasion were inhibited (FIG. 5E).
Experimental results show that the agent which interferes with NLN neurolysin can inhibit lung cancer cell proliferation, promote lung cancer cell apoptosis, inhibit lung cancer cell migration, interfere and knock down NLN neurolysin expression level can treat lung cancer or inhibit lung cancer metastasis. The experimental results further prove that NLN neurolysin is closely related to lung cancer and prognosis thereof, and the NLN neurolysin and the lung cancer have obvious correlation.
(5) Down-regulating NLN effect on mouse subcutaneous neoplasia
After the NLN conditional knockdown A549 cell model is obtained, DOX containing 1ug/ml is added into a culture medium, and after induction is carried out for 48 hours, the expression of NLN is obviously inhibited at the mRNA and protein layers, namely the NLN conditional knockdown A549-Tet-shNLN cell model is successfully constructed, and the experimental result shows that after NLN knockdown, the development of A549 subcutaneous tumor is obviously inhibited (figure 6A). As the tumor progressed, the size of the tumor volume was measured, and the results showed that NLN neutravidin down-regulation significantly inhibited tumor growth (fig. 6B), and tumor weight statistics gave the same trend (fig. 6C).
Experimental results show that the NLN neurolysin gene can be knocked down to remarkably inhibit the growth of lung cancer tumors.
(6) Targeting NLN specific small molecule inhibitor NR2 to kill multiple lung cancer cell lines
According to the prior studies, targeted NLN specific small molecule inhibitor NR2 was synthesized, the synthetic route of which is shown in FIG. 7A, and IC of different lung cancer cell lines was performed 50 The results of the assay show that the inhibitor has killing effect on various lung cancer cell lines (FIG. 7B), including A549 cells, H1299 cells, H157 cells and PC-9 cells.
(7) NR2 treatment delays progression of primary lung cancer in mice
The invention is divided into two groups for inhibitor treatment experiments after constructing a KRASG 12D/P53-/-mouse primary lung cancer model, natural mice are taken as WT mice, lung surface observation is carried out after the materials are taken, and the result shows that the number of lung surface cancer foci of the administration group is obviously reduced (figure 8A). HE staining of lung tissue sections gave the same results (fig. 8B). Micro-CT imaging was performed simultaneously. 320 views were obtained for each section. At the same time, microView software was used for pulmonary 3D reconstruction. Rendering and quantification using the volumetric and measurement tools in MicroView, it can be seen that the number of lung foci in the dosing group was significantly reduced (fig. 8C), and experiments show that NR2 can significantly treat delayed progression of primary lung cancer in mice.
Experimental results show that the NLN neurolysin inhibitor NR2 can inhibit lung cancer cell proliferation and kill various lung cancer cell lines, and the NLN neurolysin inhibitor NR2 can treat lung cancer or delay the progress of primary lung cancer of mice.
In conclusion, the invention provides a new application of NLN (N-type NLN) neurolysin in preparation of a medicine for treating lung cancer, and the invention discovers that the level of NLN-type NLN in lung cancer tissues of a lung cancer patient is obviously higher than that of other tissues. According to the invention, through gene knockout, the NLN neurolysin gene can be verified to be knocked out, so that lung cancer can be effectively prevented and treated, and a novel target spot and a treatment strategy are provided for clinical treatment of lung cancer. After the expression of NLN is inhibited by siRNA, the proliferation and migration of lung cancer cells can be obviously reduced; the targeted NLN specific small molecule inhibitor has a killing effect on lung cancer cells, and animal experiments prove that the targeted NLN inhibitor can remarkably reduce the number of lung cancer foci and can remarkably treat and delay the progress of primary lung cancer of mice.
The invention also provides a kit which can screen the risk degree of lung cancer and/or lung cancer prognosis of the crowd to be detected by detecting the lung tissue NLN neurolysin level: if the NLN neurolysin level is low, the risk of having a lung cancer and/or lung cancer prognosis is low, and if the NLN neurolysin level is high, the risk of having a lung cancer and/or lung cancer prognosis is high.
Claims (11)
- Use of an nln neurolysin inhibitor in the preparation of a medicament for treating lung cancer and/or inhibiting lung cancer metastasis.
- 2. Use according to claim 1, characterized in that: the NLN-neurolysin inhibitor is an agent for reducing the expression level of NLN-neurolysin or an agent for inhibiting the activity of NLN-neurolysin.
- 3. Use according to claim 2, characterized in that: the reagent for reducing the expression quantity of NLN neurolysin is a reagent for knocking out or knocking down NLN neurolysin genes; preferably, the knockdown or knockdown NLN neurolysin gene expression inhibitor is an siRNA having a sequence number of SEQ ID NO.1, and/or SEQ ID NO.2, and/or SEQ ID NO. 3.
- 4. The use according to claim 2, characterized in that the agent inhibiting NLN neurolysin activity is a compound:。
- 5. the use according to any one of claims 1 to 4, wherein the medicament is a medicament for inhibiting lung cancer cell migration; the medicine is used for reducing the proliferation speed of lung cancer cells; preferably, the lung cancer includes lung adenocarcinoma and lung squamous carcinoma.
- 6. A medicament for treating lung cancer and/or inhibiting lung cancer metastasis, characterized in that: the preparation is prepared by taking an NLN neurolysin inhibitor as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients;preferably, the NLN neurolysin inhibitor is a drug that knocks out the NLN neurolysin gene or reduces NLN neurolysin activity/expression level.
- 7. The application of a reagent for detecting the expression level of NLN neurolysin in preparing a lung cancer and/or lung cancer prognosis screening kit.
- 8. The use according to claim 7, wherein the reagent for detecting the expression level of NLN neurolysin comprises a reagent for enzyme-linked immunosorbent assay, a reagent for immunoblotting, a reagent for immunoelectrophoresis, a reagent for tissue immunostaining, a reagent for immunoprecipitation analysis, a reagent for radioimmunoassay, a reagent for complement fixation analysis, a reagent for fluorescence-activated cell sorting, a reagent for mass analysis, or a reagent for protein microarray.
- 9. The use of claim 8, wherein the reagent for detecting NLN neurolysin is detecting NLN neurolysin in lung tissue.
- 10. A lung cancer and/or lung cancer prognosis screening kit, characterized in that it comprises a reagent for detecting NLN neurolysin.
- 11. The kit of claim 10, wherein the reagent for detecting the expression level of NLN neurolysin comprises a reagent for enzyme-linked immunosorbent assay, a reagent for immunoblotting, a reagent for immunoelectrophoresis, a reagent for tissue immunostaining, a reagent for immunoprecipitation analysis, a reagent for radioimmunoassay, a reagent for complement fixation analysis, a reagent for fluorescence-activated cell sorting, a reagent for mass analysis, or a reagent for protein microarray.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311070241.9A CN117180430A (en) | 2023-08-23 | 2023-08-23 | New application of NLN (N-type neurolysin) in preparation of medicines for treating lung cancer |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311070241.9A CN117180430A (en) | 2023-08-23 | 2023-08-23 | New application of NLN (N-type neurolysin) in preparation of medicines for treating lung cancer |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117180430A true CN117180430A (en) | 2023-12-08 |
Family
ID=88997013
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311070241.9A Pending CN117180430A (en) | 2023-08-23 | 2023-08-23 | New application of NLN (N-type neurolysin) in preparation of medicines for treating lung cancer |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117180430A (en) |
-
2023
- 2023-08-23 CN CN202311070241.9A patent/CN117180430A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2017067454A1 (en) | Pharmaceutical use and drug product adopting lsd1 inhibitor for preventing and treating triple negative breast cancer | |
US20210137883A1 (en) | Anti-neoplastic compounds and methods targeting qsox1 | |
WO2021203798A1 (en) | Use of splicing factor prpf31 inhibitor in preparing drug | |
US10232013B2 (en) | Use of an antimicrobial peptide TP4 in treating a cancer | |
JP6262707B2 (en) | Methods and compositions for the treatment, prevention and diagnosis of cancer comprising or derived from cancer stem cells | |
Shi et al. | miR-142-3p improves paclitaxel sensitivity in resistant breast cancer by inhibiting autophagy through the GNB2-AKT-mTOR pathway | |
Qin et al. | Combination of dendrobium mixture and metformin curbs the development and progression of diabetic cardiomyopathy by targeting the lncRNA NEAT1 | |
US20200138915A1 (en) | Use of Upstream Open Reading Frame 45aa-uORF Nucleotide Sequence of PTEN Gene and Polypeptide Coded by 45aa-uORF | |
CN113230249A (en) | Application of pseudolaric acid B in serving as or preparing Hedgehog signal path inhibitor | |
CN115054605B (en) | Application of G9a inhibitor in preparation of medicines for treating uveal melanoma | |
Xu et al. | ApoM suppresses kidney renal clear cell carcinoma growth and metastasis via the Hippo-YAP signaling pathway | |
WO2020113877A1 (en) | Function and use of e2f6 inhibitor | |
CN108653737B (en) | Application of MTHFD1L inhibitor in preparation of tongue squamous carcinoma treatment drug | |
CN117180430A (en) | New application of NLN (N-type neurolysin) in preparation of medicines for treating lung cancer | |
CN109966479A (en) | Application of the EZH2 in preparation prevention or treatment polycystic kidney disease drug | |
CN110960546B (en) | Application of MicroRNAs in preparation of reinforcing agent for treating liver cancer by sorafenib | |
CN114452393A (en) | Application of PINCH-1 protein as target in preparation of medicine for treating pulmonary fibrosis | |
KR102591642B1 (en) | Targets and their applications for drug treatment of tumor metastases | |
CN111518911A (en) | Application of LINC01843 as liver cancer diagnosis and treatment marker | |
CN111557943A (en) | Application of PD0332991 and oxitinib in preparation of drug for treating NSCLC | |
CN105617401B (en) | Tumor radiation sensitization and radiation side effect weakening effects of miRNA, implementation method and application | |
CN110665007A (en) | Combined medicine for treating cancer and application thereof | |
CN108295261B (en) | Function and use of PHF14 | |
CN113521291B (en) | Application of ZNF143-MDIG-CDC6 axis in hepatocellular carcinoma | |
KR102576855B1 (en) | Composition for preventing or treating of liver cancer comprising tsp1 as an active ingredient |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |