Use of a preparation for knocking out an hdac3 gene or a preparation for knocking out an ikkα gene in the preparation of a drug for inhibiting tumor metastasis

Abstract

This invention belongs to the field of tumor immunotherapy and provides the application of Hdac3 gene knockout formulations or Ikkα gene knockout formulations in the preparation of drugs that inhibit tumor metastasis. The research results of this invention show that Hdac3 and Ikkα gene mutations significantly inhibit lung tumor metastasis and prolong the survival of mice, and this metastasis inhibition is independent of T, B, and NK cells. Simultaneously, this invention also discovers that Hdac3 and Ikkα mutations inhibit lung tumor metastasis by affecting the sensitivity of tumor cells to tumor necrosis factor, making tumor cells more susceptible to apoptosis, thereby providing a method for sustained in vivo killing of tumor cells. This invention also provides a mutant vector, which exhibits good anti-tumor metastasis effects.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy, specifically relating to the application of Hdac3 gene knockout agents or Ikkα gene knockout agents in the preparation of drugs that inhibit tumor metastasis. Background Technology

[0002] Cancer metastasis originates from the primary tumor, where genetic variations, epigenetic remodeling, and microenvironmental signals synergistically drive the metastatic phenotypic transformation. This transformation process regulates cell invasiveness, vascular permeability, circulatory system tolerance, tissue extravasation efficiency, and the potential for colonization in distant target organs, ultimately forming a cascade of pathological processes. The formation of tumor metastasis follows a series of complex cellular biological events—collectively known as the "invasion-metastasis cascade." This cascade reaction drives the primary tumor epithelial cells to complete the following key steps: (a) circulating tumor cells (CTCs) penetrate the local extracellular matrix and stromal cell layer; (b) CTCs infiltrate into the vascular lumen or lymphatic vessels; (c) they withstand the survival pressure of the circulatory system; (d) they localize and remain in distant organs and extravasate into the distant tissue parenchyma to form disseminated tumor cells (DTCs); (e) DTCs survive in the heterogeneous microenvironment and form micrometastases, restarting the proliferation program and eventually developing into a clinically detectable tumor that is visible to the naked eye (this stage is often referred to as "metastasis").

[0003] Recent meta-analyses of various human cancers indicate that HDAC3 may be one of the most frequently upregulated genes in cancer cells. HDAC3 is the third HDAC discovered in mammals and a unique and important member of the HDAC family. It is primarily located in the nucleus but can dynamically shuttle between the nucleus and cytoplasm. The role of HDAC3 in anti-tumor immunity has been extensively studied, and selective HDAC3 inhibitors are currently considered a potential therapeutic strategy against various cancers; selective HDAC3 inhibitors have already been developed.

[0004] HDAC3 also plays an important role in the progression of malignant tumors, especially in tumor cell proliferation, apoptosis, and metastasis. In metastatic breast cancer cells, HDAC3 deficiency and cAMP-response-binding protein 3 (CREB3) overexpression jointly promote cell migration. In pancreatic cancer, HDAC3 expression is found to be higher than in paired adjacent normal tissues, and high HDAC3 expression mediates the invasion of pancreatic cancer cells. Malignant melanoma is a highly aggressive and deadly skin cancer with no completely effective treatment currently available; studies have found that HDAC3 overexpression is associated with lymph node metastasis in melanoma. In ovarian cancer, HDAC3 expression is high, and this high expression enhances the migration of ovarian cancer cells by downregulating E-cadherin.

[0005] The specific mechanisms by which HDAC3 plays a role in tumor metastasis are not yet fully understood. Its substrate diversity, tissue specificity, and the complexity of its regulatory networks pose major challenges to research. Elucidating the mechanisms of action of HDAC3 in tumor metastasis will not only contribute to a deeper understanding of the molecular basis of tumor metastasis but may also provide new targets and strategies for cancer treatment. Therefore, research based on HDAC3 is of great significance in providing new strategies for cancer treatment. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide the application of Hdac3 gene knockout formulations or Ikkα gene knockout formulations in the preparation of drugs that inhibit tumor metastasis. This invention develops a method that enhances the sensitivity of tumor cells to TNF-α through Hdac3 knockout, induces TNF-α (Tumor necrosis factor-α)-mediated tumor cell apoptosis, and ultimately inhibits tumor metastasis and progression, prolonging survival. This solves the problems of poor prognosis and low overall survival rate in clinical patients with metastatic tumors.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides the use of a formulation that knocks out the Hdac3 gene or a formulation that knocks out the Ikkα gene in the preparation of a drug for inhibiting tumor metastasis.

