Part:BBa_K3638004

phage-pre-miR-155

Phage-pre-miR-155 was applied to overexpress miR-155 in cells.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

The effect of pEGFP-miR-155-sponge-1 as a monitor to detect the expression of miR-155

In order to explore whether the expression of miR-155 correlates with the GFP value of pEGFP-miR-155-sponge-1 in breast cancer cells, breast cancer cells were cotransfected with pEGFP-miR-155-sponge-1 and pre-miR-155 (overexpression of mature miR-155). After transfection, cells were examined under fluorescence microscopy (Fig. 6 A, B, C, D). The fluorescence of GFP was decreased in MDA-MB 231 cells and MDA-MB 468 cells transfected with pre-miR-155, compared with that of controls (Fig. 6). In addition, we also measured the value of GFP fluorescence by plate reader (SpectraMax i3). The down-regulation of GFP fluorescence was observed in breast cancer cells transfected with pre-miR-155, compared with controls (Fig. 7). Taken together, these results reveal pEGFP-miR-155-sponge-1 could act as a monitor to detect the expression of miR-155.

Fig 6. The images of different breast cells transfected with different plasmids.

(A). pEGFP-miR-155-sponge-1 was transfected in MDA-MB 468 cells. (B). pEGFP-miR-155-sponge-1 and pre-miR-155 were cotransfected in MDA-MB 468 cells. (C). pEGFP-miR-155-sponge-1 was transfected in MDA-MB 231 cells (D). pEGFP-miR-155-sponge-1 and pre-miR-155 were cotransfected in MDA-MB 231cells.

To further test the effect of pEGFP-miR-155-sponge-1 as a monitor to detect the expression of miR-155, cells were transfected with pEGFP-miR-155-sponge-1 with different concentration of pre-miR-155. We used the plasmid of pre-miR-155 to quantify miR-155 expression and calculate the copy numbers of miR-155 by using the formula listed below. The down-regulation of GFP fluorescence was observed in breast cancer cells transfected with different concentration of pre-miR-155 compared with normal cells (Fig. 7). Taken together, these results reveal a combination of pEGFP-miR-155 sensor and XIST. copies/ul= (6.02×1023)×(plasmids concentrations ng/ul×10-9)/(DNA length×660)

The standard curve of pEGFP-miR-155-sponge-1 was made by EXCEL (Fig8). We find that the value of fluorescence is dependent on the copy numbers of miR-155 in cells. Based on the formula, the correlation coefficient (R2 value) of pEGFP-miR-155-sponge-1 in MDA-MB 468 cells was 0.9988.

Fig 8. The standard curve of pEGFP-miR-155-sponge-1 in MDA-MB 468 cells