1.1 The expression levels of STAT 3 and PD-L1 in CT26 cells were determined by RT-PCR and Western Blot

First, to verify whether the constructed plasmids can exert biological roles. The shControl plasmid, shSTAT3 plasmid, shPD-L1 plasmid and shSTAT3/shPD-L1 recombinant plasmid were transiently transfected into mouse colon carcinoma CT26 cells and collected at 24 h after transfection, and cellular gene level expression was measured by RT-PCR; cells were collected at 48 h after transfection, and cellular protein expression was measured by Western Blot assay. The results of Figure 1 showed that the expression of STAT3 and PD-L1 mRNA was significantly reduced in the shSTAT3/shPD-L1 recombinant plasmid transfection group (P < 0.001), and STAT3 and PD-L1 protein expression were significantly decreased in the recombinant plasmid transfection group compared with the single silencing group (P < 0.001). The above results indicated that the shSTAT3/shPD-L1 recombinant plasmid was successfully transfected into CT26 cells and played biological roles.

Our shSTAT3/shPD-L1 recombinant plasmid could significantly inhibit the expression of STAT3 and PD-L1 genes!

Figure 1. Expression levels of STAT3 and PD-L1 in CT26 cells were determined by RT-PCR and Western Blot Quantification of mRNA expression of STAT3 and PD-L1 (B) Protein of STAT3 and PD-L1 Expression status (C) Quantification of protein expression of STAT3 and PD-L1（Data are mean±SEM）

1.2 Effect of recombinant plasmids on the proliferation of colon cancer CT26 cells

1.2.1 CCK-8 assay to determine the effect of recombinant plasmid on colon cancer CT26 cell proliferation

The effect of the shSTAT3/shPD-L1 recombinant plasmid on the growth status of colon cancer cells was analyzed by the CCK-8 assay. After transfected CT26 cells were spread in 96-well plates and cultured for 24,48 and 72 h, CCK-8 solution was added for 1 h to reflect cell growth by absorbance size. Figure 2 shows that the shSTAT3/shPD-L1 recombinant plasmid significantly inhibited CT26 cell growth (P < 0.001), with significant differences.

Figure 2. Cell proliferation was measured in the CCK-8 assay（Data are mean±SEM）

1.2.2 Clone formation assay to test the effect of recombinant plasmids on proliferation in colon cancer CT26 cells

To examine the effect of shSTAT3/shPD-L1 recombinant plasmid on colon cancer cell proliferation, it was analyzed by clone formation assay. As shown in Figure 3, after inoculation of CT26 cells, the number of clones formed in the shSTAT3/shPD-L1 recombinant plasmid transfection group was significantly less than that of the control group and the silent single genome (P < 0.001), indicating significant differences in statistical analysis. The above results indicated that the shSTAT3/shPD-L1 recombinant plasmid was able to significantly inhibit the clonogenic formation ability of colon cancer CT26 cells.

Figure 3. Cell proliferation was measured in the clone formation assay（A）Results of clone formation experiment diagram (B) Quantification diagram of results of clone formation experiment（Data are mean±SEM）

Our shSTAT3/shPD-L1 recombinant plasmid can significantly inhibit the proliferation of colorectal cancer cells!

2.1 Construction and characterization of a recombinant suicide plasmid for asnB-pRE112

pRE112 SaciⅠ and XbaⅠrestriction sites on both the pRE112 suicide plasmid and the asnB-arm-pUC57 plasmid. Double digestion of both, respectively, The homology arm fragment of the target gene asnB (Figure 4A) and the suicide vector fragment of 5760 bp (Figure 4B). The both the two for connection conversion. And transferred into the host bacterium χ7213 Escherichia coli. Positive clones were selected by using the chloramphenicol resistance gene. Plasmids from the positive clones were then reextracted, The digestion digestion revealed two clearly clear bands. Consistent with the fragment size before ligation (Figure 4C). Verified by PCR with the upstream and downstream primer asnB-F / R. A band of about 173 bp (Figure 4D). Suggesting the successful transfer of the asnB-pRE112 recombinant suicide plasmid into χ7213 E. coli.

Figure 4. Construction of a recombinant suicide plasmid for asnB-pRE112 M: DNA Marker; A: double digestion of aasnB-arm-pUC plasmid; B: pRE112 suicide plasmid digested C:asnB-pRE112 recombinant suicide plasmid; D: asnB-pRE112 recombinant suicide plasmid identified by PCR

2.2 Construction and characterization of the PtrcflaB-asnB-pRE112 recombinant suicide plasmid

A fragment of the flaB gene of 1303 bp in length was cut from the PtrcflaB-pUC57 plasmid using the restriction endonucleases NotI and SbfI. The pRE112 suicide vector fragment attached with a homologous arm fragment of the asnB gene was cut from the asnB-pRE112 reconstituted suicide plasmid. Length size is 6588 bp (Figure 5 A). The both the two for connection conversion. And transferred into the host bacterium χ7213 Escherichia coli. Positive clones were selected by the chloramphenicol resistance gene. Plasmids from the positive clones were then reextracted. The digestion digestion revealed two clearly clear bands. Consistent with the fragment size before ligation (Figure 5 B). Verified by PCR with the primer flaB-F / R. A band of 1280 bp was amplified (Figure 5 C). Consistent with the expectations. Show successful construction of PtrcflaB-asnB-pRE112 recombinant suicide plasmid .

