Application of prosaposin in prognosis judgment, diagnosis and treatment target of gastric cancer

Abstract

The present invention discloses the use of the Prosaposin gene / protein, belonging to the technical field of cancer precision medicine drugs. Through experiments such as ELISA and multi-color immunofluorescence, the present invention has confirmed that the Prosaposin protein is expressed in the blood and cancer tissues of gastric cancer patients, significantly higher than its expression in the blood of healthy people and benign gastric mucosa tissues, and the prognosis of patients with high expression of the Prosaposin protein is poor. The Prosaposin gene / protein is a target for precision therapy, and a targeted drug for treating diseases with high expression of the Prosaposin gene / protein such as gastric cancer, and a kit for diagnosis or prognosis judgment are prepared. The CRISPR-Cas9 system of the present invention can efficiently knock out the Prosaposin gene overexpressed in gastric cancer, inhibit the proliferation, invasion and migration of gastric cancer cells, and the system is simple to operate and has a high knockout efficiency. Therefore, the present invention has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of precision cancer medicine, and more specifically, relates to the application of Prosaposin in the prognosis judgment or treatment target of gastric cancer. Background Art

[0002] In recent years, the incidence of global malignant tumors has been showing a continuous upward trend. The incidence and mortality of malignant tumors in China, especially digestive tract tumors, are higher than the global average level. Gastric cancer is a high-incidence malignant tumor in China. The malignant phenotype of cancer cells is regulated by various related genes and their signaling pathways within the cell, as well as factors such as cell interaction in the microenvironment. With the application of high-throughput technologies such as second-generation, third-generation sequencing, single-cell sequencing, proteomics, and metabolomics, finding tumor differential genes or proteins related to clinical significance has become a common method in the forefront of clinical research. Although there have been a large number of research results on the mechanisms of gastric cancer development and prognosis, including bioinformatics databases mainly based on transcriptome data, there is an urgent clinical need for secretory protein markers that are convenient for diagnosis and treatment.

[0003] Prosaposin protein is not Prostate Acid Phosphatase (protein name abbreviation: PSAP, gene name also known as: ACP3, ACPP). The gene encoding Prosaposin protein (subsequently referred to as Prosaposin in this application) is located at position 10q22.1 of chromosome 10 and has 15 exons. Prosaposin protein is a highly conserved secretory lysosomal protein and is a common precursor of proteins required for lysosomal degradation in vivo. It is cleaved into 4 smaller saposin products in the lysosome, namely saposins A, B, C, and D required for sphingolipid hydrolysis, promoting sphingolipid catabolism. These precursor proteins exist both as secretory proteins and as integral membrane proteins. Under physiological conditions, Prosaposin mainly participates in important biological processes such as sphingolipid metabolism, lysosomal transport, autophagy, and lipid metabolism to regulate the life activities of the body.

[0004] Previous domestic and foreign literature has reported on the related research of Prosaposin in several types of tumors. The secreted Prosaposin protein is expressed at a higher level in oral squamous cell carcinoma tissues than in the surrounding benign tissues. In prostate cancer, it can promote the growth, invasion, and migration of cancer cells. High expression of Prosaposin is an independent risk factor for short progression-free survival in prostate cancer. In primary breast cancer, Prosaposin mainly exerts an anti-apoptotic effect through the ERK-1 / 2, MAPK, and PI3K-Akt pathways, promoting the proliferation of cancer cells. An elevated serum Prosaposin level can promote the invasion and migration of breast cancer cells. In gliomas, in vitro and in vivo experiments have confirmed that Prosaposin can promote the growth, invasion, and epithelial-mesenchymal transition process of glioblastoma. However, in serous ovarian cancer, exogenous Prosaposin can act as a tumor metastasis inhibitor, promoting tumor regression in xenograft models. The above studies have shown that Prosaposin plays different or even opposite roles in these tumors. However, the relationship between Prosaposin and gastric cancer is currently unclear, and its expression and specific regulatory mechanisms in gastric cancer are still unknown. Summary of the Invention

[0005] In view of the above problems existing in the prior art, the technical problems to be solved by the present invention are to provide biomarkers and kits for predicting the prognosis of gastric cancer to meet the use requirements of precise detection. Another technical problem to be solved by the present invention is to provide the application of the Prosaposin gene as a drug target in the preparation of targeted drugs for the treatment of gastric cancer to meet the use requirements of targeted drugs.

