A vector for attenuated Salmonella carrying the shSTAT3 / shPD-L1 recombinant plasmid and its application

Abstract

This invention discloses an attenuated Salmonella vector carrying the shSTAT3 / shPD-L1 recombinant plasmid and its applications, belonging to the field of biotechnology. This invention successfully constructs an attenuated Salmonella vector carrying the shSTAT3 / shPD-L1 recombinant plasmid by using two promoters to express two shRNAs in the same expression vector. The shSTAT3 / shPD-L1 recombinant plasmid can simultaneously silence the expression of STAT3 and PD-L1 in colorectal cancer cells, thereby inhibiting cancer cell proliferation and migration, promoting apoptosis, and inducing cell cycle arrest. The attenuated Salmonella carrying the shSTAT3 / shPD-L1 recombinant plasmid can significantly inhibit the growth of orthotopic colorectal cancer in C57BL / 6N mice through mechanisms such as inhibiting cell proliferation, promoting apoptosis, and increasing immune cell infiltration, exhibiting good anti-tumor effects and becoming an effective new strategy for the treatment of colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to an attenuated Salmonella vector carrying the shSTAT3 / shPD-L1 recombinant plasmid and its application. Background Technology

[0002] Colorectal cancer is a common malignant tumor of the digestive tract, most commonly occurring at the junction of the rectum and sigmoid colon. Decades ago, due to limited medical technology and a lack of public awareness about hygiene, colorectal cancer was rarely diagnosed. Today, however, it is the third leading cause of cancer death worldwide, with an incidence rate of 10.2% and a mortality rate accounting for 9.2% of all cancer deaths. Colorectal cancer not only impacts patients' quality of life, but improving treatment outcomes while reducing costs has become a key focus of research.

[0003] Standard treatments for colorectal cancer include surgery, chemotherapy, and radiation therapy. These treatments may be used in combination depending on the location and progression of the disease. Total mesorectal excision (TME) via laparoscopy and transanal surgery is often the treatment option for locally developed cancer; however, complete removal of all cancer cells is usually impossible, and some colorectal cancer patients require further adjuvant chemotherapy and radiation therapy. Because these therapies have non-specific cytotoxicity to any growing and dividing cells, they can cause numerous side effects. Furthermore, a significant proportion of patients relapse even after adjuvant therapy. Therefore, exploring effective alternative treatments is particularly important for patients with colorectal cancer. Summary of the Invention

[0004] One of the objectives of this invention is to provide a short hairpin RNA, wherein the short hairpin RNA is shSTAT3 or shPD-L1, wherein the shSTAT3 sequence is shown in SEQ ID NO.2 and the shPD-L1 sequence is shown in SEQ ID NO.3.

[0005] A second objective of this invention is to provide a recombinant plasmid containing shSTAT3 and shPD-L1 as described in claim 1.

[0006] Preferably, the recombinant plasmid contains a PLKO.1 backbone.

[0007] More preferably, the sequence of the recombinant plasmid is shown in SEQ ID NO.1.

[0008] A third objective of this invention is to provide an attenuated Salmonella vector containing a recombinant plasmid with the sequence shown in SEQ ID NO.1.

[0009] The fourth objective of this invention is to provide the use of shSTAT3, as shown in SEQ ID NO.2, or shPD-L1, as shown in SEQ ID NO.3, in the preparation of drugs for colorectal cancer and related solid tumors.

[0010] The fifth objective of this invention is to provide the application of the above-mentioned recombinant plasmid in the preparation of drugs for colorectal cancer and related solid tumors.

[0011] The sixth objective of this invention is to provide the application of the above-mentioned attenuated Salmonella carrier in the preparation of drugs for colorectal cancer and related solid tumors.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] (1) In this invention, two promoters are used in the same expression vector to express two shRNAs respectively, and an attenuated Salmonella vector carrying shSTAT3 / shPD-L1 recombinant plasmid is successfully constructed.

[0014] (2) The shSTAT3 / shPD-L1 recombinant plasmid can simultaneously silence the expression of STAT3 and PD-L1 in colorectal cancer cells, thereby causing cell cycle arrest, inhibiting cancer cell proliferation and migration, and promoting cell apoptosis;

[0015] (3) Attenuated Salmonella carrying the shSTAT3 / shPD-L1 recombinant plasmid can specifically accumulate in tumor tissues and has good tumor targeting ability;

[0016] (4) The shSTAT3 / shPD-L1 recombinant plasmid carried by attenuated Salmonella can significantly inhibit the growth of colorectal cancer in C57BL / 6N mice through mechanisms such as inhibiting cell proliferation, promoting apoptosis and increasing immune cell infiltration. It has a good anti-tumor effect and has become an effective new strategy for the treatment of colorectal cancer. Attached Figure Description

[0017] Figure 1 This is a quantitative map of STAT3 and PD-L1 mRNA expression in Example 1.

[0018] Figure 2 The results of RT-PCR and Western Blot detection of STAT3 and PD-L1 expression levels in CT26 cells in Example 1 are shown in Figure 1. (A) is a quantitative map of mRNA expression of STAT3 and PD-L1; (B) is a quantitative map of protein expression of STAT3 and PD-L1; and (C) is a quantitative map of protein expression of STAT3 and PD-L1.

[0019] Figure 3This is a diagram showing the cell proliferation results detected by the CCK-8 experiment in Example 2.

[0020] Figure 4 The images shown are cell proliferation detection diagrams from the colony formation experiment in Example 2, where (A) is a diagram of the colony formation experiment results; and (B) is a quantitative diagram of the colony formation experiment results.

[0021] Figure 5 The graph shows the proportion of apoptosis detected by flow cytometry in Example 2, where (A) is the result of apoptosis detection by flow cytometry; and (B) is the quantitative graph of apoptosis results.

