Part:BBa_K4737005
SLC7A11-shRNA
SLC7A11-shRNA is gene of the RNAi effector short hairpin RNA (shRNA) of SLC7A11 gene
Usage and Biology
As one class of cystine transporter proteins, SLC7A11 (Solute Carrier Family 7 Member 11) is also one of the most critical upstream regulators of ferroptosis. In recent years, many articles have revealed that SLC7A11 drives cell resistance to ferroptosis and plays an important regulatory role in many diseases such as tumors.
As a key regulatory protein of ferroptosis, the main function of SLC7A11 is to transport extracellular cystine into the cell. Cystine is transported into the cell and reduced to cysteines, which is involved in protein biosynthesis, such as the production of prototype glutathione (GSH), and it also regulates the synthesis rate of glutathione peroxidase 4 (GPX4). The latter substance is able to reduce toxic peroxides with GSH as a cofactor, thus inhibiting cell ferroptosis. When the expression of SLC7A11 is down-regulated, the content of cystine in tumor cells will be decreased and the cysteine metabolic pathway will also be inhibited, resulting in the synthesis rate of the GSH and GPX4 decreased or even blocked. This will lead to the accumulation of lipid peroxide, destroy membrane lipid bilayer structure and increase cell membrane’s permeability, eventually induce cell ferroptosis.
Tumor cells' metabolism is more dependent on SLC7A11 than normal cells. Based on this, we tried to silence SLC7A11 gene expression by delivering RNAi effector short hairpin RNA (shRNA) through the engineered attenuated Salmonella VNP20009 to tumor cells. In this module, we designed a functional plasmid containing a shRNA sequence used to down-regulate the SLC7A11 gene in tumor cells. When the engineered bacteria invade the tumor cells, they can release the shRNA into the cell and specifically degrade the corresponding mRNA, thereby blocking the translation of the target gene SLC7A11. Meanwhile, to improve the silencing efficiency, we added the HlyA gene encoding Listeriolysin-O protein to the functional plasmid. This pore-forming protein safeguards shRNA from degradation by allowing its rapid release from entry vesicles11, resulting in the efficiency of gene silencing greatly improved.
Figure 1. Schematic diagram of engineered bacteria expressing shRNA.
Figure 2. shRNA-mediated gene silencing and induction of ferroptosis.
1.Plasmids construction
In order to silence the SLC7A11 gene in tumor cells and induce ferroptosis, a functional plasmid pSilence-SLC7A11 was constructed. This plasmid included a short hairpin RNA (shRNA) sequence to silence the target gene SLC7A11, and the HlyA gene encoding listolysin protein was also added to the plasmid. Based on literature review, we learned that T7 promoter is often used to initiate shRNA transcription in the prokaryote to realize trans-kingdom RNAi, however the deficiency of T7 RNA polymerase in our chassis bacteria VNP20009 will be a problem. Therefore, we used a constitutive promoter to initiate T7 RNA polymerase expression in the engineered bacteria.
At the same time, in order to qualitatively verify whether the engineered bacteria can release shRNA after invading tumor cells to silence the expression of the corresponding genes in tumor cells, we constructed the plasmid pVB230-shRNA-GFP, which consists of a shRNA sequence for silencing green fluorescent protein (GFP).
The above constructed plasmids were transferred into attenuated Salmonella typhimurium VNP20009 by electric shock transformation method to obtain engineered bacteria capable of down-regulating gene expression in tumor cells.
Figure 3. Constructed pSilence-SLC7A11 plasmid to deliver shSLC7A11 to tumor cells.
Figure 4. Constructed pVB230-shRNA-GFP plasmid to deliver shGFP to tumor cells.
2.Western blot analysis
To verify the engineered bacteria can achieve the expression of HlyA, bacterial lysates were prepared from S. typhimurium strain VNP20009 carrying appropriate expression vectors for shRNA-SLC7A11 and subjected to immunoblotting analysis.HlyA expression detected by Western blotting indicates that our plasmids were successfully transferred into VNP20009 and effectively expressed (Figure 5).
Figure 5. Western blot analysis of Listeriolysin O. a. Listeriolysin O is a pore-forming protein (59kDa) encoded by gene HlyA; b. GAPGH is a reference protein in cells with a molecular weight of 36 kDa.
3.Delivery of shRNA to tumor cells by bacteria infection
3.1 Delivery of shRNA-GFP to silence the expression of GFP
We selected human gastric adenocarcinoma cells BGC823 and transiently transfected with the GFP eukaryotic expression vector. After the fluorescent protein was effectively expressed in BGC-823 24h after transfection, the cells were co-cultured with the engineered bacteria expressing shRNA-GFP.
We chose the multiplicity of infection (MOI) of cells and engineered bacteria (VNP20009-shGFP) as 1:500, at the same time, we also used VNP20009 without shRNA-GFP at an MOI of 1:500 to infect tumor cells as negative control results. We observedthe results =under fluorescence microscope after 24 hours (Figure. 6). Compared with the VNP20009 control, the tumor cells’ fluorescence was significantly weakened after VNP20009-shGFPinfection, indicating that the engineered bacteria actually delivered shRNA-GFP after entering the tumor cells, and effectively down-regulated the gene expression.