[0006] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] The biomarker for predicting the prognosis of gastric cancer is the Prosaposin protein expressed in the peripheral blood of gastric cancer patients.

[0008] The kit for predicting the prognosis of gastric cancer contains reagents for detecting the expression level of Prosaposin protein in the peripheral blood of gastric cancer patients.

[0009] The application of the Prosaposin gene (Gene ID: 5660) as a drug target in the preparation of targeted drugs for the treatment of gastric cancer.

[0010] In the above application, the targeted drug inhibits the expression of Prosaposin protein in gastric cancer cells, or knocks out the Prosaposin gene in gastric cancer cells, or silences the Prosaposin gene in gastric cancer cells.

[0011] In the above application, the targeted drug is the CRISPR-Cas9 system.

[0012] The application described above, wherein the targeted drug is a Prosaposin gene knockout system containing sgRNA with the following sequences:

[0013] Prosaposin-gRNA1:

[0014] Prosaposin-gRNA2:

[0015] Prosaposin-gRNA3:

[0016] Beneficial effects: Compared with the prior art, the advantages of the present invention are as follows: Through the peripheral blood of gastric cancer patients before and after surgery and the peripheral blood of healthy people, as well as the tissue microarray of gastric cancer clinical samples, it is confirmed by ELISA and multi-color immunofluorescence that the expression of Prosaposin protein in the blood and cancer tissues of gastric cancer patients is significantly higher than its expression in the blood of healthy people and benign gastric mucosa tissues, and the prognosis of patients with high expression of Prosaposin protein is poor; the CRISPR-Cas9 system of the present invention can efficiently knockout the overexpressed Prosaposin gene in gastric cancer, inhibit the proliferation, invasion and migration of gastric cancer cells, and this system is simple to operate and has a high knockout efficiency. The Prosaposin gene / protein is a target for precision therapy, and is used in the preparation of targeted drugs for treating diseases with high expression of Prosaposin gene / protein such as gastric cancer, and kits for diagnosis or prognosis judgment. Description of the Drawings

[0017] Figure 1 It is a graph showing the expression level of Prosaposin in the peripheral blood and tissues of gastric cancer; in the figure, A is the graph showing the expression of Prosaposin in the plasma of 6 normal people detected by DIA-MS; B is the graph showing the expression of Prosaposin in the plasma of 6 gastric cancer patients before and after surgery detected by DIA-MS, and the expression of Prosaposin in the postoperative plasma decreases; C is the standard curve graph showing the expression of Prosaposin in the sera of normal and gastric cancer patients detected by ELISA; D is the graph showing the expression of Prosaposin in the sera of 9 normal people detected by ELISA: 3.356 - 0.866 (ng / mL); E is the graph showing the expression of Prosaposin in the sera of 11 gastric cancer patients before and after surgery detected by ELISA; F is the graph showing the difference in the expression of Prosaposin in the cancer tissues and surgical margins (benign gastric mucosa epithelial tissues) of the corresponding 11 gastric cancer patients detected by immunofluorescence, and the expression of Prosaposin in the cancer tissues is significantly higher than that in the surgical margins;

[0018] Figure 2It is the result diagram of immunofluorescence detection of the expression of Prosaposin in gastric cancer and adjacent tissues; in the figure, A is the immunofluorescence staining diagram of Prosaposin in benign gastric mucosal epithelial cells; B is the fluorescence staining diagram of Prosaposin in gastric cancer tissues;

[0019] Figure 3 It is the relationship diagram between high and low expression of Prosaposin and the prognosis of gastric cancer patients;

[0020] Figure 4 It is the detection of the expression of Prosaposin in gastric cancer cells by Western Blot; in the figure, A is the expression diagram of Prosaposin in GES1 cells, HGC27 cells, AGS cells, MKN1 cells, and MKN45 cells; B is the expression diagram of Prosaposin in AGS cells and MKN1 cells after knocking out this gene; C is the expression diagram of Prosaposin in HGC27 cells and MKN45 cells after overexpressing this gene;

[0021] Figure 5 It is the vector diagram of the lentivirus-mediated CRISPR-Cas9 system;

[0022] Figure 6 It is the effect diagram of knocking out the Prosaposin gene on the proliferation, migration, and invasion functions of gastric cancer cells;

[0023] Figure 7 It is the effect diagram of overexpressing the Prosaposin gene on the proliferation, migration, and invasion functions of gastric cancer cells. Specific implementation manners

[0024] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention, but do not limit the implementation scope of the present invention.

[0025] The main reagents used in the following embodiments are:

[0026] Opal 7-color immunohistochemistry kit: PerkinElmer, USA. Rabbit anti-human PSAP monoclonal antibody (for immunofluorescence assay): abcam. Horseradish peroxidase-labeled secondary antibody (for immunofluorescence assay): PerkinElmer, USA. Antibody diluent / blocking solution: AKOYA, USA. AR6 repair solution: AKOYA, USA. Xylene, neutral balsam, etc. are provided by the pathology department. Gastric cancer cell lines are purchased from Nanjing Kebai Biotechnology Co., Ltd. 1640 medium, fetal bovine serum: gibco, USA. BCA protein assay kit: Biosharp. PVDP membrane (for Western blot assay): Bio-Rad. Rabbit anti-human PSAP monoclonal antibody (for Western blot assay): ABclonal, China. GAPDH antibody (for Western blot assay): proteintech, USA. ECL developing solution (Suzhou Xinsaimi Co., Ltd.). RPMI-1640 complete culture medium: Add RPMI-1640 and fetal bovine serum and mix well to make their final concentrations 90%, 10%, 1×, and store at 4°C. Cell cryopreservation solution: Prepare by mixing RPMI-1640 complete culture medium, fetal bovine serum, and DMSO in a ratio of 5:4:1 and store at 4°C. 1×TBST 1L: Take 2.42 g of Tris, 8.0 g of NaCl, 0.5 mL of Tween-20, mix and dissolve, make up the volume to 1L, and store at room temperature. 1× transfer membrane Buffer 1L: 14.4 g of glycine, 3.03 g of Tris, add appropriate amount of double-distilled water and stir to dissolve, then add 200 mL of anhydrous methanol, make up the volume to 1L, and mix well (prepare when in use). Blocking solution 100 mL: Take 5 g of skim milk powder, add 100 mL of 1×TBST, and mix and dissolve (prepare when needed). ELISA coating diluent: 0.05 moL / L sodium carbonate-sodium bicarbonate buffer, pH 9.6. ELISA blocking solution: 5% calf serum / PBS solution (5 mL of calf serum, 95 mL of 1*PBS (pH 7.4)). Washing solution PBST: 0.8 g of NaCl, 0.02 g of KH2PO4, 0.29 g of Na2HPO4·12H2O, 0.02 g of KCl, 0.05 mL of Tween20, 0.01 g of sodium azide, add double-distilled water to 100 mL, and adjust to pH 7.4. Sample diluent: 0.8 g of NaCl, 0.02 g of KH2PO4, 0.29 g of Na2HPO4·12H2O, 0.02 g of KCl, 0.01 g of sodium azide, add double-distilled water to 100 mL, and adjust to pH 7.4. Dilution range of enzyme-labeled secondary antibody (goat anti-rabbit): 1:5000 - 1:100000. Substrate solution A: 20 mg of TMB, 10 mL of absolute ethanol, add double-distilled water to 100 mL.Substrate solution B (0.1 moL / L citric acid - 0.2 moL / L sodium dihydrogen phosphate buffer, pH 5.0 - 5.4), 1.46 g of Na2HPO4, 0.933 g of citric acid, 0.64 mL of 0.75% urea hydrogen peroxide, add triple-distilled water to 100 mL, and adjust to pH 5.0 - 5.4. Substrates A and B are mixed in a ratio of 1:1 to form the TMB - urea hydrogen peroxide solution. Termination solution (2 moL / L H2SO4 solution) 200 mL of double-distilled water, 34 mL of concentrated sulfuric acid (slowly add dropwise with continuous stirring), add double-distilled water to 300 mL.

[0027] The main instruments used in the following examples are as follows:

[0028] Tissue microarray maker: Beecher Instruments, USA; Automatic immunohistochemical staining instrument (2D): LABVISION, USA; Inverted phase contrast microscope: Olympus, Japan; Gel imaging system: Tianneng, China; Multifunctional microplate reader: Thermo, USA; Multispectral pathology scanning system: Perkin Elmer, USA.

[0029] Example 1: Expression study of prosaposin in gastric cancer

[0030] 1. Serum specimens of gastric cancer patients

[0031] 1) Enzyme-linked immunosorbent assay (Elisa):

[0032] (1) Coating process (note setting up blank control and negative control): Dilute the antigen used to an appropriate concentration with coating diluent (generally, the required antigen coating amount is 20 - 200 μg per well), add 100 uL of antigen to each well, incubate at 37°C for 4 h, or at 4°C for 24 h; discard the liquid in the wells (to avoid evaporation, cover the plate or place the plate flat in a metal wet box with wet gauze at the bottom).

[0033] (2) Block the enzyme-labeled reaction wells: Block with 5% calf serum at 37°C for 40 min. When blocking, fill each reaction well with the blocking solution and remove the air bubbles in each well. After blocking, wash the wells full with washing solution 3 times, 3 min each time. Washing method: Aspirate the reaction solution in the wells, fill the plate wells with the washing solution, let it stand for 2 min with slight shaking, aspirate the liquid in the wells, pour out the liquid and pat dry on the absorbent paper. The number of washing times is 3 times.

[0034] (3) Add the sample to be tested (establish an appropriate concentration gradient): The test is generally performed at a dilution of 1:50-1:400. A larger dilution volume should be used to ensure that the sample volume is >20 μL. Add the diluted sample to the enzyme-labeled reaction wells. Add at least two wells per sample, 100 μL per well, and place at 37°C for 40-60 minutes. Wash the wells three times with washing solution, each time for 3 minutes.

[0035] (4) Add enzyme-labeled antibody: The enzyme-labeled antibody is diluted according to the reference working dilution provided by the enzyme conjugate provider. 37°C, 30-60 min. If it is shorter than 30 min, the results are often unstable. Add 100 μL to each well and wash as before.

[0036] (5) Add substrate solution (prepared immediately before use): TMB-hydrogen peroxide urea solution is preferred, followed by OPD-hydrogen peroxide substrate solution system. Substrate addition amount: 100 μL per well, incubate at 37°C in dark for 3-5 minutes, and add stop solution for color development.

[0037] (6) Termination of reaction: Add 50 μL of stop solution to each well to terminate the reaction and measure the experimental results within 20 min.

[0038] 2) Result judgment:

[0039] OPD color development detection requires a wavelength of 492nm, and TMB reaction product detection requires a wavelength of 450nm. Be sure to first zero the blank well system during testing. The ratio of the absorbance value of the test specimen well to the average value of a group of negative specimen wells (P / N) is used to represent the antibody titer when P / N is greater than 2 (the value depends on the specific test requirements). The results showed that the expression level of Prosaposin in the peripheral blood of patients before surgery was significantly higher than that after surgery and in healthy people, and the expression level of Prosaposin after surgery was slightly higher than that in healthy people, and the expression level of Prosaposin in the serum of gastric cancer patients before and after surgery was consistent with the plasma test results ( Figure 1 ).

[0040] 2. Immunohistochemistry specimens

[0041] Gastric cancer tissue samples were collected from the Affiliated Hospital of Nantong University between 2004 and 2009, including 337 fresh frozen gastric cancer tissues and 122 benign tissues. These tissue samples were fixed in formalin, embedded in paraffin, and graded according to the latest WHO diagnostic criteria. All cases were confirmed by pathological histology by two pathologists. The patients had not received immunotherapy, chemotherapy or radiotherapy before surgery, and the clinical case data were detailed and complete.

