Exosomes secreted by gastric cancer tissue cells with knockdown of TAGLN2 gene expression, preparation method and application, nucleic acid molecules and drugs

Abstract

The present invention belongs to the technical field of biomedicine, and discloses an exosome secreted by gastric cancer tissue cells with knocked-down TAGLN2 gene expression, a preparation method and application, a nucleic acid molecule and a drug. The exosomes secreted by gastric cancer tissue cells with knocked-down TAGLN2 gene expression provided by the present invention have the potential to delay the growth of vascular endothelial cells and regulate the normalization of vascular endothelial cell function to achieve the normalization of the vascular system of gastric cancer tissue, have high biocompatibility and low immunogenicity, and the exosomes are used in combination with tumor treatment methods (radiotherapy, chemotherapy, immunotherapy, etc.) to produce a synergistic effect, which can achieve better therapeutic effects and have good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to exosomes secreted by gastric cancer tissue cells with knocked-down TAGLN2 gene expression, a preparation method and application thereof, a nucleic acid molecule and a drug. Background Art

[0002] Tumor vascular normalization is a new tumor treatment strategy that has emerged in the clinic in recent years. It rationally uses anti-angiogenic drugs to normalize the structure and function of tumor blood vessels within a specific time window. It is used in combination with anti-tumor treatments such as chemotherapy and immunotherapy to have a synergistic mechanism. However, the time window for tumor vascular normalization is short and reversible, and is affected by drugs, tumor location and type, making it difficult to define the time window for tumor vascular normalization clinically. Therefore, finding new drugs, new targets and new pathways that effectively prolong tumor vascular normalization is one of the difficulties and hot spots in the current clinical combined anti-angiogenic treatment of solid tumors.

[0003] In recent years, studies have reported that the transfer of exosomes (mitoExo) rich in mitochondrial components such as mtDNA, mtRNA, proteins, lipids, and mitochondrial fragments can establish a new communication mechanism for donor cells to regulate bioenergetics and / or redox of target cells. However, there are few reports on the application of cell-cell communication mediated by exosomes rich in mitochondrial components in the field of tumor vascular normalization to extend the time window of tumor vascular normalization. Summary of the invention

[0004] After extensive and in-depth research, the inventors of the present application found that gastric cancer tissue BGC-823 cells containing high-abundance TAGLN2 protein can transport TAGLN2 protein to the extracellular space through exosomes and be absorbed by vascular endothelial cells Ealy926, causing the latter's intracellular TAGLN2 protein expression level to increase significantly, significantly promoting vascular endothelial cell migration and in vitro tubule formation, while upregulating the expression of vascular marker molecules such as VEGFR2 and CD31 and Tagln2 downstream molecules such as NRP1 and SEMA4D, reducing the expression levels of intercellular connection markers such as Occludin, ZO-1, and Claudin5, and changing the expression of endothelial-mesenchymal transition markers, resulting in reduced E-cadherin expression and upregulated N-cadherin expression, ultimately promoting tumor metastasis.

[0005] Based on the above creative discovery, the inventors conducted further research and found that the exosomes secreted by gastric cancer tissue BGC-823 cells with stable knockdown of TAGLN2 expression can inhibit endothelial cell proliferation, migration and in vitro tubule formation, reduce the expression of vascular marker molecules and Tagln2 downstream molecules, and promote intercellular connectivity. The exosomes have the potential to delay the growth of vascular endothelial cells and regulate the normalization of vascular endothelial cell function to achieve the normalization of the vascular system of gastric cancer tissue, and the exosomes have the characteristics of high biocompatibility and low immunogenicity. They are used in combination with tumor treatment methods (radiotherapy, chemotherapy, immunotherapy, etc.) to produce a synergistic effect to achieve better therapeutic effects. Based on this, the technical solution of this application is obtained:

[0006] The first object of the present invention is to provide the use of exosomes secreted by gastric cancer tissue cells in which TAGLN2 gene expression is knocked down in the preparation of drugs for promoting vascular normalization and drugs for treating tumors.

[0007] The second object of the present invention is to provide a nucleotide molecule.

[0008] The third object of the present invention is to provide exosomes.

[0009] The fourth object of the present invention is to provide a method for preparing exosomes.

[0010] The fifth object of the present invention is to provide exosomes prepared by the above method.

[0011] The sixth object of the present invention is to provide a medicine.

[0012] Specifically, the present invention provides the use of exosomes secreted by gastric cancer tissue cells in which TAGLN2 gene expression is knocked down in the preparation of vascular normalization drugs.

[0013] The nucleotide molecule provided by the present invention includes one or more segments of the nucleotide fragments shown in SEQ ID NOs: 1-6.

[0014] In the present invention, the specific examples of the nucleic acid molecule can be but are not limited to siRNA and / or shRNA. The siRNA is a chemically synthesized linear double-stranded RNA, which enters gastric cancer tissue cells by transfection, participates in the RNAi pathway, and silences the TAGLN2 gene. The shRNA is a short double-stranded RNA structure generated by stem-loop sequences, which is recombined with siRNA and a vector, enters cells by transfection or infection, and uses the Dicer enzyme in the cell to cut and generate the corresponding siRNA, participates in the RNAi pathway, and silences the TAGLN2 gene.

[0015] In some specific embodiments, the nucleotide molecule is preferably shRNA, and the nucleotide molecule includes a nucleotide fragment or its complementary fragment as shown in any one of SEQ ID NOs: 13 to 16. At this time, using nucleotide molecules to treat gastric cancer tissue cells can achieve a better effect of knocking down TAGLN2 expression.

[0016] The exosomes provided by the present invention are obtained by secretion from gastric cancer tissue cells in which the expression of the TAGLN2 gene is knocked down.

[0017] In the present invention, specific examples of the gastric cancer tissue cells include but are not limited to: one or more of BGC-823 cells, MGC-803 cells, SGC-7901 cells, AGS cells, HS-746T cells, NCI-N87 cells, SNU-16 cells and KATOIII cells.

