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MiR-21 antisense digonucleotides and use thereof

An oligonucleotide and mir-21 technology, which is applied in the field of preparation of tumor drugs, can solve the problems of poor curative effect, low survival rate and poor curative effect in patients with distant metastasis, and achieve growth inhibition and Malignant proliferation ability, effect of promoting cell apoptosis

Inactive Publication Date: 2009-06-17
SUZHOU GENEPHARMA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the past 30 years, although the comprehensive treatment of tumors has been very common clinically, the comprehensive treatment based on surgery and supplemented by radiotherapy and chemotherapy has not significantly improved the survival rate of cancer patients, and the 5-year overall survival rate is still low, hovering At about 30% to 55%, there is no significant improvement, and the 5-year survival rate of middle and advanced patients is even lower, about 20%.
Moreover, these methods have their own limitations, especially for the middle-advanced and relapsed patients, and the curative effect is even worse for those with distant metastasis.

Method used

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  • MiR-21 antisense digonucleotides and use thereof
  • MiR-21 antisense digonucleotides and use thereof
  • MiR-21 antisense digonucleotides and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 Inhibition of antisense nucleic acid on the expression of miR-21

[0017] The antisense nucleic acid for miR-21 (hereinafter referred to as miR-21 AS) designed and synthesized by the present invention, the number and sequence of the antisense nucleic acid molecule (after full thio modification) are as follows:

[0018] AS0 5’-UCAACAUCAGUCUGAUAAGCUA-3’ (antisense RNA sequence against the full length of miR-21)

[0019] AS1 5’-TCAACATCAGTCTGATAAGCTA-3’ (antisense DNA sequence against the full length of miR-21)

[0020] AS3 5’-AGUCUGAUAAGCUA-3’ (antisense RNA sequence targeting 14 bases at the 5’ end of miR-21)

[0021] AS5 5’-UCAACAUCAGUC-3’ (antisense RNA sequence targeting 12 bases at the 3’ end of miR-21)

[0022] AS6 5’-TCAACATCAGTCTG-3’ (antisense DNA sequence targeting 14 bases at the 3’ end of miR-21)

[0023]AS7 5’-ACAUCAGUCUGAUAAGC-3’ (antisense RNA sequence of 17 bases against the middle region of miR-21)

[0024] The sequence of the control lin4AS0 is: 5’...

Embodiment 2

[0053] Example 2 MTT method to detect cell viability

[0054] The MTT experiment was used to detect the changes in the survival rate of tongue squamous cell carcinoma cells before and after transfection of miR-21 AS0 and TPM-1 siRNA. The results showed that miR-21 AS0, miR-21 AS1, miR-21 AS3, miR-21 AS5 were transfected respectively The survival rate of tongue squamous cell carcinoma cells SCC-15 and CAL27 after miR-21 AS6 and miR-21 AS7 was significantly lower than that before miR-21 AS0 transfection; while there was no significant change in cell survival after transfection with lin4 AS0:

[0055] 1) Collect the transfected tongue squamous cell carcinoma cells and inoculate them in 96-well plates, about 5×10 3 Pcs / well, set at 37℃, 5% CO 2 Incubator culture.

[0056] 2) Calculate 72 hours after transfection, remove the culture medium in each well of the 96-well plate, add 180 μl of fresh DMEM culture medium to each well, then add 20 μl of MTT solution (5 mg / ml), and continue to c...

Embodiment 3

[0061] Example 3 Clone formation experiment to detect cell malignant proliferation ability

[0062] The clone formation experiment was used to detect the changes in the proliferation ability of tongue squamous cell carcinoma cells before and after transfection of miR-21 AS0 and TPM-1 siRNA. The results showed that the clone formation rate of tongue squamous cell carcinoma cells SCC-15 and CAL27 after being transfected with miR-21 AS0, miR-21 AS1, miR-21 AS3, miR-21 AS5, miR-21 AS6, miR-21 AS7, respectively Before miR-21 antisense nucleic acid was transfected, it was significantly reduced, and the difference was significant; while after transfection with lin4 AS0, there was no significant change in the cell clone formation rate:

[0063] 1) After transfection, the tongue squamous cell carcinoma cells are seeded in a flat-bottomed 6-well culture plate, 400 cells / well, shake the culture plate to make the cells evenly distributed, and place at 37℃, 5% CO 2 Incubator culture.

[0064] ...

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Abstract

The invention discloses an antisense oligonucleotide for inhibiting miR-21 expression in the tongue squamous cell carcinoma and its application. The antisense oligonucleotide of the invention comprises complementary sequences with at least 12 continuous nucletides in nucleotide sequences as below: 5'-UAGCUUAUCAGACUGAUGUUGA-3', and said complementary sequences can specifically bind with different regions of human miR-21. The antisense oligonucleotide of the invention can be a ribonucleotide or a deoxyribonucleotide and modify any nucleotide. The miR-21 antisense oligonucleotide with anti-tongue squamous cell carcinoma action of the invention can effectively inhibit the expression of the miR-21 in the tongue squamous cell carcinoma cell SSC-15 and CAL-27, simultaneously inhibit said two cells growth and proliferation, promote the apoptosis, thereby effectively treating the tongue squamous cell carcinoma and other miR-21 high expression tumors.

Description

Technical field [0001] The present invention relates to antisense nucleic acid and its application, and in particular to the antisense nucleic acid of miR-21 and its application in the preparation of tumor treatment drugs. Background technique [0002] Studies have found that microRNAs (also known as miRNAs or micronucleic acids) are closely related to the occurrence and development of many diseases. Among many diseases, the abnormal expression of miRNA in malignant tumors has attracted the most attention. Berezikov et al. (Berezikov E, Guryev V, van de Belt J, et al. Phylogenetic shadowing and computational identification of human microRNA genes. Cell, 2005, 120(1): 21-24) showed that more than 30% of protein-coding genes were analyzed by computer May become the target gene of miRNA. Calin et al. (Calin GA, Sevignani C, Dumitru CD, et al. Human microRNAgenes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci USA, 2004, 101(9): 299...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11A61K31/7088A61P35/00C12N15/113
Inventor 李劲松宋尔卫张佩琢
Owner SUZHOU GENEPHARMA
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