Part:BBa_K5082014
pMIR -EIF3B-HSU
Usage and Biology
Previously, it has been reported that gastric cancer (GC) tissue cells overexpress the oncogene G3BP1 [1]. Meanwhile, G3BP1 proteins have been proved to have HSU mRNA degrading activity [2]. Based on these findings, we designed and constructed the pMIR-EI3B-HSU plasmid to utilize G3BP1’s HSU mRNA-degrading activity to detect G3BP1 concentration and thereby diagnose GC.
Design
The pMIR-EIF3B-HSU plasmid is constructed by fusing the EIF3B-HSU sequence (BBa_K5082005) into the pMIR vector backbone (BBa_K5082010). The EIF3B-HSU sequence is inserted downstream the luciferase reporter gene contained in the plasmid backbone. Upon transcription, the EIF3B-HSU sequence will form a highly structured 3’UTR (HSU) structure downstream the luciferase gene. G3BP1 proteins can bind with HSU structures and recruit proteins to degrade HSU mRNA. Therefore, by detecting luciferase concentrations, we can indirectly measure G3BP1 concentration and thereby diagnose GC. The plasmid map for pMIR-EIF3B-HSU is shown in Figure 1.
Figure 1. pMIR-EIF3B-HSU plasmid map.
Results
In our experiments, we used three cell lines. We used the GES-1 cell line to simulate healthy stomach cells and used the MGC-803 and AGS cell lines to simulate GC tissue cells. After transfecting the pMIR-EIF3B-HSU plasmid into the three cell lines, we cultured the cells under identical conditions for 48 hours. Afterward, we lysed the cells and measured their luciferase activity using SpectraMax i3. The results are shown in Figure 2 and Table 1.
Figure 2. Luciferase activity of cell lines transfected with pMIR-EIF3B-HSU plasmid measured using SpectraMax i3.
Table 1. Luciferase activity of cell lines transfected with pMIR-EIF3B-HSU plasmid measured using SpectraMax i3.
As shown in Figure 2, the luciferase activity in the GES-1 cell line is much higher than that of the two GC cell lines. This trend suggests that the GES-1 cell line contains a higher luciferase protein concentration than the two GC cell lines. Given the same concentration of pMIR-EIF3B-HSU plasmids, the higher luciferase protein concentrations in the GES-1 cell line indicates more mRNA translation, longer-lasting mRNA and thus lower G3BP1 concentrations, as compared to the two GC cell lines. This is consistent with our hypothesis, validating our theory and proving that our sensor is effective.
Reference
References [1] Ge, Yidong et al. “The roles of G3BP1 in human diseases (review).” Gene vol. 821 (2022): 146294. doi:10.1016/j.gene.2022.146294 [2] Xiong, Rui et al. “G3BP1 activates the TGF-β/Smad signaling pathway to promote gastric cancer.” OncoTargets and therapy vol. 12 7149-7156. 2 Sep. 2019, doi:10.2147/OTT.S213728
Sequence and Features
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