[0009] For a long time, tumor cells have been the primary research focus in the fight against metastasis in malignant tumors. Metastasis has traditionally been considered a late-stage event in tumor progression, essentially the final stage of a complex pathological process known as the "invasion-metastasis cascade." This process endows cancer cells with specific biological characteristics, enabling them to detach from the primary tumor and migrate to distant tissues. Since cancer cells that successfully reach distant organs often develop drug resistance, clinical treatment faces even greater challenges once metastatic lesions form. Therefore, the core proposition of current medical research lies in elucidating the mechanisms by which cancer cells colonize distant organs.

[0010] To improve the clinical efficacy of tumor metastasis treatment, we present a molecular mechanism by which Hdac3 regulates the TNF-NF-κB signaling axis, thereby affecting TNF-α-mediated apoptosis sensitivity and ultimately determining tumor metastasis. This invention elucidates the mechanism of action of Hdac3 in tumor metastasis, which not only contributes to a deeper understanding of the molecular basis of tumor metastasis but may also provide new targets and strategies for tumor treatment. Developing specific inhibitors of Hdac3 to inhibit tumor metastasis and effectively suppress it could provide new strategies for cancer treatment.

[0011] Preferably, the drug inhibits lung colonization of tumor cells.

[0012] Preferably, the drug downregulates the NF-κB pathway, enhances the sensitivity of cells to TNF-α, and promotes tumor cell apoptosis.

[0013] Preferably, the tumor includes any one or a combination of at least two of melanoma, sarcoma, lymphoma, carcinoma, or leukemia.

[0014] Preferably, the cancer includes any one or a combination of at least two of brain cancer, breast cancer, liver cancer, stomach cancer, lung cancer, or colon cancer.

[0015] This invention systematically elucidates the molecular mechanism by which HDAC3 influences tumor cell apoptosis sensitivity and ultimately determines tumor metastasis by regulating the TNF-NF-κB signaling axis, thus deepening our understanding of the role of HDAC3 in tumor metastasis.

[0016] In this invention, knocking out Hdac3 and Ikkα in MCA205 cells using CRISPR-Cas9 gene editing technology can inhibit tumor metastasis and prolong the survival of mice.

[0017] In this invention, we found that subcutaneous tumor grafting of MCA205 cells and Hdac3 knockout of MCA205 cells in NSG mice inhibited tumor metastasis. Furthermore, intravenous injection of cells into NSG mice at a concentration of 1×102 cells... 3 At a certain time, Hdac3 can significantly prolong the survival of mice and inhibit lung tumor colonization.

[0018] In this invention, we found that the absence of Gzmb and Prf had no effect on lung colonization of Hdac3 knockout MCA205 cells, while the absence of Tnf allowed Hdac3 knockout MCA205 cells to colonize in the lungs of mice, indicating that the killing effect of TNF on Hdac3 knockout MCA205 cells is related to HDAC3-mediated tumor metastasis.

[0019] Secondly, the present invention provides the use of formulations that knock out the Hdac3 gene or the Ikkα gene in the preparation of reagents that inhibit lung colonization of tumor cells or promote apoptosis of tumor cells.

[0020] According to the research results of this invention, preparations that knock out the Hdac3 and Ikkα genes can promote tumor cell apoptosis at the cellular level, meaning they can be formulated as a simple experimental preparation to explore the physiological and metabolic processes of tumor cell apoptosis; and in vivo, they can inhibit tumor metastasis and prolong the survival of mice. The reagents claimed in this invention are not intended to eliminate the cause or lesion, but rather are intended for non-therapeutic purposes in the preparation of reagents that inhibit tumor cell lung colonization or promote tumor cell apoptosis.

[0021] Thirdly, the present invention provides an sgRNA, wherein the sgRNA includes an sgRNA targeting the Hdac3 gene or an sgRNA targeting the Ikkα gene.