[0022] Figure 6 The following are the results of Western blot detection of apoptosis-related protein expression in Example 2, where (A) is the result of apoptosis-related protein; (B) is the quantitative result of Bcl-2 / Bax protein; and (C) is the quantitative result of Cleaved-caspase3 protein.

[0023] Figure 7 The image shows the results of the Transwell chamber invasion assay for detecting cell migration ability in Example 2, where (A) is the result of the invasion assay; and (B) is the quantitative result of the invasion assay.

[0024] Figure 8 The image shows the results of the cell scratch assay for detecting cell migration ability in Example 2, where (A) is the result of the scratch assay; and (B) is the quantitative result of the scratch assay.

[0025] Figure 9 The images show the cell cycle detection results of the flow cytometry experiment in Example 2, where (A) is the cell cycle detection results of the flow cytometry experiment; and (B) is the quantitative cell cycle result.

[0026] Figure 10 The image shows the results of flow cytometry detection of cell cycle-related proteins in Example 2, where (A) is the cell cycle-related protein result image; and (B) is the quantitative result image of cell cycle-related proteins.

[0027] Figure 11 The graphs show the changes in tumor volume and body weight of mice during the treatment period in Example 3, where (A) shows the changes in tumor volume and (B) shows the changes in body weight.

[0028] Figure 12 This is a diagram showing the distribution of attenuated Salmonella in mice in Example 3.

[0029] Figure 13The images shown are of the mouse tumor weight results after treatment in Example 3, where (A) is a picture of the mouse tumors harvested after treatment; and (B) is a picture of the mouse tumor weight quantification after treatment.

[0030] Figure 14 The image shows the expression of tumor-related proteins in Example 3, where (A) is a protein expression diagram and (B) is a protein expression quantification diagram.

[0031] Figure 15 This is a diagram of the major organs of the mouse in Example 3.

[0032] Figure 16 This is an image of H&E staining (Bar = 50 μm) of the major organs of the mouse in Example 3.

[0033] Figure 17 This is an image of H&E staining (Bar = 50 μm) of tumor tissue from Example 3.

[0034] Figure 18 The image shows the expression of STAT3 and PD-L1 in the tumor in Example 3 (Bar = 50 μm).

[0035] Figure 19 This is a graph showing the expression of tumor proliferation-related proteins in Example 3 (Bar = 50 μm).

[0036] Figure 20 This is a graph showing the expression of tumor apoptosis-related proteins in Example 3 (Bar = 50 μm).

[0037] Figure 21 This is a diagram showing the TUNEL staining of the tumor in Example 3 (Bar = 50 μm).

[0038] Figure 22 This is a graph showing the expression of tumor cycle-related proteins in Example 3 (Bar = 50 μm).

[0039] Figure 23 This is a diagram showing the tumor-infiltrating immune cells (Bar = 50 μm) in Example 3.

[0040] Figure 24 The graph shows the changes in tumor-related cytokines IL-6, IFN-γ, and TNF-α in Example 3, where (A) is a quantitative graph of IL-6 concentration; (B) is a quantitative graph of IFN-γ concentration; and (C) is a quantitative graph of TNF-α concentration.

[0041] Figure 25 The diagram shows the modeling process and weight changes of the orthotopic colorectal cancer mouse model in Example 4, where (A) is the AOM-DSS modeling process; (B) is the mouse weight change diagram; and (C) is the mouse symptoms during the modeling period.

[0042] Figure 26 Figure 4 shows the in situ colorectal cancer tumor and tumor differences in Example 4, where (A) is the harvested tumor; (B) is the intestinal weight / body weight quantification; (C) is the colorectal length quantification; and (D) is the number of tumors in the colorectal region.

[0043] Figure 27 The image shows the results of Western Blot detection of STAT3 and PD-L1 expression levels in CT26 cells in Comparative Example 1. (A) shows the protein expression of STAT3 and PD-L1; (B) shows the quantitative protein expression of STAT3 and PD-L1.

[0044] Figure 28 The graphs shown are the proportions of apoptosis detected by flow cytometry in Comparative Example 1, where (A) is the result of apoptosis detected by flow cytometry; and (B) is the quantitative graph of apoptosis results. Detailed Implementation

[0045] The reagents used in the following examples are shown in the table below:

[0046] Table 1

[0047]

[0048]

[0049] Example 1

[0050] 1. Obtaining plasmids

[0051] The shControl plasmid, shSTAT3 plasmid, shPD-L1 plasmid, and shSTAT3 / shPD-L1 recombinant plasmid containing the PLKO.1 backbone used in this embodiment were constructed by Saixin Biotechnology. The recombinant plasmid contains an ampicillin (Amp) resistance gene to exclude interference from other bacteria. The shSTAT3 / shPD-L1 recombinant plasmid sequence is shown in SEQ ID NO.1.

[0052] The shRNA sequence information is as follows:

[0053] SEQ ID NO.2: shSTAT3(5'-GCAGCAGCTGAACAACATG-3');

[0054] SEQ ID NO. 3: shPD-L1 (5'-CCGAAATGATACACAATTCGA-3').

[0055] The attenuated Salmonella phoP / phoQ mutant strain preserved in our laboratory is the attenuated Salmonella phoP / phoQ mutant strain disclosed in the patent "CN108913690BPD-1 Specific Interference Sequence, Plasmid, Attenuated Salmonella and Its Application in Anti-tumor".

[0056] The PCR primers are shown in Table 2.

[0057] Table 2

[0058]

[0059] 2. Recombinant plasmid transfection of mouse colon cancer CT26 cell line

[0060] (1) Digest and centrifuge to collect CT26 cells with 80%-90% confluence, count the cells, and place approximately 2 × 10⁶ cells into each six-well plate. 5 Add 2 mL of culture medium to each well of a six-well plate, shake the plate in a cross shape to distribute the cells evenly, and incubate in a cell incubator for 24 hours.

