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.
Usage and Biology
Aim of experiment
Our project constructed four miRNA sensors which specific miRNA binding sites were inserted the 3’UTR of the fluorescent reporter (eGFP or mCherry) for the detection miRNA expression in different cell lines and offered a non-invasive and highly sensitive approach for early diagnosis and treatment of gastric cancer in the future. miRNA sensor, which contains multiple target sites complementary to a miRNA of interest, is used to be a inhibitor of miRNA. 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. The expression of specific miRNAs should be monitored by measuring the expression of fluorescent reporter.
Methods
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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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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