[0022] Preferably, the sgRNA targeting the Hdac3 gene is transcribed from double-stranded DNA formed by annealing sequences including those shown in SEQ ID No:1 and SEQ ID No:2.

[0023] SEQ ID No: 1: CACCGCCCAATGAAACCTCATCGCC.

[0024] SEQ ID No:2:AAACGGCGATGAGGTTTCATTGGGC.

[0025] Preferably, the sgRNA targeting the Ikkα gene is transcribed from double-stranded DNA formed by annealing a sequence including the sequence shown in SEQ ID No:3 or SEQ ID No:4.

[0026] SEQ ID No: 3: CACCGCCGTTCCCGCATCTCCCAG.

[0027] SEQ ID No: 4: AAACCTGGGAGATGCGGGAACGGCC.

[0028] Fourthly, the present invention provides a gene editing system comprising the sgRNA and Cas9 described in the third aspect.

[0029] Fifthly, the present invention provides a recombinant vector containing the sgRNA described in the third aspect, and the backbone of the recombinant vector being the lentiCRISPRv2-puro plasmid.

[0030] In a sixth aspect, the present invention provides a recombinant virus, which is obtained by co-transfecting lentiviral packaging cells with the recombinant vector described in the fifth aspect and the lentiviral packaging vectors psPAX2 and pMD2.G.

[0031] In a seventh aspect, the present invention provides a pharmaceutical composition for inhibiting tumor metastasis and progression, the pharmaceutical composition comprising any one of the gene editing system described in the fourth aspect, the recombinant vector described in the fifth aspect, or the recombinant virus described in the sixth aspect.

[0032] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) Hdac3 deficiency can inhibit tumor metastasis and prolong the survival of mice. This inhibition of tumor metastasis is mediated by TNF.

[0035] (2) Hdac3 deficiency inhibits tumor metastasis and prolongs the survival of mice by downregulating the NF-κB pathway. Attached Figure Description

[0036] Figure 1 To validate the knockout cell lines, Figure A shows the Western blot validation of Hdac3 knockout, and Figure B shows the Western blot validation of Ikkα knockout cell lines.

[0037] Figure 2 Schematic diagrams showing subcutaneous tumor growth and lung metastasis in NSG mice after MCA205 and Hdac3 knockout of MCA205 cells. Figure A shows a 5×10⁶ subcutaneous tumor in NSG mice. 5 Subcutaneous tumor growth curves of MCA205 cells and Hdac3 knockout MCA205 cells. Figure B shows in vivo imaging of mice 25 days after injection. Figure C shows the number of lung tumor nodules after lung harvesting from mice. Figure D shows H&E staining of the lungs.

[0038] Figure 3Schematic diagrams showing the survival and lung tumor growth of NSG mice after tail vein injection of different numbers of MCA205 and Hdac3 knockout MCA205 cells. Figure A shows the survival of NSG mice after tail vein injection of 1×10⁻⁶ MCA205 cells. 5 The survival time of mice after MCA205 and Hdac3 knockout of MCA205 cells. Figure B shows the tail vein injection of 1×10⁻⁶ cells into NSG mice. 4 Survival time of mice after MCA205 and Hdac3 knockout of MCA205 cells. Figure C shows the tail vein injection of 1×10⁻⁶ cells into NSG mice. 3 The survival time of mice after MCA205 and Hdac3 knockout of MCA205 cells. Figure D shows the tail vein injection of 1×10⁻⁶ cells into NSG mice. 3 H&E staining of mouse lungs after MCA205 and Hdac3 knockout of MCA205 cells.

[0039] Figure 4 To administer 1×10⁻⁶ mmol / L via tail vein injection to C57 mice and Gzmb / Prf / Tnf knockout C57 mice, respectively. 6 Schematic diagrams showing the survival time and lung tumor growth of mice after MCA205 and Hdac3 knockout of MCA205 cells. In the diagram, A shows the survival time of C57 mice, B shows the H&E staining of lung tissue from C57 mice, C shows the survival time of Gzmb knockout C57 mice, D shows the survival time of Prf knockout C57 mice, E shows the survival time of Tnf knockout C57 mice, and F shows the H&E staining of lung tissue from Tnf knockout C57 mice after lung harvesting.

