Application of ACBD3 inhibitor in preparation of medicine for treating lung cancer and gRNA for inhibiting ACBD3 gene expression

Abstract

The invention relates to application of an ACBD3 inhibitor in preparation of a medicine for treating lung cancer and gRNA for inhibiting ACBD3 gene expression, and belongs to the field of bioengineering.199 binding proteins of ACBD3 are obtained through co-immunoprecipitation and mass spectrum identification, new biological functions of ACBD3 are excavated through GO analysis and KEGG pathway analysis of the binding proteins of ACBD3, and the application of the ACBD3 inhibitor in preparation of the medicine for treating lung cancer is developed. The ACBD3 is used for regulating and controlling a KEGG pathway enrichment map of a differential gene, and the ACBD3 is found to participate in the occurrence and development of cancers; the ACBD3 is successfully knocked out by designing gRNA, and tests prove that the multiple effects of slowing down A549 lung cancer cell proliferation, inhibiting lung cancer cell migration, inhibiting A549 lung cancer cell invasion, retarding A549 cell cycle G1 phase and reducing tumorigenicity can be achieved by knocking out the ACBD3.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and specifically relates to an application of an ACBD3 inhibitor in the preparation of a drug for treating lung cancer and a gRNA for inhibiting the expression of the ACBD3 gene. Background Art

[0002] Lung cancer is the malignant tumor with the highest mortality rate. The number of new cases and deaths of lung cancer in my country remains high. At present, the occurrence and metastasis mechanism of lung cancer is still unclear, and there is a lack of effective early diagnosis standards and treatment measures in clinical practice. Conventional surgery, chemotherapy and radiotherapy have limited therapeutic effects on advanced non-small cell lung cancer, and have large toxic side effects. Molecular targeted therapy for lung cancer has the significant advantage of accurately attacking tumor cells without harming normal cells. About 69% of patients with advanced non-small cell lung cancer have potential molecular targets. Studies have found that lung cancer is associated with genetic mutations such as EGFR, ALK, and BRAF. Drug development and clinical application targeting these targets have greatly improved the quality of life of patients. However, the clinical drug resistance of currently used targeted drugs has seriously affected the treatment effect, and the mutation of tumor cells and the complex regulatory network have brought severe challenges to targeted therapy. Therefore, finding new therapeutic targets and biomarkers for early diagnosis is an urgent task before us.

[0003] Acyl-coenzyme A binding domain containing 3 (ACBD3) is a member of the acyl-coenzyme A binding protein (ACBP) family. It is composed of 528 amino acids and is a multifunctional Golgi protein. Its N-terminus contains an acyl-coenzyme A binding domain that reflects biochemical properties, and its C-terminus contains a GOLD domain that determines intracellular localization and transport capacity. ACBD3 participates in maintaining the structure of the Golgi apparatus and plays an important role in multiple cell signaling cascades after being released from the Golgi apparatus. ACBD3 is involved in regulating steroidogenesis, viral replication, embryogenesis, Huntington's disease, etc.

[0004] In recent years, studies have found that ACBD3 has a high expression trend in gastric cancer and breast cancer, which may be closely related to the occurrence and development of cancer. Targeted therapy can accurately attack cancer cells and has the characteristics of specificity and efficacy of therapeutic effects. Finding targeted genes that affect cancer treatment has positive significance for the research of therapeutic drugs for lung cancer and other cancers. Summary of the invention

[0005] In order to overcome the problems existing in the background technology, the present invention provides the use of ACBD3 inhibitors in the preparation of drugs for treating lung cancer and gRNA for inhibiting the expression of ACBD3 gene. The research of the present invention proves that ACBD3 is involved in the occurrence and development of cancer. By inhibiting the expression of ACBD3, the invasion, migration and proliferation of lung cancer cells can be effectively inhibited. The present invention also relates to the construction of gRNA targeting ACBD3, which can effectively knock out the expression of ACBD3 protein, providing a target for the development of new drugs for effectively preventing and treating ACBD3 gene, and also providing a new target gene for the research of anti-lung cancer drugs.

[0006] The first objective of the present invention is to provide an application of an ACBD3 inhibitor in the preparation of a drug for treating lung cancer.

[0007] Furthermore, the ACBD3 inhibitor is a gRNA that specifically knocks out the ACBD3 gene using the CRISPR / Cas9 system or a small interfering molecule that specifically interferes with the expression and processing of the ACBD3 gene, including small interfering RNA (siRNA) or short hairpin RNA (shRNA).

[0008] Furthermore, the ACBD3 inhibitor comprises a gRNA that inhibits the expression of the ACBD3 gene, and the sequence of the gRNA is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0009] Furthermore, the ACBD3 inhibitor comprises an expression vector or cell line constructed by gRNA; the gRNA sequence is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0010] The second object of the present invention is to provide a gRNA targeting ACBD3, the sequence of the gRNA is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0011] The second object of the present invention is to provide a gRNA expression vector targeting ACBD3, characterized in that the gRNA sequence is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0012] The third object of the present invention is to provide an expression vector or cell line comprising the above-mentioned gRNA.

[0013] The fourth object of the present invention is to provide an ACBD3 inhibitor, which comprises a gRNA that inhibits the expression of the ACBD3 gene, and the sequence of the gRNA is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0014] The fifth object of the present invention is to provide an ACBD3 inhibitor for use in the preparation of a drug for treating pathogenic Escherichia coli infection, epithelial cell bacterial invasion, Parkinson's disease, human papillomavirus infection, hypertrophic cardiomyopathy, breast cancer, diabetes or dilated cardiomyopathy.

[0015] The sixth objective of the present invention is to provide the use of ACBD3 as a target gene in the preparation of anti-lung cancer drugs.

[0016] Beneficial effects of the present invention:

[0017] The present invention obtains 199 binding proteins of ACBD3 through immunoprecipitation and mass spectrometry identification, and explores new biological functions of ACBD3 through GO analysis and KEGG pathway analysis of ACBD3 binding proteins. Through the KEGG pathway enrichment map of ACBD3-regulated differential genes, it is found that ACBD3 plays a role in multiple cell and biological regulatory processes, and ACBD3 is involved in the occurrence and development of cancer.

[0018] The present invention designs and constructs a gRNA targeting ACBD3, which can effectively knock out the expression of ACBD3 protein, provides a target for the development of new drugs that effectively prevent and treat the ACBD3 gene, and also provides a new target gene for the research of anti-lung cancer drugs.

[0019] CCK-8 detection showed that the proliferation of lung cancer cells was inhibited after ACBD3 was knocked out; flow cytometry detection showed that the cell cycle of lung cancer cells was blocked in the G1 phase after ACBD3 was knocked out; transwell experiment and scratch experiment analysis showed that the invasion and migration of lung cancer cells were inhibited after ACBD3 was knocked out; xenograft tumor experiment analysis showed that tumor formation was blocked after ACBD3 was knocked out. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The Western blot comparison of HA and ACBD3 expression, Lysate: lysate, FT: flow-through, IP: immunoprecipitation product;

[0021] Figure 2 This is the Cytoscape image of ACBD3 binding protein;

[0022] Figure 3 GO analysis and KEGG pathway analysis of ACBD3 binding proteins. Figure A shows the GO analysis classification of ACBD3 binding proteins, and Figure B shows the KEGG pathway analysis of ACBD3 binding proteins.

[0023] Figure 4 It is a Western blot comparison of ACBD3 protein expression in H1299 and KO-ACBD3 cells;

[0024] Figure 5 It is the DNA fragment deleted in H1299 lung cancer cells with KO-ACBD3;

[0025] Figure 6 The differentially expressed genes in the transcriptomes of H1299 and KO-ACBD3 cells were 122 genes up-regulated and 574 genes down-regulated;

[0026] Figure 7 GO analysis of differentially regulated genes by ACBD3;

[0027] Figure 8 KEGG pathway enrichment map of ACBD3-regulated differentially expressed genes, orange: human diseases; blue: environmental information processing; red: cellular processes; green: biological systems; yellow: metabolism;

[0028] Fig. 9 Western blot comparison of ACBD3 expression in A549 wild-type cells and ACBD3 knockout A549 cells (KO-ACBD3);

[0029] Fig.10 It is the DNA fragment deleted in A549 lung cancer cells with KO-ACBD3;

[0030] Fig.11 Comparison of proliferation of A549 and KO-ACBD3 cells;

[0031] Fig.12 The effect of ACBD3 on the cell cycle;

[0032] Fig.13 Transwell assay to analyze the effect of ACBD3 on lung cancer cell invasion, scale bar: 100 μm, *, *, ** indicate p<0.05, p<0.01 and p<0.001, respectively;

[0033] Fig.14 The wound healing assay was used to analyze the wound healing in A549 cells and KO-ACBD3 cells. Scale bar:

[0034] 200 μm;

[0035] Fig.15 Results of in vivo tumorigenicity analysis of ACBD3 knockout to inhibit lung cancer. Figures A and B are images of A549 wild-type cells and KO-ACBD3 lung cancer cell-related tumors in vitro and ex vivo; Figure C is a comparison of tumor masses in different groups; *, *, **, respectively indicate p<0.05, p<0.01, and p<0.001;

[0036] Fig.16Figure 1 shows the results of tumor volume analysis in lung cancer in vivo after knocking out the ACBD3 gene. Figures A and B show the tumor volume and body weight analysis of nude mice inoculated with A549 and KO-ACBD3 cells.

[0037] Fig.17 HE staining analysis of A549 and KO-ACBD3 tumor tissues, scale bar: 50 μm;

[0038] Fig.18 The results of immunohistochemistry identification are shown in Figure A, where Figure A is the expression of Ki-67 in tumor tissues identified by immunohistochemistry, scale bar: 200 μm; Figure B is the expression of β-catenin in tumor tissues identified by immunohistochemistry, scale bar: 200 μm; Figure C is the expression of CDK1 in tumor tissues identified by immunohistochemistry, scale bar: 200 μm; Figure D is the expression of E-cadherin in tumor tissues identified by immunohistochemistry, scale bar: 200 μm;

[0039] Fig.19 This is a diagram illustrating the regulatory mechanism of ACBD3 in lung cancer. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0041] In order to more clearly illustrate the present invention, it is described in detail through the following examples.

[0042] Example 1 ACBD3 binding protein and function identification

[0043] (1) Construction of ACBD3 high expression plasmid pcDNA5 / FRT / TO-HA-ACBD3

[0044] Total RNA was extracted from wild-type A549 cells (human alveolar basal epithelial cells of lung cancer). Human A549 lung cancer cells were subcultured to 10 cm cell culture dishes. When the cells grew to a density of 70% to 80% and were in good condition, they could be used for experiments. The culture medium of the cells was removed and washed twice with 1× PBS. 1 ml of TRIZOL was added to the culture dish, and the cell samples were collected into 1.5 ml EP tubes after gently blowing and mixing. 200 μl of chloroform was added, and the mixture was vigorously mixed for 15 seconds to fully emulsify it, and then allowed to stand at room temperature for 15 minutes, and then centrifuged at 4°C 12000g for 15 minutes. After centrifugation, the colorless supernatant was aspirated, and an equal volume of isopropanol was added, and the mixture was gently mixed and allowed to stand at room temperature for 10 minutes, and then centrifuged at 4°C 12000g for 10 minutes. The supernatant was removed, and 1 ml of 75% ethanol was added, and the mixture was centrifuged at 4°C 12000g for 5 minutes. The ethanol was evaporated in a clean place at room temperature to dry the precipitate, and finally RNase-free water was added to dissolve it. The total RNA concentration was determined and verified by agarose gel electrophoresis. The total RNA was then reverse transcribed using a reverse transcription kit to generate cDNA.

[0045] Using cDNA as a template, primers containing KpnI and NotI restriction endonuclease sites and HA tag were designed (Table 1) to amplify the target gene ACBD3.

[0046] Table 1. Primer sequences for ACBD3 high expression

[0047]

[0048] Note: The double-underlined sequence is the HA tag sequence.

[0049] The specific amplification system is as follows: 0.2μl cDNA template, 0.6μl upstream primer, 0.6μl downstream primer, 0.8μl GXL DNA polymerase, 4μl 5×GXL buffer, 1.6μl dNTP mixture, 2.5μl 40% glycerol, add 12.2μl water to a total volume of 20μl. The PCR reaction conditions are: pre-denaturation at 98℃ for 10 minutes; denaturation at 98℃ for 10 seconds, gradient annealing for 15 seconds, extension at 68℃ for 2 minutes, 30 cycles; 68℃ for 7 minutes. After amplification, perform agarose gel electrophoresis, cut out the gel fragment of the target size, and purify and recover.