[0018] In some specific embodiments, the exosomes contain high abundance of mitochondrial-related proteins, including one or more of MCU, PHB2, SAMM50, NDUFB10, TOMM40, NDUFA13, ATP5MF, HAX1, SLC25A13, FAM162A and NNT. Furthermore, the mitochondrial-related proteins mentioned above are mainly involved in biological processes such as mitochondrial configuration rotation and respiratory chain complex assembly, involving JAK-STAT signaling pathways, metabolic pathways and oxidative phosphorylation signaling pathways. The mitochondrial-related proteins in the exosomes act on vascular endothelial cells, which can increase the level of tricarboxylic acid cycle in the mitochondria of vascular endothelial cells, increase the production of more ROS products, damage mitochondria and depolarize, thereby causing oxidative stress response in cells, so as to ultimately achieve the effect of delaying the growth of vascular endothelial cells and regulating the normalization of vascular endothelial cell function.

[0019] The method for preparing exosomes provided by the present invention comprises: taking the nucleic acid molecules described above to transfect gastric cancer tissue cells to obtain gastric cancer tissue cells with knocked-down TAGLN2 gene expression; taking the gastric cancer tissue cells with knocked-down TAGLN2 gene expression to culture, and purifying to obtain the exosomes.

[0020] In some specific embodiments, the transfection method is selected from one or more of electroporation, liposome transfection, gene gun and virus-mediated method. Further, the transfection method described above is a conventional method used in the prior art, which is limited to enabling nucleic acid molecules to enter gastric cancer tissue cells and silence the TAGLN2 gene. The present invention does not impose any particular limitation on its specific process and conditions.

[0021] In some specific embodiments, the purification method is selected from one or more of ultracentrifugation / differential centrifugation, density gradient ultracentrifugation, size exclusion chromatography, polymer precipitation, immunoaffinity and microfluidics. Further, the purification method described above is a type of method commonly used in the preparation of exosomes, which is limited to being able to separate and obtain exosomes from cell culture, and the present invention does not impose any particular limitation on its specific process and conditions.

[0022] The present invention also provides exosomes prepared by the above exosome preparation method.

[0023] The drug provided by the present invention includes the above-mentioned nucleotide molecules and / or exosomes. At this time, the drug can be used in the treatment of gastric cancer, and can inhibit the expression of TAGLN2 in gastric cancer tissue cells through nucleic acid molecules, thereby causing gastric cancer tissue cells to secrete the above-mentioned exosomes; or directly affect vascular endothelial cells through the above-mentioned exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is an agarose gel electrophoresis diagram of total RNA extracted from siTAG2-BGC and siNC-BGC cells provided in Example 1 of the present invention;

[0025] Figure 2 This is a qRT-PCR test result diagram of the interference effect of the three siTAG2 sequences provided in Example 1 of the present invention on the mRNA expression of TAGLN2 gene in BGC-823 cells;

[0026] Figure 3 This is a microscope image of the shTAG2-62 BGC-823 stable transfected cell provided in Example 2 of the present invention (photographed at 10x10);

[0027] Figure 4 This is a microscope image of the shTAG2-63 BGC-823 stable cell line provided in Example 2 of the present invention (photographed at 10x10);

[0028] Figure 5 This is a microscope image of the shNC BGC-823 stable cell line provided in Example 2 of the present invention (photographed at 10x10);

[0029] Figure 6 This is a graph showing the western blot results of the Tagln2 protein expression level in four shTAG2 BGC-823 stable transfected cells in Example 2 of the present invention;

[0030] Figure 7Transmission electron microscopy images (1.0 μm and 100 nm) of exosome shTAG2-exo provided in Example 3 of the present invention;

[0031] Figure 8 Transmission electron microscopy images (1.0 μm and 100 nm) of exosome shNC-exo provided in Example 3 of the present invention;

[0032] Fig. 9 The western blot experimental results of positive and negative markers of exosomes and Tagln2 protein levels in shTAG2-exo and shNC-exo provided in Example 3 of the present invention;

[0033] Fig.10 This is a graph showing the experimental results of detecting the effects of different exosome treatments on endothelial cell proliferation using the CCK8 method provided in Example 4 of the present invention;

[0034] Fig.11 This is one of the microscope pictures of the Transwell assay provided in Example 4 of the present invention showing the effects of different treatments on the migration ability of endothelial cells (co-incubated with PBS, shot at 10x10);

[0035] Fig.12 The second microscope picture of the Transwell assay for the effect of different treatments on the migration ability of endothelial cells provided in Example 4 of the present invention (shNC-exo co-incubation, 10x10 photography);

[0036] Fig.13 The third microscopic picture of the Transwell assay for the effects of different treatments on the migration ability of endothelial cells provided in Example 4 of the present invention (shTAG2-exo co-incubation, 10x10 photography);

[0037] Fig.14 This is a statistical result diagram of the Transwell assay provided in Example 4 of the present invention for the effect of different exosome treatments on the migration ability of endothelial cells;

[0038] Fig.15 This is one of the microscope pictures of the effects of different treatments on the angiogenesis ability of endothelial cells in vitro provided in Example 4 of the present invention (PBS co-incubation, 10x4 shooting)

[0039] Fig.16 The second microscopic picture of the effect of different treatments on the in vitro angiogenesis ability of endothelial cells provided in Example 4 of the present invention (shNC-exo co-incubation, 10x4 shooting)

[0040] Fig.17The third microscopic picture of the effect of different treatments on the in vitro angiogenesis ability of endothelial cells provided in Example 4 of the present invention (shTAG2-exo co-incubation, 10x4 shooting)

[0041] Fig.18 This is a statistical result diagram of the effects of different exosome treatments on the in vitro angiogenesis ability of endothelial cells provided in Example 4 of the present invention;

[0042] Fig.19 This is one of the experimental results of detecting the effects of different treatments on mitochondrial membrane potential of endothelial cells provided in Example 4 of the present invention (PBS co-incubation, 10x10 photography);

[0043] Fig. 20 Figure 2 is the experimental result of detecting the effects of different treatments on mitochondrial membrane potential of endothelial cells provided in Example 4 of the present invention (shNC-exo co-incubation, 10x10 photography);