[0040] Figure 5 1×10⁻⁶ mmol / L was injected into the tail vein of C57 mice and Tnf knockout C57 mice. 6 MCA205 and Ikkα knockout MCA205 cells were shown. Figure A shows the survival time of mice, and Figure B shows H&E staining of mouse lung tissue.

[0041] Figure 6 Figure 1 shows the protein expression results of the NF-κB pathway in MCA205 and Hdac3 knockout MCA205 cells and two cell lines treated with TNF.

[0042] Figure 7 The figures show the cell death rates of MCA205 and Hdac3 knockout MCA205 cells after the addition of PBS or TNF. Figure A shows the apoptosis rate of MCA205 and Hdac3 knockout MCA205 cells after the addition of PBS or TNF. Figure B shows the apoptosis rate of MCA205 cells after the addition of HDAC inhibitors and PBS or TNF. Figure C shows the apoptosis rate of MCA205 and Ikkα knockout MCA205 cells after the addition of PBS or TNF. Detailed Implementation

[0043] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0044] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0045] Example 1

[0046] The Hdac3 / Ikkα knockout MCA205 cell line was constructed using CRISPR / Cas9 gene knockout technology.

[0047] 1. Construction of sgRNA and expression vector.

[0048] (1) Using the CRISPR sgRNA design tool (https: / / chopchop.cbu.uib.no / ), input the knockout gene name and species, and select a high-efficiency sequence without self-folding. Find the gene cDNA sequence in NCBI, import it into the snap gene software, mark the exon sequence, and compare the primers with the cDNA. Primers located in the middle of the cDNA are optimal.

[0049] (2) After identifying the sgRNA, Bsm BⅠ restriction sites were added to both ends, and the sgRNA was sent to Genewiz for oligo sequence synthesis. The sequence is as follows:

[0050] SEQ ID No: 1: CACCGCCCAATGAAACCTCATCGCC.

[0051] SEQ ID No:2:AAACGGCGATGAGGTTTCATTGGGC.

[0052] SEQ ID No: 3: CACCGCCGTTCCCGCATCTCCCAG.

[0053] SEQ ID No: 4: AAACCTGGGAGATGCGGGAACGGCC.

[0054] (3) Add the synthesized sgRNA to nuclease-free water and dilute it to a working concentration of 10 μM. Take 10 μL of upstream and downstream primers and add them to a 50 μL PCR tube. Shake to mix and then place it in a PCR instrument for annealing. The annealing program is set as follows: 70℃, 20 min; 37℃, 30 min; 4℃, keep warm.

[0055] (4) The lentiCRISPRv2-puro plasmid containing the U6 promoter, which was stored in the laboratory, was transformed and amplified. Single clones were selected for sequencing, sequence alignment, and plasmid extraction. The plasmid vector was then digested with Bsm BI enzyme, recovered from the gel, and the sgRNA was ligated to the digested vector with T4 ligase. The vector was then transformed, single clones were selected for sequencing, and after the sequence alignment was correct, the plasmid was extracted by shaking.

[0056] 2. Cell transfection.

[0057] (1) Lay 1×10⁻⁶ molten metal in each of the two holes of the 6-hole plate. 6 After 24 hours, when the cell density reaches 60-70%, transfection is performed on individual cells.

[0058] (2) The lentiCRISPRv2-puro-sgRNA plasmid and packaging plasmids psPAX2 and pMD2.G were mixed in a mass ratio of 4:3:1 to form a 2 μg mixture. Then, 200 μL of OPTI-MEM was added for dilution, followed by 8 μL of PEI. The mixture was shaken and mixed, and then allowed to stand for 15 min after a brief incubation period.

[0059] (3) Add the above mixture into one well of the 6-well plate.

[0060] (4) 48 hours after transfection, lentivirus was collected, filtered through a 0.22 μM filter membrane, concentrated, and resuspended in 1 mL of complete culture medium, followed by cell infection.

[0061] 3. Cell infection.

