Part:BBa_K5082015
pMIR-EIF3B-MUT
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. To validate our theory, we deleted and altered a few nucleotides in the EIF3B-HSU sequence to form the EIF3B-MUT sequence. Afterward, we constructed the pMIR-EIF3B-MUT plasmid.
Design
The pMIR-EIF3B-MUT plasmid is constructed by fusing the EIF3B-MUT sequence BBa_K5082006) into the pMIR vector backbone (BBa_K5082010). The EIF3B-MUT sequence is inserted downstream the luciferase reporter gene contained in the plasmid backbone. Upon transcription, the EIF3B-MUT sequence will not form a highly structured 3’UTR (HSU) structure downstream the luciferase gene due to the deletion and alteration of specific nucleotides. Therefore, G3BP1 proteins will not affect mRNA degradation and translation. The plasmid map for pMIR-EIF3B-MUT is shown in Figure 1.
Figure 1. pMIR-EIF3B-MUT plasmid map.
Result
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-MUT 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-MUT plasmid measured using SpectraMax i3.
Table 1. Luciferase activity of cell lines transfected with pMIR-EIF3B-MUT plasmid measured using SpectraMax i3.
As shown in Figure 2 and Table 1, there is no significant difference in luciferase activity between all three cell lines. This suggests that the different G3BP1 concentrations among the cell lines do not have an effect on mRNA without HSU regions, proving that our theory was correct.
References
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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