[0042] 1) Making tissue microarray

[0043] (1) Mark the representative cancer nest areas on the wax blocks according to the microscopic examination results of HE-stained sections; (2) Mix paraffin and beeswax at a ratio of 1:1 to make blank recipient wax blocks; Design a 10×7 hole on the wax block, with a total of 350 tissue arrays, and then use a tissue microarray instrument to make a TMA blank wax block; (3) Select the most representative cancer nest area at the marked points on the donor wax block, take a tissue block with a diameter of 2 mm, and take 1 core for each case; (4) Transfer the taken tissue cores to the holes of the recipient wax block, and take the corresponding adjacent cancer tissues as controls; (5) Heat and fuse the tissue array block in a constant temperature oven at 55 °C for 10 minutes, and let it cool to room temperature before it melts quickly, so that the recipient wax block and the donor tissue are integrated; (6) Freeze the tissue microarray at 4 °C for about 4 hours, and then use a fully automatic tissue slicer to correct the tissue array block at a speed of 20 mm / revolution until all tissue cores are completely exposed; (7) Use a slicer to slice the tissue array block, float the consecutive sections in cold water to make them unfold naturally, and then transfer the sections to warm water at 45 °C to flatten them for about 2 minutes. After unfolding, stick them on the glass slides treated with anti - detachment agent and let them dry; (8) Bake the sections in an environment at 60 °C for 3 minutes and continue to bake at 58 °C for 16 h; (9) Store the prepared tissue microarray in a slide box and keep it in the refrigerator at 4 °C for standby.

[0044] 2) Immunofluorescence staining

[0045] (1) Dewaxing and hydration: Before dewaxing, place the cut paraffin tissue microarray on a baking instrument and bake at 70°C for 1 hour, then bake at 60°C for 1 hour; immerse the dried tissue microarray in xylene twice for 5 minutes each time; after taking out, perform gradient alcohol dehydration, 100% ethanol for 5 minutes, 95% ethanol for 5 minutes, 75% ethanol for 5 minutes, and rinse the tissue microarray with distilled water; (2) Place the tissue microarray on a high-temperature resistant section rack and place it in AR6 repair solution with a pH of 6.0 for high-temperature antigen repair: heat at 100% power for 2.5 minutes, then heat at 20% power for 15 minutes; (3) After naturally cooling to room temperature, take out the microarray in distilled water, rinse it 3 times with PBS for 2 minutes each time, use an immunohistochemistry pen to draw the approximate tissue range on the tissue microarray, then add 200 μL of primary antibody blocking solution and block for 10 minutes; (4) Add 200 μL of rabbit anti-human Prosaposin monoclonal antibody working solution (dilution ratio 1:100) to the tissue microarray and incubate overnight at 4°C; (5) The next day, take out the tissue microarray, warm it up for half an hour, recover the primary antibody and then rinse it with PBS for 2 minutes, repeat 3 times, then take out and drain; (6) Add 200 μL of secondary antibody working solution to the tissue microarray and incubate at room temperature for 10 minutes, then rinse it with PBS for 2 minutes, repeat 3 times, then take out and drain; (7) Prepare the fluorescent dye with the required wavelength, add the prepared fluorescent dye to the tissue microarray, incubate in the dark at room temperature for 10 minutes, then rinse it with PBS for 2 minutes, repeat 3 times; (8) If you want to apply the second antibody next, perform high-temperature antigen repair as described above; if you no longer apply the antibody, after drying and clearing, seal the slide with DAPI.

[0046] 3) Result judgment

[0047] The immunofluorescence staining results were observed under a microscope, and staining in the corresponding parts of the cells was regarded as a positive manifestation. Each sample was captured using the Vectra 3 automated imaging software at a magnification of 20 times. The images were analyzed and scored using inFonn 4.1.0 (Perkin Elmer), and a threshold for positive or negative cells was set for each cell. The percentage of cells in each region was calculated and scored (0 - 100). The final staining score of Prosaposin was the product of the staining intensity and the stained area of positive cells. The cut-off point for the Prosaposin expression score was obtained by the X-tile software based on the survival time and survival status. The scoring was as follows: 0 - 60.17 was low expression or no expression, and 60.18 - 100 was high expression. All data were processed using the statistical software SPSS V.22.0 and STATA V.9.0. Measurement data were expressed as mean ± standard deviation. One-way analysis of variance was used for comparison between groups. The relationship between Prosaposin expression and the prognosis of gastric cancer patients was analyzed using Kaplan-Meier survival analysis. All test results with P < 0.05 were considered statistically significant. The results showed that the expression of Prosaposin in gastric cancer tissues was higher than that in normal and benign gastric mucosal epithelial cells, and patients with high expression had a shorter survival period and a poor prognosis( Figure 2 and 3 ).

