Application of APMAP Gene and Its RNAi Interference System

Abstract

The present invention discloses the application of the APMAP gene and its RNAi lentiviral interference system, which relates to the field of biomedical technology. The APMAP gene and the RNAi lentiviral interference system of the APMAP gene of the present invention are prepared for the treatment of colorectal cancer. This RNAi lentiviral interference system can efficiently knockdown the APMAP gene / protein in colorectal cancer cells, inhibit the proliferation, invasion and migration of cancer cells, with simple operation and high efficiency. The present invention also discloses using the APMAP gene / protein as a target for precision therapy to prepare a kit for the diagnosis or prognosis judgment of diseases with high expression of the APMAP gene / protein such as colorectal cancer, and it is also used to prepare targeted drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biological medicine technology. More specifically, it relates to the application of APMAP gene (Gene ID: 57136, Protein ID: Q9HDC9) and its RNAi interference system in the preparation of drugs for the treatment of colorectal cancer. Background Art

[0002] Although immunotherapy has reduced the risk of deterioration to a certain extent, only patients with microsatellite instability characteristics can benefit from immunotherapy. Despite a large number of mechanisms of colorectal cancer evolution and scientific research results related to prognosis, including bioinformatics databases mainly based on transcriptome data, there is an urgent clinical need for convenient secretory protein markers for diagnosis and treatment.

[0003] Secretory proteins are a class of proteins that are synthesized, modified, and then secreted extracellularly by exocytosis. Due to their long half-life in the blood and high specificity, etc., secretory proteins can be used as potential tumor markers and drug targets. For example, free prostate specific antigen (FPSA) can be used for the diagnosis and observation of prostate cancer, and human epididymis secretory protein 4 can be used for the follow-up of gynecological malignancies. Then, whether the high content of secretory proteins in the peripheral blood and tissues of colorectal cancer patients can be used as biomarkers for the liquid biopsy of colorectal cancer or can become a diagnosis and treatment target.

[0004] Since colorectal cancer is prone to recurrence and it is difficult to obtain biopsy cancer tissues, it is more practical to detect colorectal cancer markers using the peripheral blood of patients. In view of this, the project team randomly retrieved the preoperative and postoperative peripheral blood plasma of 6 colorectal cancer patients from the clinical samples stored in the hospital's biobank, and took the peripheral blood plasma of 6 healthy people of the same age as the control. After detection by the next-generation full-scan data-independent acquisition mass spectrometry (DIA-MS), 1035 proteins and 4825 peptide segments were identified, and 9 differentially expressed secretory proteins were screened from the quality-controlled data. Combining the experimental results of proteomic bioinformatics analysis and ELISA, it was confirmed that adipocyte plasma membrane associated protein (APMAP) was significantly higher in the preoperative peripheral blood of colorectal cancer patients than that after surgery and in healthy people; while no significant changes were observed in other digestive system tumors, such as gastric cancer, liver cancer, etc. Correspondingly, the expression of APMAP protein in colorectal cancer tissues was significantly higher than that in benign control samples.

[0005] APMAP is an integral membrane protein that can drive the differentiation of preadipocytes into mature adipocytes, thereby regulating the normal metabolic functions of adipocytes. Current research shows that APMAP can regulate inflammatory responses, tumor metastasis, and tumor immune escape. APMAP can promote the metastasis of prostate cancer or cervical cancer by inhibiting the degradation of epidermal growth factor receptor or activating β-catenin. In addition, the knockout of APMAP can promote the antibody-dependent cell phagocytosis of macrophages. Summary of the Invention

[0006] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide the application of APMAP gene and its RNAi lentiviral interference system for preparing a drug for treating colorectal cancer.

[0007] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] Application of APMAP gene in preparing a drug for treating colorectal cancer.

[0009] Further, the treatment of colorectal cancer is to inhibit cell proliferation.

[0010] Further, the treatment of colorectal cancer is to inhibit cell migration.

[0011] Further, the treatment of colorectal cancer is to inhibit cell invasion.

[0012] The biomarker for detecting the expression level of APMAP protein in the peripheral blood or cancer tissue of a patient is the APMAP gene.

[0013] The kit for detecting the expression level of APMAP protein in the peripheral blood or cancer tissue of a patient contains the APMAP gene.

