Difference between revisions of "Part:BBa K3160009"

Revision as of 11:42, 18 October 2019

pEGFP-miR-196a sensor

The expression of miR-196a was found to be significantly associated with all tumor stage of gastric cancer. We designed miR-196a sensor contain two complementary binding sites tomiR-196a1, which inhibit miR-196a expression. The sequence of miR-196a sensor was synthetised by GenScript (Shanghai, China). We inserted miR-196a sensor into the 3' end of GFP, which generated GFP-miR-196a sensor.

Usage and Biology

The expression of miR-196a is highly associated with the early stage of gastric cancer [1]. So we constructed the plasmid [pEGFP-miR-196a sensor (BBa K3160009 website)] and try to test the possibility of detecting tumor cells by using the plasmid. To detect the validity of pEGFP-miR196a sensor in cells, pEGFP–C1 (as negative controls), pEGFP-miR-196a 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). 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.

Sequence and Features