Part:BBa_K4833007

shSTAT3/shPD-L1 transcribes shSTAT3 and shPD-L1, silencing STAT3 and PD-L1 gene expression.

The shSTAT3/shPD-L1 composite element consists of four parts: the H1 promoter, shSTAT3 sequence, U6 promoter, and shPD-L1 sequence. The shSTAT3/shPD-L1 composite element is transcribed to generate shSTAT3 and shPD-L1, which silence the expression of STAT3 and PD-L1 genes in tumor cells through RNAi mechanism and inhibit tumor cell proliferation. PD-L1 is a molecule present on the surface of cancer cells that inhibits the immune pathway. Binding of PD-L1 to the programmed death receptor 1 (PD-1) on the surface of immune cells can initiate programmed cell death in T cells, inducing immune evasion of tumor cells. Silencing the PD-L1 gene can relieve the inhibition of cancer cells on immune cells, activate the immune system, and inhibit the occurrence and development of tumors. STAT3 is a member of the signal transducer and activator of transcription (STAT) family, and it is involved in various biological processes including proliferation, metastasis, angiogenesis, immune response, and chemoresistance. Sustained activation of STAT3 is frequently observed in cancer and is associated with tumor progression and poor prognosis. The STAT3 signaling pathway is involved in the development of multiple types of tumors. In colorectal cancer, constitutive activation of STAT3 increases tumor cell viability and promotes tumor cell proliferation by upregulating the expression of Cyclin D1, Survivin, and Myc. STAT3 can directly enhance VEGF expression to drive tumor angiogenesis and growth. Additionally, STAT3 can reduce the production of reactive oxygen species (ROS) by altering oxidative phosphorylation and glycolysis processes in cancer cells, which is favorable for the growth of cancer cells in the body. Furthermore, aberrant activation of STAT3 can trigger immune evasion or suppress the anti-tumor ability of dendritic cells (DCs). STAT3 can also directly bind to the promoter region of PD-L1, promoting the transcription and expression of PD-L1, suggesting that STAT3 may be involved in tumor immune evasion through PD-L1. Therefore, silencing STAT3 gene expression can inhibit tumor cell proliferation through multiple pathways. The sequence we designed can transcribe shRNA targeting STAT3 and PD-L1. In the plasmid, the shSTAT3/shPD-L1 sequence can silence the gene expression of STAT3 and PD-L1 in tumor cells through the RNAi mechanism. This sequence transcribes and produces shSTAT3 and shPD-L1 sequences, which are complementary to a part of STAT3 mRNA and PD-L1 mRNA. Inside the cell, shRNA is processed into siRNA, and the guide strand of these siRNAs is loaded into the RISC complex. The RISC complex then binds to the complementary sequences on STAT3 mRNA and PD-L1 mRNA, catalyzing the cleavage and degradation of the target mRNA. In summary, this composite element can silence the gene expression of STAT3 and PD-L1 in tumor cells, activate the immune response, and inhibit tumor cell proliferation.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 22
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 419
  

As shown in Figure 1，the shSTAT3/shPD-L1 composite part consists of four parts: the H1 promoter, shSTAT3 sequence, U6 promoter, and shPD-L1 sequence. The shSTAT3/shPD-L1 composite element is transcribed to generate shSTAT3 and shPD-L1, which silence the expression of STAT3 and PD-L1 genes in tumor cells through RNAi mechanism and inhibit tumor cell proliferation.

Fig.1 shSTAT3/shPD-L1 composite part

We placed this element in a plasmid for biological functional testing and compared it with shPD-L1 and shSTAT3.

1 RT-PCR and Western Blot were used to detect the expression levels of STAT3 and PD-L1 in CT26 cells

We used Lipo3000 transfection reagent to transiently transfect shControl plasmid, shSTAT3 plasmid, shPD-L1 plasmid, and shSTAT3/shPD-L1 recombinant plasmid into mouse colon cancer CT26 cells. Cells were collected 24 hours after transfection for RT-PCR to detect gene expression levels and 48 hours after transfection for Western blot to detect protein expression levels. As shown in Figure 2 and 3, compared with the single silencing group, the shSTAT3/shPD-L1 recombinant plasmid transfection group showed a significant decrease in STAT3 and PD-L1 mRNA expression (P<0.001). As shown in Figure 4、5 and 6, compared with the single silencing group, the recombinant plasmid transfection group showed a significant decrease in STAT3 and PD-L1 protein expression (P<0.001). These results indicate that the shSTAT3/shPD-L1 recombinant plasmid was successfully transfected into CT26 cells and exerted its biological effects.

Fig.2 Quantification of STAT3 mRNA expression levels（Data are mean±SEM）

Fig.3 Quantification of PD-L1 mRNA expression levels（Data are mean±SEM）

Fig.4 Protein expression levels of STAT3 and PD-L1.