[0050] Then, the purified target fragment and pcDNA5 / FRT / TO vector were digested with enzymes. The vector digestion system is as follows: 1.5 μl of vector, 0.5 μl of KpnI endonuclease, 0.5 μl of NotI endonuclease, 1 μl of 10×FD buffer, and 6.5 μl of water to a total volume of 10 μl. The digestion system of the ACBD3 target fragment is: 17 μl of the recovered purified fragment, 0.5 μl of KpnI endonuclease, 0.5 μl of NotI endonuclease, 2 μl of 10×FD buffer, and a total volume of 20 μl. Place the prepared digestion system in a 37°C water bath for 1 hour and 30 minutes to 2 hours.

[0051] The digested product was verified by agarose gel electrophoresis, and the gel fragments were cut off, recovered and purified, and then connected by T4 ligase. The connection system is: 1μl T4 ligase, 2μl 10×FD buffer, 1.5μl recovery vector, 15.5μl recovery fragment, total volume 20μl, placed in a metal heating module at 16°C for 12 to 16 hours. All the connection products were transformed into competent cells and plated. After growing a single clone, colony PCR verification was performed. The verification system is: 5μl 2×Taq PCR Mix, 0.3μl upstream primer, 0.3μl downstream primer, 4.4μl water, total volume 10μl, pick a single clone and mix it lightly in the mixture with a gun tip to use as a template. After successful verification, the plasmid was extracted and sent to a biological company for sequencing. The sequencing results of the biological company were compared with the ACBD3 sequence, and it was found that the sequencing results were completely consistent, that is, the pcDNA5 / FRT / TO-HA-ACBD3 plasmid was successfully constructed.

[0052] (2) Construction of HA-ACBD3 overexpressing 293T cell line

[0053] Transfect 293T cells with pcDNA5 / FRT / TO-HA-ACBD3 plasmid and pcDNA5 / FRT / TO empty vector plasmid using Lipofectamine2000:

[0054] 293T cells were cultured in a 37°C cell culture incubator containing 5% CO2. The DMEM medium used to culture the cells contained 10% FBS. Optim medium and Lipofectamine 2000 were used for transfection. The pcDNA5 / FRT / TO-HA-ACBD3 plasmid and pcDNA5 / FRT / TO empty vector plasmid were transfected according to the instructions. After 5-6 hours, the transfected cells were replaced with DMEM medium containing 10% FBS. After 24 hours of culture, the cells were digested with trypsin for expansion and subsequent verification.

[0055] Western blotting experiment verifies the expression of HA-ACBD3: Add RIPA lysis buffer to the collected cell samples and lyse on ice for 30 minutes. Add the prepared protein samples and protein markers to the corresponding lanes in turn. The specific conditions for electrophoresis are: 80V constant voltage for 30 minutes, and then adjust to 120V constant voltage for about 1 hour and 30 minutes. The transfer buffer needs to be pre-cooled in advance, and the protein on the gel after electrophoresis is transferred to the PVDF membrane. The transfer condition is 200mA constant current for 2 hours. The transferred PVDF membrane is blocked in 5% skim milk prepared with 1×TBS / T, placed on a shaker at room temperature, and treated for 1 hour and 30 minutes to 2 hours. Use 5% skim milk prepared with 1×TBS / T to prepare the primary antibody, place the corresponding PVDF membrane and antibody in the antibody incubation box, and incubate on a shaker at 4°C overnight, or incubate at room temperature for about 2 hours. The PVDF membrane incubated with the primary antibody was rinsed with 1×TBS / T for 3 times, 5 to 10 minutes each time, and then placed in the prepared secondary antibody for incubation at room temperature for about 1 hour. The PVDF membrane incubated with the secondary antibody was rinsed with 1×TBS / T for 3 times, 5 to 10 minutes each time. After rinsing, add an appropriate amount of developer to the PVDF membrane and place it in a chemiluminescence instrument for development. The HA-ACBD3 overexpression 293T cell line (such as Figure 1 ).

[0056] (3) Co-immunoprecipitation to enrich ACBD3 binding proteins

[0057] HA-ACBD3-expressing 293T cells and control cells were collected in 15 ml centrifuge tubes by cell scraping, washed twice with pre-cooled PBS, and centrifuged at 1000 RCF to collect the cell pellet in a 2 ml EP tube. Pre-cooled IP buffer containing protease inhibitors was added to the collected cell pellet, placed on a rotary instrument, and gently rotated at 4°C for 30 minutes to fully lyse the cells. Cell lysate containing 1.5 mg of total protein was incubated with 30 μl of HA beads on a rotating wheel at 4°C for 2 hours, and then the sample was centrifuged at 14,000 RPM for 2 minutes. The supernatant was aspirated and the beads were washed four times with IP buffer. Proteins were eluted from the beads using SDS loading buffer.

[0058] (4) Mass spectrometry identification of ACBD3 binding proteins

[0059] Sample enzymatic hydrolysis: Take 50μg protein sample, add 100mM DTT, place the sample tube in a 95℃ heater for 5min, and after cooling to room temperature, add 200μL 8M UA to each sample, mix and shake, transfer to ultrafiltration tube, centrifuge at 14000g for 30min at room temperature, and centrifuge 3 times in this step. Add 100μL 100mM IAM to each sample tube and keep it in the dark for 30min. Add 100μL 8M UA to each sample tube, centrifuge at 12 000g for 30min at room temperature, and centrifuge 3 times in this step. Add 100μL 25mM ABC to each tube, centrifuge at 14000g for 30min at room temperature, and centrifuge 3 times in this step. After centrifugation, add Trypsin protease, dissolve the enzyme to be added to each tube in 40μL 100mM ABC, mix and shake, add to the ultrafiltration tube, cover the lid tightly, and place it in a 37℃ water-proof constant temperature incubator for reaction for 20h. After the reaction is completed, take out the sample, wait for the sample tube to cool to room temperature, centrifuge at 14 000g for 20 minutes at room temperature. Add 40μL 25mM ABC to each sample tube, centrifuge at 14 000g for 30 minutes at room temperature. Take out the ultrafiltration tube, add 10μL 10% formic acid aqueous solution to each collection tube, mix and shake.

[0060] The peptide samples were desalted using peptide desalting centrifugal columns. The desalting column was activated with 100% acetonitrile and balanced with 0.1% formic acid. The enzymatic peptides were added and the column was washed with 0.1% TFA solution. The waste liquid was discarded to remove the salt and impurities remaining on the column. This step was repeated twice. Finally, the eluent (50% ACN, 0.1% TFA) was added for elution and the eluent was collected. The sample was dried using a vacuum centrifugal concentrator.

