Part:BBa_K3160021

MCherry-miR-21-17 sensor

The expression of miR-21 and miR-17 were found to be significantly associated with all tumor stage of gastric cancer. We designed miR-21-17 sensor contain complementary binding sites to miR-21 and miR-17 which inhibit miR-21 and miR-17 expression. The sequences of miR-21-17 sensor were synthesized by GenScript (Shanghai, China). We inserted miR-21-17 sensor into the 3’UTR of mcherry, which generated mcherry -miR-21-17 sensor.

Usage and Biology

The mcherry-miR-21-17 sensor was controlled by EF-1α promoter (EF-1 alpha -mcherry-miR-21-17). The plasmids of EF-1 alpha -mcherry-miR-21-17 were transfected into different cell lines (one is human gastric epithelial cell line. The others are gastric cancer cell lines).We measuresd the fluorescence of mcherry in different cells transfected with EF-1 alpha -mcherry-miR-21-17 for 24 h by plate reader (SpectraMax i3), which analyses the potential value of EF-1 alpha -mcherry-miR-21-17 to measure the expression of miR-21 and miR-17.

Results

The effect of mCherry-miR-21 sensor and mCherry-miR-21-17 in gastric cancer cells

The expression of miR-21 and miR-17 is associated with all stage of gastric cancer, especially metastasis stage [1, 2]. We constructed two plasmids [mCherry–miR-21 sensor (K3160020) and mCherry–miR-21-17 sensor (K3160021)] and try to test the possibility of detecting tumor cells by using these plasmids. To detect the validity of mCherry–miR-21 sensor and mCherry–miR-21-17 sensor in cells, mCherry (as negative controls), mCherry–miR-21 sensor or mCherry–miR-21-17 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 mCherry was significantly decreased in GES-1 cells transfected with mCherry–miR-21 sensor compared with controls (Fig. 1 A and B). mCherry–miR-21 sensor contains binding sites of miR-21 and mCherry–miR-21-17 sensor contains binding sites of two miRNAs. Surprisingly, mCherry–miR-21 sensor transfection induced strongest inhibition of mCherry fluorescence (Fig. 1 A, B, and C). We also measured the value of mCherry fluorescence by plate reader (SpectraMax i3) (Table 1). The similar results were also observed in two gastric cancer cells (SGC-7901 and MGC-803) (Fig. 1, 2 and Table 1). The result suggested mCherry–miR-21 sensor strongly inhibited the expression of miR-21 in cells, which imply the potential possibility of this sensor in gene therapy for gastric cancer in the future.

References

1. Song JH, Meltzer SJ. MicroRNAs in pathogenesis, diagnosis, and treatment of gastroesophageal cancers. Gastroenterology. 2012 Jul;143(1):35-47. 2. Sierzega M, Kaczor M, Kolodziejczyk P, Kulig J, Sanak M, Richter P. Evaluation of serum microRNA biomarkers for gastric cancer based on blood and tissue pools profiling: the importance of miR-21 and miR-331. Br J Cancer. 2017 Jul 11;117(2):266-273.