Fig.5 Quantification of STAT3 protein expression levels（Data are mean±SEM）

Fig.6 Quantification of PD-L1 protein expression levels（Data are mean±SEM）

Our designed shSTAT3/shPD-L1 composite part can significantly inhibit the transcription and translation of PD-L1 and STAT3 genes!

2 CCK-8 experiment was conducted to assess the impact of recombinant plasmids on the proliferation of colon cancer CT26 cells：

CCK-8 assay was performed to analyze the effect of shSTAT3/shPD-L1 recombinant plasmids on the growth status of colon cancer cells. Transfected CT26 cells were seeded in a 96-well plate and cultured for 24, 48, and 72 hours. After incubating with CCK-8 solution for 1 hour, cell growth was assessed by measuring the absorbance. Figure 7 demonstrates that compared to the control group and single gene silencing group, shSTAT3/shPD-L1 recombinant plasmids significantly inhibited the growth of CT26 cells (P<0.001), with a statistically significant difference.

Fig.7 Cell proliferation capacity detected by CCK-8 assay.(Data are presented as mean ± SEM).

Our designed shSTAT3/shPD-L1 composite part can significantly inhibit the proliferation of colon cancer cells!

3 The colony formation assay was performed to assess the effect of recombinant plasmids on the proliferation of CT26 colon cancer cells.

To investigate the effect of shSTAT3/shPD-L1 recombinant plasmids on the proliferation of colon cancer cells, a colony formation assay was conducted. As shown in Figure 8 and 9, after seeding CT26 cells, the number of colonies formed in the shSTAT3/shPD-L1 transfected group was significantly lower than the control group and the single gene silencing group (P<0.001), indicating a significant difference according to statistical analysis. These results suggest that shSTAT3/shPD-L1 recombinant plasmids can significantly inhibit the colony formation ability of CT26 colon cancer cells.

Fig.8 Results of colony formation assay

Fig.9 Quantification of colony formation assay（Data are mean±SEM）

Our designed shSTAT3/shPD-L1 composite part can significantly inhibit the proliferation of colon cancer cells！

4 Flow cytometry was used to evaluate the effect of recombinant plasmids on apoptosis in CT26 colon cancer cells.

The effect of the recombinant plasmid on CT26 cell apoptosis was quantified using flow cytometry. We collected the cells after 48h transfection and stained them with Annexin V-FITC/PI, and measured the fluorescence intensity to determine the proportion of apoptotic cells (Fig 10 and 11). The results showed that the proportion of apoptotic CT26 cells significantly increased after 48h transfection with shSTAT3/shPD-L1 recombinant plasmid, compared with the control and single-silenced groups. The results indicated that the recombinant plasmid shSTAT3/shPD-L1 promoted the apoptosis of CT26 cells.

Fig.10 Apoptosis detect by Flow cytometry assay

Fig.11 Quantified apoptosis assay（Data are mean±SEM）

Our designed shSTAT3/shPD-L1 composite part can significantly promotes apoptosis in colorectal cancer cells!

5 Western blot was performed to assess the effect of recombinant plasmids on apoptosis in CT26 colon cancer cells.

Next, we detected the expression of apoptosis-related proteins by western blotting to verify the effect of the shSTAT3/shPD-L1 recombinant plasmid on the apoptosis of colon cancer CT26 cells. Compared to the control group and the single-silenced group, Cleaved Caspase-3 protein was significantly increased in the shSTAT3/shPD-L1 recombinant plasmid transfection group, the expression level of Bcl-2 was decreased, and the ratio of Bcl-2 to Bax protein was decreased (Fig 12、13and 14). These results further illustrate that the shSTAT3/shPD-L1 recombinant plasmid promoted apoptosis in colon cancer cells by regulating the expression of apoptosis-related proteins.

Fig.12 Apoptosis-related proteins tested by Western Blot

Fig.13 Quantification of Cleaved caspase3 protein measure (Data are mean ± SEM)

Fig.14 Quantification of Bcl-2/Bax protein measure (Data are mean ± SEM)

Our designed shSTAT3/shPD-L1 composite part can promote apoptosis in CT26 cells by regulating apoptosis-related proteins！

6 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 15 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 was able to make CT26 cells undergo G1 phase arrest, thereby inhibiting cancer cell proliferation.

Fig.15 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）

7 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 16 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 may induce G1 phase cycle arrest, which may inhibit cell proliferation.

Fig.16 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）

8 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 17, 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).

Fig.17 Transwell chamber invasion assay for cell migration ability (A)Plot of invasion results (B) Quantification of invasion results（Data are mean±SEM）

9 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 18 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.

Fig.18 Cell scratch assay tested for cell migration ability (A)Results of scratch results (B) Quantification of scratch results （Data are mean±SEM）

In summary, our designed shSTAT3/shPD-L1 composite part can significantly inhibit CT26 cell proliferation, promote CT26 cell apoptosis, and suppress the migration of colorectal cancer CT26 cells.