[0061] (2) When the CT26 cell confluence is around 80%, transfect according to the Lipofectamine™ 3000 instructions. Take four autoclaved 1.5 mL EP tubes (tube A), add 125 μL of RPMI-1640 culture medium without fetal bovine serum and penicillin-streptomycin antibiotics to each tube, and then add 7.5 μL of Lipofectamine™ 3000 to each tube. Take another four autoclaved 1.5 mL EP tubes (tube B), add 125 μL of RPMI-1640 culture medium without fetal bovine serum and penicillin-streptomycin antibiotics to each tube, and add 2.5 μg of shControl plasmid, shSTAT3 plasmid, shPD-L1 plasmid, and shSTAT3 / shPD-L1 recombinant plasmid to each tube, and then add 5 μL of P3000™ to each tube. Add the reagents from tube A to tube B, incubate at room temperature for 15 min, mix gently, and drop into each of the four wells of a six-well plate. Gently shake the six-well plate to distribute the transfection reagent coated with plasmids evenly in the six-well plate. After 4-6 hours, aspirate the culture medium from the six-well plate, add 2 mL of fresh culture medium to each well, and continue culturing for 24-48 hours.

[0062] 3. qPCR detection of mRNA expression

[0063] 24 h after transfection, discard the cell culture medium. Add 0.5 mL of Trizol lysis buffer to a 6-well plate, collect the cells, and transfer the Trizol to a 1.5 mL centrifuge tube. Incubate for 5 min. Add 0.2 mL of chloroform, mix well, incubate for 10 min, and centrifuge at 12000 rpm and 4°C for 15 min. Transfer the upper aqueous phase to a new 1.5 mL centrifuge tube, add 0.5 mL of isopropanol, mix well, incubate at room temperature for 15 min, and centrifuge at 12000 rpm and 4°C for 8 min. Discard the supernatant, add 1 mL of 75% ethanol, mix well, and centrifuge at 9000 rpm and 4°C for 5 min. Discard the supernatant, dry the centrifuge tube for 10 min, dissolve the precipitate in 50 μL of sterile DEPC water, gently pipette, and place on ice. Analyze the RNA concentration.

[0064] The sample RNA, reverse transcription mix (Full Gold, AE341), and RNase-free water were mixed in proportion, incubated at 42°C for 15 min, heated at 85°C for 5 sec, and the concentration of synthesized cDNA was detected by instrument.

[0065] Mix cDNA, target gene primers, qPCR Mix (Full Gold, AQ601), and RNase-free water in the specified proportions. Amplification is performed using a two-step method: 94℃ for 30 seconds; then 94℃ for 5 seconds, followed by 60℃ for 30 seconds, for 45 cycles. See attached image for detection results. Figure 1 ,Depend on Figure 1 It can be seen that, compared with the single silencing group, the expression of STAT3 and PD-L1 mRNA in the shSTAT3 / shPD-L1 recombinant plasmid transfection group was significantly reduced (P<0.001).

[0066] 4. Cell protein extraction, concentration determination, and Western blot detection

[0067] (1) Collect the cell culture medium after 48 h of transfection in a 1.5 mL EP tube, centrifuge at 1050 rpm for 5 min and discard the supernatant. Collect the pellet and add 50 μL of RIPA (1% PMSF) cell lysis buffer to each well of a six-well plate. Use a cell scraper to scrape the adherent cells from the six-well plate and transfer them to the corresponding EP tube. After sonication, place the cells on ice for 10 min and centrifuge at 12000 rpm and 4℃ for 15 min. Collect the supernatant to a new EP tube.

[0068] (2) Protein standard (5 mg / mL): Dilute 10 μL of the stock solution with PBS 10-fold. Add the diluted protein standard to 96-well plates at concentrations of 0, 1, 2, 4, 8, 12, 16, and 20 μL, and then add PBS to bring the total volume to 20 μL. Add 2 μL of the protein sample stock solution to each well of the 96-well plate, and then add PBS to bring the total volume to 20 μL. Add 200 μL of pre-prepared BCA working solution to each well and incubate at 37°C for 30 min. Measure the absorbance at A562 using a microplate reader. Plot a protein concentration standard curve based on the protein standard values ​​and calculate the concentration of the sample to be tested.

[0069] (3) Sample preparation: Add loading to the protein sample, quantitatively prepare 20 μg / 20 μL of sample protein, and place it in boiling water at 100℃ for 10 min to deform the protein.

[0070] SDS-PAGE, buffer solutions, and antibody preparation: Prepare 8%, 10%, or 12% concentration SDS-PAGE gels according to molecular weight. Electrophoresis buffer, transfer buffer, and corresponding antibodies should be prepared in proportion and stored at 4°C for short-term use.

[0071] Electrophoresis: Remove the comb from the SDS-PAGE gel in the electrophoresis buffer, discard the edge 2 channels, load 10 μL of sample into each channel, and add marker for labeling. Set the voltage of the upper stacking gel to 75V for 30 min, and the voltage of the lower separating gel to 110V for 60 min.

[0072] Transfer: After electrophoresis, cut the gel according to the marker position, activate the PVDF membrane with methanol for 30 seconds, and attach it to the gel surface. The transfer clamp should be placed in the following order: moistened sponge, three layers of moistened filter paper, electrophoresis gel fragment, three layers of moistened filter paper, and moistened sponge, avoiding air bubbles during placement. Add freshly prepared transfer solution to the transfer tank and cover it with ice to prevent the excessive heat released during transfer from damaging the machine. Set the voltage to 100V and the time to 45 minutes.

[0073] Skim milk blocking: After the transfer is completed, the PVDF membrane is placed in 5% skim milk for 1 hour. After blocking, the PVDF membrane is washed with TBST 3 times for 5 minutes each time, and placed on a horizontal shaker during the process.

