Specific siRNA (siFX1) for inhibiting FOXO1 gene expression and application thereof
A gene expression and specific technology, applied in DNA/RNA fragments, genetic engineering, recombinant DNA technology, etc., can solve problems such as short-term, instability, and off-target effects of cell membrane permeability, and achieve strong inhibition and strong specific effect
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Embodiment 1
[0032] This example verified the inhibitory effect of siFX1 on the transcription level of the FOXO1 gene in alveolar rhabdomyosarcoma cell lines. The specific experimental steps and results are as follows:
[0033] Inoculate the representative cell line RH4 (SJ-RH4) of alveolar rhabdomyosarcoma cells in a 6-well plate or a 60mm cell culture dish, in DMEM culture medium containing 10% FBS (fetal bovine serum), but without antibiotics, Grow overnight to 60-80% cell density at 37°C, 5% CO2. Lipofectamine 2000 reagent was used to transfect siFX1 and siCON (control siRNA, siCON-s (sense strand): 5'-CUAUAGCGAACCGUGAUCUdTdT-3' (SEQ ID No.3), siCON-as (antisense strand): 5'-AGAUCACGGUUCGCUAUAGdTdT -3'(SEQ ID No.4)) sequence into the cells (siRNA final concentration is 20nM), after 6-8 hours, replace the culture solution with DMEM culture solution containing 0.3% FBS, 1% antibiotics, 37 ℃, Under the condition of 5% carbon dioxide, cultivate for 72 hours. Cells were collected, extract...
Embodiment 2
[0042] This example verifies that in the alveolar rhabdomyosarcoma cell line, siFX1 and the known specific FOXO1 siRNA sequence (siFX4, siFX4-s (sense strand): 5'-GGAGGUAUGAGUCAGUAUAdTdT-3'(SEQ ID No.9), siFX4-as (antisense strand): 5'-UAUACUGACUCAUACCUCCdTdT-3' (SEQ ID No.10); refer to "FOXO1 inhibits the invasion and metastasis of hepatocellular carcinoma by reversing ZEB2-induced epithelial-mesenchymal transition", Tianxiu Dong et al, Oncotarget, Volume 8, Issue 1, Pages 1703-1713, 20170103) the inhibitory effect on the FOXO1 gene at the protein level, the specific experimental procedures and results are as follows:
[0043] As in Example 1, RH4 cells were transfected with siFX1 or siCON, cultured for 72 hours under the condition of no serum or low concentration of fetal bovine serum (0.3%), the cells were collected, and the total protein was extracted. Take 30-50 μg of total protein for SDS-PAGE gel electrophoresis, transfer to PVDF membrane, and incubate with FOXO1, α-tub...
Embodiment 3
[0047] This example verifies the influence of siFX1 and the known positive control siFX4 on cell morphology. The specific experimental operations and results are as follows:
[0048] As in Example 1, siFX1, siFX4 siRNA and siCON were transfected into RH4 cells respectively, cultured for 60-72 hours under the condition of low concentration of fetal bovine serum (0.3% FBS), and different time points were selected to observe the cells under a microscope The difference in shape changes, and take pictures to record, the results are as follows Figure 4 shown.
[0049] Depend on Figure 4 It can be seen that the siFX1 interference sequence and the known control siFX4 interference sequence have different effects on cell morphology. After knocking down the expression of FOXO1 by transfecting siRNA sequence, siFX1 has almost no effect on cell morphology and growth under low concentration serum culture conditions, while siFX4 seriously affects its morphology and cell growth, resulting...
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