Team:YiYe-China/Improvement
Results
1.Construction of the plasmids
The miRNA “ sponge” method was introduced by several years ago as a means to create continuous miRNA loss of function in cell lines and transgenic organisms. More effective are sponges containing bulged sites that are mispaired opposite miRNA positions 9-12 (Ebert et al. 2007, Gentner et al. 2009), presumably because they form a more stable interaction with the miRNA. pEGFP-miR196a-148a 2.0(Part K3822005) was designed based on the principle of effective sponges containing bulged sites (Fig. 1). We compare pEGFP-miR196a-148a 2.0 with pEGFP-miR196a-148a 1.0 (Part K3160007,designed by a 2019 iGEM team) on monitoring the expression of miRNAs in gastric cancer cells.
Fig 1. Pairing of a miRNA with a bulged sponge site shows mismatches opposite miRNA nucleotides.
The plasmids of pEGFP-miR-196a-148a 2.0 was synthesized by Nanjing Qingke Biotechnology Corporation. The electrophoresis of the plasmids was showed in Fig 2.
Fig 2. Electrophoresis of pEGFP-miR-196a-148a 2.0.
2. The effect of pEGFP-miR-196a-148a 1.0 and pEGFP-miR-196a-148a 2.0 in gastric cancer cells
To detect the validity of pEGFP-miR-196a-148a 1.0 and pEGFP-miR196a-148a 2.0 in cells, pEGFP–C1 (as negative controls), pEGFP-miR-196a-148a 1.0 or pEGFP-miR-196a-148a 2.0 (0.5 ug plasmids for each well) was transfected into human gastric epithelial cells (GES-1 cells) in 24-well plate, respectively. After transfection, cells were examined under fluorescence microscopy (Fig 3A-C). The fluorescence of GFP was decreased in GES-1 cells transfected with pEGFP-miR-196a-148a 1.0 and pEGFP-miR-196a-148a 2.0 compared with controls (Fig 3 A-C). Because pEGFP-miR-196a-148a 2.0 contains more effective binding sites than pEGFP-miR-196a-148a 1.0, the fluorescence of GFP was further decreased in GES-1 cells transfected with pEGFP-miR-196a-148a 2.0 compared with that in cells transfected with pEGFP-miR-196a-148a 1.0 (Fig. 3 and Table 1). In addition, we also measured the value of GFP fluorescence by plate reader (SpectraMax i3) (Fig. 3-4 and Table. 1). The similar results were also observed in SGC-7901 cells (gastric cancer cell line) (Fig. 3-4, and Table 1). Compared with pEGFP-miR-196a-148a 1.0, pEGFP-miR-196a-148a 2.0 is more effective to be inhibited by the endogenous miRNAs (Fig. 3 and Table. 1).
Fig 3. The images of different gastric cells transfected with different plasmids. (A). pEGFP–C1 (control) was transfected in GES-1 cells. (B). pEGFP-miR-196a-148a 1.0 was transfected in GES-1 cells. (C). pEGFP-miR-196a-148a 2.0 was transfected in GES-1 cells. (D). pEGFP–C1 (control) was transfected in SGC-7901 cells. (E). pEGFP-miR-196a-148a sensor 1.0 was transfected in SGC-7901 cells. (F). pEGFP-miR-196a-148a sensor 2.0 was transfected in SGC-7901 cells.
Table 1. The value of GFP fluorescence in cells. 
Fig 4. The value of GFP fluorescence in cells. Cells were transfected with pEGFP–C1, pEGFP-miR-196a-148a 1.0 or pEGFP-miR-196a-148a 2.0 for 24 h. GFP fluorescence was measured by SpectraMax i3.
In order to estimate the effect of pEGFP-miR-196a-148a sensor 1.0 and pEGFP-miR-196a-148a sensor 2.0 on reflecting the different expression of miR-196a and miR-148a in gastric cancer cells compared with normal cells, we reanalyzed the data of table1. The down-regulation of GFP fluorescence was observed in gastric cancer cells transfected with miRNA sensors compared with normal cells (Fig 5). Taken together, these results reveal a possibility of detecting tumor cells by using pEGFP-miR-196a-148a sensor 2.0.
Fig 5. The value of GFP fluorescence in different cells transfected with pEGFP –C1, pEGFP-miR-196a-148a 1.0 or pEGFP-miR-196a-148a 2.0.
3. The effect of miR-196a-148a 2.0 as a monitor to detect the expression of miR-148a
To test the effect of miR-196a-148a 2.0 as a monitor to detect the expression of miR-148a, cells were transfected with pEGFP-miR-196a-148a 1.0 or pEGFP-miR-196a-148a 2.0 with different concentration of pHAGE-pre-miR-148a (overexpression of miR-148a). We used the plasmid of pHAGE-pre-miR-148a to quantify miR-148a expression and calculate the copy numbers of miR-148a by using the formula listed below. copies/ul= (6.02×1023)×(plasmids concentrations ng/ul×10-9)/(DNA length×660)
Table 2. The value of eGFP fluorescence of pEGFP-miR-196a-148a 1.0. 
Table 3. The value of eGFP fluorescence of pEGFP-miR-196a-148a 2.0 
The standard curve of miR-196a-148a sensor 1.0 was made by EXCEL (Fig. 6). We find that the value of fluorescence is dependent on the copy numbers of miR-148a in cells. Based on the formula, the correlation coefficient (R2 value) of miR-196a-148a sensor was 0.9462, the slope of it was -29851.
Fig 6. The standard curve of miR-196a-148a 1.0.
The standard curve of miR-196a-148a 2.0 was also made by EXCEL (Fig. 7). We find that the value of fluorescence is also dependent on the copy numbers of miR-148a in cells. Based on the formula, the correlation coefficient (R2 value) of miR-196a-148a sensor was 0.9578, the slope was -37508. By comparing the slope, we found that miR-196a-148a sensor 2.0 is more suitable for the detection of the expression of miR-148a.
Fig 7. The standard curve of miR-196a-148a 2.0.
Reference:
[1] Ebert et al. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells.Nat Methods. 2007 Sep;4(9):721-6.doi: 10.1038/nmeth1079. Epub 2007 Aug 12.
Improvement
This year the part K 3822005 we designed is an improvement of Part K3160007, which was designed by waorlshaper-Wuhan in 2019 iGEM competition
- 1.Construction of the plasmids
- 2. The effect in gastric cancar cells
- 3. The effect of part k3822005 as a monitor
- Reference