Figure 5. Construction of PtrcflaB-asnB-pRE112 recombinant suicide plasmid M: DNA Marker; A: 1-2 PtrcflaB-pUC57 plasmid double digestion; double digestion of 3-4 asnB-pRE112 recombinant suicide plasmid; B: PtrcflaB-asnB-pRE112 recombinant suicide plasmid; C:PtrcflaB-asnB-pRE112 recombinant suicide plasmid identification by PCR

2.3 ΔasnB χ11802 Construction and characterization of an attenuated Salmonella-engineered strain

After the successfully transformed χ7213 E. coli (asnB-pRE112) was mixed with the Salmonella χ11802 strain, and the conjugation transfer occurred, the homologous arm sequence on the recombinant suicide plasmid asnB-pRE112 underwent a homologous recombination with the homology arm sequence of the asnB gene on the Salmonella chromosome, and the correct conjugation transfer was selected The strain was cultured on LB solid medium with 10% sucrose (no NaCl, arabinose and DAP). Due to the sacB negative screening gene (sucrose sensitive gene) on the pRE112 plasmid, the second homologous recombination occurred in the process of passage. The single clone obtained after chloramphenicol resistance screening was verified by PCR with primers asnB-F / R, and only a 173 bp band appeared (Figure 6), and the resulting strain was attenuated ΔasnB χ11802.

Figure 6. A PCR characterization of the ΔasnB χ11802 Salmonella mutant strain M: DNA Marker; 1-5: ΔasnB χ11802 strain; 6: χ11802 strain

2.4 Construction and identification of the ΔasnB::PtrcflaB χ11802 attenuated Salmonella engineered strain

In a mixed culture of the successfully transformed χ7213 E. coli (PtrcflaB-asnB-pRE112) with the attenuated Salmonella engineered bacterium ΔasnB χ11802, After the conjugation transfer occurs, The homologous arm sequence on the recombinant suicide plasmid PtrcflaB-asnB-pRE112 was exchanged twice by homologous recombination with the sequence of the asnB gene on the chromosome of the ΔasnBχ11802 strain, Single clones were obtained after sucrose plates and chloramphenicol resistance screening, Valified by PCR with primers asnB-F / R, The ence of only one 1451 bp amplified band, Further verified by specific PCR with the primer flaB-F / R, A band of 1280 bp appears (Figure 7), Demondicates the mutation ofΔasnB:: PtrcflaB χ11802.

Figure 7. PCR identification of the ΔasnB::PtrcflaB χ11802 Salmonella mutant strain M: DNA Marker; 1,4: ΔasnB::PtrcflaB χ11802 strains; 2: ΔasnB χ11802strains; 3: χ11802 strains; 1-3: primer asn

3.1 TEM observation

Flagella is closely related to the motility ability of bacteria, which can enhance the deep tissue colonization ability of bacteria. In this study, transmission electron microscopy was used to observe the flagellar growth of the mutant strains. As shown in the figure, χ11802, the strain had fewer flagella (Figure 8 A-B), and insertion of the flagella flaB gene increased significantly in the mutant strain (ΔasnB::PtrcflaB χ11802) (Figure 8C-D), suggesting that the motility of the strain is likely to be significantly enhanced.

Figure 8. Transmission M graphs of Salmonella mutant strain A-B: χ11802 strain; C-D: ΔasnB::PtrcflaB χ 11802 strain

3.2 Determination of growth curves of attenuated Salmonella mutant strains

After gene editing of Salmonella, we first need to observe the growth characteristics of Salmonella. In this study, attenuated Salmonella χ11802, ΔasnB χ11802 and ΔasnB::PtrcflaB χ11802 were incubated with a starting concentration of 1× 106 CFU / mL, OD 600 was measured every two hours, and growth curves were recorded and drawn for 24 h. The results are shown in Figure 9, indicating that the ΔasnB χ11802 strain is larger than the χ11802 strain The growth rate was slower, indicating that deleting the asnB gene could affect its growth rate, but the excessive expression of flagellin in the ΔasnB χ11802 strain accelerated the growth rate, slightly faster than χ11802, but not statistically significant.

