Applications of miR-194-5p
An a-sma, proliferative technology, applied in medical preparations containing active ingredients, organic active ingredients, sensory diseases, etc., can solve the problems of loss, easy recurrence, unclear research, etc., to avoid potential risks and easy to obtain. , cheap effect
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Embodiment 1
[0035] Verification that overexpression of miR-194-5p can inhibit TGFβ1-induced cell migration
[0036] 1. Cell treatment: Cells were seeded in a 96-well plate, transfected with empty vectors psuper and psuper-194-5p 0.1ug each, and transfected with Lipo2000 transfection reagent.
[0037] 2. Scratch: Use a 10uL pipette tip to scratch the cells in the shape of a "one", then wash with PBS 3 times to wash off the floating cells. Add the corresponding treatment medium to continue culturing.
[0038] 3. Take pictures: Take pictures under the microscope at the time points of 0 hour, 24 hours and 48 hours of scratching.
[0039] 4. Data analysis: Use Image J software to measure the width of scratches to obtain width data, and then use Graphpad Prism software for statistical analysis and drawing.
[0040] Microscopic images of ARPE19 cells transfected with pSuper-miR-194-5p and ARPE19 cells with pSuper empty vector at 0 hour, 24 hours and 48 hours of scratching figure 1 , figure ...
Embodiment 2
[0042] Real-time quantitative PCR detection showed that miR-194-5p inhibited the changes of EMT-related markers induced by TGFβ1 at the mRNA level: normal control, TGFβ-treated ARPE19 cells transfected with pSuper-miR-194-5p and pSuper empty vector were treated with 1ml Trizol Collected in a 1.5ml tube for extraction of total RNA. After reverse transcription and real-time quantitative PCR analysis, it was calculated by semi-quantitative method.
[0043] The main steps are as follows:
[0044] 1. Add one-fifth of 200ul volume of chloroform to the sample collected by Trizol, mix vigorously, and centrifuge at 12000rpm at 4°C for 10 minutes.
[0045] 2. After centrifugation, transfer the supernatant to a new centrifuge tube, be careful not to get the protein layer in the middle, and add an equal volume of isopropanol.
[0046] 3. Centrifuge at 12000rpm at 4°C for 10 minutes, discard the supernatant, and wash the precipitate with 75% ethanol 1-2 times.
[0047] 4. After drying t...
Embodiment 3
[0061] Western blot detection showed that miR-194-5p inhibited TGFβ1-induced changes in EMT-related markers at the protein level: Protein extraction was performed on normal control, TGFβ-treated ARPE19 cells transfected with pSuper-miR-194-5p and pSuper empty vector .
[0062] 1. Protein extraction: Inoculate the cells in a 6cm cell culture dish, after corresponding treatment, add 150μl RIPA lysate containing Protease Inhibitor Cocktail, collect the cells into a new EP tube with a cell scraper, and incubate on ice Centrifuge at 10,000 rpm for 30 minutes at 4°C for 15 minutes, gently aspirate the supernatant into a clean centrifuge tube, and store at -80°C for later use.
[0063] 2. Determination of protein concentration: BCA quantitative method was used for protein quantification, and the specific method was as follows: prepare a standard protein solution of 2 μg / μl, and store it at -20°C for later use; The volume ratio of :1 is fully mixed, and it is prepared and used immedi...
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