[0062] (1) To explore the optimal concentration of puro (puromycin) for killing cells. 1 × 10⁶ cells were seeded into each well of a 6-well plate. 6 Cells were subjected to treatment. After 24 hours, 1 mg / mL Puro was added in a gradient. The medium was changed daily with complete culture medium containing the drug. Cell death was observed for four days. The lowest concentration that resulted in complete cell death was the optimal killing concentration.

[0063] (2) Lay 1×10⁻⁶ molten metal in each of the two holes of the 6-hole plate. 6 After 24 hours, the concentrated virus was resuspended in one well and infected for 48 hours. The culture medium was removed, the cells were washed once with 1 mL PBS, and 2 mL of DMEM complete medium containing an appropriate concentration of puromycin was added. The drug-containing DMEM complete medium was changed daily for one week.

[0064] 4. Use flow cytometry to sort monoclonal cells.

[0065] Cells screened by the above drugs were digested with trypsin, resuspended in 500 μL PBS, and single clones were sorted by flow cytometry into 96-well plates containing 200 μL of DMEM complete medium and cultured in an incubator for about 2 weeks.

[0066] 5. Identification of knockout cell lines.

[0067] (1) When the cells grow to more than 40%, they are transferred from the 96-well plate to the 12-well plate for culture. After the 12-well plate is fully grown, 1 / 5 of them are passaged. Protein is extracted from the remaining cells and Western blot is used to identify whether the knockout was successful.

[0068] (2) For cells without bands, the genome was extracted using KAPA enzyme. Then, approximately 100 bp fragments upstream and downstream of the sgRNA in the genome were amplified using primers and ligated into a seamless clone with a pcDNA3.1-GFP direct-labeled vector containing the CMV promoter digested with HindIII and BamHI. The resulting plating was then transformed and sent to Genewiz for sequencing the next day. The gene and protein translation sequences were compared to further confirm the success of the knockout.

[0069] The Hdac3 knockout identification primers are shown in SEQ ID No:5 and SEQ ID No:6.

[0070] SEQ ID No: 5: CCACTATGGTAGGGCTCAGGGTTG.

[0071] SEQ ID No: 6: GAGAGGGGAGAAGGATAGAGGGCAC.

[0072] The Ikkα knockout identification primers are shown in SEQ ID No:7 and SEQ ID No:8.

[0073] SEQ ID No:7: AGTCTCGCGAGACGGAGGACT.

[0074] SEQ ID No:8:CGAGCTGTCAGTCAAGGTGGGAG.

[0075] Figure 1 Figure A in the diagram shows the Western blot verification of HDAC3 knockout. Figure 1 Figure B in the diagram shows the Western blot validation of the Ikkα knockout cell line. From... Figure 1 It can be seen that the knockout cell line construction is complete.

[0076] Example 2

[0077] Animal experiments were conducted to observe the survival time and lung colonization of mice with subcutaneous tumor-implanted tumors and tumor cells implanted in the tail vein.

[0078] 1. Subcutaneous tumor grafting.

[0079] (1) After centrifuging the cells according to the cell passage method, resuspend them with an appropriate amount of PBS, take 10 μL of cells into a cell counting plate, and then place them in a cell counter for counting.

[0080] (2) Take 7×10 6 Transfer each cell to a 1.5 mL EP tube, centrifuge at 1100 rpm for 5 min, resuspend the cells in 700 μL PBS, and immediately place on ice.

[0081] (3) Transfer the ice box to the animal room, take the mouse to be implanted with subcutaneous tumor, shave the hair off the back of the mouse with a hair removal machine, take 100 μL of resuspended cell solution with a 1 mL insulin injection needle and inject it subcutaneously into the right back of the mouse.

[0082] 2. Tumor metastasis model created by tail vein injection of tumor cells.

[0083] (1) Cells were obtained using the subcutaneous tumor grafting method.

[0084] (2) Fix the mouse in the tail vein injection device, take 100 μL of cell suspension with a 1 mL insulin injection needle, and inject it into the mouse blood vessel along the tail vein.

[0085] (3) Observe the survival period of mice and draw mouse survival curves.

[0086] 3. In vivo imaging technology was used to detect the localization of tumor cells overexpressing luciferase in mice.

[0087] (1) Overexpress the luciferase reporter gene fragment in tumor cells to stably express the fragment in tumor cells.

[0088] (2) Subcutaneous tumor implantation or tail vein injection to introduce tumor cells into mice.

[0089] (3) After anesthetizing mice with isoflurane, inject bioluminescent liquid into the peritoneum, wait 6 minutes, and then place them in a live imaging instrument to detect the luminescent location and degree.

[0090] 4. Observe and stain the lungs of mice with H&E.

[0091] (1) After spraying the dead mouse or the mouse with the neck dislocated with disinfectant alcohol, place it in the clean bench, cut open the abdominal skin with surgical scissors, cut the dorsal aorta to bleed it, and then open the chest cavity.

[0092] (2) The heart was perfused to remove blood from the lungs. Then, 5 mL of PBS solution was drawn into the trachea and injected to inflate the lungs. The trachea was then cut and the lungs were detached.

[0093] (3) Then the trachea and heart were separated and the bloodstains were washed off with PBS.

[0094] (4) Visually observe the growth of lung tumors and take pictures. Then fix the lungs in 4% paraformaldehyde and shake slowly in a shaker at room temperature for 24 hours.

[0095] (5) After fixation, the lung was sent to Fangke Biotechnology for tissue embedding, sectioning and H&E staining.

[0096] The growth of subcutaneous tumors and lung metastasis were detected in NSG mice after subcutaneous tumor implantation of MCA205 and Hdac3 knockout of MCA205 cells, as described above. The results are as follows: Figure 2 As shown in the figure, tumor metastasis was significantly inhibited after Hdac3 was lost.

[0097] Following the above method, the survival time and lung tumor growth of NSG mice after tail vein injection of different numbers of MCA205 and Hdac3 knockout MCA205 cells were detected. The results are as follows: Figure 3 As shown in the figure, Hdac3 deletion significantly prolonged the survival of mice and significantly reduced lung tumors. This effect was observed when the injected cell amount was 1×10⁻⁶. 3 At that time, the mice did not die, and no tumor cells were colonized in their lungs.

[0098] Following the method described above, 1×10⁻⁶ ozontally was injected into the tail vein of C57 mice and Gzmb / Prf / Tnf knockout C57 mice, respectively. 6 MCA205 and Hdac3 knockout MCA205 cells, results as follows Figure 4 As shown in the figure, Hdac3 deletion significantly prolongs the survival time of C57 mice and significantly reduces lung tumors. Gzmb and Prf knockout mice have no effect on the survival time of mice, while Hdac3 deletion significantly shortens the survival time of mice when Tnf is knocked out, and tumor cells colonize the lungs of mice to form tumor nodules.

[0099] Following the method described above, 1×10⁻⁶ mmol / L was injected into the tail vein of C57 mice and Tnf knockout C57 mice. 6 MCA205 and Ikkα knockout MCA205 cells, results as follows Figure 5 As shown in the figure, Ikkα deficiency significantly prolongs the survival of mice. When TNF is not expressed in mice, Ikkα knockout MCA205 cells cannot be cleared by the body, and tumor cells colonize the lungs, leading to the death of mice.

[0100] Example 3

[0101] Western blot assays were used to detect the expression of NF-κB pathway proteins in Hdac3 knockout cell lines and MCA205 WT cell lines.

[0102] 1. Extract proteins from cells.

[0103] (1) After the cells are digested with trypsin, take an appropriate amount of cells and centrifuge at 1100 rpm for 3 min, remove the supernatant, and wash the precipitate with 1 mL PBS.

[0104] (2) Discard the PBS with a pipette, add an appropriate amount of RIPA medium lysis buffer (containing protease phosphatase inhibitor), mix well by pipetting, and place on ice for 30 min.

[0105] (3) Pre-cool the centrifuge to 4°C, put the protein solution into the centrifuge, and centrifuge at 12000 rpm for 30 min.

[0106] (4) Slowly aspirate the supernatant and transfer it to a new 1.5 mL EP tube. To prevent protein degradation, this process must be performed on ice.

[0107] (5) Add 5×SDS loading buffer to the protein supernatant and vortex mix. After a brief separation, boil in a 100°C metal bath for 10 minutes. Then, remove the water vapor from the lid and vortex mix. For short-term storage, place in a -20°C refrigerator, or for long-term storage, place in a -80°C refrigerator.

[0108] 2. Preparation of PAGE gel.

[0109] The PAGE gel kits used in the experiment were all from Amatech, and the specific operating procedures are as follows:

[0110] (1) Wash the glass plate (1.5 mm) with clean water, let it air dry or blow dry with a hair dryer, and put it on the glue-making frame.

[0111] (2) Take the PAGE gel kit out of the 4°C refrigerator and let it return to room temperature.

[0112] (3) Take 4 mL of the lower gel solution and the lower gel buffer solution into a 15 mL centrifuge tube, mix them evenly, and then add 80 μL of modified coagulant and mix evenly.

[0113] (4) Transfer the above mixed solution to a glass plate using a pipette, add anhydrous ethanol and press the separating gel flat.

[0114] (5) After 30 min, remove the anhydrous ethanol and use absorbent paper to absorb as much of the residual anhydrous ethanol as possible.

[0115] (6) Take 1 mL each of the upper gel solution and the blue upper gel buffer into a new 15 mL centrifuge tube, mix well, add 20 μL of modified coagulant, and mix well. Use a pipette to add the mixed solution to a glass plate, and insert the cleaned and dried comb into the upper gel.

[0116] (7) After 30 minutes, remove the glue and use it immediately or soak it in water and store it in a refrigerator at 4°C.

[0117] 3. Sample loading and electrophoresis.

[0118] (1) Load the prepared PAGE gel into the electrophoresis tank, add an appropriate amount of electrophoresis solution to the tank, remove the comb, and add the protein molecular weight marker and protein sample into the well in sequence.

[0119] (2) Cover the electrophoresis tank, turn on the power, maintain a constant voltage of 80 V until the sample enters the separating gel, adjust the voltage to 120 V until the sample reaches the bottom of the separating gel.

[0120] (3) Turn off the power, open the cover, take out the gel plate, rinse off the electrophoresis solution with clean water, place it in the transfer solution, and perform the transfer.

[0121] (4) Activate the PVDF membrane with a pore size of 0.22 μm in anhydrous methanol, and then soak it in the transfer solution for later use.

[0122] (5) Pour a small amount of transfer solution into the metal basin, open the transfer clamp in the basin with the black side down, and then place a sponge and a piece of moistened three-layer filter paper on it; open the large plate of the gel plate with the palm of your hand, cut off the top layer of gel, spread the gel flat on the three-layer filter paper, pour an appropriate amount of transfer solution on the gel, and then immediately shake the PVDF membrane in the transfer solution to remove air bubbles, and cover the gel along the edge so that there are no air bubbles between the gel and the membrane; then place another piece of three-layer filter paper and a sponge on it, close the transfer clamp, and transfer it to the transfer tank, with the black side of the transfer clamp corresponding to the black side of the transfer tank.

[0123] (6) Add two appropriately sized ice boxes to the transfer tank, transfer them to a basin, cover the basin, and connect the power supply. Add an appropriate amount of ice-water mixture to the basin, and then transfer the membrane at a constant current of 250 mA for 2 h.

[0124] 4. Blocking and antibody incubation.

[0125] (1) After the transfer is completed, the PVDF membrane is quickly removed and placed in 50 mL of 5% skim milk or BSA solution at room temperature for 1 h.

[0126] (2) Recover the milk or BSA and rinse the membrane with TBST solution to remove the milk or sealing liquid.

[0127] (3) Dilute the antibody with antibody diluent according to the instructions, cut the membrane according to the protein size, add antibody diluent to the membrane, and incubate overnight at 4°C with gentle shaking.

[0128] (4) Recover the primary antibody and wash the membrane three times with TBST for 15 min each time.

[0129] (5) Take the secondary antibody corresponding to the species of the primary antibody, dilute it with antibody diluent according to the instructions, add it to the membrane, and shake it slowly at room temperature for 1 h.

[0130] (6) Discard the secondary antibody and wash the membrane three times with TBST for 15 min each time.

[0131] (7) Mix developer solution A and solution B in a 1:1 ratio, add the mixture to the dried PVDF membrane, and display the protein bands on a fluorescence chemiluminescence analyzer.

