Team:XHD-WS-Wuhan-A/experiment/results/
Results
1. Construction of the plasmids
We constructed five plasmids pEGFP-miR-196a sensor, pEGFP-miR196a-148a sensor, mCherry-miR-21 sensor, mCherry-miR-21-17 sensor, and Phage-miR-148a. The plasmids of pEGFP-miR-196a sensor, pEGFP-miR196a-148a sensor, mCherry-miR-21 sensor, and mCherry-miR-21-17 sensor were used to monitor the expression of miRNAs in gastric cancer cells. We also used the plasmid of Phage-miR-148a to overexpress miR-148 in cells.
1.1 Construction of pEGFP-miR-196a sensor and mCherry-miR-21 sensor
1.1.1 Amplification of miR-196a sensor and miR-21-5p sensor fragment.
To monitor the expression level of miR-21 and miR-196a in different cells, we constructed two plasmids (pEGFP-miR-196a sensor and mCherry-miR-21 sensor) in the 3’UTR of GFP or mCherry. Firstly, we designed the miR-21 sensor contained two miR-21 binding sites based on the sequence of hsa-miR-21 according to the previous study (Ebert MS, Neilson JR, Sharp PA. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods. 2007 Sep;4(9):721-6). And the miR-196a sensor contained two miR-196a binding sites was also designed. Then, we amplified miR-196a sensor and miR-21 sensor using primers (Fig. 1). After that, we purified the PCR products by PCR Purification Kit. miR-196a sensor wasdigested by restriction enzymes Xho I and Hind III. And miR-21 sensor was digested by Asc I and Avr II.
Fig 1. Electrophoresis of PCR products of miR-196a sensor (A) and miR-21 sensor (B).
1.1.2 Digested pEGFP-C1 and pAAV-EF1a-mCherry-IRES-Flpo vector (mCherry vectors)
We know that microRNA can bind the 3 'UTR of genes to regulate the expression. So we choose EGFP and mCherry to be the detector of miR-196a and miR-21,respectively. We digested the pEGFP-C1 vectors with restriction enzymes Xho I and Hind III and digested the mCherry vectors with restriction enzymes Asc I and Avr II (Fig 2).
Fig 2. Electrophoresis of the digested mCherry(A) and pEGFP-C1(B)vector.
1.1.2 Ligation of purified miR-196a sensor fragments to pEGFP-C1 vector and miR-21 sensor fragments to mCherry vector.
The fragments of miR-196a sensor were ligated to pEGFP-C1 vector and the fragments of miR-21 were ligated to mCherry vector. Then we selected the positive clones by using sequencing and electrophoresis (Fig 3 and 4)
mCherry-miR-21 sensor
Fig 3. Sequencing of pEGFP-miR-196a sensor and mCherry-miR-21 sensor
Fig 4. Electrophoresis of mCherry-miR-21 sensor (A) and pEGFP-miR-196a sensor (B).
1.1.4 The solid plates of each plasmid constructed (Fig 5)
Fig 5. Solid plates of pEGFP-miR-196a sensor and mCherry-miR-21 sensor.
1.2 Construction of mCherry-miR-21-17 sensor and pEGFP-miR-196a-148a sensor
The plasmids of mCherry-miR-21-17 sensor and pEGFP-miR-196a-148a sensor were synthesized by Nanjing Qingke Biotechnology Corporation. The electrophoresis of these two plasmids was showed in Fig 6.
mCherry-miR-21-17 sensor
pEGFP-miR-196a-148a sensor
Fig 6. Electrophoresis of mCherry-miR-21-17 sensor
and pEGFP-miR-196a-148a sensor.
1.3 Construction of miR-148a precuser expression plasmid
1.3.1 Amplification of miR-148a precuser fragment.
To set up a model to determent the amount of miR-148a in cells, we made a construct to express miR-148a. We designed the specific primers of human miR-148aprecuser based on the sequences of miR-148a precuser (http://www.mirbase.org/cgi-bin/get_seq.pl?acc=MI0000253) and amplified 68 bp fragment by PCR. After that, we digested the purified PCR products with restriction enzymes Sal I and Not I (Fig 7).