[0014] Application of the RNAi lentiviral interference system of APMAP gene in preparing a drug for treating colorectal cancer, and the sequences of the RNAi lentiviral interference system of APMAP gene are as follows:

[0015] APMAP-shRNA1: 5′-GGTCGTCACAGACGATGATGG-3′,

[0016] APMAP-shRNA2: 5′-CACCGATTCTAGCAGCAAATG-3′.

[0017] Further, the RNAi lentiviral interference system of APMAP gene is to knockdown the APMAP gene in colorectal cancer cells.

[0018] Furthermore, the RNAi lentiviral interference system of the APMAP gene inhibits the expression of APMAP protein in colorectal cancer cells.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1) The RNAi lentiviral interference of the present invention can efficiently knockdown the APMAP gene / protein in colorectal cancer cells, inhibit the proliferation, invasion and migration of colorectal cancer cells. This system is simple to operate and has high efficiency.

[0021] 2) The APMAP gene / protein is a target for precision therapy. Kits for the diagnosis or prognosis judgment of colorectal cancer and other diseases with high expression of the APMAP gene / protein are prepared, and are also used to prepare targeted drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the expression diagram of APMAP protein in peripheral blood and tissues of colorectal cancer (A is the diagram of the difference in protein content between the plasma of 6 normal people and the plasma of 6 colorectal cancer patients detected by DIA-MS; B is the diagram of the difference in protein content of APMAP detected by DIA-MS in the preoperative plasma of 6 colorectal cancer patients; C is the intersection of high-content peptide segments and low-content peptide segments in the serum of colorectal cancer patients and its expression heat map; D is the diagram of the expression of APMAP protein in the serum of 15 normal people, 15 colorectal cancer, gastric cancer and liver cancer patients before and after surgery: the content of APMAP in normal human serum is 5.714±0.572 (ng / mL), the content of APMAP in the preoperative serum of colorectal cancer patients is 8.836±0.551 (ng / mL), and the content of APMAP in the postoperative serum of colorectal cancer patients is 6.873±0.667 (ng / mL));

[0023] Figure 2 It is the result diagram of immunofluorescence detection of the expression of APMAP protein in colorectal cancer and benign intestinal epithelial tissues (A is the fluorescence staining of APMAP in colorectal cancer tissues and benign intestinal epithelial tissues; B is the statistical chart of fluorescence intensity; C is the relationship diagram between the high and low expression groups of APMAP protein and the prognosis of colorectal cancer patients);

[0024] Figure 3 It is the expression diagram of APMAP protein in NCM460 cells, HCT116 cells, LoVo cells, SW480 cells, DLD-1 cells, SW1116 cells, HCT15 cells and SW620 cells;

[0025] Figure 4 It is the expression diagram of APMAP protein in HCT116 cells and SW1116 cells after knockdown and overexpression of this gene;

[0026] Figure 5 It is the lentiviral knockdown vector plasmid map (A) and the lentiviral overexpression vector plasmid map (B);

[0027] Figure 6 It is the expression map of APMAP protein after knocking down and overexpressing this gene in DLD-1 cells and SW620 cells;

[0028] Figure 7 It is the proliferation viability map of HCT116 cells after knocking down APMAP and the proliferation viability map of DLD-1 cells after overexpressing APMAP;

[0029] Figure 8 It is the migration ability map of HCT116 cells and SW1116 cells after knocking down APMAP and the migration ability map of DLD-1 cells and SW620 cells after overexpressing APMAP;

[0030] Figure 9 It is the invasion ability map of HCT116 cells and SW1116 cells after knocking down APMAP and the invasion ability map of DLD-1 cells and SW620 cells after overexpressing APMAP. Detailed implementation manners

[0031] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are all conventional means well known to those skilled in the art.

[0032] Paraffin tissue microarray of colorectal cancer in the biobank of Affiliated Hospital of Nantong University (surgery between 2004 and 2009, patients did not receive immunotherapy, chemotherapy or radiotherapy before surgery, and clinical case data were complete), including 120 cases of colorectal cancer tissue and 40 cases of benign tissue.

[0033] The main reagents, consumables and animal models used in the following embodiments are:

[0034] Opal 7-color immunohistochemistry kit (Perkin Elmer, USA).

[0035] Anti-human APMAP antibody (25953-1-AP, Proteintech, Wuhan, China).

[0036] Anti-CK antibody.

[0037] Colorectal cancer cell lines (HCT116, LoVo, SW480, DLD-1, SW1116, HCT15, SW620) and intestinal epithelial cell line (NCM460) were purchased from Nanjing Kebai Biotechnology Co., Ltd.

