Difference between revisions of "Part:BBa K3160021"
 
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The expression of miR-21 and miR-17 is associated with all stage of gastric cancer, especially metastasis stage [1, 2].  
 
The expression of miR-21 and miR-17 is associated with all stage of gastric cancer, especially metastasis stage [1, 2].  
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We constructed this mCherry–miR-21-17 sensor  [https://parts.igem.org/Part:BBa_K3160021 K3160021] )] plasmid 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.   
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We constructed this mCherry–miR-21-17 sensor  [https://parts.igem.org/Part:BBa_K3160021 K3160021] ] plasmid 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.   
  
  
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1. Song JH, Meltzer SJ. MicroRNAs in pathogenesis, diagnosis, and treatment of gastroesophageal cancers. Gastroenterology. 2012 Jul;143(1):35-47.
 
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.
 
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.

Latest revision as of 09:13, 21 October 2019


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

Aim of experiment

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. We inserted miR-21-17 sensor into the 3’UTR of mcherry, which generated mcherry -miR-21-17 sensor. We constructed this part mcherry-miR-21-17 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

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 this mCherry–miR-21-17 sensor K3160021 ] plasmid 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.


T--XHD-WS-Wuhan-A--miR17-21-fig_1.jpeg

File-T--XHD-WS-Wuhan-A--miR17-21-fig3.jpeg

T--XHD-WS-Wuhan-A--miR17-21-fig2.jpeg

T--XHD-WS-Wuhan-A--miR17-21-fig3.jpeg

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.