Antisense STAT3 oligonucleotide and uses thereof
An oligonucleotide and antisense technology, applied in the field of high-efficiency antisense STAT3 oligonucleotide and its application in anti-tumor drugs, can solve the problems of unsatisfactory anti-tumor effect, and improve radiation sensitivity Effect
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Embodiment 1
[0025] Example 1: Prediction of the secondary structure of STAT3 mRNA
[0026] Using the mRNA secondary structure prediction software RNAstructure 4.2 (for Windows version), the full-length sequence of STAT3 mRNA (sequence number: L29277) was retrieved from the gene library genebank, and the secondary structure of STAT3 mRNA was predicted by the software, and the predicted secondary structure of STAT3 mRNA was obtained. structure, on the basis of the predicted secondary structure, use the oligowalk module of the software to design antisense sequences with relatively low total free energy, a total of 10, the minimum free energy of each sequence is shown in Table 1, each sequence crosses Synthesized by Shanghai Sangong, modified with full thio, purified with polyacrylamide.
[0027] Table 1 STAT3 antisense nucleic acid sequence oligowalk program designed with RNAstructure 4.2 simulation software oligowalk module
[0028] No.
Embodiment 2
[0029] Example 2: Inhibitory effect of each sequence on the proliferation of lung gland A549 cells
[0030] Positive control antisense sequence (ASC-STAT3) reported in the literature: 5'-GCTCCAGCATCTGCTGCTTC-3', nonsense sequence (NS-STAT3): 5'-GCTCCAGCATCTGCTGCTTC-3', each sequence pair was detected by CCK-8 assay Proliferation inhibitory effect of lung adenocarcinoma A549 cells (American Type Culture Collection, available from ATCC). Its steps include:
[0031]2.1 Cell culture A549 cells were cultured with RPMI-1640 medium containing 10% fetal bovine serum, 100 u / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO 2 Cultured under saturated humidity conditions, the cells used in the experiment were in the exponential growth phase.
[0032] 2.2 Oligonucleotide transfection Each oligonucleotide was encapsulated with cationic liposomes (Oligofectamine purchased from Invitrogen) to transfect A549 cells, and the operation was performed according to the kit instructions....
Embodiment 3
[0036] Example 3: Inhibitory effect of different concentrations of AS10 on the proliferation of lung adenocarcinoma A549 cells
[0037] Then use the optimal sequence group AS10, the positive control group (ASC), and the nonsense group (NS) to transfect in a 96-well plate at different concentrations (50, 100, 150, 200, 250, 300, 400nmol / L), After 48 hours, the above-mentioned method was used to calculate the survival fractions of each concentration in each group, and the results are shown in Fig. 2 .
[0038] Survival fraction=(administration group-background) / (normal control group-background)×100%.
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