Application of TDP-43 inhibitor in preparation of medicine for treating lung adenocarcinoma

Abstract

The invention belongs to the technical field of biological agents, and particularly relates to application of a TDP-43 inhibitor in preparation of a medicine for treating lung adenocarcinoma. Research results show that the TDP-43 can promote lung adenocarcinoma cell proliferation and inhibit cell death. By inhibiting the expression of the TDP-43, the proliferation activity of the cells can be remarkably reduced, and the cell death rate can be improved. In addition, the TDP-43 has the function of inhibiting ferroptosis of lung adenocarcinoma cells. After the TDP-43 is silenced, the expression of the iron ion chaperonin PCBP1 is obviously reduced. The TDP-43 can be used for positively regulating and controlling the expression level of the PCBP1.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological preparations, and in particular relates to the application of a TDP-43 inhibitor in the preparation of a drug for treating lung adenocarcinoma. Background Art

[0002] Lung cancer is a common respiratory malignancy in my country, and lung adenocarcinoma is the most common histological type of lung cancer, accounting for about 40% of all lung cancers. It mainly originates from the bronchial mucosal epithelium and mucous glands, and is more likely to occur in women and smokers at a younger age. It is a peripheral lung cancer. Over the years, despite the continuous development and improvement of tumor treatment methods, lung adenocarcinoma is still one of the most invasive and short-term deterioration tumor types. Therefore, elucidating the molecular mechanism of the occurrence and development of lung adenocarcinoma and finding new therapeutic targets are of great significance for improving the prognosis of patients.

[0003] TDP-43 is a ubiquitously expressed DNA / RNA binding protein encoded by the TAR gene. It can homodimerize through its N-terminal domain and preferentially bind to UG-rich sequences through its C-terminal domain, thereby participating in the regulation of multiple processes such as RNA transcription, selective splicing, stability, transport and translation. Over the years, TDP-43 has been widely reported for its important role in neurodegenerative diseases, but recent studies have also found that it is closely related to the occurrence and development of cancer. Studies have shown that TDP-43 is abundantly expressed in various tissues such as breast cancer, liver cancer and melanoma. In breast cancer cells, TDP-43 can form a coordinated complex with the splicing factor SRSF3, promoting the metastasis of cancer cells by regulating the splicing process of target gene exons. Recent studies by our research group have found that the expression of TDP-43 in lung adenocarcinoma tissue is significantly higher than that in adjacent tissues; moreover, TDP-43 can promote lung cancer cell proliferation and inhibit cell death; however, its specific role in the malignant progression of lung adenocarcinoma remains to be explored. Among them, the full English name of TDP-43 is TARDNA-bindingprotein43.

[0004] Ferroptosis is a newly discovered type of iron-dependent cell death. Intracellular iron accumulation and lipid peroxidation are two important events that lead to ferroptosis. More and more evidence has revealed the important role of the iron chaperone protein PCBP1 in the regulation of ferroptosis. PCBP1 is a multifunctional RNA-binding protein involved in gene transcription, RNA regulation, and iron loading of ferritin. It is responsible for transporting iron to ferritin for iron metabolism, storage, and transport. There is evidence that blocking the function of PCBP1 as an iron carrier can induce an increase in the level of cellular free iron, which is prone to excessive iron accumulation and leads to ferroptosis. However, the product use of TDP-43 in the treatment of lung adenocarcinoma associated with cellular ferroptosis has not yet been found. Summary of the invention

[0005] In order to solve the problem that the use of TDP-43 in the treatment of cell ferroptosis and lung adenocarcinoma has not yet been found in the prior art, the present invention provides the use of a TDP-43 inhibitor in the preparation of a drug for treating lung adenocarcinoma.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] The preliminary RNA-seq data of the present invention showed that TDP-43 is closely related to the process of ferroptosis, and PCBP1 may be a downstream target gene of TDP-43. In response to the above scientific issues, the present invention further proposes a scientific hypothesis: TDP-43 inhibits the occurrence of cell ferroptosis by regulating the expression of PCBP1, promotes cell survival, and then promotes the malignant progression of lung adenocarcinoma. Based on this, the present invention provides the use of TDP-43 inhibitors in the preparation of drugs for the treatment of lung adenocarcinoma, wherein the lung adenocarcinoma is related to cell ferroptosis. The present invention provides TDP-43 inhibitors that can be used for the preparation of drugs for the treatment of lung adenocarcinoma, which solves the problem that the product use of TDP-43 in the treatment of lung adenocarcinoma related to cell ferroptosis has not yet been found in the prior art.

[0008] Preferably, the proliferation of lung adenocarcinoma cells is inhibited by knocking down or knocking out the TDP-43, or inhibiting the expression of the TDP-43.

[0009] The present invention also provides the use of the TDP-43 inhibitor in the preparation of a drug for promoting ferroptosis of lung adenocarcinoma cells.