[0038] RPMI-1640 medium, high-glucose DMEM medium, and fetal bovine serum (Gibco, USA).

[0039] The main instruments used in the following examples are as follows:

[0040] Multispectral pathology scanning system: Perkin Elmer, USA.

[0041] Inverted fluorescence microscope: Carl Zeiss, Germany.

[0042] Gel imaging system: Tianneng, China.

[0043] Multifunctional microplate reader: Thermo, USA.

[0044] Example 1

[0045] Direct enzyme-linked immunosorbent assay (ELISA):

[0046] Dilute the antigen used with coating diluent for blank control and negative control, and set up. Add 100 μL of antigen to each well, place it at 4°C overnight, and then discard the liquid in the wells; block with 5% calf serum at 37°C for 1 h. After blocking, wash the wells three times with washing solution, 3 min each time. After pouring out the liquid, pat it dry on the absorbent paper; add the diluted sample to the enzyme-labeled reaction wells, at least 3 replicates for each sample, 100 μL per well, incubate at 37°C for 1 h, wash the wells three times with full well, 3 min each time; the enzyme-labeled antibody is carried out according to the reference working dilution provided by the enzyme conjugate provider, at 37°C, between 30 - 60 min (results are often unstable when shorter than 30 min), add 100 μL to each well, wash as before; add TMB-hydrogen peroxide urea solution, 100 μL per well, place at 37°C in the dark for 3 - 5 minutes; add 50 μL of stop solution to each well to terminate the reaction, measure the experimental results within 15 min, detect the absorbance value at 450 nm wavelength, read the data, and calculate.

[0047] The results are as Figure 1 shown. The content of APMAP protein in the preoperative peripheral blood of colorectal cancer patients was significantly higher than its expression in postoperative and healthy human peripheral blood, and was consistent with the change trend of APMAP protein in colorectal cancer plasma detected by mass spectrometry.

[0048] Example 2

[0049] After the tissue microarray was baked on a baking instrument, it was dewaxed in xylene and then dehydrated with gradient ethanol. After rinsing with distilled water, it was placed in AR9 repair solution with a pH of 6.0 on a high-temperature-resistant section rack for high-temperature antigen repair. After natural cooling to room temperature, it was rinsed with PBS, and a primary antibody blocking solution was added and blocked for 10 min. 200 μL of anti-human APMAP antibody working solution (25953-1-AP) with a dilution ratio of 1:100 was added dropwise to the tissue microarray and incubated overnight at 4°C. The tissue microarray was taken out, rewarmed for 0.5 h, and then rinsed with PBS. 200 μL of secondary antibody working solution was added dropwise to the tissue microarray, rinsed with PBS after 10 min at room temperature. The prepared fluorescent dye was added dropwise to the tissue microarray, incubated in the dark at room temperature for 10 min, and then rinsed with PBS. If the second antibody was to be incubated next, high-temperature antigen repair was performed as described above. If no more antibodies were to be incubated, after drying and making it transparent, it was sealed with DAPI.

[0050] The staining results were observed under a fluorescence microscope, and staining in the corresponding parts of the cells was considered a positive manifestation. Each sample was captured at a magnification of 20 times using Vectra 3 automatic imaging software. The images were analyzed and scored using inForm 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 cut-off point of the APMAP protein expression score was obtained by X-tile software based on the survival time and survival status. The scoring was as follows: 0 - 58.73 was low expression or no expression, and 58.74 - 100 was high expression. All data were processed using statistical software SPSS V.22.0, the chi-square test was used for inter-group comparison, and the Cox proportional hazards regression analysis was used to analyze the prognostic factors of the patients; the Kaplan-Meier method and log-rank test were used for univariate analysis of the patient prognosis. All test results with P < 0.05 were considered statistically significant. The results were as Figure 2 shown, the expression of APMAP protein in colorectal cancer tissues was higher than that in benign intestinal epithelial cells, and the survival period of patients with high expression was short and the prognosis was poor.

[0051] Example 3

[0052] 1. Colorectal cancer cell lines: DLD-1 cells, SW1116 cells, HCT116 cells, and HCT15 cells were cultured in RPMI-1640 complete medium; LoVo cells, SW480 cells, and SW620 cells were cultured in DMEM high-glucose complete medium, and intestinal epithelial cells NCM460 cells were cultured in RPMI-1640 complete medium. The temperature was maintained at 37°C and the CO2 saturation humidity was 5% in the incubator. Routine passage culture was carried out in the incubator, and cells in the logarithmic growth phase were selected for the experiment.