[0074] Hybridization: The PVDF membrane was incubated with the appropriate primary antibody dilution and placed in a 4°C refrigerator overnight. On the second day, the PVDF membrane was washed three times with TBST for 5 min each time, then blocked with the appropriate secondary antibody dilution for 1 h, and washed three times with TBST for 5 min each time.

[0075] ECL development: Prepare the development solution fresh each time, and keep it away from light. Incubate the PVDF membrane and immediately place it in the ECL imaging instrument for development.

[0076] Protein band quantification: ImageJ software was used to quantify protein bands and determine differences in protein expression levels.

[0077] See results Figure 2 Compared with the single silencing group, the expression of STAT3 and PD-L1 mRNA was significantly reduced in the shSTAT3 / shPD-L1 recombinant plasmid transfection group (P<0.001); compared with the single silencing group, the expression of STAT3 and PD-L1 proteins was significantly reduced in the recombinant plasmid transfection group (P<0.001). These results indicate that the shSTAT3 / shPD-L1 recombinant plasmid was successfully transfected into CT26 cells and exerted its biological effects.

[0078] Example 2

[0079] 1. Electroporation-attenuated Salmonella phoP / phoQ mutant strain

[0080] (1) Preparation of electroconversion competent cells: On the first day, 1 μL of attenuated Salmonella phoP / phoQ mutant bacterial culture was placed in 50 mL of LB liquid medium and cultured at 37 °C and 200 rpm for 14-16 h. On the second day, 1 mL of bacterial culture was placed in 100 mL of fresh culture medium, and the OD value of the bacterial culture was monitored periodically. When the OD value reached 0.3-0.4, the culture was pre-cooled on ice for 30 min. The bacterial culture was added to two 15 mL centrifuge tubes, centrifuged at 4200 rpm and 4 °C for 10 min to collect the precipitate, the supernatant was discarded, the precipitate was resuspended in ddH2O, centrifuged at 4200 rpm and 4 °C for 10 min to collect the precipitate, the two tubes were combined into one tube, the precipitate was resuspended in ddH2O, centrifuged at 4200 rpm and 4 °C for 10 min to collect the precipitate, the supernatant was discarded, the precipitate was resuspended in 10% glycerol, transferred to a 1.5 mL EP tube, frozen in liquid nitrogen for 1 min, and stored at -80 °C.

[0081] (2) Electroporation procedure: Take 1 μL of plasmid with a concentration of not less than 500 ng / μL and place it in a 1.5 mL EP tube. Pre-cool the tube and place it on ice with a 0.1 cm electrode cup. Transfer 100 μL of thawed competent cells to this 1.5 mL EP tube, mix thoroughly, and place on ice for 10 min. Adjust the voltage of the electroporator to 2.5 kV, 25 μF, and 200 Ω. Transfer the mixture to the electrode cup and gently tap the electrode cup to ensure the mixture is evenly distributed to the bottom. Place the electrode cup in the electroporator. After the beep, the electroporation will end. Immediately add 1 mL of autoclaved LB liquid medium to the electrode cup, resuspend the liquid, transfer it to a 1.5 mL EP tube, and place it in a shaker at 37°C and 250 rpm for 1 h to recover. Spread 200 μL of the transformation product on solid LB medium containing Amp and incubate overnight at 37°C.

[0082] (3) Preservation: After overnight culture, check the solid LB plate and pick out the single colonies into new liquid LB medium (containing Amp). Incubate at 37℃ and 200rpm for 14-16h. Take a portion of the bacterial solution and add 25% glycerol at a ratio of 1:1. Mix thoroughly and store in a -80℃ refrigerator.

[0083] 2. Identification of Salmonella containing recombinant plasmids

[0084] The attenuated Salmonella bacterial culture after electroporation was sent to Sangon Biotech for sequencing to detect the presence of the target gene fragment and obtain successfully transfected attenuated Salmonella phoP / phoQ mutant strains.

[0085] 3. CCK-8 assay for cell proliferation

[0086] On day 1, 7000 CT26 cells were added to each well of a 96-well plate with 100 μL of culture medium per well. On day 2, according to the experimental groups, recombinant plasmids were transiently transfected into CT26 tumor cells using transfection reagents. 24 h after transfection, 10 μL of CCK-8 solution was added to each well, and the cells were incubated in a cell incubator for 1 h. The absorbance was then measured using an A450 microplate reader. The above steps were repeated after 48 h and 72 h of transfection. Results are shown below. Figure 3 ,Depend on Figure 3 It can be seen that, compared with the control group and the single silent genome, the shSTAT3 / shPD-L1 recombinant plasmid can significantly inhibit the growth of CT26 cells (P<0.001), and the difference is statistically significant.

[0087] 4. Cell colony formation assay to detect cell proliferation capacity

[0088] Add 500 CT26 cells transfected with plasmid for 24 hours to each well of a six-well plate and incubate for 7-9 days. Discard the culture medium and stop culturing when cell cloning is observed. Wash once with PBS solution, blow-dry the bottom of the six-well plate with a hairdryer to accelerate drying, fix with formaldehyde for 20 minutes, aspirate the formaldehyde, stain with 0.1% crystal violet for 5-10 minutes, wash away residual crystal violet with ddH2O, and photograph for record-keeping. See results below. Figure 4 ,Depend on Figure 4 It was found that after inoculation of CT26 cells, the number of clones formed in the shSTAT3 / shPD-L1 recombinant plasmid transfection group was significantly less than that in the control group and the group with a single silent genome (P<0.001), and the difference was statistically significant. These results indicate that the shSTAT3 / shPD-L1 recombinant plasmid can significantly inhibit the clonogenic ability of colon cancer CT26 cells.