[0048] Example 2: Functional study of the effect of Prosaposin on gastric cancer cells

[0049] 1. sgRNA design

[0050] For the Prosaposin gene sequence, Guangzhou Funeng Gene Co., Ltd. was commissioned to prepare sgRNA. The DNA sequences corresponding to the gRNAs specifically targeting the Prosaposin gene are shown below:

[0051] Prosaposin-gRNA1:

[0052] Prosaposin-gRNA2:

[0053] Prosaposin-gRNA3:

[0054] 2. Culture of gastric cancer cell lines

[0055] Gastric cancer cell lines, including: HGC27 cells, AGS cells, MKN45 cells, MKN1 cells, were all cultured in RPMI1640 complete medium. The temperature was maintained at 37°C and the C02 saturation humidity was 5% in the incubator. Regular subculture was carried out in the incubator, and the medium was changed once every 1 - 2 days. Cells in the logarithmic growth phase were selected for the experiment.

[0056] 3. Expression of Prosaposin in Gastric Cancer Cell Lines

[0057] 1) Extraction of Total Cellular Proteins

[0058] (1) All kinds of gastric cancer cells were cultured in an incubator at 37°C with a saturated humidity of 5% CO2 and cultured with RPMI1640 complete medium. The medium was changed every 1 - 2 days and subcultured according to the corresponding density; (2) Gastric cancer cells were collected, the medium was discarded, and the cells were washed 2 times with pre-cooled PBS. The PBS was discarded, and the remaining PBS solution was aspirated clean with a pipette to avoid diluting the cellular proteins; (3) According to the size of the cell culture flask and the growth density of the cells, different volumes of 1×SDS cell lysis buffer were added, and then the cells were scraped clean with a cell scraper and transferred to a clean EP tube; (4) The scraped cellular proteins were fully lysed on ice for 30 minutes; (5) Centrifuged at 4°C (12000r × 10min); (6) The supernatant was retained, the concentration of the cellular proteins was measured with a UV spectrophotometer, and then loading buffer was added and mixed well by pipetting. The mixture was boiled at 95°C for 10 minutes and stored in a -80°C refrigerator for later use.

[0059] 2) Protein Immunoblotting Assay (Western Blot)

[0060] (1) Prepare polyacrylamide gels (5% stacking gel, 10% separating gel); (2) Clean the glass plates, let them dry tilted, assemble the glass plates, check for leaks with deionized water. After the leak check, add the separating gel to the assembled glass plates, filling it to about 2 cm from the upper end of the glass plates. Immediately add isopropanol for liquid sealing, and let it stand for 30 min. After the separating gel solidifies, gently pour off the upper layer of isopropanol, then add the stacking gel to the top of the glass plates, immediately insert the comb, and let it stand for 1 h until the stacking gel solidifies; (3) Place the prepared gel into the electrophoresis tank, fill it with electrophoresis buffer. After loading the Maker and the extracted protein samples, add the remaining electrophoresis buffer, connect the power supply, adjust the voltage to 80 V. After the marker is separated, adjust the voltage to 100 V. After completion, take out the gel and cut out the target protein; (4) Cut a PVDF membrane of a certain size, polarize it in methanol for about 5 min, and then place it in the transfer buffer for about 10 min. At the same time, take sponges and filter papers, soak them in the transfer buffer for about 20 min, install the transfer device, and the discharge order is: cathode carbon plate + sponge + filter paper + gel + PVDF membrane + filter paper + sponge + anode carbon plate; Place the transfer device into the transfer tank, add ice packs, and add the transfer buffer until full; Connect the power supply, adjust the constant current to 300 mA for electrotransfer for 1.5 h. The electrotransfer needs to be carried out in an ice box; (5) After the electrotransfer is completed, place the PVDF membrane into the blocking solution (5 g of skim milk powder dissolved in 100 mL of TBST), and block it at room temperature on a shaker at a rotation speed of 80 r / min for 2 h; (6) After blocking, prepare the primary antibody dilution solution with the blocking solution according to the primary antibody dilution ratio, evenly drip the diluted primary antibody on the PVDF membrane, and incubate overnight at 4 °C; (7) The next day, wash the membrane 3 times with TBST, 15 min each time; After the membrane washing is completed, prepare the secondary antibody dilution solution with TBST according to the secondary antibody dilution ratio, evenly drip the diluted secondary antibody on the PVDF membrane, and incubate at room temperature for 1.5 h; After the incubation is completed, wash the membrane 3 times with TBST, 15 min each time; (8) After the membrane washing is completed, blot the PVDF membrane dry with filter paper, lay it flat at the corresponding position of the imaging instrument. Mix solution A and solution B of the ECL luminescent solution in equal proportions before use, dilute it with TBST, and evenly drip it on the PVDF membrane. Take a photo and save it with a gel imaging system.