[0044] Fig.21 Figure 3 is the experimental result of detecting the effects of different treatments on mitochondrial membrane potential of endothelial cells provided in Example 4 of the present invention (shTAG2-exo co-incubation, 10x10 photography);

[0045] Fig. 22 This is one of the results of a western blot test of the effects of different exosome treatments on the expression of endothelial cell angiogenesis marker molecules provided in Example 4 of the present invention;

[0046] Fig.23 Figure 2 is a western blot test result showing the effects of different exosome treatments on the expression of endothelial cell intercellular junction marker molecules provided in Example 4 of the present invention;

[0047] Fig.24 This is a graph of the experimental results of cell immunofluorescence staining of the effects of different exosome treatments on the expression of VE-cadherin and tight junction proteins in endothelial cells provided in Example 4 of the present invention (scale bar is 10 μm);

[0048] Fig.25 This is the targeted metabolomics profile of endothelial cells before and after co-incubation with shTAG2-exo provided in Example 4 of the present invention.

[0049] Fig.26 This is one of the in vivo treatment experiment results of shTAG2-exo enhancing the chemotherapy sensitivity of gastric cancer tissue provided in Example 5 of the present invention (tumor growth curve).

[0050] Fig. 27Figure 2 (ex vivo tumor tissue) is the in vivo treatment experimental result of shTAG2-exo enhancing the chemotherapy sensitivity of gastric cancer tissue provided in Example 5 of the present invention.

[0051] Fig.28 Figure 3 (tumor weight) is the in vivo treatment experimental result of shTAG2-exo enhancing the chemotherapy sensitivity of gastric cancer tissue provided in Example 5 of the present invention. DETAILED DESCRIPTION

[0052] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0053] Example 1

[0054] This example is used to illustrate the screening of siRNA for inhibiting the expression of TAGLN2 gene, which specifically includes:

[0055] (1) Recovery and culture of BGC-823 cells: Transfer the cryopreserved tube of BGC-823 cells to a 15 mL centrifuge tube, add 10 mL of RPMI-1640 medium (BioInd, catalog number 01-100-1ACS) containing 10% fetal bovine serum (Sigma, catalog number f8313) and 1% penicillin / streptomycin (MeilunBio, catalog number MA0110), centrifuge at 300 × g for 5 min, remove the supernatant, resuspend the cells and divide into RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and culture in a CO2 incubator with 5% CO2, saturated humidity and 37°C;

[0056] After the cells are fully grown, wash them three times with PBS, then add 1 mL of Trypsin-EDTA solution (Meilunbio, catalog number MA0233) and shake well; aspirate the Trypsin-EDTA solution, culture in a 37°C incubator for 3 min, tap the wall of the culture flask to make most of the cells fall off, and when the cells are observed to be granular under an inverted microscope, add an appropriate amount of RPMI-1640 medium containing serum to stop digestion.

[0057] (2) Transfection of BGC-823 cells: 5×10 4 The cells were inoculated at a density of 100 cells / well and cultured in a CO2 incubator at 5% CO2, saturated humidity and 37°C until the cell confluence reached 40%;

[0058] Take siTAG2 mother solutions 1 to 3 and comparison siNC mother solutions and mix them with Opti-MEM to obtain A solutions 1 to 9 and comparison A solutions 1 to 3 with a final volume of 500 μL and a final concentration of 25, 50 and 100 nM, respectively; take 5 μL of the transfection reagent Lipofectamine TM RNAiMAX was gently mixed with 500 μL of Opti-MEM culture medium to obtain a final volume of 505 μL of liquid B; liquid A 1-9 and comparison liquid A 1-3 were respectively mixed with liquid B and allowed to stand for 20 minutes, added to the above-mentioned culture dish, and incubated in a CO2 incubator with 5% CO2, saturated humidity and 37°C for 6 hours, the culture medium was aspirated, washed twice with PBS, replaced with RPMI-1640 culture medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and cultured for a further 24 hours, and samples were collected for testing and analysis, thereby constructing siTAG2-BGC cell lines and siNC-BGC cell lines.

[0059] Among them, siTAG2 mother solution 1 contains the siTAG2-61 fragment whose nucleotide sequence is shown in SEQ ID NO: 1 and 2; siTAG2 mother solution 2 contains the siTAG2-62 fragment whose nucleotide sequence is shown in SEQ ID NO: 3 and 4; siTAG2 mother solution 3 contains the siTAG2-63 fragment whose nucleotide sequence is shown in SEQ ID NO: 5 and 6; the comparison siNC mother solution includes the siNC fragment whose nucleotide sequence is shown in SEQ ID NO: 7 and 8; and the siRNA contained in A solutions 1 to 9 and comparison A solutions 1 to 3 and their concentrations are specifically shown in Table 1.

[0060] Table 1.

[0061]

[0062]

[0063] (3) Extraction of total RNA: siTAG2-BGC and siNC-BGC cells were collected by centrifugation, and 1 mL of Trizol solution was added, which was mixed by pipetting and allowed to stand for 5 min to allow the cells to fully lyse. Then, 200 μL of chloroform was added, which was mixed vigorously for 30 s, allowed to stand at room temperature for 2 min, and then centrifuged at 4°C and 14,000 × g for 15 min to collect the upper aqueous phase containing RNA. The mixture was transferred to a new RNase-free EP tube, and an equal volume of isopropanol was added. The mixture was gently mixed and allowed to stand at room temperature for 10 min to precipitate RNA. The mixture was centrifuged at 4°C and 14,000 × g for 10 min, and the supernatant was removed to collect the precipitate. The precipitate was washed twice with 75% ethanol, and then 60 μL of DEPC water was added to dissolve the precipitate to obtain a crude sample.