Fig 7. Electrophoresis of miR-148a precouser PCR product. The lanes 1-3 showed the miR-148a precouser bands.
1.3.2 Digested pHAGE-puro vector
For eukaryotic expression of has-miR-148a in cells, we digested the eukaryotic expression vector pHAGE-puro with the same restriction enzymes Sal I and Not I (Fig 8).
Fig 8. Electrophoresis of the digested pHAGE-puro vector.
1.2.3 Ligation of purified miR-148a-precuser fragment to pHAGE-puro vector
To sequence the miR-148a precuser cloning we got, the purified miR-148a precuser fragment was ligated to pHAGE-puro vector. Then we selected the positive clones by PCR and sequencing (Fig 9 and 10).
Fig 9. Electrophoresis of the PCR identified production of pHAGE- pre-miR-148a vector.
Fig 10. Electrophoresis of pHAGE-pre-miR-148a vector.
2. The effect of pEGFP-miR-196a sensor and pEGFP-miR196a-148a sensor in gastric cancer cells
The expression of miR-196a and miR-148a is highly associated with the early stage of gastric cancer. So we constructed two plasmids [pEGFP-miR-196a sensor (kkb website) and pEGFP-miR196a-148a sensor (kkb website)]and try to test the possibility of detecting tumor cells by using these plasmids. To detect the validity of pEGFP-miR-196a sensor and pEGFP-miR196a-148a sensor in cells, pEGFP–C1 (as negative controls), pEGFP-miR-196a sensor or pEGFP-miR-196a-148a 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. 14 A, B, and C). 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. 14 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. 14 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 15). The similar results were also observed in two gastric cancer cells (SGC-7901 and MGC-803) (Fig 14; Fig 15 and Table 1). The result suggested pEGFP-miR-196a sensor and pEGFP-miR-196a-148a sensor can show the different expression of miRNAs in cells.
Fig 14 . The images of different gastric cells transfected with different plasmids.
(A). pEGFP–C1 was transfected in GES-1 cells. (B). pEGFP-miR-196a-sensor was transfected in GES-1 cells. (C). pEGFP-miR-196a-148a sensor was transfected in GES-1 cells. (D). pEGFP –C1 was transfected in MGC-803 cells. (E). pEGFP-miR-196a-sensor was transfected in MGC-803 cells. (F). pEGFP-miR-196a-148a sensor was transfected in MGC-803 cells. (G). pEGFP–C1 was transfected in SGC-7901 cells. (H). pEGFP-miR-196a-sensor was transfected in SGC-7901 cells. (I). pEGFP-miR-196a-148a sensor was transfected in SGC-7901 cells.
Table 1. The value of GFP fluorescence in cells.
|
Fluorescence value of GFP |
Average |
SD |
||||
|
GES-1 |
control |
301563.7 |
359556.7 |
344156.7 |
335092.4 |
24527.8 |
|
miR-196a sensor |
301741.7 |
336146.7 |
239592.7 |
292493.7 |
39956.8 |
|
|
miR-196a-148a sensor |
323108.7 |
303552.7 |
290000.7 |
305554.0 |
13590.2 |
|
|
MGC-803 |
control |
390604.4 |
312486.1 |
304467.1 |
335852.5 |
38853.6 |
|
miR-196a sensor |
227681.3 |
262490.9 |
169285.8 |
219819.3 |
38454.8 |
|
|
miR-196a-148a sensor |
162634 |
64496.59 |
140128.1 |
122419.6 |
41975.6 |
|
|
SGC-7901 |
control |
335652.3 |
309507.9 |
309919.9 |
318360.0 |
12228.6 |
|
miR-196a sensor |
74471.35 |
115007.4 |
101757.4 |
97078.7 |
16876.2 |
|
|
miR -196a-148a sensor |
78326.9 |
83842.4 |
85962.85 |
82710.7 |
3218.4 |
|
Fig 15. The value of GFP fluorescence in cells.
Different cells were transfected with pEGFP –C1, pEGFP-miR-196a-sensor or pEGFP-miR-196a-148a sensor for 24 h.
