Team:Nanjing NFLS/Demonstrate

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• Home
• Project
  • Description
  • Background
  • Design
  • Experiment
  • Demonstrate
  • Notebook
  • Vision
• Parts
• Safety
• Model
• Team
  • Team Members
  • Attributions
  • Collaborations
• Human Practices
  • Overview
  • Integrated Human Practices
  • Public Engagement
• Judging

Demonstrate

• Tumor-specific promoter function verification
• miRNA inhibition verification
• “AND” GATE function verification

Tumor-specific promoter function verification

Tumor-specific promoters are used to regulate the expression of the gene of HBsAg in tumor cells, which is an important means to achieve target therapy. In order to verify the performance of two tumor-specific promoters we constructed, we put hTERT and HULC into eukaryotic expression vector PGL3 which contains the reporter luciferase. The luciferase reporter gene vector PGL3 provides an analytical basis for quantifying the activity of various factors that may regulate mammalian gene expression. This detection method with the dual-luciferase reporter gene largely improves the detection sensitivity. Therefore, we can evaluate and compare the effectiveness of hTERT and Hulc promoters by measuring the expression of Luc in human HepG2 cells and normal cultured cells - 293T.

Table1 Relative promoter activity of hTERT, HULC and SV40 promoters

Vector	HepG2	293T
PGL3-Basic	22.02	1.32
PGL3-hulc	237.38	130.39
PGL3-htert	597.56	2.30
PGL3-Control	2782.88	3318.18

The data is the mean of the relative Luc activity RL/RL (%) measured.

The PGL3-Basic is the negtive control group while the PGL3-Control is the positive control group. From the table shown above, the PGL3-Basic group without any promoter showed a low relative Luc activity, indicating that the entire detection system exhibited no interference. The PGL3-Control group with SV40 promoter has good priming performance in both engineered HEK293T cell line and tumor cell HepG2 cells: maintains a good value of 3000% of relative Luc activity. However, it does not have the tumor-specific selection ability. We found that the Hulc promoter's ability to initiate tumor cells is twice that in normal cells, indicating that Hulc is an ideal promoter in the AND GATE system.

The hTERT promoter works better. First, in terms of specificity, its relative Luc activity was 597.56% in tumor cells, apparent compared to 2.30% in 293T cells, which showed perfect specific priming ability. In addition, the hTERT promoter has higher activity than Hulc. In HepG2 cells, the activity of the hTERT promoter is 2.52 times that of Hulc, which is equivalent to  21% of SV40.

In summary, both Hulc and hTERT have tumor-specific activity and the hTERT promoter performance is significantly stronger than Hulc. Therefore, we chose hTERT as the promoter in HBsAg expression system and Hulc in specific regulation system.

 

miRNA inhibition verification

MiRNA can silence the expression of HBsAg and takes a core role in safety regulation. Therefore, verification of miRNA function is necessary and significant. We transfected the plasmids containing miR-HBsAg-70, miR-HBsAg-95, or miR-HBsAg-125 into HepG2 cells, and observed the expression of EmGFPs under a fluorescence microscope after 36 hours. To determine the nuclear position, we used DAPI dye to stain the nuclear DNA. All EmGFPs were taken with the same parameter settings to ensure the comparability of the experimental results.

Figure 1 Each of the EmGFP images uses ISO400 and 10s exposure time length to ensure that each picture is consistent.

A: pCDNA6.2-hTERT- HBsAg EmGFP-miR-Control

B: pCDNA6.2-hTERT-HBsAg-EmGFP-miR-70

C: pCDNA6.2-hTERT-HBsAg-EmGFP-miR-95

D: pCDNA6.2-hTERT-HBsAg-EmGFP-miR-125

From the above data, we found that the fluorescence intensity of the control group without inhibitory miR-HBsAg component maintains at a relatively normal level. Since mRNA at the upstream HBsAg fragment (Trojan horse) is not inhibited by miRNA, the EmGFP at downstream can normally express. When inhibitory parts miR-HBsAg-70, miR-HBsAg-95, miR-HBsAg-125 existed, we could clearly see the decrease in fluorescence brightness. The low fluorescence intensities of downstream EmGFPs indicated the regulatory part inhibited the mRNA of HBsAg (Trojan horse), resulting in a decrease in fluorescence brightness.

Therefore, the verification experiment proved our plasmid could work as expected. All the miRNA we predicted could regulate the expression of HBsAg. In other words, it can switch the Trojan Horse expression system to the "off" state.

 

“AND” GATE function verification

"AND" GATE is one of the core designs of CITHA. It can regulate Trojan Horse expression in order to prevent appearance of HBsAg in normal tissue cells. Therefore, the verification of "AND" GATE is of significance and necessity. We transfected the plasmids containing respective miR-HBsAg-70, ceR-HBsAg-70; miR-HBsAg-95, ceR-HBsAg-95, and miR-HBsAg-125, ceR-HBsAg-125 into HepG2 cells, and observed the expression of EmGFPs under a fluorescence microscope after 36 hours. To determine the nuclear position, we used DAPI dye to stain the nuclear DNA. All EmGFPs were taken with the same parameter settings to ensure the comparability of the experimental results.

Figure 2 Each of the EmGFP images uses ISO400 and 10s exposure time length to ensure that each picture is consistent.

A: pCDNA6.2-hTERT-HBsAg-EmGFP-miR-70

B: pCDNA6.2-hTERT-HBsAg-EmGFP-miR-70 co-transformed with pCDNA3.1(+)-Hulc-CeR-70

C: pCDNA6.2-hTERT-HBsAg-EmGFP-miR-95

D: pCDNA6.2-hTERT-HBsAg-EmGFP-miR-95 co-transformed with pCDNA3.1(+)-Hulc-CeR-95

E: pCDNA6.2-hTERT-HBsAg-EmGFP-miR-125

F: pCDNA6.2-hTERT-HBsAg-EmGFP-miR-125 co-transformed with pCDNA3.1(+)-Hulc-CeR-125

For the data above, we can see that the green fluorescent protein EmGFP was inhibited by miRNA in A, C, and E so the fluorescence brightness was dark. Each group exhibited a different state after the co-transfection of "on" part CeR-HBsAg. In B and F, we co-transfected pCDNA3.1(+)-Hulc-CeR-70 and pCDNA3.1(+)-Hulc-CeR-125. CeRNA plays a competitive binding role for hTERT-HBsAg-EmGFP-miR-70 and pCDNA6.2-hTERT-HBsAg-EmGFP-miR-125, and thus, miRNA can binds to the corresponding ceRNA like sponge adsorption. As a result, ceRNA reduced miRNA regulation ability for HBsAg (Trojan horse). Since it reduced the inhibition of miRNA, the expression system switched to the "on" state, or be activated. The fluoresecence intensity in B and F was significantly higher than in Figure A and E, indicating that "AND" GATE system works normally and the "Trojan Horse" expressed as expected.

In Figure D, though we co-transfected plasmid pCDNA6.2-hTERT-HBsAg-EmGFP-miR-95 and pCDNA3.1(+) -Hulc-CeR-95, the fluorescent intensity of EmGFP was not significantly improved, which showed relatively low efficiency of CeR-HBsAg-95. It exhibited that the ceR-95ceRNA was not strong enough for competitive binding so the expression system was still in the "off" state. The result matches the prediction in our model: binding sites 70 and 125 are more powerful than 95. Therefore, in subsequent experiments, we will select CeR-HBsAg-70 and CeR-HBsAg-125 components for experiments. We successfully verified the effectiveness of the "AND" GATE, which ensured safety.