Part:BBa_K5304006

T7::sh-PGC1α

The T7 promoter::sh-PGC1α composite part utilizes the T7 promoter to drive the expression of short hairpin RNA (shRNA) targeting the PGC1α gene, a key regulator of mitochondrial biogenesis linked to 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. PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1 alpha) plays a critical role in mitochondrial energy metabolism and is implicated in cancer cell survival and resistance to chemotherapy. Its overexpression has been shown to protect tumor cells from chemotherapy-induced apoptosis by enhancing mitochondrial biogenesis. Targeting PGC1α through RNA interference offers a potential therapeutic strategy to reduce drug resistance and promote cell death in resistant cancer cells.

Usage and Biology

The T7 promoter::sh-PGC1α composite part is designed to reduce the expression of the PGC1α gene through the production of shRNA in systems expressing T7 RNA polymerase. The shRNA specifically targets PGC1α mRNA for degradation, leading to a decrease in PGC1α protein levels in tumor cells. By knocking down PGC1α, the part aims to enhance chemotherapy sensitivity by disrupting mitochondrial biogenesis, thus promoting apoptosis in resistant cancer cells. The T7 promoter ensures high-level production of shRNA when used in conjunction with T7 RNA polymerase, providing robust and specific gene silencing.

Design

This composite part consists of the T7 promoter linked to an shRNA sequence targeting PGC1α. The T7 promoter requires T7 RNA polymerase for activation, ensuring controlled and efficient shRNA production. The shRNA is designed to target PGC1α mRNA, reducing its expression in chemotherapy-resistant cancer cells. The sequence was incorporated into a suitable plasmid for experimental validation.

Figure 1. plasmid pSilencer-PGC1α.

Validation of PGC1α Knockdown by χ11803/pSilencer-PGC1α

We used SKOV3/CDDP and A2780/CDDP cells for further validation of the system's effects. The sensitivity of the tumor cells to chemotherapy drugs was assessed using CCK-8 assays. The χ11803/pSilencer-PGC1α treated group displayed significantly lower IC50 values for cisplatin (CDDP) in both SKOV3/CDDP and A2780/CDDP cells, indicating enhanced chemotherapy sensitivity.

Figure 2. CCK-8 Assay to Assess SKOV3/CDDP and A2780/CDDP Cells Sensitivity to Cisplatin.

Apoptosis was measured using Annexin V/PI staining and flow cytometry. Tumor cells treated with χ11803/pSilencer-PGC1α and cisplatin showed a higher apoptosis rate compared to other groups, demonstrating the knockdown's effect on promoting apoptosis.

Figure 3. Annexin V/PI Staining to Assess the Effect on Cell Apoptosis.

Western blot analysis showed that the significant increase in caspase-9, cleaved-caspase-3, and BAX expression, along with the decrease in BCL-2 expression, was primarily observed when χ11803/pSilencer-PGC1α was combined with cisplatin. This further highlights the synergistic effect of PGC1α knockdown and cisplatin in inducing apoptosis in cisplatin-resistant ovarian cancer cells.

Figure 4. Western Blot Analysis of Apoptosis-Related Protein Expression.

These results confirm that our system targeting PGC1α successfully reduces chemotherapy resistance by promoting apoptosis in drug-resistant ovarian cancer cells.

Conclusion and Outlook

In conclusion, the outcomes of PGC1α knockdown by χ11803/pSilencer-PGC1α was successfully validated through various methods, demonstrating that the system successfully enhanced chemotherapy sensitivity and increased apoptosis rates. The significant reduction in PGC1α 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]