siRNA for inhibiting gene ADAMTS-5 and application of siRNA
A DNA molecule, methoxy modification technology, applied in the field of siRNA, can solve problems such as differences in silencing effects
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Embodiment 1
[0154] Embodiment 1, the screening of the effective oligomeric nucleic acid that suppresses ADAMTS-5 gene mRNA expression
[0155]1. Perform siRNA design to determine siRNA targeting ADAMTS-5, and perform bioinformatic screening to ensure that the sequence is specific to the ADAMTS-5 sequence and not specific to sequences from any other gene. The target sequence was checked against the sequence in GenBank using the BLAST search engine provided by NCBI. After preliminary experiments, 8 effective siRNAs were screened out, named siRNA-RB-01, siRNA-RB-02, siRNA-RB-03, siRNA - RB-04, siRNA-RB-05, siRNA-RB-06, siRNA-RB-07, siRNA-RB-08. The above siRNAs are siRNAs designed for different positions of the ADAMTS-5 gene sequence.
[0156] 2. Cell transfection
[0157] The experiment was divided into 10 groups, namely siRNA-RB-01 to siRNA-RB-08 experimental groups, No target (NTC) negative control group, and NC blank control group.
[0158] The setting method of siRNA-RB-01 to siRNA-R...
Embodiment 2
[0179] Embodiment 2, the inhibition of oligomeric nucleic acid to inflammatory factor
[0180] 1. The experiment is divided into the following groups:
[0181] hFLS-siRNA-RB-04 experimental group: primary culture hFLS cells to 6-well plates, when the cell density was about 50%, transfect hFLS cells with siRNA-RB-04 at a final concentration of 50nM according to the Lipofectamine2000 kit instructions.
[0182] 293T-siRNA-RB-04 experimental group: primary culture 293T cells to 6-well plates, when the cell density was about 50%, transfect cells with siRNA-RB-04 at a final concentration of 50nM according to the instructions of the Lipofectamine2000 kit.
[0183] hFLS-No target (NTC) negative control group: the siRNA in the hFLS-siRNA-RB-04 experimental group was replaced with random non-specific siRNA, and the rest of the steps were the same as in the hFLS-siRNA-RB-04 experimental group. Among them, the random non-specific siRNA is not specific to the siRNA designed for the target...
Embodiment 3
[0210] Embodiment 3, homologous oligomeric nucleic acid is to the verification of ADAMTS-5 gene inhibitory effect
[0211] In order to verify the influence of the homology ratio on the effect of siRNA-RB-04 on inhibiting ADAMTS-5 gene, the following three sets of experiments were carried out:
[0212] 1. The first set of experiments
[0213] The antisense strands of the first group of siRNAs are all "5'-AUGAUGCCCACAUAAAUCC-3'", and the sense strands are the homologous sequences of "5'-GGAUUUAUGUGGGCAUCAU-3'", as shown in Table 1.
[0214] Table 1 Antisense strand group
[0215]
[0216]
[0217] Note: S=sense strand, AS=antisense strand. Choose 15nt, 11nt, 23nt, 27nt, and mismatches for the sense strands, respectively.
[0218] According to the method of Example 1, each siRNA shown in Table 1 was transfected into hFLS cells, and its inhibitory efficiency on ADAMTS-5 gene mRNA expression was detected.
[0219] 2. The second set of experiments
[0220] The sense stran...
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