[0061] Dissolve the peptide freeze-dried powder in 20 μL formic acid water (0.1% FA), and take 1 μg of sample for liquid mass spectrometry. The mobile phase is an acetonitrile-water-formic acid system, in which mobile phase A is 0.1% formic acid aqueous solution and phase B is 0.1% formic acid acetonitrile solution (acetonitrile is 84%). After the chromatographic column is equilibrated with 95% phase A, the sample is directly loaded onto the chromatographic column by an automatic sampler, and then separated by gradient separation of the chromatographic column. After chromatographic separation, the sample is analyzed by mass spectrometry using a Q Exactive HF-X mass spectrometer. The detection method is data-dependent scanning mode (DDA), the parent ion scanning range is 360–1350 m / z, the primary mass spectrometry resolution is 60,000 (200 m / z), and the AGC (Automatic gain control) target is 3×e 6, the maximum injection time (Maximum IT) is 50 ms, and the dynamic exclusion time (Dynamic exclusion) is 45.0 s. The mass-to-charge ratio of peptides and peptide fragments was collected according to the following method: MS2Activation Type is HCD, the secondary mass spectrometry resolution is 15,000 (200 m / z), and the Normalized Collision Energy is 28 eV.

[0062] The mass spectrometry data were used for protein qualitative analysis using the GCF_000001405.39_GRCh38.p13_protein protein sequence database. The DDA data were analyzed using Thermo Scientific Proteome Discoverer3.1. The main software parameters were set as follows: the enzyme was trypsin; the maximum missing cleavage was 2; the peptide mass tolerance was ±20ppm; the fragment mass tolerance was 0.1Da; the fixed modification was aminomethyl (C); the dynamic modification was oxidation (M) and acetylation (protein N-terminus). All reported data are based on 99% protein identification confidence and a false discovery rate (FDR) of ≤1%. 293T cells expressing HA-ACBD3 identified 3541, 3793 and 3864 binding proteins in three independent experiments, respectively. Three independent experiments in control cells identified 3635, 3693 and 3794 binding proteins, respectively (Table 2).

[0063] Table 2. Statistics of proteins bound to ACBD3

[0064] Grouping Peptides Identification of binding proteins ACBD3-1 18678 3541 ACBD3-2 21418 3793 ACBD3-3 21988 3864 Con-1 18936 3635 Con-2 20519 3693 Con-3 21510 3794

[0065] By analyzing the binding proteins of HA-ACBD3-expressing 293T cells and control cells, a total of 199 ACBD3-binding proteins were obtained. The STRING database (https: / / string-db.org / ) was used for protein interaction analysis. In order to evaluate the proteins in the database that bind to ACBD3, we input ACBD3 into STRING for interacting protein search, set the interaction score threshold to be greater than 0.4, and derived a total of 6 ACBD3-binding proteins: TBC1D22A, SLC35A1, TBC1D22B, BLZF1, PI4KB, and GOLGB1. The 6 ACBD3-binding proteins derived from the STRING database were analyzed with the 199 ACBD3-binding proteins we identified. It was found that among the 199 ACBD3-binding proteins we identified, 3 proteins (BLZF1, PI4KB, and TBC1D22A) have been reported, and 196 proteins are newly discovered ACBD3-binding proteins. Use Cytoscape to intuitively draw the results (Appendix Figure 2 ), Figure 2 The black line in the middle represents the binding proteins discovered in this study, and the red line represents the binding proteins reported in the STRING database.

[0066] GO is a database established by the Gene Ontology Consortium (Gene Ontology, http: / / geneontology.org / ). The identified proteins are assigned corresponding GO numbers through ID correspondence or sequence annotation, and the GO numbers can be used to correspond to GO terms, i.e., functional categories or cellular localizations. The protein lists and their quantities under each GO term for all proteins and the entire species background proteins are annotated and calculated, and then the Pvalue (significance level) of these proteins is evaluated using the Fisher exact test method. The standard for significant enrichment is set at a Pvalue of less than 0.05. In this way, the GO terms for the identified proteins that are significantly enriched compared to the entire proteome background are identified, thereby determining the specific categories and characteristics of these proteins. The results are shown in Figure 2. Figure 3 Figure A shows that ACBD3-binding proteins are enriched in the positively regulated Ras protein signaling pathway, Rab protein signaling pathway, epidermal growth factor receptor signaling pathway, insulin receptor signaling pathway, etc.

[0067] ClusterProfiler was used to perform KEGG enrichment analysis. The protein list and protein number of each pathway were calculated using the genes / proteins annotated in the KEGG pathway. The P value was then calculated using the hypergeometric distribution method to find the KEGG pathways in which the identified proteins were significantly enriched compared to the entire proteome background, thereby determining the main biological functions of the identified proteins. The results are shown in Figure 2. Figure 3 Figure B shows that ACBD3 is involved in the occurrence of pathogenic Escherichia coli infection, bacterial invasion of epithelial cells, Parkinson's disease and other diseases, and is involved in the regulation of multiple signaling pathways.

[0068] Example 2 Differential genes regulated by ACBD3

[0069] (1) Construction of ACBD3 knockout plasmid (PX459-ACBD3)

[0070] In the construction of ACBD3 knockout plasmid, we used PX459 plasmid. The gRNA of ACBD3 was designed through the online gRNA design website chopchop (https: / / chopchop.cbu.uib.no / #). Enter the Gene ID of ACBD3 (64746) in the corresponding option group, select Homo Sapines (hg38 / GRCh38) for species, select CRISPR / Cas9 for method, and select knock out for mode. Among all the gRNA sequences displayed, a pair of gRNAs targeting the ACBD3 gene was selected based on the targeting efficiency (higher than 60%) and the exon location of the gRNA (located in the first exon).

[0071] Table 3 gRNA sequences targeting ACBD3

[0072]

[0073] Note: The underlined sequence is the sticky end of the gRNA connected to the PX459 plasmid.

