Part:BBa_K5304005
T7 promoter::sh-CLDN6
The T7 promoter::sh-CLDN6 composite part utilizes the T7 promoter to drive the expression of short hairpin RNA (shRNA) targeting the CLDN6 gene, a key player in chemotherapy resistance. This system is designed for use with T7 RNA polymerase, enabling efficient shRNA production for gene silencing.
Background
The T7 promoter is a well-known strong promoter derived from the T7 bacteriophage and is specifically activated by T7 RNA polymerase. This promoter-RNA polymerase system is widely used for high-level expression of genes in prokaryotic systems, allowing for precise control of gene expression. CLDN6 (Claudin-6) is a member of the claudin family of proteins, which play crucial roles in maintaining tight junctions between cells. Emerging research has linked CLDN6 to chemotherapy resistance in certain cancers. Overexpression of CLDN6 has been associated with the protection of cancer cells against drug-induced apoptosis, making it a promising target for RNA interference-based therapies aimed at overcoming drug resistance in tumors.
Usage and Biology
The T7 promoter::sh-CLDN6 composite part is designed to reduce the expression of the CLDN6 gene through the production of shRNA in systems expressing T7 RNA polymerase. The shRNA specifically targets CLDN6 mRNA for degradation, leading to a decrease in CLDN6 protein levels in tumor cells. By knocking down CLDN6, the part aims to enhance the sensitivity of cancer cells to chemotherapy, addressing the challenge of drug resistance that often develops during treatment. The T7 promoter ensures high-level production of shRNA when used in conjunction with T7 RNA polymerase, providing strong and specific gene silencing.
Design
This composite part consists of the T7 promoter linked to an shRNA sequence targeting CLDN6. The T7 promoter requires T7 RNA polymerase for activation, ensuring controlled and efficient shRNA production. The shRNA is designed to target CLDN6 mRNA, reducing its expression in chemotherapy-resistant cancer cells. The sequence was incorporated into the plasmid pSilencer-CLDN6 for experimental validation.
Figure 1. plasmid pSilencer-CLDN6.
Validation of CLDN6 Knockdown by χ11803/pSilencer-CLDN6
To validate the knockdown efficiency of χ11803/pSilencer-CLDN6 targeting the CLDN6 gene, Western blot analysis was performed to detect changes in CLDN6 expression. We used two types of cell lines: a CLDN6-overexpressing cell line (MCF-7/CLDN6) and a drug-resistant cell line (MCF-7/MDR). The bacteria were co-cultured with the tumor cells for 5 hours, allowing the system to deliver shRNA targeting CLDN6. After incubation, protein lysates were collected from the tumor cells, and Western blot analysis was conducted to examine CLDN6 expression levels. As shown in the figure, the expression of CLDN6 was significantly reduced in the χ11803/pSilencer-CLDN6 treated group compared to the control groups, including the mock, χ11803, and si-Scramble groups.
Figure 2. Western Blot Analysis of CLDN6 Knockdown in MCF-7/CLDN6.
Figure 3. Western Blot Analysis of CLDN6 Knockdown in MCF-7/MDR.
We used MCF-7/MDR cells for further validation of the system’s effects. The sensitivity of tumor cells to chemotherapy drugs was tested. The χ11803/pSilencer-CLDN6 treated group showed a significantly lower IC50 for ADM, 5-FU, and DDP compared to the control group, indicating enhanced chemotherapy sensitivity.
Figure 4. Western Blot Analysis of CLDN6 Knockdown in MCF-7/MDR.
Western blot data showing reduced expression levels of drug resistance-related proteins, including BCRP, P-gp, and MRP-1 in MCF-7/MDR cells.
Figure 5. Western Blot Analysis of Drug Resistance-Related Protein.
Annexin V/PI staining and flow cytometry was used to measure apoptosis rates. Tumor cells treated with χ11803/pSilencer-CLDN6 and cisplatin (DDP) showed a higher apoptosis rate compared to other groups.
Figure 6. Annexin V/PI Staining to Assess the Effect on Cell Apoptosis.
The expression of cleaved-PARP and cleaved-caspase-9, markers of apoptosis, was significantly increased in the χ11803/pSilencer-CLDN6 treated group, especially when combined with ADM, further validating the effect of CLDN6 knockdown on promoting apoptosis in MCF-7/MDR cells.
Figure 7. Western Blot Analysis of Apoptosis-Related Protein.
These results confirm the potential of our system to reduce chemotherapy resistance associated with drug resistance-caused gene overexpression.
Conclusion and Outlook
In conclusion, the outcomes of CLDN6 knockdown by χ11803/pSilencer-CLDN6 was successfully validated through various methods, demonstrating that the system successfully enhanced chemotherapy sensitivity and increased apoptosis rates. The significant reduction in CLDN6 expression highlights the potential of this system to combat chemotherapy resistance linked to drug resistance-caused gene overexpression. Looking forward, the T7 expression system in combination with Salmonella represents a versatile platform with applications beyond shRNA-mediated gene silencing. By leveraging the T7 expression cassette, the system can be adapted to express a wide range of gene products, including cytotoxic proteins, immune modulators, or anticancer peptides. This modularity enables not only the silencing of drug resistance genes but also the potential to directly kill tumor cells or enhance immune responses. This part provides a valuable tool for the iGEM community, offering a flexible and expandable approach for bacterial-based therapies. Its broad applicability could contribute to advances in cancer treatment, allowing future iGEM teams to build upon this work and explore new therapeutic strategies that combine gene silencing with direct antitumor effects or immune modulation.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |