Part:BBa_K3638003

pEGFP-miR-155-sponge-1 sensor

lncRNAs could act as sponges to compete miRNAs. miRNA Sponges contain complementary binding sites to a miRNA of interest, which inhibit miRNA activity [1]. When perfectly matched, RNA sponge site will bind to the target miRNA preventing it from binding to their target mRNAs and to perform mRNA silencing.

We designed this part with GFP to monitor the expression of miR-155 in cells.the GFP value of cells transfected with pEGFP-miR-155-sponge-1 plasmid could show the different expression of miRNAs in cells.What we expect to see is that the fluorescence value decreases as the expression of miRNA increases.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1028
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

result of BBa_K3638003

effect of pEGFP-miR-155-sponge-1 in breast cancer cells

MicroRNA-155 (miR-155) is a well-known oncogenic miRNA overexpressed in many human cancers, including breast cancer[5]. So we constructed pEGFP-miR-155-sponge-1 and try to test the possibility of detecting miR-155 expression by using this plasmid. In order to explore whether XIST correlates with pEGFP-miR-155 sensor in breast cancer cells with potential target sites. pEGFP–C1 (as negative controls), pEGFP-miR-155-sponge-1 (0.8 ug plasmids for each well) was transfected into human breast cells (MDA-MB 231 cells and MDA-MB 468 cells) in 24-well plate, respectively. After transfection, cells were examined under fluorescence microscopy (Fig.4 A, B, C, D and Table 1). The fluorescence of GFP was decreased in MDA-MB 231 cells and MDA-MB 468 cells transfected with pEGFP-miR-155-sponge-1 compared with controls (Fig. 5). In addition, we also measured the value of GFP fluorescence by plate reader (SpectraMax i3). The endogenous miR- 155 could inhibit the expression of GFP in cells transfected with pEGFP-miR-155-sponge-1 (Table 1 and Fig5). The results suggested the GFP value of cells transfected with pEGFP-miR-155-sponge-1 could show the different expression of miRNAs in cells.

Fig 4. The images of different breast cells transfected with different plasmids.

(A). pEGFP–C1 was transfected in MDA-MB 468 cells. (B). pEGFP-miR-155-sponge-1 was transfected in MDA-MB 468 cells. (C). pEGFP–C1 was transfected in MDA-MB 231 cells. (D).pEGFP-miR-155-sponge-1 was transfected in MDA-MB 231 cells.

Fig5. The value of GFP fluorescence in cells.

Different cells were transfected with pEGFP-C1 or pEGFP-miR-155-sponge-1 for 48 h.

Conclusion：

The above results show that compared with the control group, the expression of part in the cells is significantly down-regulated and it can work as expected.

Reference:

1. Ebert MS, Sharp PA. MicroRNA sponges: progress and possibilities. [J]RNA,2010,16(11):2043-50.