[0064] Take the crude sample and digest DNA at 37°C for 30 minutes, then inactivate at 65°C for 10 minutes; then add an equal volume of phenol, mix well, centrifuge at 10000rpm for 5 minutes, and take the supernatant; add an equal volume of chloroform to the supernatant, mix well, centrifuge at 10000rpm for 10 minutes, and take the supernatant; add an equal volume of isopropanol to the supernatant, mix well, let stand at -20°C for 15 minutes, centrifuge at 4°C and 10000×g for 10 minutes, remove the supernatant, and obtain total RNA, wash twice with 75% ethanol, and dry in a clean hood; add 40μL of DEPC water to dissolve the precipitate; measure the OD of the extracted total RNA 260 / OD 280 The ratio is greater than 1.8, the total RNA is relatively pure and has no protein contamination; 1 μL of total RNA was taken for gel electrophoresis, and the results were as follows Figure 1 As shown. Figure 1 It can be seen that the 5s rRNA, 18s rRNA and 28s rRNA bands of the total RNA are intact, indicating that the total RNA extraction is complete and subsequent tests are carried out.

[0065] The reaction system for DNA digestion includes: 20 μL of crude sample, 20 μL of DNase I, 10 μL of 10× buffer, 0.5 μL of RNase inhibitor and 49.5 μL of nuclease-free water.

[0066] (4) Reverse transcription of total RNA: A reverse transcription kit (manufacturer: Vazyme, catalog number: R423) was used to perform in vitro reverse transcription on total RNA according to the instructions to obtain cDNA.

[0067] (5) qRT-PCR detection: qRT-PCR detection of cDNA was performed using a qRT-PCR kit (manufacturer: Vazyme, catalog number: Q121) according to the instructions, and 18srRNA was used as the internal reference fragment to measure the Ct value. The inhibition efficiency of each siRNA was calculated according to the following formula. The results are shown in Table 2 and Figure 2 shown.

[0068] Inhibition efficiency (%) = (x1-x0) / x0×100%;

[0069] x1 is the relative expression level of TAGLN2 gene in siTAG2-BGC cell line, and x0 is the relative expression level of TAGLN2 gene in siNC-BGC cell line obtained by treatment with corresponding concentrations of siNC fragments;

[0070] Relative expression level = 2 -ΔΔCt ;

[0071] ΔCt = average value of (target gene Ct - internal reference Ct); ΔΔCt = average value of (target gene ΔCt in the test sample - target gene ΔCt in the reference sample); relative expression level = (2 -ΔΔCt The target gene was TAGLN2, and the reference gene was 18s.

[0072] The primer system for qRT-PCR includes: primers Tagln2-F and Tagln2-R whose nucleotide sequences are shown in SEQ ID NOs: 9 and 10; and primers 18srRNA-F and 18srRNA-R (internal reference gene) whose nucleotide sequences are shown in SEQ ID NOs: 11 and 12.

[0073] Table 2. Inhibition efficiency of siRNA

[0074]

[0075]

[0076] From Table 2 and Figure 2 The test results show that siTAG2-61, siTAG2-62 and siTAG2-63 fragments all have good interference efficiency. Using them to transfect gastric cancer tissue cells can inhibit the expression of TAGLN2 gene, and the inhibition efficiency of siTAG2-62 and siTAG2-63 fragments is better than that of siTAG2-61.

[0077] Example 2

[0078] This example is used to illustrate the construction of a shTAG2-BGC cell line. Based on the siTAG2-62, siTAG2-63 and siNC fragments obtained in Example 1, shTAG2-62-F, shTAG2-62-R, shTAG2-63-F and shTAG2-63-R with nucleotide sequences as shown in SEQ ID NOs: 13 to 16 were designed; and shTAG2 recombinant plasmid and shNC control recombinant plasmid were constructed, and shTAG2-BGC-823 cells and shNC-BGC-823 cells were constructed and detected, specifically including:

[0079] 1. Construction of shTAG2-62 and shTAG2-63 recombinant plasmids

[0080] (1) Take 15 μL of PLV2-U6-PURO vector (KEMOBio, catalog number KM1056864), 3 μL of EcoR I enzyme, 5 μL of 10× PCR buffer and 27 μL of ddH2O and mix them evenly. React at 37°C for 4 h, and use gel electrophoresis and DNA gel recovery kit (Magen, catalog number D2110-02) to recover the reaction solution according to the instructions to obtain the enzyme digestion product.

[0081] (2) shTAG2-62-F, shTAG2-62-R, shTAG2-63-F, shTAG2-63-R, shNC-F and shNC-R were mixed with an equal volume of nuclease-free water, boiled in a boiling water bath for 5 min, then placed at 72°C for 15 min, and naturally cooled to room temperature for 30 min to obtain shRNA substrates.

[0082] (3) Take 4 μL of shRNA to be ligated, 3 μL of enzyme digestion product, 4 μL of 5×CE MultiS Buffer, 2 μL of Exnase MultiS2 and 7 μL of ddH2O and mix them evenly. After reacting at 37°C for 30 min, the ligation product was obtained, and the ligation product was immediately cooled on ice.

[0083] (4) In an ice bath, take 5 μL of the ligation product and add it to 50 μL of DH5α competent cell culture medium, gently rotate to mix, and place in an ice bath for 30 min; heat shock in a 42°C water bath for 90 s, then quickly transfer to an ice bath and place on ice for 2 min; then add 200 μL of LB medium and mix, and shake and culture at 37°C and 200 rpm for 1 h; evenly spread the bacterial liquid on an LB plate containing ampicillin (100 μg / mL) and place at room temperature until the liquid is absorbed; then invert the plate and culture overnight at 37°C.

[0084] (5) Several single clones were picked from the plate and added to 3 mL of LB medium (10 g of Tryptone, 5 g of Yeast Extract, and 10 g of NaCl). The culture was shaken overnight at 37°C and 200 rpm. The plasmid was extracted using a high-purity plasmid mini-preparation kit (Magen, catalog number P112-02) and sent for sequencing.

[0085] In this example, shTAG2-62 and shTAG2-63 recombinant plasmids were constructed. Among them, the shTAG2-62 and shTAG2-62-2 recombinant plasmids include the shTAG2-62-F and shTAG2-62-R fragments shown in the nucleotide sequences of SEQ ID NOs: 13 and 14, respectively, and the shTAG2-63 and shTAG2-63-2 recombinant plasmids include the shTAG2-63-F and shTAG2-63-R fragments shown in the nucleotide sequences of SEQ ID NOs: 15 and 16, respectively.

