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Antisense oligonucleotide for tiny RNA-21 seed sequence and application thereof

A technology of RNA-21 and antisense oligonucleotide, which is applied in the field of drug preparation, can solve problems such as toxic and side effects, and achieve the effect of low toxic and side effects and high transfection efficiency

Active Publication Date: 2013-09-25
广州安镝声生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Chronic myelogenous leukemia and multiple myeloma are malignant proliferative diseases. Traditional chemical drugs have certain toxic and side effects, so it is urgent to find new drugs

Method used

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  • Antisense oligonucleotide for tiny RNA-21 seed sequence and application thereof
  • Antisense oligonucleotide for tiny RNA-21 seed sequence and application thereof
  • Antisense oligonucleotide for tiny RNA-21 seed sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Design and synthesis of embodiment 1 antisense oligonucleotide

[0023] The human microRNA-21 seed sequence was obtained from the microRNA Families database, and the antisense oligonucleotide sequence (t-antimiR-21) targeting the seed sequence was designed according to the principle of sequence complementarity, and the antisense oligonucleotide sequence was determined by BLAST software analysis sequences and random control sequences (such as figure 1 ). Antisense oligonucleotide sequence (t-antimiR-21): 5'-ATAAGCTA-3'; random sequence (scramble, SCR): 5'-TCATACTA-3', all synthesized by Shanghai Bioengineering Co., Ltd., all sulfur Generation modification, HPLC purification.

Embodiment 2K562

[0024] Embodiment 2K562 cell culture

[0025] K562 cells were inoculated in RPMI-1640 medium containing 10% neonatal bovine serum and antibiotic-free, and RPMI-8266 cells were inoculated in RPMI-1640 medium containing 10% fetal bovine serum and antibiotic-free. base, placed in 37 °C, 5% CO 2 Incubator, cultivated under saturated humidity. Change the medium every 2-3 days for passage. Cells in the logarithmic growth phase with a mass fraction of 0.2% trypan blue and an exclusion rate of >95% were selected for the experiment.

Embodiment 3

[0026] Embodiment 3 MTT method screens the optimal action concentration of antisense oligonucleotide

[0027] This experiment was divided into t-antimiR-21 group, random control group (SCR) and blank control group, and each group had 5 replicate wells. The final nucleic acid concentrations of t-antimiR-21 in the t-antimiR-21 group were 0.2, 0.3, 0.4, 0.5 and 0.6 μmol / l, and the random sequences in the random control group were 0.2, 0.3, 0.4, 0.5 and 0.6 μmol / l. K562 cells and RPMI-8266 cells in the logarithmic growth phase were taken, and the cells in each group were divided into 1×10 5 Cells / mL were inoculated in 96-well plates, 50 μL per well, and the final volume of transfection was 100 μL (for the transfection method, refer to Invitrogen Lipofectamine TM2000 instructions), the blank control group was added 50 μL of serum-free Opti-MEM medium. Six hours after transfection, 100 μL of RPMI-1640 medium containing 20% ​​fetal bovine serum was added to each well to make the f...

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Abstract

The invention discloses an antisense oligonucleotide for tiny RNA-21 seed sequence and an application thereof and belongs to the field of medicine preparation. According to the research, tiny antisense nucleic acid t-antimiR-21 of 8 basic groups is designed and synthetized for miR-21 seed sequence. It shows through a t-antimiR-21 transfection cell experiment that t-antimiR-21 targets miR-21 of K562 cell and RPMI-8266 cell strain, cell growth is inhibited and cell apoptosis is obviously increased. It shows that lymphoma cell growth can be effectively inhibited through interfering with miRNA-21 expression by t-antimiR-21. Meanwhile, it shows that miRNA-21 might be used as a potential target for lymphoma gene therapy. t-antimiR-21 can be used in the preparation of an antineoplastic medicine, especially in the preparation of a medicine against chronic granulocytic leukemia and multiple myeloma.

Description

technical field [0001] The invention belongs to the field of medicine preparation, and in particular relates to an antisense oligonucleotide targeting microRNA-21 seed sequence and its application. Background technique [0002] MicroRNA (miRNA) is a kind of endogenous non-coding single-stranded small molecule RNA with a length of about 22 nucleotides, which is involved in physiological processes such as cell differentiation, proliferation and apoptosis, individual development, and body metabolism. The "seed sequence" at the 5' end of the miRNA (7-8 nucleotide sequences at the 5' end of the miRNA) performs base complete complementary pairing with the 3' untranslated region (3' untranslated region, 3'UTR) of the target gene mRNA, promoting the target gene mRNA degrades or inhibits mRNA translation, thereby post-transcriptionally regulating the expression level of target genes. [0003] As of August 2012, 21264 entries of miRNA sequences have been recorded in the human genome ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61P35/00A61P35/02
Inventor 费嘉
Owner 广州安镝声生物医药科技有限公司
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