[0074] The PX459 plasmid was selected to construct the knockout plasmid of ACBD3. The designed and synthesized pair of ACBD3 gRNA (Table 3) was diluted with RNase-free water to a working concentration of 100 μM. The pair of gRNAs (gRNA-F and gRNA-R) were phosphorylated and annealed by a PCR instrument. The specific ratio was as follows: 1 μl of gRNA-F, 1 μl of gRNA-R, 1 μl of 10×T4 ligase buffer, 1 μl of T4 polynucleotide kinase, 6 μl of RNase-free water, a total of 10 μl. The above mixture was placed in a PCR instrument, 37°C for 30 min, 95°C for 5 min, and then reduced to 25°C at 5°C / min.

[0075] The phosphorylated and annealed product in the previous step was diluted 200 times with RNase-free water, and the product in the previous step was connected with the PX459 plasmid vector according to the following ratio: 2μl of the diluted product in the previous step, 1μl of PX459 plasmid, 2ul of 10×Tango buffer, 0.5ul of 20mM DTT, 0.4μl of 25mM ATP, 1μl of BbsI restriction enzyme, 0.5μl of T7 ligase, and 12.6μl of RNase-free water to a total volume of 20μl. The mixed mixture was placed in a PCR instrument, and the reaction conditions were 37℃5min, 21℃5min, and 6 cycles of reaction.

[0076] The digested and connected products were treated with PlasmidSafe exonuclease to hydrolyze some linear DNA. The specific reaction system is as follows: 11μl of the previous step reaction product, 1.5μl of 10×PlasmidSafe buffer, 1.5μl of 10mM ATP, 1μl of PlasmidSafe exonuclease, and the total volume is 15μl. The mixed liquid was placed in a PCR instrument, and the reaction conditions were 37℃30min, then 70℃30min.

[0077] The partially hydrolyzed product was used for the E. coli DH5α transformation experiment. The operation was performed in a clean bench. The specific transformation process was as follows: take out the competent cells from the -80℃ low-temperature refrigerator and place them on ice until they are completely thawed; take 50μl of competent cells and add the partially hydrolyzed product, gently mix and place on ice for 30 minutes; quickly and gently place it in a 42℃ water bath for heat shock for 45 seconds; then place it on ice for 2 minutes and 30 seconds; then add 800μl of non-resistant LB liquid culture medium; place the EP tube on a 37℃ shaker at 250rpm for 50 to 60 minutes. After taking it out, centrifuge it at 6000rpm for 3 minutes, remove 700μl of supernatant, mix the remaining about 100μl of liquid culture medium with the bottom sediment, and evenly apply it to the LB solid plate containing ampicillin with a coating rod; after the surface of the plate is dry, invert the plate and place it in a bacterial incubator at 37℃ for 12 to 16 hours. When a single clone grows on the plate, pick the single clone and expand it in LB liquid culture medium containing ampicillin; extract the plasmid and send it to a biological company for sequencing, and retain the plasmid with correct sequencing results for subsequent experiments.

[0078] (2) Construction of H1299 lung cancer cell line with ACBD3 knockout

[0079] The PX459-ACBD3 plasmid with the correct sequencing results was used to construct a stable knockout lung cancer cell line of ACBD3. The H1299 lung cancer cells were required to be in good culture state and free of contamination. The cells with a growth density of more than 80% and normal morphology were subcultured and plated into a 6-well cell culture plate. When the cells grow to a density of 70% to 80%, they can be transfected. Before transfection, remove the cell culture medium in the 6-well plate and replace it with Optim culture medium for transfection. Take a 1.5ml EP tube, add 250μl of Optim culture medium, add 5μl of transfection reagent Lipofectamine 2000, mix well and let stand for 5 minutes. Take another 1.5ml EP tube, add 250μl of Optim culture medium, add a total of 2000ng of PX459-ACBD3 plasmid, and mix well. Mix the mixture in the two 1.5ml EP tubes and let stand for 20 minutes. Add the mixture to the 6-well plate. 5 to 6 hours after transfection, replace the cells in the 6-well plate with normal cell culture medium containing FBS. On the second day after transfection, digest the cells in the 6-well plate, transfer them to a 15cm cell culture dish, and continue to culture. On the third or fourth day after transfection, when the cells in the cell culture dish are growing well, add puromycin to the culture medium for screening (H1299 cell screening concentration 2μg / ml). Change the medium every 3 to 4 days, and the screening and changing of the medium can be performed 4 to 5 times. After the screening obtains a single clone, pick the single clone by dilution method or cloning cup method. Pay attention to observation. After the monoclonal cells are full, samples can be collected for subsequent western blotting verification of protein levels and PCR amplification sequencing of DNA levels.

[0080] Western blotting verification results (see attached Figure 4 ) showed that H1299 monoclonal lung cancer cells suspected of knocking out ACBD3 at the protein level were obtained, and the corresponding cell samples were collected for genome extraction. After genome extraction of wild-type H1299 cells and cells suspected of knocking out ACBD3, verification primers were designed for PCR amplification (Table 4).

[0081] Table 4. PCR primer sequences for ACBD3 knockout validation

[0082]

[0083] The amplified product was verified by agarose gel, and the gel fragments of the correct molecular weight were cut out, purified and recovered, and sent to a biological company for sequencing. The sequencing results showed that the ACBD3 knockout H1299 lung cancer cell line (the missing DNA fragment is as shown in the attached Figure 5 shown).

[0084] (3) Transcriptomic analysis of differentially regulated genes in H1299 lung cancer cells with ACBD3 knockout

[0085] Trizol reagent was used to extract mRNA from wild-type H1299 lung cancer cells and ACBD3 knockout H1299 lung cancer cells. mRNA was enriched with mRNA Capture Beads magnetic beads. After magnetic bead purification, mRNA was fragmented at high temperature. The fragmented mRNA was used as a template to synthesize the first strand of cDNA in a reverse transcriptase mixed system. While synthesizing the second strand of cDNA, end repair and A tailing were completed. Then, the adapter was connected and Hieff DNA SelectionBeads magnetic beads were used to purify and select the target fragments; PCR library amplification was then performed, and finally Illumina Novaseq XPlus was used for detection to obtain the differentially expressed genes in the transcriptome of H1299 and KO-ACBD3 cells (see attached). Figure 6 ).

[0086] The library construction quality detection standards are as follows:

[0087] (1) Agarose gel electrophoresis: analyze the integrity of sample RNA and whether there is DNA contamination;

[0088] (2) NanoPhotometer spectrophotometer: detect RNA purity (OD260 / 280 and OD260 / 230 ratio);

[0089] (3) Qubit2.0 Fluorometer: Accurate quantification of RNA concentration;

[0090] (4) Agilent 2100 bioanalyzer: Accurately detect RNA integrity.