[0086] 2. Construction of shTAG2-BGC cells

[0087] (1) According to 5×10 4 293T cells were inoculated into DMEM medium (Meilunbio, catalog number MA0212) containing 10% fetal bovine serum and 1% penicillin / streptomycin at a cell density of 5×10 cells / well, shaken well, and cultured in a CO2 incubator at 5% CO2, saturated humidity, and 37°C. Cells were passaged when the cell confluence reached 90%; 5×10 6 293T cells were re-seeded in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at a cell density of 100 cells / dish, and cultured in a CO2 incubator at 5% CO2, saturated humidity and 37°C for 24 h until the cell density reached 70%-80%;

[0088] Take 40 μL of Lipo2000 and 1.46 mL of Opti-MEM culture medium (invitrogen, catalog number 31985070) and mix to obtain liquid A; take shTAG2-62 recombinant plasmid, shTAG2-62-2 recombinant plasmid, shTAG2-63 recombinant plasmid, shTAG2-63-2 recombinant plasmid, shNC control recombinant plasmid and shNC-2 control recombinant plasmid and packaging plasmid pMD2.G, pSPAX2 at a mass ratio of 1:1:2 (total mass of 40 μg), and add Opti-MEM culture medium to a total volume of 1.5 mL to obtain liquid B; after letting liquid A and liquid B stand for 5 minutes respectively, slowly add liquid B dropwise to liquid A, gently mix and let stand for 20 minutes;

[0089] 2 h before transfection, the culture medium of 293T cells was replaced with DMEM medium containing 10% fetal bovine serum, and then the precipitation mixture was evenly added dropwise, and cultured in a CO2 incubator with 5% CO2, saturated humidity and 37°C. The virus culture supernatant was collected 48 to 72 h after transfection, centrifuged at 1000 rpm for 5 min, the supernatant was filtered through a 0.45 μm syringe filter, and high-speed centrifuged at 4°C and 25000×g in an ultrahigh-speed refrigerated centrifuge for 2 h to obtain the virus precipitate. The virus precipitate was fully dissolved with 1 mL of PBS and filtered through a 0.22 μm syringe filter to obtain a virus concentrate.

[0090] (2) The virus concentrate was preheated to 37°C, and the virus concentrate was diluted to an MOI of 125 in 10% fetal bovine serum and 1% penicillin / streptomycin 1640 medium (BioInd, catalog number 01-100-1ACS) containing 6 μg / mL polybrene to obtain a virus dilution solution; 2×10 5 BGC-823 cells were inoculated at a cell density of 100 μL / well and cultured in a CO2 incubator at 5% CO2, saturated humidity and 37°C until the cell confluence reached 50%. The culture medium was aspirated and the virus dilution solution was added at an amount of 200 μL / well. The cells were cultured overnight in a CO2 incubator at 5% CO2, saturated humidity and 37°C. The virus-containing culture medium was aspirated and replaced with 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. The cells were cultured in a CO2 incubator at 5% CO2, saturated humidity and 37°C. Culture, and select single clones for cell amplification and interference efficiency detection on the third day of infection. Use 6 μg / mL puromycin (meilunbio, catalog number MA0318) to continue screening and culturing the single clones with high interference efficiency for 1 month, and construct shTAG2-62-BGC cells, shTAG2-62-2-BGC cells, shTAG2-63-BGC cells, shTAG2-63-2-BGC cells, shNC-BGC cells and shNC-2-BGC cells.

[0091] Figures 3 to 5 These are microscopic observation pictures of shTAG2-62-BGC cells, shTAG2-63-BGC cells, and shNC-BGC cells. As can be seen from the pictures, the constructed shTAG2-62-BGC cells, shTAG2-63-BGC cells, and shNC-BGC cells have a typical cobblestone morphology and are in good growth condition.

[0092] 3. Detection of shTAG2-BGC-823 cells

[0093] (1) According to the method provided in Example 1, total RNA was extracted, reverse transcribed and qRT-PCR was performed on shTAG2-62-BGC cells, shTAG2-62-2-BGC cells, shTAG2-63-BGC cells, shTAG2-63-2-BGC cells, shNC-BGC cells and shNC-2-BGC cells, and the relative expression level of the TAGLN2 gene of each cell was calculated, as shown in Table 3.

[0094] Table 3. Relative expression of TAGLN2 gene

[0095]

[0096] From the test results shown in Table 3, it can be seen that the expression of TAGLN2 gene in shTAG2-63-BGC cells and shTAG2-63-2-BGC cells was significantly inhibited, showing a good inhibitory effect, and the inhibition efficiency of shTAG2-63-2 recombinant plasmid on TAGLN2 gene expression was as high as 77%, that is, the shTAG2-BGC cell line was successfully constructed in this embodiment.

[0097] (2) Take 1 mL of RIPA lysis buffer (Meilunbio, catalog number MA0152), 10 μL of PMSF (Merck, catalog number 78830), and 10 μL of Cocktail (Merck, catalog number P8340) and mix them with the culture medium of shTAG2-BGC cells and shNC-BGC cells (cell density is 10 4 / mL), shake and lyse on ice for 30 minutes, centrifuge at 4°C and 14000rpm for 5 minutes, take the supernatant to obtain a protein solution; mix the protein solution with 5× loading buffer (Meilunbio, catalog number MA0003), boil for 10 minutes, slowly return to room temperature, and perform protein sample quantitative SDS-PAGE electrophoresis, protein transfer and immunoblotting.