[0089] 5. Flow cytometry detection of apoptosis

[0090] CT26 cells were collected 24 hours after transfection, with 1-5 × 10⁶ cells collected.5 Cell apoptosis was detected using the Annexin V-FITC / PI apoptosis detection kit (Shenyang Wanlei, WLA001c): Cells were resuspended in 500 μL binding buffer; 5 μL Annexin V-FITC was added to resuspend the cells, followed by 10 μL PI. After incubation in the dark for 10 min, cell staining was detected by flow cytometry. The results are shown below. Figure 5 As shown, 48 hours after transfection of CT26 cells with the shSTAT3 / shPD-L1 recombinant plasmid, the proportion of apoptotic cells significantly increased compared to the control group and the single-silence group (P<0.001). These results indicate that the shSTAT3 / shPD-L1 recombinant plasmid has a significant pro-apoptotic effect on CT26 cells.

[0091] 6. Western blot analysis of the effect of recombinant plasmids on apoptosis in colon cancer CT26 cells.

[0092] The expression of apoptosis-related proteins was detected by Western blot assay to verify the effect of the shSTAT3 / shPD-L1 recombinant plasmid on apoptosis in CT26 colon cancer cells. Results are as follows: Figure 6 As shown, compared with the control group and the single silencing group, the shSTAT3 / shPD-L1 recombinant plasmid transfection group showed a significant increase in cleaved caspase-3 protein (P<0.01), a significant decrease in Bcl-2 expression level (P<0.001), and a decrease in the Bcl-2 to Bax protein ratio (P<0.01). These results further demonstrate that the shSTAT3 / shPD-L1 recombinant plasmid promotes CT26 cell apoptosis by regulating apoptosis-related proteins.

[0093] 7. Transwell assay to detect cell migration ability

[0094] CT26 cells were collected 24 hours after transfection, with 1-2 × 10⁶ cells collected. 5 Cells were transferred to Transwell chambers, and 200 μL of serum-free culture medium was added. The chambers were placed in 24-well plates, with serum-containing culture medium added to the lower chamber. The plates were then incubated. After 48 hours, the chambers were transferred to new 24-well plates, the culture medium was aspirated, and 600 μL of 4% paraformaldehyde was added for fixation for 20-30 minutes. The fixative was discarded, and the cells were stained with 0.1% crystal violet for 5-10 minutes. The cells were washed three times with PBS to remove unbound crystal violet. Cells on the upper side of the chamber were gently wiped away with a cotton swab. Microscopic images were taken for preservation. The crystal violet dye was eluted with 3% acetic acid, and the OD value (570 nm) of the eluent was measured on a microplate reader to indirectly reflect the difference in the number of migrating cells between groups. Results are shown below. Figure 7 ,Depend on Figure 7It can be seen that the shSTAT3 / shPD-L1 recombinant plasmid transfection group had the least staining and the fewest number of migrating cells. By destaining with crystal violet dye and quantitatively detecting the difference in absorbance between the groups at a wavelength of 570 nm, the absorbance value of the shSTAT3 / shPD-L1 recombinant plasmid transfection group was the lowest, indicating that it had the fewest migrating cells, and the difference was statistically significant (P<0.001).

[0095] 8. Cell scratch assay to detect cell migration ability

[0096] Before cell transfection, a straight line of cells was scraped off from each well of a six-well plate using a 1 mL pipette tip. Non-adherent cells were removed, and images were taken under a microscope. Twenty-four hours after transfection, images were taken again to observe the wound healing process. Results are shown below. Figure 8 ,Depend on Figure 8 It can be seen that, compared with the single silencing group, the scratch healing distance of the shSTAT3 / shPD-L1 recombinant plasmid transfection group was the shortest (P<0.001), indicating that the shSTAT3 / shPD-L1 recombinant plasmid can significantly inhibit the migration ability of CT26 colon cancer cells.

[0097] 9. Flow cytometry for detecting cell cycle changes

[0098] Forty-eight hours after cell transfection, cells were collected, washed once with PBS, and fixed with 500 μL of pre-chilled 70% ethanol, then incubated overnight at 4°C. The next day, the cells were centrifuged at 1050 rpm for 5 min to collect the pellet. The supernatant was discarded, and the cell pellet was resuspended in 100 μL of RNase A solution, then 400 μL of PI staining solution was added and mixed well. The mixture was incubated in the dark for 30 min before analysis. Results are shown below. Figure 9 As shown in Figure 9, compared with the control group, the number of cells in the G1 phase of the recombinant plasmid group was significantly increased (P<0.001), and the number of cells in the S phase was significantly decreased (P<0.05), with statistically significant differences. These results indicate that the shSTAT3 / shPD-L1 recombinant plasmid can induce G1 phase arrest in CT26 cells, thereby inhibiting cancer cell proliferation.

[0099] 10. Western blot analysis of the effect of recombinant plasmids on cell cycle-related proteins in CT26 colon cancer cells.

[0100] To investigate the effect of the shSTAT3 / shPD-L1 recombinant plasmid on cell cycle arrest in CT26 cells, Western blot was used to detect changes in the expression of cell cycle-related proteins in CT26 cells after plasmid transfection. Figure 10The results showed that, compared with the single silencing group, the expression of Cyclin D1, a protein related to G1 phase arrest, was significantly downregulated after transfection of cells with the shSTAT3 / shPD-L1 recombinant plasmid (P<0.01). Western blot experiments indicate that the recombinant plasmid may induce G1 phase cell cycle arrest by regulating Cyclin D1 protein expression, thereby inhibiting cell proliferation.

[0101] Example 3

[0102] 1. Construction of a subcutaneous tumor-bearing model of colon cancer

[0103] CT26 cells in the logarithmic growth phase were collected, counted, and then subcutaneously injected into each C57BL / 6N mouse with 100 μL of a solution containing 3 × 10⁻⁶ cells. 5 A suspension of CT26 cells was injected into the left back area, where hair was removed before the experiment. Treatment was initiated when the tumor reached the size of a soybean, using an intraperitoneal injection of 100 μL of attenuated Salmonella phoP / phoQ mutant strain at a concentration of 10%. 7 CFU. Tumor volume and mouse weight were measured every two days during the period, and curves showing changes in tumor volume and mouse weight were plotted. The results are as follows: Figure 11 As shown, by Figure 11 It was found that, compared with the single silencing group, the attenuated Salmonella carrying the shSTAT3 / shPD-L1 recombinant plasmid showed the most significant decrease in tumor volume after 12 days of treatment in mice, and the difference was statistically significant (P<0.05); compared with the PBS treatment group, the mice in the shSTAT3 / shPD-L1 group showed the greatest increase in body weight, and the difference was statistically significant (P<0.05).

[0104] Formula for calculating mouse tumor volume: Tumor volume = (major axis) × (minor axis) 2 / 2.

[0105] Under aseptic conditions, 80 mg each of tumor, liver, spleen, and lung tissue from mice treated for 4 days were collected. The tissues were minced and ground, diluted 4-fold with pre-cooled PBS, and 50 μL was inoculated into solid culture medium containing Amp. The medium was incubated overnight at 37°C. The number of single colonies was calculated the next day. Results are shown below. Figure 12 ,Depend on Figure 12 It was found that 2170±122 monoclonal antibodies formed in the tumor sample, 17±4 monoclonal antibodies formed in the spleen, 12±2 monoclonal antibodies formed in the liver, and no monoclonal antibodies formed in the lung. The number of monoclonal antibodies in the tumor tissue was significantly different from that in other organs (P<0.001). The above results indicate that attenuated Salmonella can target and accumulate at the tumor site.

[0106] On day 12 of treatment, all mice were sacrificed, and tumors were collected and weighed. The results are as follows: Figure 13As shown, compared with other groups, the shSTAT3 / shPD-L1 group of mice had the lightest tumor weight (P<0.05), and the difference was statistically significant. These results indicate that attenuated Salmonella carrying the shSTAT3 / shPD-L1 recombinant plasmid had the most significant inhibitory effect on colon cancer tumor growth.

[0107] 2. Western blot detection of tumor tissue-related protein expression

[0108] To investigate the effect of attenuated Salmonella carrying recombinant plasmids on tumor growth, mice were sacrificed after 12 days, and Western blot analysis was used to detect apoptosis and cell cycle-related protein expression in tumor tissues. The results are as follows: Figure 14 As shown, attenuated Salmonella carrying the shSTAT3 / shPD-L1 plasmid significantly reduced the expression of the target genes STAT3 and PD-L1 (P<0.001). Compared with the single silencing group, the shSTAT3 / shPD-L1 group showed significantly decreased expression of the anti-apoptotic protein Bcl-2 (P<0.001), significantly increased expression of the activated apoptotic protein Cleaved-caspase3 (P<0.001), and significantly decreased expression of the cyclin Cyclin D1 (P<0.001). These results indicate that attenuated Salmonella carrying the shSTAT3 / shPD-L1 recombinant plasmid inhibits tumor progression by regulating apoptosis and cyclin expression.

[0109] 3. Immunohistochemical staining detection

[0110] To investigate the effects of attenuated Salmonella treatment on mice, mice were sacrificed after 12 days, and H&E staining was performed on the heart, liver, spleen, lungs, and kidneys to observe any toxic side effects in the mouse organs. Figure 15 The results showed no significant changes in the mouse organs upon visual inspection. Figure 16 The H&E staining results showed no significant differences in the major organs of mice in the attenuated Salmonella treatment group compared to the control group. These results indicate that the attenuated Salmonella treatment had no significant toxic side effects on the organs of mice.

[0111] 4. To detect the cytotoxic effect of Salmonella carrying the shSTAT3 / shPD-L1 plasmid on tumor tissue, mice were sacrificed after 12 days, and H&E staining was performed on the tumor tissue. The results are as follows: Figure 17 As shown, under a light microscope, the tumor tissue area in the shSTAT3 / shPD-L1 group was significantly reduced, with numerous pyknosis and rupture of cell nuclei, which appeared bluish-black, while the cytoplasm was pale red. These results indicate that attenuated Salmonella carrying the shSTAT3 / shPD-L1 recombinant plasmid has a significant killing effect on tumor cells.

[0112] 5. To detect whether the plasmid carried by attenuated Salmonella could exert RNA interference function in vivo, mice were sacrificed after 12 days, and immunohistochemical staining was performed to detect the expression of STAT3 and PD-L1 genes. The results are as follows: Figure 18 As shown, compared with the control group and the single-gene transfection group, the expression of STAT3 and PD-L1 in tumors of the shSTAT3 / shPD-L1 group was significantly reduced. These results indicate that the plasmid carried by attenuated Salmonella can exert its biological function by reducing the expression of STAT3 and PD-L1 in tumor tissues.

[0113] 6. After euthanizing mice on day 12, tumor tissue was subjected to histochemical staining for proliferation-related proteins. The results are as follows: Figure 19 As shown, the expression of Ki67 in tumors was significantly reduced in the shSTAT3 / shPD-L1 group, indicating that the attenuated Salmonella carrying the shSTAT3 / shPD-L1 recombinant plasmid inhibits tumor growth by affecting the expression of Ki67 in tumors.

[0114] 7. After euthanizing mice on day 12, tumor tissue was subjected to histochemical staining for apoptosis-related proteases. The results are as follows: Figure 20 As shown, the expression of Caspase3 in tumor-activated cells was significantly increased in the shSTAT3 / shPD-L1 group, indicating that attenuated Salmonella carrying the shSTAT3 / shD-L1 recombinant plasmid promotes tumor apoptosis by activating Caspase3.