[0091] In order to ensure data quality, the raw data should be filtered before information analysis to reduce the analysis interference caused by invalid data. First, we use fastp to perform quality control on the raw reads off the machine, filter low-quality data (remove reads containing adapters; remove reads with N ratio greater than 10%; remove reads that are all A bases; remove low-quality reads, the number of bases with quality value Q≤20 accounts for more than 50% of the entire read) to obtain clean reads.

[0092] After the data is filtered, we will analyze the composition and quality distribution of the bases to visually display the data quality. The more balanced the base composition, the higher the quality, and the more accurate the subsequent analysis.

[0093] The differentially expressed genes were mapped to each term in the GO database (http: / / www.geneontology.org / ), and the number of genes for each term was calculated, thereby obtaining a gene list with a certain GO function and a gene number statistics (the results are shown in the attached Figure 7 ). The differentially expressed genes regulated by ACBD3 are involved in: transcriptional regulator activity, structural molecule activity, ATP-dependent activity, and cytosolic motility activity. In addition, the differentially expressed genes are involved in bioadhesion, immune system processes, viral processes, and multiple signaling pathways.

[0094] The differentially expressed genes of ACBD3 were subjected to KEGG pathway analysis, and it was found that the differentially expressed genes involved the MAPK signaling pathway, PI3K-Akt signaling pathway, Notch signaling pathway, Rap1 signaling pathway, and Wnt signaling pathway. In addition, these genes were found to be significantly enriched in human diseases, such as human papillomavirus infection, hypertrophic cardiomyopathy, breast cancer, diabetes, and dilated cardiomyopathy (results are shown in the attached Figure 8 ). Therefore, it is suggested that ACBD3 may play a role in multiple cellular and biological regulatory processes and that ACBD3 is involved in the development and progression of cancer.

[0095] Example 3: Effect of ACBD3 on the proliferation ability of lung cancer cells

[0096] Construct an A549 lung cancer cell line with knockout ACBD3. The PX459-ACBD3 plasmid with the correct sequencing results was used to construct an ACBD3 stable knockout lung cancer cell line. The A549 lung cancer culture state is required to be good and there should be no contamination. The cells with a growth density of more than 80% and normal morphology were subcultured and plated into a 6-well cell culture plate. When the cells grow to a density of 70% to 80%, they can be transfected. Before transfection, remove the cell culture medium in the 6-well plate and replace it with Optim culture medium for transfection. Take a 1.5ml EP tube, add 250μl of Optim culture medium, add 5μl of transfection reagent Lipofectamine 2000, mix well and let stand for 5 minutes. Take another 1.5ml EP tube, add 250μl of Optim culture medium, add a total of 2000ng of PX459-ACBD3 plasmid, and mix well. Then mix the mixture in the two 1.5ml EP tubes and let it stand for 20 minutes. Add the mixture to a 6-well plate. 5 to 6 hours after transfection, replace the cells in the 6-well plate with normal cell culture medium containing FBS. On the second day after transfection, digest the cells in the 6-well plate, transfer them to a 15 cm cell culture dish, and continue to culture. On the third or fourth day after transfection, when the cells in the cell culture dish are growing well, add puromycin to the culture medium for screening (A549 cell screening concentration 0.5 μg / ml). Change the medium every 3 to 4 days, and the screening and changing of the medium can be performed 4 to 5 times. After the single clone is screened, pick the single clone by dilution method or cloning cup method. Pay attention to observation. After the monoclonal cells are full, samples can be collected for subsequent western blotting verification of protein levels and PCR amplification sequencing of DNA levels.

[0097] Western blotting verification results (see attached Fig. 9 ) showed that A549 monoclonal lung cancer cells suspected of knocking out ACBD3 at the protein level were obtained, and the corresponding cell samples were collected for genome extraction. After genome extraction of wild-type A549 cells and cells suspected of knocking out ACBD3, verification primers were designed for PCR amplification (Table 4). The amplified products were verified by agarose gel, and gel fragments of the correct molecular weight were cut out. After purification and recovery, they were sent to a biological company for sequencing. The sequencing results showed that the ACBD3 knockout A549 lung cancer cell line was successfully constructed (the missing DNA fragment is as shown in the attached Fig.10 shown).

[0098] A549 wild-type lung cancer cells and A549 lung cancer cells with ACBD3 knockout were used for CCK-8 proliferation experiments. The counted cells were evenly plated into a 96-well plate according to a system in which 2000 cells were contained in 100 μl of culture medium per well. Each experimental group should have at least 3 parallel wells. The 96-well plate with cells was placed in a cell culture incubator for normal culture. After about 24 hours of plating, when the cells were completely attached to the wall, the 96-well plate was removed from the cell culture incubator, and 10 μl of CCK-8 reagent was added to the wells of the first group. After addition, it was returned to the incubator for 1 hour. The measurement was performed using an ELISA reader at 450 nM, and the value measured at this time was the measurement result of the first day. Thereafter, the other experimental groups were measured at the same time every day, using the same measurement method as the first day, for a total of 4 days (see results). Fig.11 ). 1×PBS was added to the non-experimental wells at the edge of the 96-well plate to prevent the volatilization of the culture medium due to the edge effect, which would cause inaccurate measurement data. The measurement data of each day were statistically analyzed, and finally the data were analyzed using GraphPadPrism 6 software. Knockout of ACBD3 inhibited the proliferation of A549 lung cancer cells.

[0099] Example 4. Flow cytometry detection of the effect of ACBD3 on lung cancer cell cycle

[0100] (1) Cell collection: Discard the supernatant of A549 wild-type lung cancer cells and A549 lung cancer cells with ACBD3 knockout in culture, add 1 mL of PBS to wash once, discard the supernatant, and then add 1 mL of 0.25% trypsin to digest the cells. When the cells become round and some cells are suspended, add culture medium to terminate digestion. Use a pipette to gently blow the cells to suspend the cells. Transfer the cell suspension into a 1.5 mL centrifuge tube and centrifuge at 1500 rpm for 5 min to collect the cells.

[0101] (2) Ethanol fixation: Slowly add 1 mL of -20°C precooled 75% ethanol to the pellet, resuspend the cells, and incubate at 4°C overnight.

[0102] (3) Wash cells with PBS: Centrifuge the cells fixed overnight at 1500 rpm for 5 min and discard the supernatant. Add 1 mL of PBS and mix well. Centrifuge at 1500 rpm for 5 min to collect the cells. Add 100 μL of PBS to resuspend the cells.