[0098] The protein transfer and immunoblotting specifically include: placing the gel obtained by quantitative SDS-PAGE electrophoresis on filter paper to form a gel transfer stacking layer with a filter paper-gel-PVDF membrane-filter paper structure, treating it at low temperature and 100V constant voltage for 22 minutes, taking out the hybridization membrane and rinsing it with TBST for 5 minutes, then adding 5% skim milk powder solution to block it at room temperature for 1 hour; taking out the hybridization membrane and rinsing it with TBST for 10 minutes; adding Tagln2 primary antibody (Proteintech Cat#15508-1-Ap, 1:1000 dilution) and incubating it at 4°C overnight; taking out the hybridization membrane and rinsing it with TBST for 10 minutes, rinsing it 4 times; adding secondary antibody (Southern Biotech, catalog number 4050-05, 1:20000 dilution) was incubated at 37°C for 1 hour, the hybridized membrane was taken out and rinsed with TBST for 10 minutes, rinsed 4 times, the hybridized membrane was placed on a transparent plastic plate, and the chemiluminescent substrate (Millipore, catalog number WBKLS0500) was evenly added to the surface of the hybridized membrane for 5 minutes, the excess substrate solution on the membrane surface was absorbed by filter paper, and the membrane was placed in a dark box for development to obtain the gray value of TACLN2 and GAPDH proteins. The results of SDS-PAGE electrophoresis are shown in Figure 6 The gray value results obtained by immunoblotting are shown in Table 4.

[0099] Table 4. Grayscale values ​​of TAGLN2 and GAPDH proteins

[0100]

[0101] Depend on Figure 6 As shown in the results in Table 4, the TAGLN2 gene expression knockdown effect of shTAG2-63-2-BGC cells was the best, and the TAGLN2 protein expression inhibition rate was 72.20%. A TAGLN2 expression stable knockdown cell line was constructed.

[0102] In the following examples, the shTAG2-BGC cells are the shTAG2-63-2-BGC cells provided in Example 2, and the shNC-BGC cells are the shNC-BGC cells described in Example 2.

[0103] Example 3

[0104] This example is used to illustrate a method for extracting exosomes rich in mitochondrial components from shTAG2-BGC cells, which specifically includes:

[0105] (1) Isolation and extraction of exosomes: shTAG2-BGC cells and shNC-BGC cells were added to 10 mL of RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and cultured in a CO2 incubator with 5% CO2, saturated humidity and 37°C until the cell confluence reached 80%. The RPMI-1640 medium was replaced and cultured for 48 h under the same conditions, and the supernatant was collected;

[0106] Centrifuge at 4°C and 300×g for 10 min, collect the supernatant; centrifuge at 4°C and 3000×g for 15 min, collect the supernatant; centrifuge at 4°C and 2000×g for 30 min, collect the supernatant; centrifuge at 4°C and 10000×g for 45 min, collect the supernatant, then filter the supernatant with a 0.45μm filter, centrifuge at 4°C and 100000×g for 70 min, remove the supernatant; resuspend the collected precipitate with PBS, centrifuge again at 4°C and 100000×g for 70 min, collect the precipitate, and obtain exosomes.

[0107] Figure 7 and 8 Transmission electron micrographs of exosomes shTAG2-exo and shNC-exo extracted from shTAG2-BGC cells and shNC-BGC cells, respectively. Figure 7 and 8 It can be seen that the exosomes shTAG2-exo and shNC-exo have clear saucer-like structures, and the average particle size of shTAG2-exo is 77.83nm, and the average particle size of shNC-exo is 78.32nm.

[0108] (2) Protein identification of exosomes: Exosomes shTAG2-exo and shNC-exo were subjected to protein extraction, BCA quantification, enzymatic hydrolysis, LC-MS / MS analysis, database retrieval, and data analysis.

[0109] Among them, protein extraction includes: grinding the exosome shTAG2-exo and shNC-exo respectively with liquid nitrogen, then adding SDT lysis solution (4% SDS, 100mM Tris-HCl, 1mM DTT, pH=7.6), treating in a boiling water bath for min, ultrasonically breaking for 2min, and then centrifuging at 4°C and 16000×g for 20min to collect the supernatant. Sequencing-grade modified trypsin (Promega, Madison, WI) was used for enzymatic hydrolysis at 37°C. The reagents and conditions used in LC-MS / MS analysis refer to the reagents and conditions commonly used in the relevant test methods in the prior art, and the reagents and conditions used for exosome shTAG2-exo and shNC-exo are consistent.

[0110] Fig. 9 The western blot results of positive and negative markers of exosomes and Tagln2 protein levels in exosomes shTAG2-exo and shNC-exo are shown. Fig. 9 It can be seen that both exosomes positively expressed exosomal markers TSG101, CD9 and CD81 and negatively expressed endoplasmic reticulum protein Calnexin, and the content of Tagln2 protein in exosome shTAG2-exo was significantly lower than that in exosome shNC-exo.

[0111] In the LC-MS / MS analysis, a total of 38,061 peptides were identified in exosomal shTAG2-exo and shNC-exo, corresponding to 4,456 proteins. The specific differential proteins of exosomal shTAG2-exo compared with shNC-exo are shown in Table 5.

[0112] Table 5. Differential proteins in exosomes shTAG2-exo compared with shNC-exo

[0113]

[0114]

[0115] As shown in Table 5, there are 63 differential proteins between exosome shTAG2-exo and shNC-exo, of which 41 are up-regulated proteins. Among the up-regulated proteins, 11 mitochondrial proteins, including MCU, PHB2, SAMM50, NDUFB10, TOMM40, NDUFA13, ATP5MF, HAX1, SLC25A13, FAM162A and NNT, account for 26.8%. The above 11 mitochondrial proteins include related proteins such as mitochondrial inner membrane, outer membrane and matrix proteins, which are mainly involved in biological processes such as mitochondrial configuration rotation and respiratory chain complex assembly, involving JAK-STAT signaling pathway, metabolic pathway and oxidative phosphorylation. In other words, the exosome shTAG2-exo secreted by gastric cancer tissue cells with knockdown of TAG2 gene expression is rich in mitochondrial components, and its packaging components are significantly changed.

[0116] Example 4

[0117] This example is used to illustrate the ability of the exosome shTAG2-exo provided in Example 3 to inhibit endothelial cell angiogenesis and promote vascular normalization, specifically including: taking 5 μg of exosome shTAG2-exo and adding it to Ealy926 cells grown to 80% confluence in 10 mL of RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and co-incubating in a CO2 incubator at 5% CO2, saturated humidity and 37°C, and using exosome shNC-exo as a control and PBS as a blank control to perform the following tests:

[0118] (1) CCK8 assay for cell proliferation: Collect cells at 0 h, 24 h, 48 h, 72 h, and 96 h of incubation, replace the culture medium, and mix 10 μL of CCK8 assay solution (MeilunBio, catalog number MA0218) with 100 μL of culture medium and incubate for 4 h. Use an ELISA reader to read the OD value of the culture medium. 490 The corresponding cell proliferation rate (%) was calculated according to the following formula. The results are shown in Figure 8 shown.

[0119] Cell proliferation rate (%) = (A i -A0) / A0×100%

[0120] Among them, A i is the OD during incubation 490 A0 is the OD at 0h of incubation. 490 value.

[0121] Depend on Fig.10It can be seen that compared with the PBS group, co-incubation of exosomes shNC-exo and Ealy926 cells significantly promoted the proliferation of endothelial cells, while exosomes shTAG2-exo significantly inhibited the proliferation of Ealy926 cells.

[0122] (2) Transwell assay for cell migration ability: cells were collected at 0 h and 48 h of incubation, and 1×10 5 The cells were resuspended in 100 μL of RPMI-1640 medium and added to the upper chamber of the Transwell chamber, and 600 μL of complete medium was added to the lower chamber. The upper chamber of the Transwell cell culture plate was cultured in a CO2 incubator with 5% CO2, saturated humidity and 37°C for 48 h. The chamber was taken out, the cells in the upper chamber were wiped with a cotton swab, and then fixed with 4% paraformaldehyde for 20 min, washed once with PBS, stained with crystal violet for 10 min, washed once with PBS, observed under a microscope, and photographed and counted. The results are shown in the figure. Figures 11 to 14 shown.

[0123] Depend on Figures 11 to 14 It can be seen that compared with the co-culture of Ealy926 cells with PBS and exosome shNC-exo, exosome shTAG2-exo significantly inhibited the migration of Ealy926 cells.

[0124] (3) Angiogenesis assay: Matrigel (BD Biosciences, catalog number 354230) was added to the culture plate at a volume of 200 μL / well and evenly spread over the bottom of the plate. After observing that there were no bubbles, the plate was placed in a 37°C incubator for 2 h until solidified. Then, 2 × 10 4 Ealy926 cells were added at a concentration of 100 μg / well and cultured in a CO2 incubator with 5% CO2, saturated humidity and 37°C for 6 h. The angiogenesis was observed and counted. The results are shown in the figure. Figures 15 to 18 shown.

[0125] Depend on Figures 15 to 18 It can be seen that compared with the co-incubation of Ealy926 cells with PBS and exosome shNC-exo, exosome shTAG2-exo significantly inhibited the formation of in vitro tubules by Ealy926 cells.

[0126] The results of CCK8 assay for cell proliferation, Transwell assay for cell migration and angiogenesis assay showed that exosomal shTAG2-exo has a moderate effect on endothelial cells, mainly delaying growth and regulating normal function, avoiding the problem of short and difficult-to-define time window for vascular normalization transformation caused by high cytotoxicity and massive killing in a short period of time, and has higher clinical application value.

[0127] (4) Mitochondrial membrane potential detection: Ealy926 cells were detected using a mitochondrial membrane potential detection kit (JC-1, Meilunbio, catalog number MA0338) according to the instructions and observed under a fluorescence microscope. The results are as follows: Figures 19 to 21 shown.

[0128] Depend on Figures 19 to 21 It can be seen that compared with the co-incubation of Ealy926 cells with PBS, the Ealy926 cells co-incubated with exosomes shNC-exo had a strong orange fluorescence signal, indicating that treatment with shNC-exo can enhance the aggregation of JC-1 in healthy mitochondria of cells and promote the formation of JC-1 polymers; and the Ealy926 cells co-incubated with exosomes and shTAG2-exo had a strong green fluorescence signal, indicating that JC-10 diffused out of the mitochondria and returned to the monomer form, that is, the co-incubation of shTAG2-exo caused the mitochondria in Ealy926 cells to be damaged and depolarized.

[0129] (5) Western blot detection of marker molecules: The marker molecules in the culture medium of Ealy926 cells were detected by referring to the method provided in Example 3. The results are as follows: Fig. 22 and 23 shown.

[0130] Among them, the antibodies used are: anti-VEGFR2 (R&D, catalog number MAB3571-100), CD31 (Santa Cruz, catalog number sc-376764), SEMA4D (R&D, catalog number MAB74701), NRP1 (R&D, catalog number AF3870), Occludin (Proteintech, catalog number 66378-1-1g), ZO-1 (Proteintech, catalog number 21773-1-Ap), Claudin 5 (Abcam, catalog number ab15106), E-cadherin (R&D, catalog number AF748), N-cadherin (R&D, catalog number AF6426) and VE-cadherin (Santa Cruz, catalog number sc-9989).

[0131] Depend on Fig. 22 and 23It can be seen that compared with the co-incubation of Ealy926 cells with PBS and exosomes shNC-exo, the co-incubation of Ealy926 cells with exosomes shTAG2-exo significantly downregulated the expression of vascular markers such as VEGFR2 and CD31, as well as pro-angiogenic molecules such as Tagln2 and Tagln2 downstream NRP1 and SEMA4D, while significantly upregulated the expression of intercellular junction markers such as Occludin, ZO-1, and Claudin5, and enhanced the expression of E-cadherin and reduced the expression of N-cadherin. Even in the shTAG2-exo incubation group, the expression of endothelial cell vascular markers and Tagln2 downstream molecules showed lower expression levels than the PBS group, and inhibited endothelial-mesenchymal transformation, significantly increased intercellular junctions, and reduced the permeability of the endothelial cell layer.

[0132] (6) Cell immunofluorescence staining: Ealy926 cells were fluorescently stained using a confocal culture dish (Millipore, catalog number: EZ Slide 4-wel) according to the instructions. Fig.24 shown.

[0133] Depend on Fig.24 It can be seen that compared with the co-incubation of Ealy926 cells with PBS and exosomes shNC-exo, stronger fluorescence signals of intercellular junction molecules were detected after co-incubation of Ealy926 cells with exosomes shTAG2-exo, indicating that the cell connections between single endothelial cell layers were tighter.