[0115] 8. After euthanizing mice on day 12, tumor tissues from each group were collected for TUNEL staining to observe the effect of attenuated Salmonella carrying the shSTAT3 / shPD-L1 recombinant plasmid on tumor cell apoptosis. Results are as follows: Figure 21 As shown, compared with the control group and the single silence group, the number of TUNEL-positive tumor cells in the shSTAT3 / shPD-L1 group was significantly increased, indicating that the number of apoptotic tumor cells in the shSTAT3 / shPD-L1 group increased, thereby inhibiting tumor growth.

[0116] 9. After euthanizing the mice on day 12, cell cycle-related proteochemical staining was performed on the tumor tissue, and the results were as follows: Figure 22 As shown, the expression of Cyclin D1 in tumors of the shSTAT3 / shPD-L1 group was significantly reduced, indicating that the attenuated Salmonella carrying the shSTAT3 / shPD-L1 recombinant plasmid inhibited tumor progression by reducing Cyclin D1 expression.

[0117] 10. To investigate the effects of attenuated Salmonella carrying different plasmids on tumor-infiltrating immune cells, mice were sacrificed after 12 days, and CD4+ staining was performed using immunohistochemical staining. + CD8 + T cells, results as follows Figure 23As shown, attenuated Salmonella treatment can promote the infiltration of tumor immune cells, with the most significant increase observed in the group treated with attenuated Salmonella carrying the shSTAT3 / shPD-L1 recombinant plasmid, resulting in a substantial increase in immune infiltration cells. These results indicate that attenuated Salmonella carrying the shSTAT3 / shPD-L1 recombinant plasmid can promote the infiltration of CD4+ cells from tumor immune cells. + CD8 + T cells infiltrate and thus exert their own immune system function to kill tumors.

[0118] 11. Detection of mouse serum cytokines

[0119] To investigate the effects of attenuated Salmonella carrying different shRNA plasmids on mouse serum cytokines, mice were sacrificed after 12 days, and the concentrations of tumor-associated cytokines IL-6, IFN-γ, and TNF-α were measured using an ELISA kit. The results are as follows: Figure 24 As shown, compared with other groups, the shSTAT3 / shPD-L1 group showed a significant decrease in IL-6 concentration (P<0.001), an increase in IFN-γ concentration (P<0.001), and a decrease in TNF-α concentration (P<0.001) after treatment, with statistically significant differences. These results indicate that attenuated Salmonella carrying the shSTAT3 / shPD-L1 recombinant plasmid inhibits tumor progression by regulating related cytokines.

[0120] Example 4

[0121] Construction of an orthotopic model of colorectal cancer:

[0122] Each mouse was intraperitoneally injected with AOM at a dose of 10 mg / kg. One week later, they were fed 3% DSS in drinking water for 7 days, followed by normal drinking water for 14 days, then 1% DSS in drinking water for 7 days, and normal drinking water for 14 days. This cycle of 1% DSS in drinking water for 7 days and normal drinking water for 14 days was repeated until the model was established. Treatment was administered on days 60 and 67 after intraperitoneal injection of AOM by intraperitoneal injection of attenuated Salmonella phoP / phoQ mutant strain at a dose of 100 μL and a concentration of 5 × 10⁻⁶. 5 CFU. All mice were sacrificed on day 74, and samples were taken from the same subcutaneous tumor-bearing model. Figure 25 (A) shows the process of modeling colorectal cancer in situ and treating with attenuated Salmonella. Figure 25 (B) shows that after mice were injected with AOM, their body weight tended to decrease, but began to increase again after 5 days. During the period of feeding with 3% DSS drinking water, their body weight dropped sharply. After switching to normal water, their body weight began to gradually increase again. The concentration of DSS in the feeding water was reduced to 1%. The mice were fed DSS water for 7 days and normal water for 14 days. This was one cycle. The cycle was repeated. During this period, their body weight grew slowly. Figure 25(C) shows that, in addition to changes in body weight, mice exhibited perianal redness, swelling, and bloody stools during each DSS water-feeding modeling period. As the modeling time increased, the bloody stools became more severe, with significant weakness in the hind limbs, a noticeable tendency for the buttocks to fall to the ground, and severe rectal prolapse. Pathological examination results showed that, compared with adjacent tissues, HE staining of the tumor site indicated significant proliferation of tumor cells. These phenomena indicate that the orthotopic colorectal cancer mouse model was successfully established.

[0123] After treatment, we harvested and evaluated the colorectal tissue of the mice. Direct observation of the colorectal tissue, as shown in Figure 26(A), revealed that attenuated Salmonella carrying recombinant plasmids significantly inhibited the growth of in situ colorectal carcinoma. Intestinal weight / body weight was used as an indicator of the severity of in situ colorectal carcinoma; the mice were weighed, along with their intestinal weight. Figure 26 The results in (B) showed that the shSTAT3 / shPD-L1 group had the lowest tumor ratio (P<0.05) compared to the single-silence group, indicating the lowest severity. In addition, we also analyzed the length of the colorectal region and the number of tumors in the mice. Figure 26 Figure 26(C) shows that there was no difference in colorectal length among mice treated with different methods, indicating that the treatment had no effect on colorectal length. The number of tumors in the colorectal region is shown in Figure 26(D). Compared with the single-silence group, the shSTAT3 / shPD-L1 group had the fewest tumors (P<0.001), indicating that attenuated Salmonella carrying the shSTAT3shPD-L1 recombinant plasmid can significantly inhibit tumor progression.