Figure 9. Growth curves of the Salmonella mutant strain

3.3 Determination of the migratory ability of the attenuated Salmonella mutant strain.

Next, the mobility of the mutant strains in soft agar plates was observed. Three bacterial solutions were added to the center of the soft agar plate, and the cultures were incubated for 8 h to observe the straight size of the sphere. The results are shown in Figure 10, showing that the ΔasnB χ11802 strains almost lost their motility, while the ΔasnB::PtrcflaB χ11802 strain had the most motility, 133% of the χ11802 strain, and they were statistically significant between all groups (P < 0.01).

Figure 10. Migrability determination of Salmonella mutant strain

3.4 Determination of the biofilm-forming capacity of the attenuated Salmonella mutant strains

Biofilm formation contributes to the development of antibiotic resistance and resistance to the external environment. In this study, the biofilm formation of the three strains was detected by crystal violet. The results are shown in Figure 11, compared with the χ11802 strain, theΔasnB χ11802 strain was reduced (P < 0.01), indicating that knocking down of Salmonella asnB gene can destroy the integrity of the bacterial outer membrane to some extent. However, the biofilm formation capacity was recovered after the insertion of the flagellin gene flaB (P < 0.01).

Figure 11. Biofilm formation assay of a Salmonella mutant strain

3.5 Determination of polymyxin B susceptibility in attenuated Salmonella mutant strains

Polymyxin B has strong antibacterial effects against most Gram-negative bacilli. To further determine whether the mutant strain had altered the external antibiotic pressure, the sensitivity of the mutant strain to polymyxin B was observed in this study. Take three strains with a concentration of 1× 106 CFU / mL and add polymyxin B to a final concentration of 0. 1 μg/mL. Take three strains with the same concentration without antibiotics, dilute the coating plate for 1 h, and the colony average value of each dilution plate was calculated on the second day. As shown in Figure 12, χ11802 strainΔasnB χ11802 was more sensitive to polymyxin B (P < 0.001). This result was consistent with the biofilm formation experiment. Similarly, the resistance of theΔasnB::PtrcflaBχ11802 strain to polymyxin B returned to similar levels in the χ11802 strain, statistically significant compared to the ΔasnB χ11802 strain (P < 0.01).

Figure 12. The susceptibility experiments of Salmonella mutant strains to polymyxin B

3.6 Adhesion invasion of attenuated Salmonella mutant strains

The adhesive and invasive effects of bacteria can reflect their ability to invade and colonize the organism. Human small intestinal epithelial cells were infected with INT4072 h with three strains, free bacteria were washed away, cells were lysed with 1% Triton-×100, and the number of colonies reflected the adhesion of bacteria. As shown in Figure 13, the adhesion rate of ΔasnBχ11802 strain was 16.6% of χ11802, while the adhesion rate of ΔasnB::PtrcflaB χ11802 strain increased significantly to 172.4% (P < 0.001). After the addition of gentamicin, the lysed bacteria were subjected to plate counting, and the number of colonies reflected the invasion of the bacteria. As shown in Figure 13 (A, C), the invasion rate of ΔasnB χ11802 strain was only 1.2% of 11802. However, the invasion rate of the ΔasnB::PtrcflaB χ11802 strain increased to 151.6% (P < 0.001). The results showed that the insertion of flagellin flaB gene effectively enhances the adhesion and invasion of Salmonella.

4.1 Identification of attenuated Salmonella strains after electrotransformation

To test whether the shSTAT3/shPD-L1 recombinant plasmid was successfully electrotransferred into the attenuated Salmonella χ11802 mutant strain, the electrotransferred bacteria were expanded in culture and some of the fluids were taken for sequencing. Figure 14 shows that the STAT3 and PD-L1 gene fragments were detected in the bacterial solution, indicating the successful electrotransfer of the shSTAT3/shPD-L1 recombinant plasmid into the attenuated Salmonella χ11802 mutant strain.

Figure 14. Gene sequencing of χ11802 mutant strains of attenuated Salmonella sp (A) Sequencing results of shSTAT3 plasmid. (B) Results of shPD-L1 plasmid (C) Results of the electroconversion group of the shSTAT3 / shPD-L1 plasmid

4.2 Effect of a recombinant plasmid carried by Salmonella on apoptosis in colon cancer CT26 cells

4.2.1 The effect of recombinant plasmids on the apoptosis of colon cancer CT26 cells was determined by flow cytometry

The capacity of the recombinant plasmid for proapoptotic capacity in CT26 cells was quantified by flow cytometry. Cells were first collected 24 h after transfection, and the proportion of apoptotic cells was determined by computer with Annexin V-FITC / PI staining and fluorescence intensity. Figure 15 shows that the fraction of apoptotic cells increased 48h with the shSTAT3/shPD-L1 recombinant plasmid compared to the control and single silent CT26 cells (P < 0.001).The above results demonstrate that the shSTAT3/shPD-L1 recombinant plasmid carried by Salmonella has a significant pro-apoptotic capacity in CT26 cells.