[0053] 2. Extraction of total cell protein

[0054] All kinds of colorectal cancer cells were cultured in complete medium containing 10% fetal bovine serum at 37°C under 5% CO2 conditions; the colorectal cancer cells were collected, the medium was discarded, and the cells were washed twice with pre-cooled PBS; according to the size of the cell culture flask and the growth density of the cells, different cell lysis buffers were added, and then the cells were scraped clean with a cell scraper and transferred to a clean EP tube; the scraped cell proteins were lysed fully on ice for 30 min; centrifuged at 4°C to collect the supernatant, and the concentration of cell proteins was measured by the BCA method and a multifunctional microplate reader, and stored in a -20°C refrigerator for later use.

[0055] 3. Western blot

[0056] Prepare polyacrylamide gels (5% stacking gel, 10% separating gel); after loading protein Marker and the extracted protein samples, adjust the running voltage to 100 V, and after completion, take out the gel for membrane transfer (PVDF membrane); transfer at a constant current of 300 mA for 1.5 h, and the membrane transfer needs to be carried out in an ice box; after the membrane transfer is completed, put the PVDF membrane into the blocking solution and block at room temperature for 2 h; prepare the primary antibody dilution solution with the blocking solution, evenly drip the diluted primary antibody on the PVDF membrane, and incubate overnight at 4°C; after washing the membrane, prepare the secondary antibody dilution solution with TBST, evenly drip the diluted secondary antibody on the PVDF membrane, and incubate at room temperature for 1.5 h; after washing the membrane, lay the PVDF membrane flat at the corresponding position of the imaging instrument, dilute the ECL luminescent solution with TBST, evenly drip it on the PVDF membrane, and take a photo and save it with a gel imaging system.

[0057] The results are as Figure 3 shown, APMAP is relatively highly expressed in HCT116 and SW1116 colorectal cancer cells, and relatively lowly expressed in DLD-1 and SW620 colorectal cancer cells.

[0058] Example 4

[0059] 1. Screening of gene knockout positive clones

[0060] 1) For the APMAP gene sequence, the gene sequences of the RNAi lentiviral interference system specifically targeting the APMAP gene are as follows, and a lentivirus-mediated RNAi interference system was constructed:

[0061] APMAP-shRNA1: 5′-GGTCGTCACAGACGATGATGG-3′,

[0062] APMAP-shRNA2: 5′-CACCGATTCTAGCAGCAAATG-3′.

[0063] 2) Select an appropriate viral infection concentration, perform lentiviral infection on the target cells, and add a co-staining reagent; after 12 - 16 h of infection, change the medium and continue culturing, and at the same time observe whether there are any abnormalities in the cell state; after 72 - 96 h of infection, observe the fluorescence under an inverted fluorescence microscope, and perform drug screening on the infected cells to collect more successfully infected cells.

[0064] 3) Dilute the cells by the limiting dilution method into 10 96-well plates; observe the monoclonal growth after one week, and transfer the grown monoclonal colonies to 48-well plates for expansion culture after about two weeks; sequentially transfer the grown monoclonal colonies to 24-well plates and 12-well plates for expansion culture; when each monoclonal colony has been 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 knockdown.

[0065] The results are as Figure 4 shown. Compared with the control group, the relative protein expression levels of APMAP in HCT116 cells and SW1116 cells treated with the RNAi lentiviral interference system were significantly decreased, indicating that the protein expression of APMAP in HCT116 cells and SW1116 cells was effectively inhibited.

[0066] 2. Construction of overexpression system and screening of stable strains

[0067] 1) Amplify the open reading frame region of APMAP mRNA using forward and reverse primers, and use gene recombination technology to recombine the APMAP expression fragment into the pCDH-CMV-MCS-EF1-EGFP-2A-puro vector ( Figure 5 ).

[0068] 2) After packaging into virus, select an appropriate viral infection concentration, perform lentiviral infection on the target cells, and add a co-staining reagent; after 12 - 16 h of infection, change the medium and continue culturing; after 72 - 96 h of infection, observe the fluorescence under an inverted fluorescence microscope, and perform drug screening on the infected cells to collect more successfully infected cells.

