Oligo-nucleic acid for suppressing tumor growth and application thereof
An oligomeric nucleic acid and tumor technology, applied in the field of oligomeric nucleic acid, to achieve long-effect half-life, enhance anti-tumor effect, and prevent tumor growth
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Embodiment 1
[0036] Embodiment 1, the preparation of oligomeric nucleic acid
[0037] The sense strand of the oligomeric nucleic acid sequence described in the present invention is: 5'-UGGCAGUGUCUUAGCUGGUUGU-3' (SEQ ID No.1); the antisense strand is 3'-UCCCGUCAUAUGAACGACUAAC-5' (SEQ ID No.2) or 3'-UUACCGUCACAGAAAUCGACCAA -5' (SEQ ID No. 3). A random sequence was selected as a negative control (NC). The sense strand sequence of NC is: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID No.4), and the antisense strand sequence is: 3'-TTAAGAGGCUUGCACAGUGCA-5' (SEQ ID No. .5). A, G, C, and U in the oligonucleotide and NC sequences represent adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides and uracil ribonucleotides, and T represents thymine deoxynucleotides.
[0038] The oligonucleotide and NC were commissioned by Shanghai GenePharma Pharmaceutical Technology Co., Ltd. to synthesize and perform any of the following chemical modifications: 2'methoxy (2'-OMe), 2'fluoro (2'-F ), thi...
Embodiment 2
[0050] Effect of embodiment 2, 2'-OMe-oligonucleotide on tumor cell proliferation
[0051] The experimental procedure is as follows: 2'-OMe-miR-34a, 2'-OMe-miR-34a* (abbreviated as miR-34a and miR-34a* in the figure) obtained in Example 1 and 2'-OMe-NC (Fig. (hereinafter referred to as NC) powder was dissolved in RNase-free sterile water to prepare three kinds of oligonucleotide solutions with a final concentration of 20 pmol / L. Collect logarithmic phase HCT116 intestinal cancer cells (purchased from Peking Union Medical College Cell Bank), adjust the concentration of cell suspension, and divide them into 24-well plates with 10,000 cells per well, add IMDM culture medium containing 2.5% fetal bovine serum (purchased from GIBCO) at 37°C with 5% CO 2 Incubate in an incubator for 16-24 hours. For transfection, 2 μl of oligonucleotide solution and 1 μl of lipofectamine 2000 (Invitrogen) were diluted in 50 μl of serum-free medium (Opti-MEM) (purchased from GIBCO), and incubated a...
Embodiment 3
[0054] Example 3, Half-life test detection of 2'-OMe-oligonucleotide inhibiting tumor cell expression CCND1
[0055] Using Invitrogen's lipofectamine TM 2000 liposomes were used for transfection, and all operations were performed according to the operating procedures provided by Invitrogen. Inoculate HCT 116 cells into a 6-well plate (200,000 cells / well), culture with IMDM medium containing 2.5% fetal bovine serum (purchased from GIBCO) for 16-24 hours, then replace the cell culture medium with antibiotic-free IMDM medium. Dissolve miR-34a*, 2′-OMe-miR-34a*, NC and 2′-OMe-NC powders in RNase-free sterile water to prepare 4 kinds of oligonucleotides with a final concentration of 20pmol / L solution. For transfection, take 10 μl of the above oligonucleotide solution and 5 μl of lipofectamine 2000 and dilute them in 250 μl of serum-free culture medium (Opti-MEM), and incubate at room temperature for 5 minutes, then the above oligonucleotide solution Mix with lipofectamine 2000 ...
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