Figure 15. The percentage of cell apoptosis was determined by flow cytometry (A) Quantification of apoptosis results detected by flow cytometry (B) Quantification of apoptosis results（Data are mean±SEM）

4.2.2 Western blot Effect of recombinant plasmid on apoptosis in colon cancer CT26 cells

The expression of apoptosis-related proteins was assessed by Western blot experiments to verify the effect of shSTAT3/shPD-L1 recombinant plasmid on apoptosis in colon cancer CT26 cells. In Figure 16, relative to the control and single silenced groups, the Cleaved Caspase-3 protein in the shSTAT3/shPD-L1 recombinant plasmid transfection significantly increased (P < 0.01), the Bcl-2 expression level decreased (P < 0.001), and the Bcl-2 to Bax protein ratio decreased (P < 0.01). The above results further illustrate that the shSTAT3/shPD-L1 recombinant plasmid carried by Salmonella promotes apoptosis in CT26 cells by regulating apoptosis-related proteins.

Figure 16. Western blot analysis for apoptosis-related protein expression(A) Results of apoptosis-related proteins (B) Quantification of Bcl-2 / Bax protein results (C) Quantification plots of the Cleaved-caspase3 protein results（Data are mean±SEM）

4.3 Recombinant plasmid carried by Salmonella induces cell cycle arrest in colon cancer CT26 cells

4.3.1 The effect of recombinant plasmids on the cell cycle of colon cancer CT26 was determined by flow cytometry

The effect of recombinant plasmids on the cell cycle of colon cancer CT26 was examined using flow cytometry. Cells transfected for 48 h were harvested for PI staining. Figure 17 shows that the number of G1 cells in the recombinant plasmid group was significantly increased (P < 0.001) and S phase cells decreased (P < 0.05), with a statistically significant difference. The above results demonstrated that the shSTAT3/shPD-L1 recombinant plasmid carried by Salmonella was able to make CT26 cells undergo G1 phase arrest, thereby inhibiting cancer cell proliferation.

Figure 17. Cell cycle was determined by flow cytometry experiments(A) Cell cycle results diagram of flow cytometry experiments (B) Quantification diagram of cell cycle results（Data are mean±SEM）

4.3.2 Western blot To test the effect of recombinant plasmid on cell cycle-related proteins of CT26 in colon cancer

To explore the effect of shSTAT3/shPD-L1 recombinant plasmid on cell cycle arrest in CT26 cells, CT26 cell cycle-related protein expression changes after plasmid transfection were determined by Western blot assay. Figure 18 shows that the expression of G1 phase arrest-related protein Cyclin D1 was significantly downregulated compared with the single silenced group (P < 0.01). Western blot The experiment show that the recombinant plasmid carried by Salmonella may induce G1 phase cycle arrest, which may inhibit cell proliferation.

Figure 18. Cell Cycle-related proteins were detected by flow cytometry (A) Results of cell cycle related proteins (B) Quantification of cell cycle related protein results（Data are mean±SEM）

4.4 Effect of recombinant plasmids carried by Salmonella on the migratory capacity of colon cancer CT26 cells

4.4.1 Transwell Detection of the effect of recombinant plasmid on the migration ability of colon cancer CT26 cells

CT26 cell migration capacity was measured by quantifying the Transwell compartment bottom membrane across cell numbers. In Figure 19, the crystal violet staining of cells crossing the bottom membrane of the Transwell chamber showed that the shSTAT3/shPD-L1 recombinant plasmid transfection group had the smallest staining degree and the least number of migrated cells; the absorbance of the shSTAT3/shPD-L1 recombinant plasmid transfection group, indicating that the least number of migrated cells and the difference was statistically significant (P < 0.001).

Figure 19. Transwell chamber invasion assay for cell migration ability (A)Plot of invasion results (B) Quantification of invasion results（Data are mean±SEM）

4.4.2 Cell scratch assay examined the effect of recombinant plasmid on the migratory ability of CT26 cells in colon cancer

To test the effect of recombinant plasmids on cell migration ability, the scratch healing distance was observed after cell transfection at 24 h by cell scratch assay. Figure 20 shows that the shSTAT3 / shPD-L1 recombinant plasmid group had the shortest scratch healing distance (P < 0.001), indicating that the shSTAT3 / shPD-L1 recombinant plasmid carried by Salmonella could significantly inhibit the migration ability of colon cancer CT26 cells.

Figure 20. Cell scratch assay tested for cell migration ability (A)Results of scratch results (B) Quantification of scratch results