[0132] Figure 6 Protein expression of the NF-κB pathway was investigated in MCA205 cells and two cell lines treated with TNF, with MCA205 and Hdac3 knockouts. Figure 6 It can be seen that the NF-κB pathway is significantly downregulated after Hdac3 is lost, and the pathway is slightly upregulated after the addition of TNF, but not significantly.

[0133] Example 4

[0134] Flow cytometry for the detection of apoptosis.

[0135] Test method:

[0136] (1) The cells of the drug-treated and control groups were digested with trypsin, and after digestion was stopped by centrifugation in complete culture medium, the cell pellet was obtained.

[0137] (2) Wash the cell pellet once with PBS buffer, then wash twice with 1× binding buffer, and transfer it to a flow cytometer.

[0138] (3) After removing the supernatant, add 90 μL of binding buffer to resuspend the sample, then add the staining reagent from the apoptosis kit and stain in the dark for 30 minutes.

[0139] (4) Add 500 μL of binding buffer to the tube, centrifuge to wash away the dye, resuspend in 400 μL of binding buffer, and transfer to a new flow cytometry tube after passing through a 40 μL filter.

[0140] (5) Detection of cell apoptosis using flow cytometer.

[0141] Figure 7To illustrate cell death in MCA205 and Hdac3 knockout MCA205 cells after the addition of PBS and TNF, Figure 7 It can be seen that Hdac3-deficient tumor cells are sensitive to TNF and undergo significant cell death; the same result can be obtained after using HDAC inhibitors; downregulation of NF-κB also leads to cell sensitivity to TNF and cell death.

[0142] In summary, this invention uses CRISPR-Cas9 gene editing technology to knock out the Hdac3 and Ikkα genes in MCA205 cells, verifying that the deletion of Hdac3 and Ikkα genes can play a role in inhibiting tumor metastasis. The role of Hdac3 and Ikkα deletion in tumor metastasis was systematically evaluated, and the results show that the deletion of Hdac3 and Ikkα may make tumor cells more easily recognized and eliminated by the immune system, inhibiting tumor cell lung metastasis and colonization.

[0143] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. Application of Hdac3 gene knockout formulations or Ikkα gene knockout formulations in the preparation of drugs that inhibit tumor metastasis.

2. Use according to claim 1, characterized in that, The drug inhibits lung colonization of tumor cells; Preferably, the drug downregulates the NF-κB pathway, enhances the sensitivity of cells to TNF-α, and promotes tumor cell apoptosis.

3. Use according to claim 1 or 2, characterized in that, The tumor includes any one or a combination of at least two of the following: melanoma, sarcoma, lymphoma, carcinoma, or leukemia; Preferably, the cancer includes any one or a combination of at least two of brain cancer, breast cancer, liver cancer, stomach cancer, lung cancer, or colon cancer.

4. Application of Hdac3 gene knockout agents or Ikkα gene knockout agents in the preparation of reagents that inhibit tumor cell lung colonization or promote tumor cell apoptosis.

5. An sgRNA, characterized in that, The sgRNA includes sgRNA targeting the Hdac3 gene or sgRNA targeting the Ikkα gene.

6. The sgRNA of claim 5, wherein, The sgRNA targeting the Hdac3 gene is transcribed from double-stranded DNA formed by annealing sequences including those shown in SEQ ID No:1 and SEQ ID No:2; Preferably, the sgRNA targeting the Ikkα gene is transcribed from double-stranded DNA formed by annealing sequences including those shown in SEQ ID No:3 and SEQ ID No:

4.

7. A gene editing system, characterized in that, The gene editing system includes the sgRNA and Cas9 as described in claim 5 or 6.

8. A recombinant vector, characterized in that, The recombinant vector contains the sgRNA as described in claim 5 or 6, and the backbone of the recombinant vector is the lentiCRISPRv2-puro plasmid.

9. A recombinant virus, wherein, The recombinant virus was obtained by co-transfecting lentiviral packaging cells with the recombinant vector described in claim 8 and the lentiviral packaging vectors psPAX2 and pMD2.G.

10. A pharmaceutical composition for inhibiting metastasis and progression of a tumor, characterized by, The pharmaceutical composition comprises either the gene editing system of claim 7 or the recombinant virus of claim 9.

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