Figure 6. Results of shGFP delivery by bacterial infection 24 hours later. Both the engineered bacteria and VNP20009 infections were at the MOI of 1:500. Control was BGC-823 not transfected with the GFP transient plasmid.
3.2 Delivery of shRNA-SLC7A11 to silence tumor cells’ expression of SLC7A11
After verifying the feasibility of engineered bacteria to deliver shRNA-SLC7A11, we used engineered bacteria (shRNA-SLC7A11) to infect BGC-823. Bacteria in early log phase were washed, diluted in DMEM and added at the desired MOI from 1:1500 to 1:5000. We used VNP20009 without functional plasmid to infect cells as the negative control (MOI = 1:5000). Meanwhile, we also used siRNA transfection with BGC-823 as a positive control to further verify the efficiency of the delivery of shRNA by the engineered bacteria.
After 24h of co-culture infection and siRNA transfection, we quantified gene expression level to verify VNP20009-shSLC7A11 mediates SLC7A11 gene silencing in BGC823 gastric adenocarcinoma cells. Total RNA from infected cells was extracted and cDNA was synthesized using random primers. qRT-PCR was performed with AceQ® Universal SYBR Green qPCR Master Mix (Vazyme, Nanjing, China) in a QuantStudio(TM) 6 Flex System (Applied Biosystems, America) to determined mRNA level.. For both siRNA transfection (Figure 7a) and engineered bacteria carrying shRNA-SLC7A11 (Figure 7b), SLC7A11 gene expression in tumor cells was significantly down-regulated compared with control cells. This demonstrates the feasibility of using engineered bacteria to conduct gene silence by delivering shRNA, and this system operates efficiently in our project.
Figure 7. qPCR results after bacteria infection and siRNA-SLC7A11 transfection. a. The SLC7A11 expression level of BGC-823 after siRNA transfection, control was BGC-823 without any treatment, and siRNA-contol was siRNA transfected with other genes. Results were significant between siRNA-SLC7A11 and control groups (*** p <0.001). b. The SLC7A11 expression level of BGC-823 after engineered bacteria infection with different MOI. VNP20009 without functional plasmids at an MOI of 1:5000 was used as control group. The results of VNP-shSLC7A11 (1:3000), VNP-shSLC7A11 (1:5000) and VNP-control groups have significant differences (* p <0.05, ** p <0.01)
4.Verification the functions of SLC7A11 protein in the induction of ferroptosis
Based on the above experiments, the results have shown that the cross-kingdom RNAi delivered from VNP20009-shSLC7A11 have been successfully achieved .. Next, we will verify that SLC7A11 inhibition promotes lipid peroxidation in tumor cells and accelerate the occurrence of ferroptosis, including the viability of tumor cells and the content of Glutathione (GSH) in tumor cells after bacterial infection. The results of the above several modules could be used as indicators to determine whether the down-regulation of SLC7A11 could promote the ferroptosis of tumor cells.
4.1 Cell viability detection
Cell viability was measured using the CCK-8 reagent after bacterial infection. BGC-823 cells were cultured in 96-well plates, and engineered bacteria was added to it with MOI at 1:500, 1:1500 and 1:3000 respectively. Bacteria and BGC-823 cells were co-cultured at MOI of 1:3000 as negative control, and siRNA transfection as positive control. We used microplate reader to measure light absorption values at 450nm, and then calculated the percentage of surviving cells in each well. The results are shown that after siRNA-SLC7A11 transfection and engineered bacterial infection, the proportion of surviving cells were below 50% compared to the control cells, indicating that the down-regulation of SLC7A11 could significantly inhibit the cellular activity of tumors (Figure 8.).
Figure 8. Results of cell viability test after bacteria infection. The siRNA-contol is the siRNA transfected with the other genes. The results between siRNA-SLC7A11 and siRNA-contol groups have significant differences; and results between engineered bacteria with different MOI and VNP20009 (1:3000) infection have significant differences (****p<0.0001).
4.2 Detection of intracellular GSH content in tumor cells
To verify whether the down-regulation of SLC7A11 has an effect on the Xc- system /GSH/GPX4 pathway in tumor cells, we detected the intracellular GSH content after bacterial infection. To achieve this goal, we firstly established a standard curve of GSH (Figure 9a). According to this standard curve, we calculated the GSH content in the supernatant after lysis of the experimental and control cells by combining the absorptive value of the treated samples at 412 nm. Compared with the control group infected with VNP20009 without functional plasmid, the GSH content within BGC-823 after VNP20009-shSLC7A11 infection was significantly reduced (Figure 9b). This indicates that the metabolic pathway of cysteine and other substances in tumor cells is effectively inhibited by the down-regulation of SLC7A11 expression.
Figure 9. Results of the GSH detection. a. Standard curve of the GSH content varying with the absorbance value; b. The intracellular GSH content in BGC-823 cells after VNP20009 and engineered bacteria infections
The above results all indicate that the engineered VNP20009 carrying functional plasmids can invade tumor cells and deliver shRNA into tumor cells to silence the expression of SLC7A11. Down-regulation of SLC7A11 will inhibit Xc- system /GSH/GPX4 pathway, leading to the accumulation of intracellular lipid peroxides, and ultimately promote ferroptosis of tumor cells. This suggests that the use of engineered bacteria-mediated RNAi is an effective way to silence intracellular gene expression, which will provide a new approach for cancer treatment.
Sequence and Features
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