Part:BBa_K3160009

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.

Aim of experiment

The expression of miR-196a is highly associated with the early stage of gastric cancer. Here, we proposed miRNA sensor could be a monitor to reflect the change of miRNA expression in cells. After transfection of the miRNA sensors, miRNA sensors can attract specific miRNAs, inhibiting the expression of fluorescent reporter. We constructed this part pEGFP-miR-196a 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

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

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)] 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.

In order to estimate the effect of pEGFP-miR-196a sensor on reflecting the different expression of miR-196a 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 3). Taken together, these results reveal a possibility of detecting tumor cells by using pEGFP-miR-196a sensor.

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]

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.