[0103] (4) Removal of RNA: Add 2 μL of 10 mg / mL RNase A to each tube of sample and incubate in a 37°C drying oven for 30 min.

[0104] (5) Fluorescence labeling: Add 100 μL of 100 μg / mL PI staining solution and stain for 10 min in the dark.

[0105] (6) Flow cytometry: The excitation wavelength was 488 nm and the emission wavelength was 585 ± 21 nm. The cell cycle was analyzed using Modfit software to determine the cell cycle distribution. The results are shown in the attached figure. Fig.12 As shown, the results showed that ACBD3 was significantly associated with cell cycle progression. ACBD3 knockout A549 cells were arrested in the G1 phase compared with wild-type A549 cells.

[0106] Example 5. Transwell invasion assay to detect the effect of ACBD3 on lung cancer cell invasion

[0107] (1) Preparation of crystal violet staining solution: Dissolve 0.05 g of crystal violet in methanol to make a 0.5% crystal violet solution, and then use PBS solution at a ratio of 1:5-8 to make a 0.1% crystal violet staining solution.

[0108] (2) Preparation of transwell invasion wells: Dilute BD matrigel and serum-free medium at a ratio of 1:8, pipette 80 μl into the upper chamber of the transwell, and place in an incubator for at least 3 h.

[0109] (3) Cell centrifugation: The cells were digested, centrifuged, resuspended without serum, and diluted to 5×10 5 / ml cell suspension.

[0110] (4) Add culture medium to the lower chamber: Add 500 μl of complete culture medium containing 20% ​​FBS to the Transwell plate and place the chamber into the plate.

[0111] (5) Inoculating cells in the upper chamber: Add 200 μl of serum-free cell suspension into the transwell chamber and incubate the transwell plate in a CO2 (5%) incubator at 37°C for 24 h.

[0112] (6) Staining: discard the upper chamber, take out the small chamber, wash away the culture medium with PBS (800ul), fix with 4% paraformaldehyde for 25min, add PBS to the upper and lower chambers to ensure that the chambers are covered, wash twice with PBS, 200ul for the upper chamber and 800ul for the lower chamber, and stain with crystal violet for 25min (protect from light); wash twice with PBS to remove the crystal violet on the surface, wipe the cells on the inoculated side of the upper chamber with a cotton swab, wash twice with PBS again, and take pictures of the non-cell inoculated side under a microscope. Data analysis was performed using GraphPad Prism 6 software, and the results are shown in the attached figure. Fig.13 As shown, the results showed that knockout of ACBD3 inhibited the invasion of A549 lung cancer cells.

[0113] Example 6. Scratch assay to detect the effect of ACBD3 on lung cancer cell migration

[0114] Place the wound healing plug from ibidi in a 6-well plate and allow it to adhere to the bottom of the 6-well plate. Add 200 μl of resuspended culture medium containing 30,000 wild-type A549 cells and ACBD3-knocked A549 cells to each small grid of the plug, and add 1 ml of normal culture medium outside the scratch plug in the 6-well plate. Place the 6-well plate with cells in the incubator and continue culturing until the cells are completely attached. On the second day, take out the 6-well plate, gently pull out the scratch plug with tweezers, aspirate the original culture medium, add culture medium containing 20 μg / ml mitomycin C and continue culturing for 2 hours, then replace the medium with normal culture medium, take pictures under a 10x microscope, and record it as 0 hours. Use the same method to take pictures at 24 hours and 36 hours to observe the healing of the scratches. The pictures obtained were processed with Image J software and then analyzed with GraphPad Prism6 software. The results are shown in the attached figure. Fig.14 As shown, the results showed that knocking out ACBD3 slowed down the migration of A549 lung cancer cells and inhibited the migration of lung cancer cells.

[0115] Example 7 Xenograft Tumor Experiment

[0116] (1) Twelve female Balb / c-Nude mice were purchased and housed in IVC cages in a barrier SPF animal room with 6 mice / cage. The room temperature was 22±2℃, the relative humidity was 40%-60%, and water and food were freely available. The experiment was started after 3 days of adaptive feeding.

[0117] (2) Preparation of cell suspension. On the day before inoculation, A549 wild-type lung cancer cell suspension and ACBD3 knockout A549 lung cancer cell suspension were prepared and the mice were divided into two groups. The A549 wild-type lung cancer cell suspension and ACBD3 knockout A549 lung cancer cell suspension were inoculated respectively. The inoculation density of each mouse was 5×10 7 , inoculation volume 150 μl.

[0118] (3) Cell inoculation: After capturing the mouse, wipe and disinfect the right armpit with an alcohol cotton pad, draw up 150ul of cell suspension with an insulin syringe, and inject an appropriate amount of cell suspension subcutaneously under the right armpit. After the injection is completed, slowly withdraw the needle, and after confirming that there is no leakage, return the mouse to its original cage for feeding.

[0119] (4) After tumor cell inoculation, the effect of tumor growth on the normal behavior of the animals was routinely monitored, including the activity of the experimental animals, food and water intake, weight gain or loss, eyes, fur and other abnormalities. Data were measured starting 7 days after inoculation, and the weight and tumor size of the mice were measured every 3 days. Tumor size calculation formula: Tumor volume (mm 3 ) = 1 / 2 × (long diameter of tumor × short diameter of tumor 2 ).

[0120] (5) 33 days after inoculation, the mice were killed and the tumors were surgically removed and weighed.

[0121] Attached Fig.15 Figures A, B and C show the tumor formation in mice inoculated with A549 wild-type cells and KO-ACBD3 cells, respectively. The results of in vitro and ex vivo images and comparison of tumor masses in different groups showed that the tumor weight of mice inoculated with KO-ACBD3 cells was significantly reduced.

[0122] Attached Fig.16 Figures A and B show the tumor volume and body weight analysis of mice inoculated with A549 wild-type cells and KO-ACBD3 cells, respectively. The results show that compared with mice inoculated with A549 wild-type cells, the tumor volume of mice inoculated with KO-ACBD3 cells was significantly reduced, but there was no significant difference in the weight changes of the two groups of mice. ACBD3 gene knockout can inhibit the occurrence of lung cancer.

[0123] Example 8. HE staining

[0124] (1) Dewaxing of paraffin sections: Place the sections in environmentally friendly dewaxing solution I for 20 min, then in environmentally friendly dewaxing solution II.