[0124] First, this invention constructed a recombinant plasmid of shSTAT3 / shPD-L1 and verified its function in the mouse colon cancer cell line CT26 using qPCR and Western blotting experiments. The results showed that the silencing efficiency of both mRNA and protein expression of the target gene in CT26 was above 50%. We found that the recombinant plasmid had a higher silencing efficiency than the single silencing group, and while silencing STAT3 expression, it also reduced PD-L1 expression. Increasing evidence also indicates that PD-L1 expression is mediated by oncogenic activation of signaling pathways and is also regulated by tumor microenvironment factors. The effect of silencing PD-L1 on upstream STAT3 may be through reducing PI3K and AKT pathways, thereby reducing STAT3 expression.

[0125] Next, the recombinant plasmid was electroporated into attenuated Salmonella using electroporation. The successfully electroporated colonies were then expanded and cultured for subsequent animal experiments. A small portion of the bacterial culture was sequenced, and the target gene fragment was detected, confirming the success of the electroporation.

[0126] Next, we observed CT26 cells transfected with the recombinant plasmid. The CCK-8 assay showed that the recombinant plasmid inhibited tumor proliferation more effectively than the single-gene transfection group. Cell colony formation assays also confirmed the inhibitory effect of the recombinant plasmid on CT26 cell proliferation. Cell scratch assays and Transwell assays demonstrated the effect of the recombinant plasmid on the migration ability of colon cancer cells. The recombinant plasmid showed better efficacy, partly due to the tumor-suppressive effect of STAT3 expression silencing (reports of the combined effects of shSTAT3 and other treatments are numerous); and partly because shPD-L1 may increase the positive feedback to STAT3. Further research is needed on the non-immune aspects of immunosuppressive molecules. There are many ways to inhibit proliferation. We first used flow cytometry to detect the proportion of early and late apoptotic cells. The results showed that the recombinant plasmid significantly promoted the proportion of late apoptosis in colon cancer cells. STAT3 is closely related to apoptosis in cancer; inhibiting STAT3 leads to the inhibition of Bcl-2, thereby promoting apoptosis. shSTAT3 also increases cleaved-caspase 3 and decreases Cyclin D1 expression, a fact confirmed by our results. The recombinant plasmid had the same effect as the shSTAT3 group, and its effect was even more pronounced. However, when using flow cytometry to detect cell cycle changes, we found that the recombinant plasmid group had significant G1 phase arrest compared to the control group, but the proportion of cell cycle arrest was lower than that of the single silencing group. Simultaneously, Western blotting of the cyclin Cyclin D1 showed that the recombinant plasmid histone expression was the lowest. We speculate that the flow cytometry results are related to the state of CT26 cells, or may be influenced by other unconfirmed mechanisms, which remains to be discussed. The effect of recombinant plasmids on the cell cycle requires further investigation.

[0127] The success of our in vitro experiments prompted us to move to in vivo animal experiments. We successfully constructed subcutaneous tumor-bearing models and orthotopic colorectal cancer models. Our results showed that attenuated Salmonella carrying the shSTAT3 / shPD-L1 recombinant plasmid exerted a synergistic anti-tumor effect. On the one hand, the attenuated Salmonella could exert oncolytic activity; on the other hand, its function as a carrier targeting the tumor site allowed the recombinant plasmid to accumulate at the tumor site, and shPD-L1 successfully activated the immune system. In the orthotopic colorectal cancer model, we examined changes in the proportion of spleen immune cells. Unfortunately, the results were not statistically significant, but we look forward to further research to investigate the greater impact of the recombinant plasmid on the immune system.

[0128] Comparative Example 1

[0129] Except for the shPD-L1 sequence, the shSTAT3 / shPD-L1 recombinant plasmid provided in this comparative example is the same as that in Example 1. The shPD-L1 sequence used in this comparative example is GACGAUAAGCAGUGUUGAA (SEQ ID NO.10).

[0130] After transfection of CT26 cells with the recombinant plasmid, compared with the shControl group, the expression of STAT3 and PD-L1 proteins in the Comparative Example 1 shSTAT3 / shPD-L1 recombinant plasmid group showed no change and no statistically significant difference; however, the shSTAT3 / shPD-L1 recombinant plasmid group in Example 1 effectively inhibited the expression of STAT3 and PD-L1, and the difference was statistically significant. See the comparison results below. Figure 27 , Figure 27 The shControl, shSTAT3, and shPD-L1 in this comparative example are the same as in Example 1, and the shSTAT3 / shPD-L1 recombinant plasmid is the recombinant plasmid constructed in this comparative example.

[0131] 24 hours after transfection of CT26 cells with the recombinant plasmid, compared with the shSTAT3 group, there was no statistically significant difference in the proportion of apoptotic and necrotic cells in the shSTAT3 / shPD-L1 recombinant plasmid group in Example 2; however, the number of apoptotic cells in the shSTAT3 / shPD-L1 recombinant plasmid group in Example 1 was significantly reduced, and the difference was statistically significant. See the comparison results below. Figure 28 , Figure 28 The shControl, shSTAT3, and shPD-L1 in this comparative example are the same as in Example 1, and the shSTAT3 / shPD-L1 recombinant plasmid is the recombinant plasmid constructed in this comparative example.

[0132] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A recombinant plasmid, characterized in that, The recombinant plasmid contains shSTAT3 as shown in SEQ ID NO.2 and shPD-L1 as shown in SEQ ID NO.

3.

2. The recombinant plasmid according to claim 1, characterized in that, The recombinant plasmid contains the PLKO.1 backbone.

3. The recombinant plasmid according to claim 1, characterized in that, The sequence of the recombinant plasmid is shown in SEQ ID NO.

1.

4. A vector for attenuated Salmonella, characterized in that, The attenuated Salmonella vector contains the recombinant plasmid as described in claim 1.

5. The use of the recombinant plasmid according to any one of claims 1 to 3 in the preparation of a drug for treating colorectal cancer.

6. The use of the attenuated Salmonella carrier according to claim 4 in the preparation of drugs for treating colorectal cancer.

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