[0061] Extract the proteins of 4 kinds of gastric cancer cells respectively according to the above method. Detect the expression of Prosaposin in 4 kinds of gastric cancer cells by Western blot, and screen out the high- and low-expression cells. The results show that the expression of Prosaposin is relatively high in AGS and MKN1 gastric cancer cells and relatively low in HGC27 and MKN45 gastric cancer cells ( Figure 4 ).

[0062] 4. Screen positive gene knockout clones

[0063] (1) sgRNA Design: For the Prosaposin gene sequence, Genecopoeia Co., Ltd. in Guangzhou was commissioned to prepare sgRNA. The DNA sequence corresponding to the gRNA that specifically targets the Prosaposin gene is shown above, and a lentivirus-mediated CRISPR-Cas9 system was constructed, that is, a DNA-targeted recognition and cleavage complex composed of sgRNA and Cas9 nuclease ( Figure 5 ). (2) Explore the optimal drug screening concentration for cells: Seed the target cells to be infected in a 12-well plate. Wait until the cells are completely spread out and the confluence is about 60-70%. Query the minimum lethal concentration of the target cells from relevant literature and set several concentration gradients near this concentration. After 72 hours, select the lowest lethal concentration as the drug screening concentration. (3) Lentiviral infection of cells: Seed the target cells to be infected in a 6-well plate. Wait until the cells are completely spread out, the confluence is about 60-70%, and the cell state is good, then start to transfect the cells with the virus. Calculate the volume of the lentivirus stock solution to be added during infection according to the lentivirus stock solution titer, add the co-infection reagent Polybrene (10 μg / ml), and gently mix on the workbench. After 12-16 hours of infection, change the medium and continue culturing. At the same time, observe whether there are any abnormalities in the cell state, observe the fluorescence and take pictures under an inverted fluorescence microscope. After 72-96 hours of infection, perform drug screening on the infected cells to collect more successfully infected cells. (4) Preparation and growth of monoclonal cells: Dilute the cells by the limiting dilution method into 10 96-well plates and incubate them statically in a 37°C, CO2 incubator. Observe the growth of monoclonal cells after one week. Transfer the grown monoclonal cells to 48-well plates for expansion culture after about two weeks. Sequentially transfer the grown monoclonal cells to 24-well plates and 12-well plates for expansion culture. (4) When each monoclonal cell is expanded to two 12-well plates, take out the cells from one well, lyse them to extract proteins, and use Western blot to detect the monoclonal strains with gene knockout.

[0064] Figure 4 Quantitative analysis results showed that compared with the untreated group, the relative protein expression level of Prosaposin was significantly decreased after treatment with the Crispr / cas9 system, indicating that the expression of Prosaposin protein was effectively inhibited.