In order to estimate the effect of pEGFP-miR-196a sensor and pEGFP-miR-196a-148a sensor on reflecting the different expression of miR-196a and miR-148a 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 16). Taken together, these results reveal a possibility of detecting tumor cells by using pEGFP-miR-196a sensor or pEGFP-miR-196a-148a sensor.
Fig 16. The value of GFP fluorescence in different cells transfected with pEGFP –C1, pEGFP-miR-196a-sensor or pEGFP-miR-196a-148a sensor.
2. The effect of mCherry-miR-21 sensor and mCherry-miR-21-17 in gastric cancer cells
The expression of miR-21 and miR-148a is associated with all stage of gastric cancer, especially metastasis stage. We constructed two plasmids (mCherry–miR-21 sensor and mCherry–miR-21-17 sensor) 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. 17 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. 17 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. 17 A, B, and C). We also measured the value of mCherry fluorescence by plate reader (SpectraMax i3) (table 2). The similar results were also observed in two gastric cancer cells (SGC-7901 and MGC-803) (Fig. 17, 18 and table 2). 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.
Fig 17. The images of different gastric cells transfected with different plasmids.
A. mCherry was transfected in GES-1 cells B. mCherry-miR-21 sensor was transfected in GES-1 cells. C. mCherry -miR-21-17 sensor was transfected in GES-1 cells. D. mCherry was transfected in MGC-803 cells. E. mCherry-miR-21 sensor was transfected in MGC-803 cells. F. mCherry -miR-21-17 sensor was transfected in MGC-803 cells. G. mCherry was transfected in SGC-7901 cells. H. mCherry -miR-21 sensor was transfected in SGC-7901 cells. I. mCherry-miR-21-17 sensor was transfected in SGC-7901 cells.
Table 2. The value of mCherry fluorescence in different cells.
|
Fluorescence value of mCherry |
Average |
SD |
||||
|
GES-1 |
control |
49702.5 |
55794.5 |
55997.5 |
53831.5 |
2920.8 |
|
miR-21 sensor |
10446.5 |
11367.5 |
13422.5 |
11745.5 |
1244.0 |
|
|
miR-21-17 sensor |
47367.7 |
33459.7 |
33662.7 |
38163.4 |
6509.0 |
|
|
MGC-803 |
control |
60776.5 |
54626.5 |
55948.5 |
57117.2 |
2643.2 |
|
miR-21 sensor |
3563.5 |
5513.5 |
4530.5 |
4535.8 |
796.1 |
|
|
miR-21-17 sensor |
14218.7 |
5032.7 |
19087.7 |
12779.7 |
5827.5 |
|
|
SGC-7901 |
control |
47367.7 |
43459.7 |
53662.7 |
48163.4 |
4203.2 |
|
miR-21 sensor |
8963.5 |
9137.5 |
7145.5 |
8415.5 |
900.8 |
|
|
miR-21-17 sensor |
14218.7 |
5032.7 |
19087.7 |
34568.7 |
3475.8 |
|
Fig 18. The value of mCherry fluorescence in cells. Different cells were transfected with mCherry, mCherry-miR-21 sensor or mCherry-miR-21-17 sensor for 24
In order to estimate the effect of mCherry–miR-21 sensor and mCherry–miR-21-17 sensor on reflecting the different expression of miR-21 and miR-17 in gastric cancer cells compared with normal cells, we reanalyzed the data of table2. The strongest inhibition of mCherry–miR-21 sensor was observed in three cell lines (Fig. 19). mCherry–miR-21 sensor and mCherry–miR-21-17 did not reflect the expression of miRNAs in cancer cells compared with normal cells. These results suggested mCherry–miR-21 sensor and mCherry–miR-21-17 are not good sensors to detect tumor cells.
Fig 19. The value of mCherry fluorescence in different cells transfected with mCherry, mCherry-miR-21 sensor or mCherry-miR-21-17 sensor for 24 h.
Address
XHD-WS-Wuhan-A
Wuhan High School
No.538,Gongxiu Road,Xiaoshan District,
Wuhan,Hubei Province,China