[0134] Depend on Figures 22-24 It can be seen that exosome shTAG2-exo can significantly increase intercellular connectivity, reduce the permeability of the endothelial cell layer, and promote vascular normalization while inhibiting endothelial-mesenchymal transition and angiogenesis.

[0135] (7) Targeted metabolomics: Sample collection, metabolite extraction, sample LC-MS / MS mass spectrometry analysis, data processing and quantitative analysis were performed in sequence, including: 1000 μL of pre-cooled 80% methanol aqueous solution was added to each tube of cell sample, vortexed to mix, sonicated in an ice bath for 20 minutes, allowed to stand at -20°C for 2 hours, centrifuged at 16,000×g and 4°C for 20 minutes, and the supernatant was evaporated by a high-speed vacuum concentrator centrifuge. The precipitate was redissolved in 100 μL of 50% methanol aqueous solution, centrifuged at 20,000×g and 4°C for 15 minutes, and the supernatant was taken and LC-MS / MS analysis was performed with reference to the reagents and conditions commonly used in the relevant test methods in the prior art, and the reagents and conditions used for Ealy926 cells co-incubated with or without exosome shTAG2-exo were kept consistent. The results are as follows Fig.25 shown.

[0136] Fig.25 The asterisks in the middle indicate metabolites that are significantly differentially expressed in Ealy926 cells after co-incubation with exosomes shTAG2-exo. After matching the significantly differentially expressed metabolites with the metabolic pathways, it can be seen that after co-incubation with exosomes shTAG2-exo, the metabolic pattern of endothelial cells is reshaped, which significantly increases the level of tricarboxylic acid cycle in the mitochondria of endothelial cells. On the one hand, more ROS products are produced, causing oxidative stress response in cells; on the other hand, it reverses the aerobic glycolysis pattern that tumor vascular endothelial cells are highly dependent on, and instead promotes the normalization of vascular endothelial cells.

[0137] Example 5

[0138] This example is used to illustrate that the exosome shTAG2-exo provided in Example 3 is combined with chemotherapy drugs to increase the sensitivity of gastric cancer tissue to chemotherapy drugs and promote the therapeutic effect. Specifically, BALB / c Nude mice (male, 4 weeks old, SPF-grade breeding conditions) are used as experimental animals. The needle is inserted subcutaneously on the right back of the nude mouse, and the needle sneaks about 5 mm subcutaneously to the right back of the nude mouse near the armpit. 0.2 mL of BGC-823 cell suspension (2×10 cells per nude mouse) is injected. 6 After the injection, the needle was rotated and retracted, and the needle hole was pressed with a cotton swab until no liquid flowed out. The subcutaneous inoculation of cells was completed and recorded as Day 0. The feeding continued, and the treatment was carried out according to the following groups starting from Day 10:

[0139] Experimental group 1 was treated with intratumoral injection of PBS, experimental group 2 was treated with intratumoral injection of shNC-exo, and experimental group 3 was treated with intratumoral injection of sh63-exo. The specific treatment conditions included: a single injection volume of 50 μL (exosome dosage of 30 μg), 4 mice in each group, once every 3 days, for a total of 4 injections; starting from Day 15, 0.2 mL of 4 mg / Kg cisplatin was injected intraperitoneally once every 4 days. The tumor volume was measured once every 3 days. The results are shown in the figure. Figures 26-28 shown.

[0140] Depend on Figures 26-28 The test results shown show that compared with the PBS group, the subcutaneous transplanted tumors in the shNC-exo pretreatment group grew faster, and the tumor volume and weight were larger, indicating that sh-NC-exo promoted the tolerance of transplanted tumors to cisplatin treatment. On the contrary, compared with the PBS group and the shNC-exo group, the subcutaneous transplanted tumors in the shTAG2-exo pretreatment group grew slower, and the tumor volume and weight were reduced. The results were statistically significant, indicating that shTAG2-exo enhanced the sensitivity of transplanted tumors to cisplatin treatment, increased the killing effect of chemotherapeutic drugs, and had a synergistic effect.

[0141] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

[0142] The nucleotide sequences involved in the above examples are shown in the following table:

[0143]

[0144]

Claims

1. Application of exosomes secreted by gastric cancer tissue cells with knockdown of TAGLN2 gene expression in combination with cisplatin in the preparation of drugs for the treatment of gastric cancer.

2. An exosome, It is characterized in that The exosomes are obtained by secretion from gastric cancer tissue cells in which TAGLN2 gene expression is knocked down.

3. The exosome according to claim 2, It is characterized in that The exosomes contain high-abundance mitochondrial-related proteins, which include mitochondrial calcium ion uniporter, mitochondrial inner membrane autophagy receptor 2, member 50 of the SAM complex, NADH oxidoreductase coenzyme 10, mitochondrial import receptor subunit TOM40 homolog, auxiliary subunit of mitochondrial complex I, mitochondrial ATP synthase f subunit, HCLS1-associated protein X-1, calcium-binding aralar2 protein of human mitochondrial carrier protein, FAM162A protein and one or more of human NNT recombinant protein.

4. A method for preparing exosomes, It is characterized in that The preparation method comprises: Transfecting gastric cancer tissue cells with a nucleic acid molecule including a nucleotide fragment with a sequence as shown in SEQ ID NO:5 and / or a nucleotide fragment with a sequence as shown in SEQ ID NO:6 to obtain gastric cancer tissue cells in which TAGLN2 gene expression is knocked down; The gastric cancer tissue cells in which the TAGLN2 gene expression is knocked down are taken for expression, and the exosomes are obtained by purification.

5. The method for preparing exosomes according to claim 4, It is characterized in that The transfection method is selected from one or more of electroporation, liposome transfection, gene gun method and virus-mediated method.

6. The method for preparing exosomes according to claim 4, It is characterized in that The purification method is selected from one or more of differential centrifugation, density gradient ultracentrifugation, size exclusion chromatography, polymer precipitation, immunoaffinity and microfluidics.

7. Exosomes prepared by the method for preparing exosomes according to any one of claims 4 to 6.

8. A drug, It is characterized in that Comprising the exosomes of claim 2, 3 or 7.

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