Team:SCUT-China B/Protumor
Tumor Specific Apoptosis
3.1 Tumor specific promoter：the Switch of Our System
Tumor specific promoters hold high activity in tumor cells , but very low or no activity in normal cells, respectively.
Therefore we applied tumor specific promoter as the switch of the project. By searching the literature, we identified two candidate promoters including the survivin[8] , the human telomerase reverse transcriptatse(hTERT)[9].
Survivin is a protein that belongs to the inhibitor of apoptosis family. The survivin protein functions to disrupt cells apoptosis and increase tumor cells growth. It is reported that survivin is expressed far more in tumor cells than in normal cells, that means survivin promoter shows high activity in tumor cells not the normal cells. Telomerase reverse transcriptase (denoted as hTERT in humans) is a catalytic subunit of the enzyme telomerase. Besides, the number of times a cell can divide mainly depends on the telomerase activity. In fact, there is a strong correlation between telomerase activity and most cancer cells, especially for lung cancer. Lung cancer is strongly associated with telomerase.
And the core promoter of hTERT includes 330 base pairs upstream of the translation start site (ATG), as well as 37 base pairs of exon 2 of the hTERT gene. The hTERT promoter is GC-rich and lacks TATA and CAAT boxes but contains many sites for several transcription factors giving indication of a high level of regulation by multiple factors in many cellular contexts.
Comparing the activities of these promoters in A549 cell line, we found that survivin promoter has higher potential to be used for our targeted lung cancer therapy.
3.2 Amplification of the expression under tumor specific promoter
3.2.1 Design Considerations
(1)Gene expression under tumor specific promoter, like survivin promoter, is not promising than other strong promoter in tumor cells.
(2)Direct activation of tumor specific promoter might lead the loss of its tumor-specifcity.
3.2.2 Design
We used tumor specific promoter, survivin promoter to express the transcription factor dCas9-VP64.
However, most of these promoters are much weaker than commonly used viral promoters like Cytomegalovirus (CMV) and early promoter and SV40 early promoter.
To increase efficiency of promoter, we applied the two-step transcription amplification system, which contains a switch and an amplifier.
3.2.2.1 survivin promoter：The Switch
According to the literature, the tumor-specificity of survivin promoter is induced by
Figure 1. dCas9-VP64-GFP expression under survivin promoter.
3.2.2.2 Bax-1 promoter: The Amplifier
Figure 2. Left: dCas9-VP64-GFP expression under Bax-1 promoter.
Right: Bax-sgRNA-1 expression under U6 promoter.
Figure 3. Up：Bax-1promoter: three protospacers for dCas9-VP64-bax-sgRNA1 targeting.
Down：The sequence of the Bax-1 promoter, characters in yellow is the protospacers complementary to the Bax-sgRNA-1.
In this system, the first step was to use tumor specific promoter, survivin promoter, to express dCas9-VP64.
The second step was to form the dCas9-VP64-sgRNA complex. And the last step was to target Bax-1 promoter which contains 3 repeated protospacer, to activate more dCas9-VP64, which was also regulated by the minimal promoter.
In our project, we gained the 980 bp survivin promoter from Genome PCR, and synthetized the Bax-1 promoter.
3.2.3 Transfection
We co-transfected the dCas-VP64-GFP expression under Bax-1 minimal promoter, and under survivin promoter, and Bax-sgRNA-1 into the A549 cells. We analyzed the fluorescence by image pro plus 6.0. The mass ratio of Bax-1-dCas9-VP64-GFP to survivin-dCas9-VP64-GFP is 1:1.
Figure 3.Left: dCas9-VP64 expression under survivin promoter and Bax-1 promoter
(B+S).Right: dCas9-VP64 expression under Bax-1 promoter(B).
Figure4. Left: dCas9-VP64 expression under survivin promoter(S). Right: dCas9-VP64 expression under EF1α promoter(E).
Figure 3. The expression of dCas9-VP64-GFP is analyzed by image pro plus 6.0 after 48h.
The dCas9-VP64-GFP expression under survivin promoter(S) was about 5% compared to EF1α promoter(E), and the expression under Bax-1 promoter(B) was about 50% compared to EF1α promoter(E). It shows that our amplifier works as expect.
And the leakage expression of Bax-1 promoter(B) is relative high compared to the expression under EF1α(B+S).
Future Work
To reduce the leakage expression of our tumor specific promoter amplifier, we plan to modify the Bax-1 promoter by reducing the number of the protospacers upstream the minimal promoter. This enables us to gain enough dCas9-VP64 while keeping the tumor-specificity of our system.
4. Discussion
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[9].Hu Z, Robbins JS, Pister A, Zafar MB, Zhang ZW, & Gupta J, et al. (2010). A modified htert promoter-directed oncolytic adenovirus replication with concurrent inhibition of tgfbeta signaling for breast cancer therapy. Cancer Gene Therapy, 17(4), 235-43.
