CN113699240A - Medical application of NRK in lung cancer treatment and prognosis diagnosis - Google Patents

Abstract

The invention discloses medical application of NRK in lung cancer treatment and prognosis diagnosis. In particular to the application of NRK in preparing products for diagnosing lung cancer prognosis and medicines for treating lung cancer; and the application of the NRK neutralizing antibody in preparing products for diagnosing lung cancer and medicines for treating lung cancer. The invention utilizes RNA-seq and a comprehensive bioinformatics method to analyze CAFs and NFs from LUAD stages I, II and III, finds that NRK is specifically expressed in lung cancer tumor-related fibroblasts, can be used as a clinical diagnosis biomarker of lung cancer, and can provide a better diagnosis and prognosis index; NRK in LUAD-related CAFs is remarkably increased, and the NRK can be used as a target of LUAD combined treatment.

Description

Medical application of NRK in lung cancer treatment and prognosis diagnosis

Technical Field

The invention relates to the technical field of biological medicines, in particular to medical application of NRK in lung cancer treatment and prognosis diagnosis.

Background

The incidence and mortality of lung cancer are the first of all in the world. Lung cancer is classified into Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC), with NSCLC accounting for 85% of all lung cancers. NSCLC is largely divided into adenocarcinoma (LUAD), squamous cell carcinoma (luxc), and large cell carcinoma, with LUAD being the major subtype of NSCLC accounting for approximately 50% of NSCLC. Although the rapid development of clinical therapies such as surgery, chemotherapy, targeted therapy and immunotherapy has significantly improved the prognosis of lung cancer patients, including LUAD, the prognosis of these patients is still poor due to poor drug resistance or poor drug treatment response, with an average 5-year survival rate of only 15%. In addition, most lung adenocarcinoma patients are diagnosed at the middle and advanced stage and are accompanied by metastatic dissemination, and tumor recurrence or metastasis and the like can occur even after treatment remission, so that the adverse factors not only influence the life quality of the patients, but also greatly limit the survival rate of the lung adenocarcinoma patients. Therefore, a potential new target point for treating the lung adenocarcinoma is searched, and the condition of improving the survival prognosis of the patient is urgent.

Although cancer drug development has traditionally focused on cancer, recent emphasis has been on the Tumor Microenvironment (TME) to seek new therapeutic and prophylactic strategies, as TME may contribute to tumor recurrence and therapeutic resistance. Among all stromal cell populations of TME, tumor-associated fibroblasts (CAFs) are the most abundant and are closely associated with the development and progression of cancer. It has been discovered that CAFs modulate the biological properties of cancer cells and other stromal cells through cell-cell communication, remodel the extracellular matrix (ECM) or basement membrane through the release of cytokines or chemokines, modulate signaling pathways, and modulate anti-tumor immune responses. Thus, targeting CAFs offers an effective way to overcome cancer by remodeling TME, reducing the incidence of immunosuppression rather than killing cancer cells directly. Indeed, a number of preclinical studies have shown that CAFs can be selected as emerging targets for cancer therapy, including anti-tumor immunotherapy. For example, dendritic cells infected with recombinant adenoviral vectors containing the FAP- α gene can induce protective anti-tumor immunity and prolong the overall survival time of immunized mice, suggesting that FAP- α may be a potential target for targeting CAF and developing immunogenic tumor vaccines. In addition to the direct depletion of CAFs, vitamin D and vitamin a reprogramming CAFs, resetting the activation state of tumor-promoting CAFs to a resting state is of concern in pancreatic ductal adenocarcinoma and colon cancer. In addition, growth of LUAD cells is promoted by fibroblast growth factor 9(FGF9) secreted by CAFs, and inhibition of Fibroblast Growth Factor Receptors (FGFRs) in vivo results in a significant reduction in tumor size and number, although not sufficient to completely eliminate the tumor, providing evidence for a combination strategy for the treatment of lung cancer. Notably, targeting CAFs may enhance anti-tumor effects in addition to cancer cells. For example, combining oxaliplatin with the small molecule dipeptidyl peptidase inhibitor PT-100 (inhibition of CAFs by targeting Fibroblast Activation Protein (FAP) can improve the chemotherapeutic response, reduce the recruitment of immunopotentiating tumor cells and angiogenesis in mouse colon cancer xenograft models. however, CAFs are heterogeneous cell populations that serve both tumor support and tumor suppression in solid malignancies, which is considered a major obstacle to the development of new CAFs for targeted diagnosis and treatment.

NRK (NIK-related kinase) is a protein kinase encoded by the NRK gene in the x chromosome, belonging to the Germinal Center Kinase (GCK) subfamily, and involved in 13 activation MAPK cascades. It was first cloned from mice with NRK and was found primarily in skeletal muscle during embryogenesis in mice. This is consistent with the embryonic origin of most fibroblasts. NRK is also likely to be expressed in the human brain. In addition, NRK reportedly regulates trophoblast cell proliferation and placental development by modulating AKT phosphorylation. Furthermore, NRK is one of highly expressed genes of Dermal Mesenchymal Stem Cells (DMSCs) in patients with psoriasis, and regulates migration of peripheral blood-derived monocytes (PBMCs) to DMSCs. Recent studies have found that NRK is strongly expressed in human blood vessels and is associated with the induction of Matrix Metalloproteinases (MMPs) and chemokines (e.g., MMP8, MMP12, CCL8, CXCL9), suggesting that NRK may be an anti-inflammatory factor in atherosclerosis. In addition to the above functions, NRK is positively correlated with survival in Triple Negative Breast Cancer (TNBC) patients. NRK deficiency during pregnancy causes mice to be prone to breast cancer. However, the expression of NRK in other tissues or cells, its substrates, and the mechanisms that regulate its kinase activity and regulate the signaling cascade have not been elucidated. At present, the related medical application of NRK in the treatment and prognosis diagnosis of lung cancer is not found.

Notably, NRK is involved in processes such as cell migration, apoptosis, skeletal protein rearrangement in humans. According to the report of the literature, the overexpression of NRK in African green monkey kidney fibroblast (COS7) can phosphorylate downstream Cofilin protein, induce the F-actin polymerization around the nucleus, and regulate the migration activity of the cell. Cofilin can regulate actin and tubulin, the active form (non-phosphorylation) of Cofilin can depolymerize actin, Cofilin is in an inactivation transition state once being phosphorylated, phosphorylated Cofilin (p-Cofilin) loses the activity of depolymerizing protein, actin microfilament polymerization is enhanced, and the motility of cells is improved. Cell migration is a cellular process requiring F-actin polymerization and actin skeleton stabilization, with increased F-actin polymerization and enhanced cell migration ability. Tumor cells spontaneously approach to the CAFs in the migration and invasion processes and continuously move along with the CAFs, the CAFs serve as a 'leading cells' to open up a path for the movement of tumors, and the tumor cells can invade along the path of the CAFs. The ability of CAFs to migrate is enhanced, and the ability of tumor cells following CAFs to migrate and invade is also enhanced correspondingly. Furthermore, NRK can inhibit the release of CXCL5 in human vascular endothelial cells. CXCL5 can promote malignant progression of tumors in cancers such as gastric cancer, colorectal cancer, and pancreatic cancer. Interestingly, in the lung microenvironment CXCL5 is considered to be a chemokine that inhibits tumor growth, and CXCL5 inhibits tumor cell colonization and growth in the lung by attracting neutrophils from the lung and establishing a neutrophil-infiltrating lung immune microenvironment.

Disclosure of Invention

The invention aims to provide medical application of NRK in treatment and prognosis diagnosis of lung cancer.

In one aspect, the invention provides the use of NRK in the preparation of a product for diagnosing lung cancer prognosis.

In particular, the product uses NRK as a diagnostic marker.

More specifically, the high expression of NRK is indicative of a poor prognosis in a patient with lung adenocarcinoma.

Specifically, the product is a real-time fluorescent quantitative PCR detection kit or diagnostic test paper.

More specifically, the real-time fluorescent quantitative PCR detection kit contains a primer for detection, and the specific sequence is as follows:

forward primer, 5'-CAGCAGGTTCGGTCACTGATGTAG-3';

reverse primer, 5'-CAGCAGGTTCGGTCACTGATGTAG-3'.

On the other hand, the invention also provides application of NRK in preparing a medicine for treating lung cancer.

In particular, the drug product has NRK as a therapeutic target.

On the other hand, the invention also provides the application of the NRK neutralizing antibody in preparing a product for diagnosing lung cancer.

Specifically, the product is a diagnostic test paper or kit, and the diagnostic sample of the product is lung secretion or lung tissue.

In particular, the product uses NRK neutralizing antibodies as diagnostic markers.

On the other hand, the invention also provides application of the NRK neutralizing antibody in preparing a medicine for treating lung cancer.

The invention utilizes RNA-seq and a comprehensive bioinformatics method to analyze CAFs and NFs from LUAD I, II and III stage tissues, finds that NRK is specifically expressed in lung cancer tumor-related fibroblasts, can be used as a clinical diagnosis biomarker of lung cancer, and can provide a better diagnosis and prognosis index; NRK in LUAD-related CAFs is remarkably increased, and the NRK can be used as a target of LUAD combined treatment.

Drawings

FIG. 1 is a morphology diagram of CAFs and NFs.

FIG. 2 is the expression of CAFs, NFs and A549.

FIG. 3 is the expression of CAFs, NFs and A549 in IF experiments and WB experiments.

FIG. 4 is a diagram of transcriptional analysis of CAFs and NFs.

Fig. 5 is a digs volcano plot in which the red dots (upper light grey dots) represent 650 up-regulated genes and the blue dots (lower dark black dots) represent 1499 down-regulated genes.

Figure 6 is a log2(Fc) heatmap.

Fig. 7-12 are Gene Ontology (GO) analyses, where fig. 7-9 are BP (biological process), MF (molecular function), and CC (cellular component), respectively, for up-regulated pegs, and fig. 10-12 are BP, MF, and CC, respectively, for down-regulated pegs.

Fig. 13 and 14 are KEGG analysis of up-regulated and down-regulated DEGs, respectively.

FIG. 15 is an overlapping DEG obtained in public data sets (E-MTAB-6149 and E-MTAB-6653) of RNA-Seq data, LncRNA microarray and Arrayexpress, where | Fc | >2, P value < 0.05.

FIG. 16 is a histogram of ten overlapping DEGs genes.

FIG. 17 is the expression levels of NRK in CAFs, NFs, BEAS-2B, A549, and H1299.

FIG. 18 shows the results of Western Blot expression of NRK proteins in CAFs, NFs, BEAS-2B, A549 and H1299.

FIG. 19 is the expression of NRK in the nucleus and cytoplasm of CAF, NFs, BEAS-2B, A549 and H1299.

Figure 20 is a NRK expression heatmap.

FIG. 21 is an NRK overall survival diagram.

FIG. 22 shows the identification of interference of CAFs cell lentiviruses on NRK expression by qRT-PCR and WB.

FIG. 23 is qRT-PCR, WB identifying NFs expression of over-expressed NRK from the cell plasmid.

FIG. 24 shows the effect of Transwell on the migration of A549 and H1299 cells in CAF-shNRK-CM.

FIG. 25 shows the effect of Transwell on A549 and H1299 cell invasion by CAF-shNRK-CM.

FIG. 26 is a graph showing that the effect of NF-OE-CM on A549 and H1299 cell migration was examined by Transwell.

FIG. 27 is a graph showing that the effect of NF-OE-CM on A549 and H1299 cell invasion was examined by Transwell.

Figure 28 is the content of CXCL5 in ELISA assay conditioned media.

FIG. 29 shows the effect of Transwell detection interference NRK on the migration of CAFs.

FIG. 30 is a graph showing the effect of Transwell detection of over-expression of NRK on NFs migration.

FIG. 31 is a graph of the effect of WB detection of interference with NRK on p-Cofilin protein expression in CAFs.

FIG. 32 shows WB assay of the effect of over-expression of NRK on p-Cofilin protein expression in NFs.

FIG. 33 shows the results of F-actin staining of CAF, CAF-shNC and CAF-shNRK cells.

FIG. 34 shows the results of F-actin staining of NF, NF-NC and NF-OE cells.

Figure 35 is the result of fibroblast "priming" a549 cell motility.

FIG. 36 is the result of fibroblast "priming" H1299 cell movement.

Detailed Description

In order that the above objects, features and advantages of the present invention can be more clearly understood, there will now be described in detail, with reference to the accompanying drawings, a non-limiting detailed description of the present invention.

Materials and methods

Lung adenocarcinoma tissue and its corresponding non-malignant lung tissue (at least 5cm from the tumor) were prospectively collected from lung adenocarcinoma patients diagnosed as primary at the affiliated tumor hospital of Guangxi medical university. The selection criteria include: 1) pathologically diagnosed as LUAD; 2) patients did not receive chemotherapy or radiation therapy prior to surgery; 3) patients had no history of other types of tumors; 4) the patient has a complete medical record. Patients lacking AJCC staging information, lacking histological information, and dying within 30 days post-surgery or dying due to other disease causes were excluded from the analysis. Human tissues for study were reviewed and approved by the ethical review committee at the university of medical science, Guangxi, involving human subject studies.

Secondly, the separation, identification and culture of cells

Isolation, characterization and culture of CAFs cells

CAFs were separated from normal fiber cells (normal fibrates) NFs. Fresh tissue is washed by PBS and cut into 1-2 mm³Then at 25cm²The tissue culture flask of (1.5 ml) DMEM medium (Gibco, USA), 10% FBS (Gibco, USA) and 1% penicillin-streptomycin (Solarbio, Beijing, China) were added, and 5-6 tissue blocks were distributed at 25cm²In a tissue culture flask of (1). After 24h the medium was removed and 2ml of fresh medium supplemented with 10% fetal bovine serum was added to the tissue culture flask. Approximately seven days later, when the fibroblasts appeared to protrude, the medium was replaced again. After cell fusion reached 50%, the tissue was removed and fibroblasts were cultured in DMEM medium containing 10% fetal bovine serum.

Culture of LUAD cell lines

The normal human lung bronchial epithelial cell line BEAS-2B was obtained from Kunming division cell Bank, Chinese academy of sciences. Human LUAD cell lines a549 and H1299 were provided by the chinese academy of sciences stem cell bank. BEAS-2B was cultured on specific BEGM (Lonza co., USA), A549 and H1299 in RIPM-1640(Gibco, USA) medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

Third, RNA extraction

Total RNA from CAFs and NFs was extracted using TRIZOL reagent (Invitgen, USA). The quantity and quality of RNA were assessed using an Agilent BioAnalyzer2100(Agilent Technologies, USA), a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA), and a Qubit 2.0(Invitgen, USA). The RNA Integrity Number (RIN) of all samples was greater than 9.0.

Fourth, library preparation and sequencing

Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (New England Biolabs (NEB), USA) and an index code was added to the attribute sequence for each sample. mRNA was purified from total RNA using poly-T oligonucleotide-linked magnetic beads. Lysis was performed with divalent cations at high temperature in NEBNext first strand synthesis reaction buffer (5X). First strand cDNA was synthesized using random hexamer primers and M-MuLV reverse transcriptase. Second strand cDNA synthesis is then performed with DNA polymerase I and ribonuclease H, and the 3' end of the DNA fragment is adenylated and then ligated with a NEBNext linker having a hairpin loop structure in preparation for hybridization. To select cDNA fragments with a length of preference to 240bp, the library fragments were purified using the AMPure XP system (Beckman Coulter, USA). Mu.l of custom enzyme (New England Biolabs (NEB), USA) was then used to react with the size-selected adaptor-ligated cDNA at 37 ℃ for 15min, then at 95 ℃ for 5min, followed by PCR using Phusion High-Fidelity DNA polymerase, universal PCR primers and index (X) primers. PCR amplification was then performed with Phusion high fidelity DNA polymerase, universal PCR primers, and index (X) primers. The PCR products were finally purified (AMPure XP system) and the quality of the library was evaluated on the Agilent BioAnalyst 2100 system.

Fifthly, using Deseq2 to perform bioinformatics analysis and differential expression analysis of the CAFs.

Deseq2 found that genes with adjusted P values <0.05 and | fold change | >2 were classified as differentially expressed genes. The number of exon Fragments Per Kilobase (FPKM) was then used to estimate gene expression levels. Gene Ontology (GO) analysis was performed by the GO SEQ R software package, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was based on the KEGG database (http:// www.genome.jp/KEGG /). According to the RNAseq data, the public data sets (E-MTAB6149 and E-MTAB6653) of LncRNA microarray and Arrayexpress (https:// www.ebi.ac.uk/ArrayExpress /) selected DEG with an | fold change | >2 and adjusted P value <0.05 as the coverage DGE.

And sixthly, verifying the RNA-Seq data by using qRT-PCR.

qRT-PCR verified mRNA expression levels of 10 overlapping DEG in CAFs, NFs, BEAS-2B and LUAD cells (A549, H1299). Total RNA was extracted from cells using an RNA isolation kit (Axygen, USA). Was synthesized by reverse transcription using PrimeScriptTMRT Master Mix Kit (Takagaku Co., Ltd.). qRT-PCR was performed using Power SYBR Green PCR Master Mix (Invitgen, USA) and ABI 7500 real-time PCR system (applied biosystems, USA). By 2^-ΔΔCTThe relative expression levels of these DEGS were determined. Table 1 lists specific primers used in the qRT-PCR assay.

TABLE 1 qRT-PCR primer sequences

Seventh, survival analysis

"NRK" was retrieved in CCLE database (https:// portal. broadassociation. org/cle/about) and expression data of NRK in lung cancer cell lines were downloaded, verifying NRK expression on CCLE and Kapan-Meier plotters. A total of 192 cell lines were selected. PC-14 cell lines were reported as contaminating or identifying errors in the International cell line identification Committee, the cross-contamination or misidentification cell line database (http:// iclac. org/databases/cross-contaminations /), and were therefore excluded from this study. A total of 191 cell lines were collected for further analysis. In addition, a heatmap based on nrk expression in lung cancer cell lines was created using HEMI (heatmap Illustrator, version 1.0; http:// HEMI. biochakoo. org /). The 191 cell lines showed high (red) or low (blue) expression of NRK to varying degrees. Survival analysis was performed on a Kapan-Meier plotter (https:// kmplot. com/analysis /).

Eighth, Western Blot (WB) detection

Proteins were extracted from cells with RIPA buffer (Beyotime, china, shanghai) and quantified with BCA protein detection kit (Beyotime, china, shanghai). 20. mu.g of denatured protein were resolved on a 10% SDS-polyacrylamide gel and transferred to PVDF membrane (MilliPore; Merck KGaA, Darmstadt, Germany). The membranes were then sealed in 5% skim milk and incubated with primary antibody overnight at 4 ℃. The following primary antibodies were used: rabbit anti-alpha-SMA monoclonal antibody (1: 1000; cell Signal technology, USA), rabbit anti-alpha-GAPDH monoclonal antibody (1: 1000; Abcam, England Kanbu); rabbit anti-Vimentin monoclonal antibody (1: 1000; cell Signal technology, USA); anti-E-cadherin monoclonal antibody (1: 1000; cell signaling technologies, USA); rabbit anti-NRK (1: 500; Invitgen; Thermo Fisher Science, USA); rabbit anti-GAPDH mAb (1: 5000; cell Signal technology, USA), murine anti-Cofilin (1: 200; Santa Cruz; USA). All membranes were washed 3 times in PBST and then incubated with anti-rabbit IgG secondary antibody (DyLightTM 8004 XPEG conjugate, Cst, usa) for 1h at room temperature. The membrane bands were then imaged using the infrared fluorescence imaging system, Oddeson (LI-COR, USA).

Nine, Immunofluorescence (IF) detection

Cells were washed 3 times with PBS, fixed with 4% paraformaldehyde (Solarbio, beijing, china) at room temperature for 1h, and gently washed 3 times with PBS. Cells were then blocked with 5% goat serum (Solarbio, beijing, china) for 30 min. The blocked cells were incubated with rabbit anti- α -SMA monoclonal antibody (1: 200; cell signaling technology, USA), rabbit anti-FAP- α (1: 200; Canbam, UK), rabbit anti-NRK (1: 25; Origene, USA) anti-NRK, and then with secondary antibody (1: 200; Abcam, UK) at room temperature for 1 h. The immunolabeled cells were counterstained with 25. mu.l of DAPI (Solarbio, Beijing, China) for 8min at room temperature. Cell samples were observed under confocal microscopy (Leica Microsystems CMS GmbH/Model DMi8, Germany).

Ten, CAFs cell lentivirus transfection experiment

(1) The CAFs cells which are in good growth state and in logarithmic growth phase are digested, inoculated into a 24-well plate, and 5000 cells per well are added with 500 mu L DMEM medium and placed in an incubator overnight.

(2) The next day, get the dropDegree of 1X 10⁸TU/mL virus solution 50. mu.L was added to 450. mu.L DMEM medium and gently mixed. The old medium was aspirated away with a sterile pasteur pipette, the cells were washed 3 times with PBS, and then diluted virus solution was added and placed in an incubator for 24 hours. NRK interfering lentivirus sequences are shown in the following table:

TABLE 1 Lentiviral interference sequences

Table 1 Lentiviral Interference Sequence

(3) And (3) discarding the diluted virus solution, adding 500 mu L of fresh DMEM medium to culture for 3-4 days, adding 2 mu g/mL puromycin to screen transfected cells if the cell state is good, and adding 1-2 drops of FBS to each cell to adjust the cell state if the cell state is not good. Puromycin can be used for screening when the cell state is good. During the screening process, liquid change or passage is carried out according to the growth state of the cells.

(4)2 mu g/mL puromycin is continuously screened for 2 weeks, and the CAFs stable transformant with the expression of the NRK knocked down can be obtained.

Eleven, NFs cell plasmid transient transfection

(1) Before use, a DNA powder sample with high plasmid purity is centrifuged (10000r/min, 10-15 seconds) and then carefully opened, a proper amount of ddH2O (the final concentration of the plasmid is 500 ng-1 mug/muL) is added, a tube cover is screwed, the DNA powder sample is fully dissolved by vortex oscillation and then can be used for cell transfection, and the dissolved plasmid powder is stored in a refrigerator at the temperature of-20 ℃.

(2) Four groups are set, a blank control group (control), a transfection reagent control group (mock transfection), a negative control group (negative control), and a target gene experiment group, and each group is provided with three duplicate wells. NFs cells which had been grown well and were in logarithmic phase were digested, inoculated into 24-well plates at 5000 cells per well, and 500. mu.L of DMEM medium was added and left in the incubator overnight.

(3) Lipofectamine2000 was allowed to equilibrate at room temperature and the plasmid was dissolved on an ice box.

(4) Two sterile 1.5mL centrifuge tubes were labeled tube A and tube B, respectively. Add appropriate volume of serum free DMEM medium and Lipofectamine2000 to tube A, mix well (24 well plates, 50. mu.L of medium per well, 2. mu.L Lipofectamine2000), incubate for 5 minutes at room temperature.

(5) Tubes B were filled with appropriate volumes of serum-free DMEM medium and plasmid (24-well plates with 50. mu.L of medium per well, 0.4. mu.g of plasmid).

(6) The liquids in the tube A and the tube B are mixed uniformly and are placed for 20 minutes at room temperature. Discarding the old culture medium in the 24-well plate, adding a proper amount of serum-free DMEM culture medium into each well, dropwise adding the transfection mixture into the well plate, uniformly mixing, and incubating in an incubator for 4-6 hours.

(7) The transfection solution was aspirated off, replaced with complete medium, and cells were collected after 48 hours incubation in an incubator.

(8) To construct stable transformants, 500. mu.g/mL of G418 was added to the complete medium for 3 weeks of selection.

Twelve, fibroblast conditioned Medium Collection

(1) NFs and CAFs cells with the same growth generation number are taken, counted after trypsinization, and 2 x 106 NFs and CAFs cells are respectively inoculated into different T75 culture bottles and placed in a 5% CO2 cell culture box at 37 ℃ overnight.

(2) The original culture medium is discarded, two groups of cells are washed by PBS for 1-2 times respectively, 10mL of serum-free DMEM culture medium is added, and the cells are placed in a cell culture box with the temperature of 37 ℃ and the content of 5% CO2 for culture for 72 hours.

(3) Collecting supernatant after 72 hours, putting the collected fibroblast supernatant into a 4 ℃ centrifuge, centrifuging for 10min at 5000r/min, and taking the supernatant as CAFs conditioned medium (CAF-CM) and NFs conditioned medium (NF-CM).

Thirteen, Transwell experiment

1Transwell migration experiment

(1) A549 in logarithmic growth phase and good growth state is taken, and H1299 cells are starved for 12 hours in advance.

(2) The cells were digested with 0.25% trypsin digestion solution, centrifuged to remove the supernatant, 1mL of serum-free medium was aspirated with a 1mL sterile blue tip and aspirated to mix well, the cells were counted, and the density of the single cell suspension was adjusted to 4X 105/mL. 100 μ L of single cell suspension of each group of cells was added to the upper chamber of the Transwell chamber.

(3) 600. mu.L of DMEM medium containing 20% fetal bovine serum, NF-CM, CAF-CM were added to the lower chamber of the Transwell. The cells were cultured in a 5% CO2 cell culture chamber at 37 ℃ for 12 hours.

(4) The well plate was removed, the cells that did not migrate in the upper chamber were gently wiped off with a cotton swab, 4% paraformaldehyde solution was added, fixed for 30 minutes at room temperature, and washed 3 times with PBS.

(5) Stained with 0.1% crystal violet solution for 1 hour and washed with tap water. The cells were photographed by placing them under a fully automatic cell imager. The field of 5 non-duplicate cells was selected at 200 x magnification and the cells were counted using Image J, 3 duplicate wells per group and independent experiments were repeated 3 times.

2 Transwell invasion test

(1) Items such as 200 mu L of sterile yellow suction head, 1.5mL of sterile centrifuge tube, serum-free DMEM medium and the like required by the experiment need to be pre-cooled in advance.

(2) Matrigel gel: serum-free DMEM gel is diluted according to the proportion of 1:8, and after the serum-free DMEM gel and the serum-free DMEM gel are fully and uniformly mixed, 80 mu L of diluted Matrigel gel is added into an upper chamber of a Transwell chamber. And (3) placing the mixture in a cell culture box for 4-6 hours to fully solidify the Matrigel gel.

The rest of the procedure was the same as 1Transwell migration experiment.

Fourteen, ELISA experiments

(1) The sample to be tested was added to a well plate of the ELISA kit, and 100. mu.L of the sample was incubated at 37 ℃ for 90 minutes.

(2) The well was decanted, 100. mu.L of biotinylated antibody/antigen working solution was added, and incubated at 37 ℃ for 60 minutes.

(3) The well plates were washed 3 times with wash solution in the ELISA kit, and 350. mu.L of wash solution was added to each well.

(4) Add 100. mu.L of enzyme-bound working solution and incubate at 37 ℃ for 30 min.

(5) Wash 5 times, add 350. mu.L of wash solution per well.

(6) 90 μ L of substrate solution was added and incubated at 37 ℃ for 15 minutes.

(7) Add 50. mu.L of stop solution and measure OD at 450nm wavelength with microplate reader.

Fifteen, F-actin cytoskeletal protein staining

(1) Putting an alcohol lamp, PBS (phosphate buffer solution), a culture medium, 0.25% pancreatin digestive juice, a 6-pore plate special circular cell slide, an alcohol lamp, tweezers and other articles in a super clean bench. And turning on the ultra-clean bench ultraviolet lamp for 30 minutes.

(2) Digesting the CAFs and NFs cells respectively by pancreatin, blowing and beating the cells to a single cell suspension by a Pasteur pipette, clamping the cell slide by tweezers, and putting the cell slide into a 6-well plate.

(3) Cell counting is carried out on the CAFs and NFs single cell suspension, cell slide is manufactured according to the concentration of 5000 cells/hole, and the cell slide is placed in a 5% CO2 cell culture box at 37 ℃ overnight.

(4) The next day, the corresponding 6-well plate was removed, the cells were washed 3 times with PBS, the PBS was blotted dry, 1mL of 4% paraformaldehyde solution was added to each well, and the cells on the slide were fixed for 30 minutes at room temperature. PBS rinse 3 times for 5 minutes each.

(5) Add 500. mu.L of 0.2% Triton X-100 solution per well to allow for 30min and rinse 3 times with PBS for 5min each.

(6) Add 1mL of 1% BSA solution to each well and block for 1 hour at room temperature, pipette dry blocking solution. Then 50. mu.L of diluted F-actin staining diluent is added, and the mixture is sealed for 60 minutes at room temperature in a dark place.

(7) Pipette off the staining diluent on the slide, rinse 3 times with PBS for 5 minutes each.

(8) 50 μ L of DAPI solution was added dropwise at room temperature in the dark, incubated for 8min, and the PBST was rinsed 3 times for 5min each. A10. mu.L white pipette tip dipped 1 drop of the anti-fluorescence quencher onto the slide, and the circular cell slide was carefully removed with the cell side down for mounting. And (5) placing the covered climbing film in a light-proof wet box for preservation.

(9) The confocal laser scanning microscope scans the fluorescence of each cell slide. Independent experiments were repeated 3 times.

Sixteen and sixteen CAFs guide cancer cell movement experiment

(1) GFP fluorescent protein marks A549 and H1299 cells.

(2) The pancreatin digests CAFs and NFs cells with good growth state respectively, and inoculates the CAFs and NFs cells in a 24-well plate, wherein each well contains 5000 cells. After 24 hours, 1. mu.L of Dil solution (Dil solution concentration 5. mu.g/. mu.L) was added to each well.

(3) After adding the Dil, putting the pore plate into an incubator at 37 ℃ for incubation for 5 minutes, then putting the pore plate into a refrigerator at 4 ℃ for incubation for 15 minutes (preventing the Dil dye from being endocytosed by cells and avoiding the cells from being incapable of being marked with red fluorescence), finally putting the pore plate into the incubator at 37 ℃ for incubation for 8 hours, and observing the condition that the fibroblasts are marked with red fluorescence by using a fluorescence microscope.

(4) The red fluorescent marked fibroblasts are trypsinized and inoculated on a 6-well plate, after about 4 hours, the fibroblasts are attached to the wall, and GFP marked A549 and H1299 cells can be added.

(5) Cells were observed by fluorescence microscopy and photographed (5 random non-repetitive fields).

Seventeen, experimental results

1. Clinical characteristics of the patient

A total of 13 samples of LUAD patients were enrolled for qRT-PCR validation, and all LUAD patients were at stage I, II, or III at diagnosis. The clinical characteristics of the patients are shown in table 2.

TABLE 2 clinical characteristics of patients with tumor and paracancerous tissues for isolation of CAFs and NFs

2. Identification of CAFs

As shown in fig. 1, CAFs are characterized by typical spindle-shaped, flattened, and fibroblast-like morphology, similar to NFs. A pair of CAFs and NFs was randomly selected and specific biomarkers for activated fibroblasts were identified by WB and IF analysis. As shown in fig. 2, CAFs strongly express α -SMA and FAP- α, whereas NFs and a549 weakly express the two biomarkers of activated fibroblasts. In addition, both CAFs and NFs strongly expressed the mesenchymal marker Vimentin, but not the epithelial marker E-cadherin. However, the LUAD cell line A549 strongly expressed E-cadherin and weakly expressed Vimentin (FIG. 2). Expression of these markers in the IF assay was consistent with expression in the WB assay (FIG. 3). The results show that the CAFs and NFs were successfully isolated without contamination by epithelial or cancer cells.

3. Identification of DEGS from RNA-Seq

Transcriptional analysis of CAFs and NFs showed that a total of 35,259 genes were detected. The Principal Component Analysis (PCA) result shows that CAFs and NFs are respectively clustered into one class, and the clustering rate of PC1 is as high as 45.9% (figure 4), which shows that the two classes have different biological characteristics and are suitable for the next differential analysis. The standard for the upregulation of DEGS is fold difference (Fc) >2, adjusted P value < 0.05; the standard for the down-regulation of DEGs was FC < -2, adjusted P value < 0.05. A total of 1799 DEGs were identified, including 650 upregulated DEGs and 1499 downregulated DEGs. The distribution of the up-and down-regulated DEGs is shown by the volcano plot (fig. 5). Up-regulated DEG is represented in red and down-regulated DEG is represented in blue. The log2(Fc) heatmap showed a different expression pattern in the CAFs compared to NFs (fig. 6).

4. GO analysis of DEG in CAFs compared to the function of DEG in non-CAF

To determine the function of DEG in the CAFs. In contrast to NFs, the GO analysis classified potential functions (adjusted P value < 0.05). There are 188 classes for BP, 83 classes for MF and 40 classes for CC. As shown in fig. 7-12, the first 15 significantly rich sets of up and down regulation DEGs in BP, MF, CC categories are shown, respectively. In the BP category, most of the up-regulated DEGS was concentrated on the development of the skeletal system (GO: 0001501), and was also associated with organelle division (GO: 0048285) and nuclear division (GO: 0000280) (FIG. 7). Furthermore, upregulated DEGs are also associated with extracellular matrix structural components (GO: 0005201), G protein-coupled amine receptor activity (GO: 0008227) and single chain ATPase activity (GO: 0043142) of the MF class (FIG. 8), and chromosomal regions (GO: 0098687), extracellular matrix (GO: 0031012) and collagen-containing extracellular matrix (GO: 0062023) of the CC class (FIG. 9). Meanwhile, the down-regulated DEGs are rich in extracellular structures (GO: 0043062), positive regulation of blood vessel diameter (GO: 0097755) and muscular system protrusion (GO: 0003012) in a BP class (figure 10), rich in receptor regulation activity (GO: 0030545), receptor ligand activity (GO: 0048018) and extracellular matrix components (GO: 0005201) in an MF class (figure 11), and rich in extracellular matrix (GO: 0031012), collagen-containing extracellular matrix (GO: 0062023) and neuronal cells (GO: 0043025) in a CC class (figure 12). Taken together, our results indicate that up-and down-regulated DEG is primarily involved in ECM function

KEGG pathway analysis in CAFs found that up-regulated DEGs are most often involved in 5 signaling pathways related to glycolysis/gluconeogenesis, pentose phosphate pathway, galactose metabolism, fructose and mannose metabolism, ascorbic acid and uronic acid metabolism (fig. 13). On the other hand, the 8 signaling pathways involved in the down-regulation of deg expression are glycolysis/gluconeogenesis, galactose metabolism, fatty acid biosynthesis, ascorbic acid and uronic acid metabolism, pentose and glucuronic acid interconversions, fatty acid elongation, pentose phosphate pathway and fructose and mannose metabolism, respectively (fig. 14). Thus, the metabolic pathways, especially the glycolytic/gluconeogenic pathway, enrich the DEGs, suggesting that CAFs may regulate the development of LUAD primarily through metabolic pathways.

5. Validation of overlapping DEGS by qRT-PCR

Overlapping DEG's with 10. sup. Fc | >2 and adjusted P value <0.05, including 6 up-regulated DEG's and 4 down-regulated DEG's (FIG. 15, Table 3), were obtained from the public datasets (E-MTAB-6149 and E-MTAB-6653) of RNA-Seq data, LncRNA microarrays and Arrayexpress (FIG. 15, Table 3), 6 of which were up-regulated DEG's and 4 down-regulated DEG's were public datasets (E-MTAB-6149 and E-MTAB-6653) from Arrayexpress (FIG. 15, Table 3). Consistent with the RNA-seq data, 6 overlapping DEGs (ZNF93, ZNF827, NRK, DPYSL4, HES4, LYPD6B) were significantly up-regulated in CAFs, while FCGBP and CFI were significantly down-regulated in CAF. In contrast, SETBP1 and HMCN1 expression were elevated in CAF with low RNA-seq expression. As shown in fig. 16, NRK was confirmed as the most significant up-regulated gene among the ten overlapping DEGs.

TABLE 3 comparison of CAFs with NFs, there were 10 intersection differences DEGs in CAFs

6. Expression levels of NRK in CAF of LUAD patients and their relationship to prognosis

The mRNA levels of NRK in CAFs were significantly higher than the corresponding NFs, normal lung epithelial cell BEAS-2B and two LUAD cell lines a549 and H1299 (fig. 17). WB results showed that NRK protein was strongly expressed in CAF, but weakly in NFs, BEAS-2B, A549 and H1299 (FIG. 18). Furthermore, the results showed that the NRK protein is mainly expressed in the nucleus and cytoplasm of CAF, but hardly expressed in NFs, BEAS-2B, A549 and H1299 (FIG. 19). In addition, 191 lung cancer cell lines expressing NRK in a CCLE database were selected. Each cell line had a corresponding NRK expression value. The NRK expression heatmap of 191 lung cancer cell lines showed a majority of bands in blue, indicating low NRK expression in lung cancer (figure 20). Only 32 cell lines NRK are highly expressed, wherein the LUAD cell line 13, the LUSC cell line 1, the large cell carcinoma cell line 1, the small cell lung carcinoma cell line 3 and other subtype lung carcinoma cell lines 14 are included. There were 159 cell lines with low NRK expression, including 42 LUAD cell lines, 6 LUSC cell lines, 3 large cell cancer cell lines, 30 small cell lung cancer cell lines, 78 other subtype lung cancer cell lines (see table below, fig. 20). Furthermore, high expression of NRK mRNA in LUAD patients is predictive of shorter overall survival (logrank P ═ 0.0093) (fig. 21). The results indicate that mRNA of NRK is weakly or not expressed in most LUAD cell lines, but is better expressed in CAFs within the TME of LUAD and is associated with low survival of LUAD patients.

7. CAFs cell lentivirus interference NRK outcomes

As shown in FIG. 22, the results of qRT-PCR showed that three lentiviral sequences interfering NRK, shNRK-770, shNRK-3629 and shNRK-3847, all reduced the mRNA expression level of NRK in CAFs compared with CAFs, and the relative mRNA expression levels of NRK in CAF-shNC, CAF-sh770, CAF-sh3629 and CAF-sh3847 cells were 1.047 + -0.079, 0.936 + -0.045, 0.559 + -0.016 and 0.226 + -0.016 respectively. WB results showed that relative expression levels of NRK protein in CAF-shNC, CAF-sh770, CAF-sh3629 and CAF-sh3847 cells were 1.036. + -. 0.072, 0.779. + -. 0.047, 0.499. + -. 0.045 and 0.244. + -. 0.125, respectively, as compared with NFs. The result shows that the shNRK-3847 interference sequence has the highest NRK gene silencing efficiency, which reaches about 80%. Therefore, in subsequent experiments, a shNRK-3847 interference sequence is selected to interfere the NRK with high CAFs expression.

8. NFs cell plasmid transient NRK results

As shown in FIG. 23, the qRT-PCR results show that the relative mRNA expression levels of NRK in NF-NC and NF-OE cells are 1.15 +/-0.14 and 3.43 +/-0.42 respectively compared with NFs, the mRNA expression level of NRK in NF-OE is obviously higher than NFs (P <0.05), and the mRNA expression level of NRK in NF-NC and NFs have no significant difference. WB results show that compared with NFs, the relative expression levels of NRK protein in NF-NC and NF-OE cells are respectively 1.097 +/-0.097 and 3.78 +/-0.30, the expression level of NRK protein in NF-OE is obviously higher than NFs (P <0.05), and the expression level of NRK protein in NF-NC and NFs have no significant difference.

9. Effect of CAF-CM interfering with NRK (CAF-shNRK-CM) on migration of lung adenocarcinoma cells

As shown in FIG. 24, the number of cells migrated from A549 cells under the conditions stimulated by CAF-CM, CAF-shNC-CM and CAF-shNRK-CM was 354. + -. 12, 330. + -. 20 and 223. + -. 17, respectively. The number of the cells migrated by the H1299 cells under the conditions of CAF-CM, CAF-shNC-CM and CAF-shNRK-CM stimulation is 560 +/-35, 542 +/-15 and 304 +/-8 respectively. Compared with CAF-shNC-CM, the cell migration number of A549 and H1299 is reduced under the stimulation of CAF-shNRK-CM, namely the CAF-shNRK-CM can reduce the migration capacity of lung adenocarcinoma cells A549 and H1299 (P < 0.05).

10. Effect of CAF-conditioned Medium (CAF-shNRK-CM) interfering with NRK on Lung adenocarcinoma cell invasion

As shown in FIG. 25, the numbers of A549 cells invading cells were 311. + -. 15, 296. + -.7 and 178. + -.13 respectively under the stimulation conditions of CAF conditioned medium (CAF-CM), CAF conditioned medium interfering with empty vector (CAF-shNC-CM) and CAF-shNRK-CM. The number of the H1299 cells invading the cells under the conditions of CAF-CM, CAF-shNC-CM and CAF-shNRK-CM stimulation is 505 +/-21, 498 +/-9 and 316 +/-8 respectively. Compared with CAF-shNC-CM, the cell invasion number of A549 and H1299 is reduced under the stimulation of CAF-shNRK-CM, namely the CAF-shNRK-CM can reduce the invasion capacity of lung adenocarcinoma cells A549 and H1299 (P < 0.05).

11. Effect of NF-conditioned Medium overexpressing NRK (NF-OE-CM) on migration of Lung adenocarcinoma cells

As shown in FIG. 26, the number of migrating cells of A549 cells was 108. + -. 8, 115. + -. 12, and 264. + -. 18 under the stimulation of NF-conditioned medium (NF-CM), NF-conditioned medium over-expressing empty vector (NF-NC-CM), and NF-OE-CM, respectively. The number of the migrating cells of the H1299 cells under the stimulation of NF-CM, NF-NC-CM and NF-OE-CM is 189 +/-12, 170 +/-20 and 312 +/-18 respectively. Compared with NF-CM, the A549 and H1299 have reduced cell migration number under the stimulation of NF-OE-CM, namely the NF-OE-CM can increase the migration capability of the A549 and H1299 of lung adenocarcinoma cells (P < 0.05).

12. Effect of NF-CM overexpressing NRK (NF-OE-CM) on Lung adenocarcinoma cell invasion

As shown in FIG. 27, the numbers of the A549 cells invading the cells were 130 + -10, 135 + -8 and 256 + -16 respectively under the stimulation of NF-CM, NF-NC-CM and NF-OE-CM. The number of the H1299 cells invading under the stimulation condition of NF-CM, NF-NC-CM and NF-OE-CM is 170 +/-15, 183 +/-11 and 320 +/-21 respectively. Compared with NF-CM, A549 and H1299 have reduced cell migration number under the stimulation of NF-OE-CM, namely the NF-OE-CM can improve the invasive ability of the lung adenocarcinoma cells A549 and H1299 (P < 0.05).

13. Effect of CAF-CM, NF-CM interfering with or overexpressing NRK on CXCL5

The content of CXCL5 in CAF-CM, CAF-shNC-CM and CAF-shNRK-CM was 10.49. + -. 0.71ng/mL, 9.93. + -. 0.58ng/mL and 23.89. + -. 1.09ng/mL, respectively. The contents of CXCL5 in NF-CM, NF-NC-CM and NF-OE-CM were 17.76. + -. 0.78ng/mL, 17.90. + -. 0.27ng/mL and 13.13. + -. 0.42ng/mL, respectively. The above results show that CXCL5 is higher in NF-CM and CAF-shNRK-CM than in CAF-CM (P <0.05), and CXCL5 is higher in CAF-shNRK-CM than in NF-CM (P < 0.05). However, the content of CXCL5 in NF-OE-CM was lower than that in NF-CM (P < 0.05). As shown in fig. 28, the ELISA results suggest that NRK in CAFs inhibits the release of CXCL 5.

14. Effect of interfering NRK on the motion capability of CAFs

As shown in FIG. 29, in the results of the Transwell migration experiment, the number of CAF-shNC migrations was 67. + -.5, the number of CAF-shNC migrations was 70. + -.7, and the number of CAF-shNRK migrations was 28. + -.7. Compared with CAF, the migration number of CAF-shNRK is reduced (P < 0.05).

15. Effect of over-expressing NRK on NFs locomotor ability

As shown in FIG. 30, the results of the Transwell migration experiment revealed that the number of NF-migrated was 22. + -.4, the number of NF-NC migrated was 28. + -.2, and the number of NF-OE migrated was 41. + -.5. The number of NF-OE migrations was increased compared to NF (P < 0.05).

16. Interference on the influence of NRK on p-Cofilin proteins in CAFs

As shown in FIG. 31, the WB results showed that the relative expression amounts of P-Cofilin protein in CAF-shNC and CAF-shNRK were 0.972. + -. 0.069 and 0.004. + -. 0.003, compared with CAF, and that the expression amount of P-Cofilin protein in CAF-shNRK was significantly decreased (P <0.05) compared with CAF, while there was no significant difference in the expression of P-Cofilin protein in CAF-shNC.

17. Effect of over-expression of NRK on p-Cofilin protein in NFs

As shown in FIG. 32, WB results showed that the relative expression amounts of P-Cofilin protein in NF-NC and NF-OE were 0.978. + -. 0.003 and 3.31. + -. 0.15, and the expression amount of P-Cofilin protein in NF-OE was significantly increased (P <0.05) compared to NF, while there was no significant difference in the expression of P-Cofilin protein in NF-NC.

18. Interference with the Effect of NRK on CAF framework protein (F-actin)

As shown in FIG. 33, the staining of F-actin in CAF-shNRK was reduced as compared with CAF, and the amount of F-actin microfilament protein accumulated in the cell membrane by CAF-shNRK was reduced, and the number of lamellipodia and filopodia was reduced.

19. Effect of over-expression of NRK on NF backbone protein (F-actin)

As shown in FIG. 34, F-actin expressed in NF-OE cells was increased as compared with NF, and F-actin microfilament proteins accumulated on cell membranes by NF-OE were increased in number, and lamellipodia and filopodia were increased.

20. Experimental results of CAFs guiding movement of lung adenocarcinoma cells

When CAFs and A549 cells were co-cultured, A549 cells were uniformly distributed around CAFs, NFs, CAF-shNC, and CAF-shNRK at 0h (FIGS. 35A-D). After 24h, a549 moved significantly along the path of CAFs movement compared to NFs (fig. 35E-F). Compared with CAFs and CAF-shNC, A549 does not move obviously along the path of CAF (CAF-shNRK) interfering with NRK (FIGS. 35G-H).

21. When CAFs and H1299 cells were co-cultured, H1299 cells were evenly distributed around CAFs, NFs, CAF-shNC, and CAF-shNRK at 0H (FIGS. 36A-D). After 24H, H1299 has moved significantly along the path of movement of the CAFs compared to NFs (FIGS. 36E F). Compared with CAFs and CAF-shNC, H1299 does not move significantly along the path of CAF (CAF-shNRK) interfering with NRK (FIGS. 36G-H).

From the above results, it was found that NRK is highly expressed specifically in CAFs by analyzing CAFs and NFs derived from LUAD stages I, II and III using RNA-seq and a comprehensive bioinformatics method. In addition, the results of cell experiments show that the NRK with high expression of CAFs can promote invasion and migration of lung adenocarcinoma cells through two ways: firstly, NRK with high expression of CAFs inhibits CXCL5 release, and possibly improves the invasion and migration capacity of lung adenocarcinoma cells. Secondly, NRK with high CAFs expression increases F-actin polymerization and enhances CAFs movement capacity through phosphorylation Cofilin, thereby possibly inducing the enhancement of invasion and migration capacity of lung adenocarcinoma cells. Therefore, NRK can be used as a clinical diagnosis biomarker of lung cancer, and can provide a better diagnosis and prognosis index; NRK in LUAD-related CAFs is remarkably increased, and the NRK can be used as a target of LUAD combined treatment.

It should be noted that the above-mentioned preferred embodiments are merely illustrative of the technical concepts and features of the present invention, and are intended to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

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<120> medical application of NRK in lung cancer treatment and prognosis diagnosis

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Claims (10)

1. Use of NRK in the preparation of a product for the diagnosis of prognosis of lung cancer.
2. 2. Use according to claim 1, characterized in that: the product uses NRK as a diagnostic marker.
3. 3. Use according to claim 2, characterized in that: the high expression of NRK is predictive of poor prognosis in patients with lung adenocarcinoma.
4. 4. Use according to claim 1, characterized in that: the product is a real-time fluorescent quantitative PCR detection kit or diagnostic test paper.
5. Application of NRK in preparing a medicine for treating lung cancer.
6. 6. Use according to claim 5, characterized in that: the drug product has NRK as a therapeutic target.
7. Use of an NRK neutralizing antibody in the manufacture of a product for diagnosing lung cancer.
8. 8. Use according to claim 7, characterized in that: the product is a diagnostic test strip or kit and the diagnostic sample of the product is lung secretions or lung tissue.
9. 9. Use according to claim 7, characterized in that: the product uses NRK neutralizing antibodies as diagnostic markers.
10. Use of an NRK neutralizing antibody in the manufacture of a medicament for the treatment of lung cancer.

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