[0010] Preferably, the TDP-43 inhibitor comprises any one or both of siRNA and sgRNA-TDP-43.

[0011] The nucleotide sequence of si-TDP-43#1 in the siRNA is shown in SEQ ID NO.1: GCTTCGCTACAGGAATCCA; the nucleotide sequence of si-TDP-43#2 in the siRNA is shown in SEQ ID NO.2: GGAGAGGACTTGATCATTA; the nucleotide sequence of sgRNA-TDP-43 is shown in SEQ ID NO.3: TAGATTGCGCAGTCTCTTTG.

[0012] Preferably, the TDP-43 inhibitor is used to promote ferroptosis of lung adenocarcinoma cells.

[0013] Preferably, the drug contains the TDP-43 inhibitor as an active ingredient and is supplemented with pharmaceutically acceptable excipients.

[0014] The concentration of the TDP-43 inhibitor in the drug is 100 nM to 200 nM.

[0015] Preferably, the auxiliary material comprises any one or more of water, saline, buffer, glycerol and ethanol.

[0016] Preferably, the drug includes oral dosage forms and injection dosage forms. The oral dosage form is a solid or liquid preparation formed by combining the TDP-43 inhibitor with excipients and then taken orally, which is convenient to carry and take.

[0017] The injectable dosage forms include normal saline injection and sterile TDP-43 inhibitor powder for injection, which are rapidly absorbed and effective.

[0018] The present invention can prepare the TDP-43 inhibitor into the following specific pharmaceutical dosage forms, but is not limited thereto: any pharmaceutical dosage form that can be used to prevent and treat lung adenocarcinoma can be prepared by the field of pharmaceutical preparations.

[0019] Preferably, the drug is a solution, and the solvent in the solution dosage form is water.

[0020] The concentration of the TDP-43 inhibitor in the solution is 100nM to 200nM. The solution drug has a large dispersion, fast absorption, rapid action, and good bioavailability; the active ingredients are evenly dispersed and can be accurately measured and used, especially suitable for children and elderly patients; the dosage size is easy to adjust and control; it can reduce the local irritation of certain soluble drugs; and it can increase the stability and safety of certain drugs. Therefore, the dosage form of the above-mentioned drug provided by the present invention is a solution.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides an application of a TDP-43 inhibitor in the preparation of a drug for treating lung adenocarcinoma. The present invention provides an application of a TDP-43 inhibitor for the preparation of a drug for treating lung adenocarcinoma, which solves the problem that the product use of TDP-43 in the treatment of lung adenocarcinoma related to cell ferroptosis has not been found in the prior art. The research results of the present invention prove that: TDP-43 can promote the proliferation of lung adenocarcinoma cells and inhibit cell death; moreover, TDP-43 can promote the expression of the iron ion chaperone protein PCBP1 and inhibit the occurrence of cell ferroptosis process. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The expression of TDP-43 in lung adenocarcinoma and its correlation with poor prognosis of patients in the present invention, wherein: Figure 1 Figure A shows the difference in TDP-43 expression between lung adenocarcinoma tissue and normal tissue; Figure 1Figure B shows the expression difference of TDP-43 in tissues of lung adenocarcinoma patients at different clinical stages; Figure 1 Figure C shows the difference in expression of TDP-43 in cancer and adjacent tissues of lung adenocarcinoma patients; Figure 1 Figure D shows the relationship between TDP-43 expression level and survival rate of patients with lung adenocarcinoma; Figure 1 Figure E in the figure shows the expression level of TDP-43 in normal lung epithelial cell lines and different lung cancer cell lines; Figure 1 Figure F in the figure shows the expression of TDP-43 in the cancer and adjacent tissues of lung adenocarcinoma patients detected by Western blot experiment; Figure 1 The G graph in Figure 1 Quantitative results of F graph in ; Figure 1 Figure H in the figure shows the difference in the expression of TDP-43 in lung adenocarcinoma and adjacent tissues detected by immunohistochemistry; immunohistochemistry scale: 20 μm. The data shown are mean ± SD (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001.

[0024] Figure 2 The effect of TDP-43 overexpression on the proliferation and death of lung adenocarcinoma cells in the present invention, wherein: Figure 2 Figure A in the figure shows the overexpression efficiency of TDP-43 detected by RT-qPCR experiment; Figure 2 Figure B in the figure is a Western blot experiment to detect the overexpression efficiency of TDP-43; Figure 2 Figure C shows the effect of overexpression of TDP-43 on cell viability detected by MTT assay; Figure 2 Figure D in the figure is an EdU experiment to evaluate the effect of TDP-43 overexpression on cell proliferation ability; Figure 2 Panel E is a quantitative analysis of panel D, scale bar: 25 μm; Figure 2 Figure F shows the cell clone formation experiment to detect the effect of TDP-43 overexpression on the number of clones formed; Figure 2 Figure G shows the effect of overexpression of TDP-43 on the cell death rate of A549 cells detected by flow cytometry.

[0025] Figure 3 The effect of TDP-43 knockdown on the proliferation and death of lung adenocarcinoma cells in the present invention, wherein: Figure 3 Figure A shows the effect of knocking down TDP-43 on the viability of NCl-H1299 cells detected by MTT assay; Figure 3 Figure B shows the effect of knocking out TDP-43 on the viability of NCl-H1975 cells detected by MTT assay; Figure 3 Figure C in the figure is Figure 3 Quantitative analysis of D graph in; Figure 3Figure D in the figure is the effect of knocking down TDP-43 on the proliferation ability of NCl-H1299 cells detected by EdU experiment; Figure 3 Figure E in the figure shows the effect of knocking out TDP-43 on the proliferation ability of NCl-H1975 cells detected by EdU experiment; Figure 3 The F diagram in Figure 3 Quantitative analysis of the E graph in; Figure 3 Figure G shows the effect of knocking down TDP-43 on the number of NCl-H1299 cell clones and the quantification thereof by using clone formation assay; Figure 3 Figure H in the figure is the effect and quantification of the knockout of TDP-43 on the number of clones formed by NCl-H1975 cells using a clone formation experiment; Figure 3 Figure I in the figure is the effect of knocking out TDP-43 on the cell death rate of NCl-H1975 cells detected and quantified by flow cytometry; Figure 3 Figure J in the figure shows the effect and quantification of TDP-43 knockdown on the cell death rate of NCl-H1299 cells detected by flow cytometry; *P<0.05, **P<0.01, ***P<0.001.

[0026] Figure 4 In the present invention, RNA-seq combined with RT-qPCR screened and confirmed the effect of TDP-43 on the ferroptosis pathway and the expression of related target genes, wherein: Figure 4 Figure A shows the relative number of differentially expressed genes after TDP-43 knockout in NCl-H1975 cells; Figure 4 Figure B is a volcano plot of related differentially expressed genes after TDP-43 knockout in NCl-H1975 cells; Figure 4 Figure C in the figure is a cluster heat map of related differentially expressed genes after knocking out TDP-43 in NCl-H1975 cells; Figure 4 Figure D in the figure is a bubble diagram of GO biological process enrichment data; Figure 4 Figure E in the figure is a bubble diagram of KEGG pathway enrichment data;

[0027] Figure 4 Figure F in the figure shows the GSEA analysis results of the ferroptosis signaling pathway; Figure 4 Figure G shows the expression changes of ferroptosis-related genes SAT2, ACSL4, ACSL5, SLC39A14, NCOV4, PCBP1, PCBP2, SLC7A11, SLC3A2, GPX4, HMOX1, and SLC11A2 after knockdown of TDP-43 by RT-qPCR experiment; the data shown are mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

[0028] Figure 5The effect of TDP-43 on the ferroptosis phenotype of lung adenocarcinoma cells in the present invention, wherein: Figure 5 Figure A shows the effect of knocking down TDP-43 on ROS levels in NCl-H1299 cells detected by ROS analysis kit; Figure 5 Figure B in the figure is Figure 5 Quantitative analysis of Figure A in ; Figure 5 Figure C shows the effect of knocking out TDP-43 on ROS levels in NCl-H1975 cells detected by ROS analysis kit; Figure 5 Figure D is a quantitative analysis of Figure C; Figure 5 Figure E in the figure is Figure 5 Quantitative analysis of the F graph in; Figure 5 Figure F shows the effect of overexpression of TDP-43 on ROS level in A549 cells detected by ROS analysis kit; Figure 5 Figure G shows the effect of overexpression of TDP-43 on MDA level in A549 cells detected by MDA analysis kit; Figure 5 Figure H in the figure shows the effect of knocking out TDP-43 on the MDA level of NCl-H1975 cells detected by MDA analysis kit; Figure 5 Figure I in the figure shows the effect of knocking down TDP-43 on the MDA level of NCl-H1299 cells detected by using the MDA analysis kit; Figure 5 The J diagram in the figure shows the application of Fe 2+ The analysis kit was used to detect the effect of TDP-43 knockout on Fe 2+ The impact of levels; Figure 5 The K graph in Figure 5 Quantitative analysis of J graphs in; Figure 5 The L diagram in the figure is the application of Fe 2+ The analysis kit detects the effect of knocking down TDP-43 on Fe 2+ The impact of levels; Figure 5 The M diagram in Figure 5 Quantitative analysis of L graphs in Figure ; Scale bar: 20 μm. *P<0.05, **P<0.01, ***P<0.001.

[0029] Figure 6 The regulatory effect of TDP-43 on PCBP1 expression level in the present invention, wherein: Figure 6 Figure A shows the relationship between PCBP1 expression and survival rate of lung adenocarcinoma patients analyzed through the GEPIA database; Figure 6 Figure B shows the relationship between PCBP1 level and overall survival rate of patients with lung adenocarcinoma analyzed by Kaplan-MeierPlotter database; Figure 6Figure C shows the expression correlation between PCBP1 and TDP-43 analyzed through the GEPIA database; Figure 6 Figure D shows the effect of overexpression of TDP-43 on PCBP1 protein level in A549 cells detected by Western blot experiment; Figure 6 Figure E in the figure is a Western blot experiment to detect the effect of knocking down TDP-43 on PCBP1 protein level in NCl-H1975 cells; Figure 6 Figure F shows the effect of knocking out TDP-43 on PCBP1 protein level in NCl-H1299 cells detected by Western blot experiment; Figure 6 Figure G shows the effect of knocking out TDP-43 on PCBP1 protein level in NCl-H1975 cells detected by Western blot experiment; *P<0.05.**P<0.01.***P<0.001. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0031] The experimental materials, reagents and instruments used in the following examples are as follows:

[0032] RNA-protein immunoprecipitation kit: P0101, Gisai Biotechnology, China.

[0033] 293T cells were obtained from ATCC cell bank.

[0034] The virus packaging plasmid pMD2.G was obtained from Shanghai Yuanmu Biotechnology Co., Ltd.

[0035] Plasmid psPAX2 was obtained from Shanghai Yuanmu Biotechnology Co., Ltd.

[0036] The plasmid containing the guide RNA targeting TDP-43 was derived from Yunzhou Biotechnology (Guangzhou) Co., Ltd.

[0037] The immunodeficient mice used in the experiment were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0038] PBS was obtained from Sevier Biotechnology Co., Ltd., with a concentration of 0.01 M and a pH of 7.4.

[0039] EdU detection kit: C0071S, Biotech.

[0040] PMSF: Blue sky.

[0041] RIPA buffer: Biotin.

[0042] SDS-PAGE gel: Solebol.

[0043] PVDF membrane: GE Healthcore Life Sciences.

[0044] Enhanced chemiluminescence solution: ECL kit, NCM Biotech.

[0045] Image Lab instrument: Azure Biosystems.

[0046] Example 1

[0047] 1. Methods

[0048] 1. Ethylene-2-deoxyuridine staining test

[0049] Follow the instructions of the EdU detection kit to perform ethynyl-2-deoxyuridine staining detection. The specific steps are as follows:

[0050] First, the EdU working solution was added to the cell culture plates of A549 cells, NCl-H1299 cells, and NCl-H1975 cells, respectively, and incubated in a 37°C constant temperature incubator in the dark for 2 hours, then the A549 cells, NCl-H1299 cells, and NCl-H1975 cells were fixed with 4% paraformaldehyde, and then the A549 cells, NCl-H1299 cells, and NCl-H1975 cells were permeabilized with 0.3% Triton X-100, and then the Click reaction solution was prepared and incubated in the dark for 30 minutes, and finally the cells were sealed with a sealing agent containing DAPI.

[0051] After the sealing operation is completed, the cells were allowed to stand at room temperature for 10 minutes, then observed and photographed using a laser confocal microscope, and finally the ImageJ software was used to count the cell positive rate.

[0052] Among them, the English abbreviation of ethynyl-2-deoxyuridine staining detection is EdU.

[0053] 2. Tetramethyl azodiazole colorimetric method

[0054] A549 cells, NCl-H1299 cells, and NCl-H1975 cells were cultured in 96-well plates, and 3*10 320 μL of 5 mg / mL MTT was added to each cell at 0, 24, 48, and 72 hours, respectively. The culture was terminated after incubation at 37°C in the dark for 4 hours. Then 150 μL of DMSO was used to dissolve the crystals, and the absorbance of the dissolved samples was detected at 490 nm.

[0055] Among them, the English abbreviation of methyl tetrazolium colorimetry is MTT.

[0056] 3. CRISPR / Cas9

[0057] The present invention utilizes CRISPR / Cas9 technology to mediate the stable knockout of TDP-43 in the lung cancer cell line NCl-H1975 cells. The specific steps are as follows:

[0058] First, the viral packaging plasmid pMD2.G, psPAX2, and a plasmid containing a guide RNA targeting TDP-43 were transfected into 293T cells simultaneously. The supernatant was collected 48 hours after transfection, and a second transfection was performed to further enrich the virus. The enriched virus was used to infect primary NCl-H1975 cells. After 48 hours of infection, 2 μg / mL of purine was added to 1640 complete medium for screening. After three screenings, the knockdown efficiency was verified by protein immunoblotting, and NCl-H1975 cells were continuously cultured in a medium containing 1 μg / mL of purine in subsequent cell culture.

[0059] Before transfection, take out the cell culture dish with a growth density of 60%, discard the culture medium and replace it with 4mL fresh DMEM culture medium, and put it into the incubator for further culture. The transfection method is as follows: take two EP tubes and mark them as tube 1 and tube 2, add 250μL DMEM culture medium and 15μL transfection reagent to tube 1; add 250μL DMEM culture medium and three plasmids to tube 2, mix gently and let stand for 5 minutes; then take out 250μL from tube 1 and add it to tube 2, mix gently and let stand for 20 minutes; finally, slowly add the mixture to the cells prepared in advance, and replace it with 5mL fresh DMEM complete culture medium after 6 hours.

[0060] Among them, the three plasmids are pMD2.G: 1μg, psPAX2: 2μg, and target plasmid: 2μg.

[0061] 4. Protein immunoblotting

[0062] The cells were lysed with RIPA buffer containing 1 nmol / L PMSF to extract total protein. Proteins of different molecular weights were separated by SDS-PAGE gel and then transferred to PVDF membranes. After blocking with 5% skim milk at room temperature for 2 hours, the primary antibody was hybridized overnight at 4°C, and then incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour. Finally, enhanced chemiluminescence solution was added to obtain immunoblot bands using Image Lab instrument and quantitatively analyzed by ImageJ software.

[0063] 5. High-throughput transcriptome sequencing analysis

[0064] The present invention uses RNA-seq to identify genes regulated by TDP-43 in NCl-H1975 cells. The cells were plated in T25 bottles, and the corresponding cells were collected when NCl-H1975-NC and NCl-H1975-KD-TDP-43 cells were in the logarithmic growth phase, with 3 parallel replicates per group. 1 mL of TRIzol was added to each T25 bottle, and after lysing on ice for 20 minutes, the cells were collected in EP tubes, and then quickly frozen with liquid nitrogen and delivered to BGI Sequencing Company. Subsequent sequencing services were provided by BGI Sequencing Company.

[0065] Among them, the English abbreviation for high-throughput transcriptome sequencing analysis is RNA-seq.

[0066] 6. Clone formation experiment

[0067] After A549 cells and NCl-H1299 cells were cultured in 24-well plates containing RPMI 1640 medium with a mass percentage concentration of 10% FBS for 24 hours, the corresponding siRNA was transfected. After the transfection time reached 48 hours, the cells in each well were digested with trypsin, and the cells were resuspended in serum-free RPMI 1640 medium and then evenly spread into six-well plates. The culture was continued for 20 days, and the culture medium was changed every three days. Among them, NCl-H1975-NC and NCl-H1975-KD-TDP-43 cells do not need to be transfected and can be directly cultured in the same way as above.

[0068] After the cell colonies were formed, the culture medium was discarded and the cells were washed twice with PBS, fixed with 4% paraformaldehyde by volume for 30 minutes, then stained with 1% crystal violet solution by volume for 15 minutes, washed three times with water, dried naturally and photographed for observation.

[0069] 7. Cell culture

[0070] The human normal lung bronchial epithelial cell line BEAS-2B and lung cancer cell lines A549, NCl-H1299, NCl-H1975 and HCC827 were purchased from Beina Chuanglian Biotechnology Co., Ltd.

[0071] The above cells were cultured in 1640 medium containing 10% fetal bovine serum by mass; the culture conditions were: 5% CO2, 37°C.

[0072] 8. Flow cytometry

[0073] The cell volume is 6*10 4 A549 cells and NCl-H1299 cells were evenly plated in 24-well plates, and after corresponding transfection treatment, the cells were collected after digestion with EDTA-free trypsin. NCl-H1975-NC and NCl-H1975-KD-TDP-43 were directly and evenly plated in six-well plates, cultured to a cell density of 90%, and the cells were collected as described above. Subsequently, the cells were washed with pre-cooled PBS, resuspended with binding buffer, and stained with 5μL AnnexinV-FITC and 5μL AnnexinV-PI. Within 1 hour, the cell death rate was detected by flow cytometry.

[0074] Among them, V-PI: KeyGEN, BioTECH.

[0075] 9. Ferrous ion detection

[0076] The transfected A549 cells and NCl-H1299 cells were evenly inoculated in fluorescent culture dishes and cultured overnight in an incubator at 37°C and 5% CO2. Subsequently, the culture medium was discarded and the cells were washed 3 times with HBSS. FerroOrange working solution at a concentration of 1 μmol / l was added to each well and cultured in an incubator for 30 minutes. Subsequently, the fluorescence in each well was observed by taking photos with a fluorescence microscope, and the differences in fluorescence signals of each group were analyzed using Image J software.

[0077] Among them, overnight culture refers to a culture time ≥ 12h.

[0078] 10. Superoxide free radical detection

[0079] The transfected A549 cells and NCl-H1299 cells were evenly inoculated in a six-well plate. DCFH-DA was diluted with serum-free culture medium at a volume ratio of 1:5000 to a final concentration of 5 μmol / L. The cell culture medium was removed, 1.5 mL of diluted DCFH-DA was added, and the cells were incubated in a 37°C cell culture incubator for 20 minutes. Subsequently, the cells were washed three times with serum-free cell culture medium, and the fluorescence in each well was observed using a fluorescence microscope, and the differences in fluorescence signals of each group were analyzed using Image J software.

[0080] 11. Malondialdehyde detection

[0081] When the transfected A549 cells and NCl-H1299 cells were cultured to a density of 95%, the cells were scraped and transferred to EP tubes. After adding 0.5 mL of reagent five to each group, mix well to make a suspension, and then ultrasonically disrupt. Take 0.1 mL of cell suspension, add it to a new EP tube, and then add 1 mL of working solution to it. After mixing, place it in a 100°C water bath for 40 minutes. After cooling and centrifugation, take 0.25 mL of the supernatant to the ELISA plate. Measure the OD value at 532 nm on the ELISA instrument, and calculate the final malondialdehyde content of each group according to the formula.

[0082] There were 3 replicate wells in each group during cell culture.

[0083] The centrifugation condition was 1078×g for 10 minutes.

[0084] 12. Immunohistochemistry

[0085] The collected patient lung adenocarcinoma samples were embedded in paraffin, and the prepared sections were used for TDP-43 staining analysis. After the sections were dewaxed and rehydrated, they were immersed in EDTA reducing solution for high-temperature antigen retrieval. After cooling, the sections were treated with 3% H2O2 by volume. Then, the sections were blocked with 10% goat anti-rabbit serum at room temperature for 30 minutes. Subsequently, the sections were incubated with TDP-43 antibodies at 4°C overnight. The next day, the sections were washed three times with PBS and incubated with secondary antibodies at 37°C for 50 minutes. After washing with PBS, diaminobenzidine was used for color development; then, hematoxylin was counterstained, dehydrated and sealed, and the staining results were analyzed using image J.

[0086] Among them, overnight refers to a time ≥12h.

[0087] TDP-43 antibody: abcam, ab190963.

[0088] Secondary antibody: Servicebio, GB23303.

[0089] Diaminobenzidine: Servicebio, G1211.

[0090] 13. Real-time fluorescence quantitative PCR

[0091] According to the manufacturer's instructions, total RNA was extracted from transfected A549 cells and NCl-H1299 cells, NCl-H1975-NC and NCl-H1975-KD-TDP-43 cells using Trizol, and then reverse transcribed into cDNA. Subsequently, according to the instructions for use of SYBR Green MasterMix reagent, the PCR system was prepared and RT-qPCR was performed using the Applied Biosystems Quantstudio 3 Real-time PCR system. Using the expression level of GAPDH as a reference, 2 -ΔΔCt The specific sequences of the primers used in the present invention are shown in Table 1.

[0092] Among them, the English abbreviation of real-time fluorescence quantitative PCR is RT-qPCR.

[0093] Trizol:Takara.

[0094] SYBR Green MasterMix Reagent: Thermo Fisher Scientific.

[0095] Table 1 RT-qPCR related primer sequences

[0096]

[0097]

[0098] 14. Cell transfection

[0099] NCl-H1299 cells and A549 cells in good growth state were evenly plated in six-well plates. When the cell density grew to about 60%, siRNAs specifically targeting TDP-43 and eukaryotic expression plasmids of TDP-43 were transferred into the above cells using lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 48 hours, cells were collected to extract RNA or protein samples to detect the transfection success rate.

[0100] 15. Patient samples

[0101] The 17 lung adenocarcinoma patient tissue samples and 8 lung adenocarcinoma tissue samples used to detect the difference in TDP-43 expression between lung adenocarcinoma and normal tissues were obtained from Shanghai Pulmonary Hospital affiliated to Tongji University. The patients had signed a statement agreeing to use lung adenocarcinoma samples for scientific research. Figure 1 F. 8 lung adenocarcinoma tissue samples Figure 1 H.

[0102] 16. Data statistics and analysis

[0103] The correlation between the expression levels of TDP-43 and PCBP1 and the overall survival rate of patients was analyzed using the online analysis website GEPIA and Kaplan-Meier Plotter, respectively; the difference in the expression of TDP-43 in normal tissues and lung adenocarcinoma tissues was analyzed by the UALCAN website and the GEPIA website. The expression correlation of TDP-43 and PCBP1 in lung adenocarcinoma tissues was analyzed by the website GEPIA. Other data were statistically analyzed using Graph Pad Prism 6.0 and EXCEL software, and the statistical significance was calculated based on the standard deviation mean (±SD). The Student′st test method was used for analysis, and P<0.05 was set as a significant difference. *P<0.05, **P<0.01, ***P<0.001.

[0104] Among them, the website GEPIA: http: / / gepia.cancer-pku.cn / .

[0105] Website Kaplan-Meier Plotter: https: / / kmplot.com / analysis / index.php?cancer =liver_rnaseq&p=service.

[0106] UALCAN website: https: / / ualcan.path.uab.edu / .

[0107] 2. Results

[0108] 1. TDP-43 is highly expressed in lung adenocarcinoma and is closely associated with poor prognosis of lung adenocarcinoma patients

[0109] To determine the role of TDP-43 in lung adenocarcinoma, we performed an online database analysis. Figure 1 Figure A in Figure 1 Figure D in .

[0110] Figure 1 The results of Figure A in Figure 2 show that TDP-43 is expressed at a higher level in lung cancer patients compared to the control group. Figure 1 The results of Figure B show that the expression level further increases with the improvement of the patient's clinical stage. Figure 1 The results of Figure C in Figure 2 show that TDP-43 is significantly overexpressed in cancer tissues compared with adjacent tissues of lung adenocarcinoma patients. Figure 1 The results in Figure D showed that its expression level was negatively correlated with the patient survival rate.

[0111] The expression levels of TDP-43 in different cell lines were detected by Western blot experiment. Figure 1As shown in Figure E: We found that the expression level of TDP-43 in lung cancer cell lines was significantly higher than that in human normal lung epithelial cell line BEAS-2B. Subsequently, we collected 17 pairs of lung adenocarcinoma case tissue samples and tested them through Western blot and immunohistochemistry experiments. The results are shown in Figure E. Figure 1 As shown in Figures F and H, TDP-43 is highly expressed in cancer tissues.

[0112] 2. Overexpression of TDP-43 promotes lung cancer cell proliferation and inhibits cell death

[0113] To further explore the effect of TDP-43 on the malignant progression of lung cancer, we first overexpressed TDP-43 in A549 cells, where the expression of TDP-43 was relatively low. Western blot and RT-qPCR experiments confirmed that TDP-43 was successfully overexpressed. Figure 2 Figure A and Figure 2 As shown in Figure B. We then tested the effect of TDP-43 on cell proliferation through MTT, EdU, and colony formation assays. Overexpression of TDP-43 increased cell viability and the number of clone-forming cells. The experimental results all confirmed that TDP-43 upregulated cell proliferation, such as Figure 2 Figure C in Figure 2 As shown in Figure F. Subsequently, we used flow cytometry to detect the level of cell death using the Annexin-FITC / PI double staining method. The results are shown in Figure Figure 2 As shown in Figure G: Overexpression of TDP-43 significantly downregulated the cell death rate.

[0114] 3. Silencing TDP-43 inhibits lung cancer cell proliferation and promotes cell death

[0115] On the contrary, in order to specifically silence the expression of TDP-43, we transiently transfected two different siRNAs into NCl-H1299 cells with relatively high expression levels: labeled as si-TDP-43#1 and si-TDP-43#2 respectively; and based on NCl-H1975 cells, we used CRISPR / Cas9 technology to construct a TDP-43 stable knockout cell line, labeled as KD-TDP-43.

[0116] The experimental results are shown in Figure 3 . Figure 3 The results further confirmed that silencing TDP-43 significantly reduced cell viability, such as Figure 3 Figure A in Figure 3 As shown in Figure F; the number of clones formed is Figure 3 The G graph and Figure 3 As shown in Figure H; and increased cell death rate as shown in Figure 3 I graph and Figure 3 As shown in Figure J.

[0117] The above experimental results show that TDP-43 can promote lung cancer cell proliferation and inhibit cell death.

[0118] 4. TDP-43 is closely related to ferroptosis

[0119] In order to further explore how TDP-43 affects the development of lung cancer and identify its potential downstream target genes, we performed high-throughput sequencing using the control group and NCl-H1975 cells with stable knockdown of TDP-43. Analysis of differential genomic expression profiles showed that compared with the control group, after stable knockdown of TDP-43, a total of 4509 genes were significantly upregulated and 4845 genes were significantly downregulated. The volcano plot more intuitively shows the status of these genes, such as Figure 4 The cluster heat map shows the genes with the most significant expression differences, such as Figure 4 As shown in Figure C. The English abbreviation of high-throughput sequencing is RNA-seq.

[0120] In order to further determine the main biological functions of these differentially expressed genes, GO enrichment analysis showed that the differentially expressed genes were involved in regulating multiple biological functions, such as Figure 4 As shown in Figure D. Further KEGG pathway enrichment analysis and GSEA data analysis showed that the ferroptosis signaling pathway strongly responded to the expression changes of TDP-43, such as Figure 4 As shown in Figure F.

[0121] The above data suggest that TDP-43 is likely to regulate the ferroptosis process of cells.

[0122] To further verify whether TDP-43 is involved in the occurrence of ferroptosis, we found 14 ferroptosis-related genes among the differentially expressed genes obtained by RNA-seq; and then used RT-qPCR experiments to detect the effect of TDP-43 on the expression levels of the above genes. Figure 4 As shown in Figure G, after TDP-43 silencing, the expression of iron chaperone protein PCBP1 was significantly downregulated.

[0123] 5. TDP-43 inhibits ferroptosis in lung adenocarcinoma cells

[0124] We tested ferroptosis-related indicators in different cell lines, including superoxide radical levels, malondialdehyde, and ferrous ion content. Figure 5 Among them, the English abbreviation of superoxide radical is ROS. The English abbreviation of malondialdehyde is MDA. The chemical name of ferrous ion is Fe 2+ .

[0125] Figure 5 The results showed that when TDP-43 expression was inhibited, ROS, MDA and Fe 2+ On the contrary, after overexpression of TDP-43, ROS and MDA were significantly inhibited, and the experimental results were consistent with expectations.

[0126] The above results indicate that TDP-43 has the function of inhibiting ferroptosis of lung cancer cells.

[0127] in, Figure 5 Figure A in Figure 5 Figure D shows the ROS results when the expression of TDP-43 is inhibited; Figure 5 The H graph and Figure 5 Figure I in the figure shows the MDA result when the expression of TDP-43 is inhibited; Figure 5 J picture in Figure 5 Figure M shows the Fe after TDP-43 expression is inhibited. 2+ Horizontal results.

[0128] Figure 5 Figure E and Figure 5 Figure F shows the ROS results after overexpression of TDP-43; Figure 5 Panel G shows the MDA results after overexpression of TDP-43.

[0129] 6. TDP-43 promotes the expression of iron chaperone protein PCBP1, and high expression of PCBP1 is closely related to the poor prognosis of patients with lung adenocarcinoma.

[0130] RT-qPCR experimental results are as follows Figure 4 The G graph in Figure 2 shows that the expression level of the iron chaperone protein PCBP1 was significantly inhibited after silencing TDP-43. Therefore, we selected it as a subsequent target gene for screening.

[0131] First, we found through online database analysis that patients with high PCBP1 expression had a significantly reduced overall survival rate, e.g. Figure 6 Figure A and Figure 6 As shown in Figure B. In addition, there is a significant positive correlation between the expression of TDP-43 and PCBP1, as shown in Figure Figure 6 As shown in Figure C.

[0132] Subsequently, to further confirm the regulation of PCBP1 expression by TDP-43, we found that PCBP1 expression was significantly upregulated after overexpression of TDP-43 in A549 cells. Figure 6 As shown in Figure D; after inhibiting TDP-43 expression in NCl-H1299 and NCl-H1975, it was found that the expression level of PCBP1 was significantly downregulated, as shown in Figure Figure 6 Figure E in Figure 6 As shown in Figure G.

[0133] The above results indicate that TDP-43 can indeed positively regulate the expression level of PCBP1.

[0134] From the above experimental results, it can be seen that TDP-43 promotes cell proliferation and inhibits cell death in lung adenocarcinoma; in-depth research shows that TDP-43 can promote the expression of iron ion chaperone protein PCBP1 and inhibit the occurrence of cell ferroptosis.

[0135] The present invention provides application of a PCBP1 targeted inhibitor in preparing a drug for treating lung adenocarcinoma, and provides a new method for treating lung adenocarcinoma.

[0136] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0137] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0138] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of TDP-43 inhibitors in the preparation of drugs for the treatment of lung adenocarcinoma.

2. Use of the TDP-43 inhibitor according to claim 1 in the preparation of a drug for promoting ferroptosis of lung adenocarcinoma cells.

3. The use according to claim 2, characterized in that: The TDP-43 inhibitor includes any one or both of siRNA and sgRNA-TDP-43; The nucleotide sequence of si-TDP-43#1 in the siRNA is shown in SEQ ID NO.1; the nucleotide sequence of si-TDP-43#2 in the siRNA is shown in SEQ ID NO.2; The nucleotide sequence of the sgRNA-TDP-43 is shown in SEQ ID NO.

3.

4. The use according to claim 2, characterized in that: The drug has the TDP-43 inhibitor as an active ingredient and is supplemented with pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that: The concentration of the TDP-43 inhibitor in the drug is 100 nM to 200 nM.

6. The use according to claim 4, characterized in that: The auxiliary material includes any one or more of water, saline, buffer, glycerol and ethanol.

7. The use according to claim 4, characterized in that: The medicine includes oral dosage form and injection dosage form.

8. The use according to claim 7, characterized in that: The drug is a solution, and the solvent in the solution dosage form is water; The concentration of the TDP-43 inhibitor in the solution is 100 nM to 200 nM.

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