[0069] 3) Preparation and growth of monoclonal colonies: Dilute the cells by the limiting dilution method into 10 96-well plates; observe the monoclonal growth after one week, and transfer the grown monoclonal colonies to 48-well plates for expansion culture after about two weeks; sequentially transfer the grown monoclonal colonies to 24-well plates and 12-well plates for expansion culture. When each monoclonal colony has been 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 overexpressed APMAP.

[0070] The results are as Figure 6As shown, compared with the control group, the relative protein expression level of APMAP in the cells after expression treatment was significantly increased, indicating that the protein expression of APMAP in DLD-1 cells and SW620 cells was effectively upregulated.

[0071] 3. Cell proliferation (CCK-8)

[0072] Digest and collect the cells of each group 48 h after transfection, centrifuge and set aside; resuspend the cells with complete medium, and adjust the cell density to 20,000 cells / mL; add 100 μL of cell suspension to each well, set 5 replicates for each group, gently tap the 96-well plate to make the cells evenly distributed; 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 detect the absorbance value at 450 nm on the microplate reader, paying attention to the linear range of the microplate reader; statistically process the measured data with Graphpad prism and draw a line graph.

[0073] The results are as Figure 7 shown. After knocking down APMAP by the RNAi lentivirus interference system, the proliferation activity of HCT116 cells decreased (A), while the proliferation activity of DLD-1 cells significantly increased after overexpressing APMAP (B).

[0074] 4. Cell migration (Transwell chamber method)

[0075] Digest and collect the cells of each group with stable transfection, centrifuge and set aside; resuspend the cells with basal medium and adjust the cell density to 3×10 4 / mL; add 800 μL of complete medium to a 24-well plate, place the chamber, fully soak it, take 100 μL of cell suspension and add it to the upper chamber of the Transwell chamber; after culturing routinely for 24 - 48 h, take it out, wash it 2 times with 1×PBS, fix it with 4% paraformaldehyde for 20 min, and wash it 2 times with 1×PBS; add 500 μL of crystal violet staining solution to the 24-well plate, place the chamber, take it out after 10 min, wash it 2 times with 1×PBS, invert the chamber, and gently wipe off the cells that did not pass through the upper chamber with a cotton swab;

[0076] The results are as Figure 8 shown. Observe the results with an inverted microscope. The results show that after knocking down APMAP by the RNAi lentivirus interference system, the migration ability of HCT116 cells and SW1116 cells decreased, while the migration ability of DLD-1 cells and SW620 cells increased after overexpressing APMAP.

[0077] 5. Cell invasion (Transwell chamber method)

[0078] Prepare the hydrogel first (50 μL of hydrogel is mixed in 350 μL of diluent, and then 50 μL of basal medium mixture is added), add it to the upper chamber of the transwell insert, 100 μL for each, and avoid generating air bubbles; digest and collect the cells in each group of stable transfection, and centrifuge for later use; resuspend the cells with basal medium and adjust the cell density to 5×10 4 / mL; add 800 μL of complete medium to a 24-well plate, place the insert in it, and soak it thoroughly. Take 100 μL of cell suspension and add it to the upper chamber of the transwell insert; after culturing routinely for 24 - 48 h, take it out, wash it twice with 1×PBS, fix it with 4% paraformaldehyde for 20 min, and wash it twice with 1×PBS; add 500 μL of crystal violet staining solution to the 24-well plate, place the insert in it, take it out after 10 min, 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;

[0079] The results are as Figure 9 shown. Observe the results with an inverted microscope. The results show that the invasion ability of HCT116 cells and SW1116 cells decreases after knocking down APMAP by the RNAi lentivirus interference system, while the invasion ability of DLD-1 cells and SW620 cells increases after overexpressing APMAP.

Claims

1. Use of an RNAi lentiviral interference system for APMAP gene in the preparation of a drug for treating colorectal cancer, characterized in that, The sequences of the RNAi lentiviral interference system for the APMAP gene are as follows: APMAP-shRNA1: 5'-GGTCGTCACAGACGATGATGG-3', APMAP-shRNA2: 5'-CACCGATTCTAGCAGCAAATG-3'.

2. The use according to claim 1, characterized in that, The RNAi lentiviral interference system for the APMAP gene is to knockdown the APMAP gene in colorectal cancer cells.

3. The use according to claim 1, characterized in that, The RNAi lentiviral interference system for the APMAP gene is to inhibit the expression of APMAP protein in colorectal cancer cells.

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