[0065] 5. Cell Proliferation Experiment (CCK-8 Experiment)

[0066] (1) Digest and collect the cells of each group 48 h after transfection, and centrifuge for later use; (2) Resuspend the cells with complete medium and adjust the cell density to 30,000 cells / mL; (3) Add 100 μL of cell suspension to each well, set 5 replicate wells for each group, gently tap the 96-well plate to make the cells evenly distributed; (4) After the cells adhere to the wall (about 6 - 8 h), add CCK-8 reagent (10 μL per well) at 0, 24, 48, 72, and 96 h respectively, gently tap the 96-well plate, take it out after incubating in the incubator for 2 h, and measure the absorbance value at 450 nm on the microplate reader, paying attention to the linear range of the microplate reader; (5) Statistically process the measured data with Graphpad prism and draw a line graph. The results show that the proliferation vitality of AGS cells and MKN1 cells decreases after knocking out Prosaposin by the Crispr / cas9 system, while the proliferation vitality of HGC27 cells and MKN45 cells increases significantly after overexpressing Prosaposin ( Figure 6 and 7 ).

[0067] 6. Cell migration experiment (scratch method)

[0068] (1) Prepare a ruler and a marker pen and irradiate them with ultraviolet light for 30 min before operation; (2) First, use the marker pen to draw horizontal lines evenly on the back of the 6-well plate with the ruler, about one line every 0.5 - 1 cm, crossing the wells horizontally, and at least 3 lines should cross each well; (3) Add about 5×10 5 cells to the wells (the specific number varies depending on the cells, and it should be mastered so that it can cover the well overnight); (4) The next day, use the pipette tip to scratch the lines as perpendicular as possible to the horizontal lines on the back with the ruler, and the pipette tip should be perpendicular and not tilted; (5) After scratching, remove the medium in the wells, wash twice with PBS to remove the scratched cells, and add 1% complete medium; (6) Place it in a 37 °C 5% CO2 incubator for culture; Take pictures at 0, 24, and 48 hours.

[0069] The results show that the migration ability of AGS cells and MKN1 cells decreases after knocking out Prosaposin by the Crispr / cas9 system, while the migration ability of HGC27 cells and MKN45 cells increases after overexpressing Prosaposin ( Figure 6 and 7 ).

[0070] 7. Cell invasion experiment (transwell chamber method)

[0071] (1) First, prepare the hydrogel (50 μL of hydrogel is mixed with 350 μL of diluent, and then 50 μL of basal medium mixture is added). Add 100 μL of the prepared mixture to the upper chamber of each Transwell insert, avoiding the formation of air bubbles. (2) Digest and collect the cells of each group 48 hours after transfection, and centrifuge for later use. (3) Resuspend the cells with 1640 basal medium and adjust the cell density to 5×104 / mL. (4) Add 800 μL of complete medium to a 24-well plate, place the Transwell insert into it, and let it soak thoroughly. Then, take 100 μL of the cell suspension and add it to the upper chamber of the Transwell insert. (5) After culturing routinely for 24 - 48 hours, take out the insert, wash it twice with 1×PBS, fix it with 4% paraformaldehyde for 20 minutes, and then wash it twice with 1×PBS. (6) Add 500 μL of crystal violet staining solution to the 24-well plate, place the Transwell insert into it, take it out after 10 minutes, wash it twice with 1×PBS, invert the insert, and gently wipe off the cells that did not pass through the upper chamber with a cotton swab.

[0072] Observe the results under an inverted microscope. The results show that after knocking out Prosaposin by the Crispr / cas9 system, the invasion ability of AGS cells and MKN1 cells decreases ( Figure 6 ), while after overexpressing Prosaposin, the invasion ability of HGC27 cells and MKN45 cells increases ( Figure 7 ).

Claims

1. Use of prosaposin gene as a drug target in the preparation of a targeted drug for treating gastric cancer, characterized in that, The described targeted drug knocks out the Prosaposin gene in gastric cancer cells; The described targeted drug is the CRISPR-Cas9 system, a Prosaposin gene knockout system of sgRNA containing the following sequences: Prosaposin-gRNA1: 5′-TTCTTGGGTAGTTTGCAG-3′, Prosaposin-gRNA2: 5′-TTCTTGGGTAGTTTGCAG-3′, Prosaposin-gRNA3: 5′-TTCTTGGGTAGTTTGCAG-3′.

Images