[0125] 20min—anhydrous ethanol I 5min—anhydrous ethanol II 5min—75% alcohol 5min, wash with tap water.

[0126] (2) Pretreatment: The sections were treated in high-definition constant stain pretreatment solution for 1 min.

[0127] (3) Hematoxylin staining: Stain the sections with hematoxylin solution for 3-5 min, wash with tap water, differentiate with differentiation solution, wash with tap water, reblue with bluing solution, and rinse with running water.

[0128] (4) Eosin staining: Dehydrate the sections in 95% alcohol for 1 min and then stain them in eosin solution for 15 s.

[0129] (5) Dehydration and sealing: The sections were placed in anhydrous ethanol I for 2 min, anhydrous ethanol II for 2 min, anhydrous ethanol III for 2 min, n-butanol I for 2 min, n-butanol II for 2 min, xylene I for 2 min, and xylene II for 2 min, and then sealed with transparent neutral gum.

[0130] (6) Microscope examination, image acquisition and analysis.

[0131] The results are attached Fig.17 As shown, the results showed that ACBD3 knockout tumor tissues presented cell necrosis and apoptosis, indicating that ACBD3 knockout A549 cells had reduced tumorigenicity in vivo.

[0132] Example 9. Immunohistochemistry

[0133] (1) Dewaxing of paraffin sections: Place the sections in environmentally friendly dewaxing solution I for 10 min, then in environmentally friendly dewaxing solution II for 10 min.

[0134] 10min-Environmentally friendly dewaxing liquid III 10min-Anhydrous ethanol I 5min-Anhydrous ethanol II 5min-Anhydrous ethanol III 5min-Wash with distilled water.

[0135] (2) Antigen retrieval: During this process, the buffer should be prevented from evaporating excessively and the slides should not be dried. After natural cooling, the slides were placed in PBS (PH7.4) and washed on a decolorizing shaker for 3 times, 5 minutes each time.

[0136] (3) Blocking endogenous peroxidase: The sections were placed in 3% hydrogen peroxide solution and incubated at room temperature in the dark for 25 min. The slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 min each time.

[0137] (4) Serum blocking: Add 3% BSA in the tissue circle to evenly cover the tissue and block at room temperature for 30 minutes. (If the primary antibody is from goat, use rabbit serum to block; if it is from other sources, use BSA to block)

[0138] (5) Add primary antibody: Gently shake off the blocking solution, add primary antibody prepared in a certain ratio with PBS on the slices, and incubate the slices flat in a humidified chamber at 4°C overnight.

[0139] (6) Adding secondary antibody: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times, 5 minutes each time. After the slices are slightly dried, add the secondary antibody (HRP-labeled) of the same species as the primary antibody in the circle to cover the tissue and incubate at room temperature for 50 minutes.

[0140] (7) DAB color development: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times, 5 min each time. After the slices are slightly dried, add freshly prepared DAB color development solution in the circle. The color development time is controlled under a microscope. The positive color is brown-yellow. Rinse the slices with tap water to stop the color development.

[0141] (8) Re-staining of cell nuclei: Re-stain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, re-blue with hematoxylin bluing solution, and rinse with running water.

[0142] (9) Dehydration and sealing: Place the slices in 75% alcohol for 5 min, 85% alcohol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, n-butanol for 5 min, and xylene I for 5 min to dehydrate and make them transparent. Take the slices out of xylene, let them dry slightly, and seal them with sealing glue.

[0143] (10) Microscopic examination: Place the sample under a white light microscope to interpret the results.

[0144] Immunohistochemical identification of Ki-67 expression in tumor tissues Fig.18 As shown in Figure A; Immunohistochemical identification of β-catenin expression in tumor tissues is shown in the attached figure. Fig.18 As shown in Figure B; Immunohistochemical identification of CDK1 expression in tumor tissues is shown in the attached Fig.18 As shown in Figure C; Immunohistochemical identification of E-cadherin expression in tumor tissues is shown in the attached figure. Fig.18 As shown in Figure D. Immunohistochemistry results showed that knocking out ACBD3 downregulated the positive rate of Ki-67 in tumor tissues; compared with wild-type cells, the protein levels of β-catenin and CDK1 in xenograft tumors of ACBD3 knockout cells decreased. The protein level of E-cadherin increased. These data indicate that ACBD3 regulates lung cancer cells through EMT and Wnt signaling pathways.

[0145] Attached Fig.19 This is the regulatory mechanism of ACBD3 in lung cancer.

[0146] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. Application of ACBD3 inhibitors in the preparation of drugs for the treatment of lung cancer.

2. The use according to claim 1, characterized in that: The ACBD3 inhibitor is a gRNA that specifically knocks out the ACBD3 gene using the CRISPR / Cas9 system or a small interfering molecule that specifically interferes with the expression and processing of the ACBD3 gene, including a small interfering RNA or a short hairpin RNA.

3. The use according to claim 1 or 2, characterized in that: The ACBD3 inhibitor comprises a gRNA that inhibits the expression of the ACBD3 gene, and the sequence of the gRNA is shown in SEQ ID NO.1 and SEQ ID NO.

2.

4. The use according to claim 3, characterized in that: The ACBD3 inhibitor comprises an expression vector or a cell line constructed by gRNA; the gRNA sequence is shown in SEQ ID NO.1 and SEQ ID NO.

2.

5. A gRNA targeting ACBD3, characterized in that The sequences of the gRNA are shown in SEQ ID NO.1 and SEQ ID NO.

2.

6. A gRNA expression vector targeting ACBD3, characterized in that: The gRNA sequences are shown in SEQ ID NO.1 and SEQ ID NO.

2.

7. A gRNA expression vector or cell line containing the gRNA as described in claim 5.

8. An ACBD3 inhibitor, characterized in that The ACBD3 inhibitor comprises a gRNA that inhibits the expression of the ACBD3 gene, and the sequence of the gRNA is shown in SEQ ID NO.1 and SEQ ID NO.

2.

9. Use of the ACBD3 inhibitor according to claim 8 in the preparation of a drug for treating pathogenic Escherichia coli infection, bacterial invasion of epithelial cells, Parkinson's disease, human papillomavirus infection, hypertrophic cardiomyopathy, breast cancer, diabetes or dilated cardiomyopathy.

10. Application of ACBD3 as a target gene in the preparation of anti-lung cancer drugs.

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