Engineering Success

Ras-GTPase-activating protein SH3 domain binding protein 1 (G3BP1) is a protein overexpressed in gastric cancer (GC) tumor cells [1]. Meanwhile, G3BP1 proteins have the activity to degenerate highly structured 3’UTR (HSU) structures in mRNA molecules [2].

To verify that G3BP1 is overexpressed in GC cells, we conducted a qRT-PCR test to measure the mRNA level of G3BP1 in healthy stomach mucosal cell line GES-1 and GC cell lines AGS and MGC-803. We found that G3BP1 mRNA levels were significantly higher in GC cell lines as compared to healthy stomach mucosal cell lines. The results are shown below in Figure 1.

In this project, we constructed two plasmids containing different reporter genes with HSU regions at 3’UTR, and their control counterparts without HSU regions. The plasmid maps for the above four plasmids are shown in Figure 2.

To amplify the plasmids, we transformed them into DH5α bacteria. We successfully grew bacterial colonies on the ampicillin plates, proving that the bacterial cells contained Ampicillin resistance genes and that our plasmid transfection was successful. The bacteria cultures are shown in Figure 3.

To verify that the plasmid was successfully constructed, we picked out single bacterial colonies and conducted DNA sequencing. The sequencing results were shown in Figure 4.

The expression of G3BP1 is highly associated with the early stage of GC [3]. So, we constructed plasmids [pCMV-EGFP-EIF3B-HSU (kkb website)] and tried to test the possibility of detecting tumor cells by using the plasmid. The pCMV-EGFP-EIF3B-MUT (kkb website) without HSU structure and the plasmid backbone pCMV-EGFP (kkb website) have been used as negative controls. The pCMV-EGFP-EIF3B-HSU plasmid was transfected into the three cell lines: normal gastric cell line-GES-1, gastric cancer cell line-AGS, and gastric cancer cell line-MGC-803 in 24-well plates with 0.8 ug of plasmid for each well. As negative controls, the pCMV-EGFP-EIF3B-MUT and pCMV-EGFP plasmids were also transfected into all three cell lines at the same concentration. After transfection, cells were cultured for 48 hours and examined under fluorescence microscopy (Fig. 5A-I). Because of low G3BP1 expression level in GES-1 cells, there was no significant difference between GES-1 cell lines transfected with all three plasmids (Fig. 5A-C). Because high expression of G3BP1 in GC cells, the fluorescence of GFP was significantly decreased in AGS and MGC-803 cells transfected with pCMV-EGFP-EIF3B-HSU compared with cells transfected with pCMV-EGFP and pCMV-EGFP-EIF3B-MUT plasmids (Fig. 5D-I). In addition, we also measured the value of GFP fluorescence by plate reader (SpectraMax i3) (Table 1 and Fig. 6). The result suggested that the pCMV-EGFP-EIF3B-HSU plasmid could show the different expression of G3BP1 in gastric cancer cells.

We also constructed the pMIR-EIF3B-HSU plasmid containing luciferase gene (another reporter) for our experiment. The pMIR-EIF3B-MUT without HSU structure and the plasmid backbone pMIR have been served as controls. pMIR-EIF3B-HSU (kkb website), pMIR-EIF3B-MUT (kkb website) (as negative control), or pMIR (kkb website) (as negative control) (0.8 ug plasmids for each well) was transfected into cells in 24-well plate, respectively. After transfection for 48 h, cells had been collected and lysed. The value of luciferase activities were measured by plate reader (SpectraMax i3). Consistent with our previous data, luciferase activity remained unchanged in GES-1 cells transfected with pMIR-EIF3B-HSU compared with controls (Table 2 and Fig. 7). Luciferase activity was significantly decreased in AGS and MGC-803 cells transfected with pMIR-EIF3B-HSU compared with cells transfected with pMIR and pMIR-EIF3B-MUT (Table 2 and Fig. 7). These data indicated pMIR-EIF3B-HSU can monitor the different expression of G3BP1 in gastric cancer cells.

To test the effect of pCMV-EGFP-EIF3B-HSU as a “sensor” to detect G3BP1 expression, cells were transfected with the same concentration of the pCMV-EGFP-EIF3B-HSU plasmid and different concentrations of pHAGE-G3BP1 plasmids which overexpress G3BP1 proteins. The plasmid map of pHAGE-G3BP1 is shown in Figure 8. We used the plasmid of pHAGE-G3BP1 to quantify G3BP1 expression and calculate the copy numbers of G3BP1 by using the formula listed below.

copies/μl= (6.02×1023)×(plasmids concentrations ng/μl×10-9)/(DNA length×660)

The relationship between fluorescence value and pHAGE-G3BP1 plasmid concentration are shown in Table 3. The standard curve of pCMV-EGFP-EIF3B-HSU sensor was made by Excel (Fig. 9). We found that the fluorescence value of cells had a strong negative correlation with G3BP1 concentration. Based on the formula, the correlation coefficient (R2 value) of pCMV-EGFP-EIF3B-HSU sensor was 0.9893. Therefore, fluorescence values can reflect G3BP1 concentration. As luciferase and GFP are affected by G3BP1 proteins through similar measures, luciferase activity, presumably, can also reflect G3BP1 concentration.