Part:BBa_K3160011

pEGFP-miR196a-148a sensor

The expression of miR-196a and miR-148a were found to be significantly associated with early tumor stage of gastric cancer. We designed miR-196a miR-148a sensor contain two complementary binding sites to miR-196a miR-148a which inhibit miR-196a and miR-148aexpression. The sequence of miR-196a and miR-148asensor wrere synthetised by GenScript (Shanghai, China). We inserted miR-196a miR-148a sensor into the 3' end of GFP, which generated GFP- miR-196a miR-148a sensor.

Aim of experiment

The expression of miR-196a and miR-148a were found to be significantly associated with early tumor stage of gastric cancer. We designed miR-196a-148a sensor contain complementary binding sites to miR-196a and miR-148a which inhibit miR-196a and miR-148 expression. Wo we constructed this part pEGFP-miR-196a-148a sensor and try to test the possibility of detecting tumor cells by using this part.

Methods

more information please link our wiki website https://2019.igem.org/Team:XHD-WS-Wuhan-A/experiment/results/

Results

K3160011 

1．The effect of pEGFP-miR196a-148a sensor in gastric cancer cells

The expression of miR-196a and miR-148a is highly associated with the early stage of gastric cancer [1]. So we constructed two plasmids [pEGFP-miR-196a sensor (BBa K3160009) and pEGFP-miR196a-148a sensor (BBa K3160011)] and try to test the possibility of detecting tumor cells by using these plasmids. To detect the validity of pEGFP-miR196a-148a sensor in cells, pEGFP–C1 (as negative controls), pEGFP-miR-196a sensor or pEGFP-miR-196a-148a sensor (0.8 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. 1 A, B, and C). The fluorescence of GFP was decreased in GES-1 cells transfected with pEGFP-miR-196a sensor or pEGFP-miR-196a-148a sensor compared with controls (Fig. 1 A, B, and C). Because pEGFP-miR-196a sensor contains binding sites of miR-196a and pEGFP-miR-196a-148a sensor contains binding sites of two miRNAs, the fluorescence of GFP was further decreased in GES-1 cells transfected with pEGFP-miR-196a-148a sensor compared with cells transfected with pEGFP-miR-196a sensor (Fig. 1 and table 1). In addition, we also measured the value of GFP fluorescence by plate reader (SpectraMax i3). The endogenous miR-196a and miR-48a could inhibit the expression of GFP in cells transfected with pEGFP-miR-196a sensor or pEGFP-miR-196a-148a sensor (Table 1 and Fig 2). The similar results were also observed in two gastric cancer cells (SGC-7901 and MGC-803) (Fig 1; Fig 2 and Table 1). The result suggested pEGFP-miR-196a-148a sensor can show the different expression of miRNAs in cells.

References

1. Zheng G, Xiong Y, Xu W, Wang Y, Chen F, Wang Z, Yan Z. A two-microRNA signature as a potential biomarker for early gastric cancer. Oncol Lett. 2014 Mar;7(3):679-684.

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]