New precursor miRNA and applications of new precursor miRNA in tumor treatment

A precursor and carrier technology, applied in the field of tumor treatment

Active Publication Date: 2016-12-07
JIANGSU MICROMEDMARK BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cell proliferation and apoptosis are often abnormal in tumors, so it is speculated that the abnormal deletion, mutation or overexpression of miRNA will lead to the occurrence of human diseases

Method used

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  • New precursor miRNA and applications of new precursor miRNA in tumor treatment
  • New precursor miRNA and applications of new precursor miRNA in tumor treatment
  • New precursor miRNA and applications of new precursor miRNA in tumor treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] Example 1: Anti-miR-214 overexpression vector construction

[0136] MicroRNA vectors can express pre-miRNA quickly, efficiently and continuously in many mammalian cells by using the CMV promoter, and form small hairpin pre-miRNAs (about 70 nt) through the action of Drosha (RNase III), and then form mature microRNA (about 70 nt) through the action of Dicer 22nt), act on target mRNA and play a role in expression regulation. The optimized vector is used to simulate the expression module of anti-miR-214, which effectively avoids the problem of -3p and -5p generated when some endogenous miRNAs are expressed.

[0137] 1. Anti-miR-214-5p sequence

[0138] >anti-miR-214-5p

[0139] 5'-ACUGCCUGUCUGUGCCUGCCUGU-3' (SEQ ID NO.:2)

[0140] 2. anti-miR-214 carrier

[0141] 2.1. Design and synthesis of Oligo DNA

[0142] Design and synthesize 2 pairs of complementary oligo DNA according to the gene sequence (please refer to Article 6 of the product instruction for the oligo desig...

Embodiment 2

[0167] Example 2: Cell experiments verify the efficiency of overexpressing anti-miR-214

[0168] The anti-miR-214 plasmid was transfected in A549 cells (purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), and the total RNA was unified using anti-miR-214-5p, anti-miR- 214-3p, miR-214 primers Real-time-PCR detection of anti-miR-214-5p, anti-miR-214-3p, miR-214 levels. The results are represented by the Ct value of the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold value. The number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold value, wherein, each template There is a linear relationship between the Ct value and the logarithm of the initial copy number of the template, the greater the initial copy number, the smaller the Ct value; the smaller the initial copy number, the larger the Ct value. Table 4 shows ...

Embodiment 3

[0172] Example 3: In vivo experiments verify the efficiency of overexpressing anti-miR-214

[0173] Inject the anti-miR-214 plasmid into the tail vein of C57 / BL6 mice, perfuse after 24 hours, collect blood, heart, liver, spleen, lung, kidney, brain, and muscle tissue, and unify the total RNA, and then perform q-PCR to detect anti-miR-214 Levels of miR-214-5p, anti-miR-214-3p, miR-214. The results are shown in Table 5.

[0174] Table 5. Expression levels of anti-miR-214-5p and anti-miR-214-3p

[0175] (Ct)

anti-miR-214-5p

anti-miR-214-3p

Blank mouse blood

38.80±0.4171

30.80±0.8066

0.1mg plasmid mouse blood

24.11±0.4588

30.74±0.1667

0.01mg plasmid mouse blood

28.09±0.9880

30.50±0.1404

Blank mouse liver

39.37±0.8329

30.61±0.1288

0.1 mg plasmid mouse liver

20.52±0.3156

30.67±0.1311

0.01mg plasmid mouse liver

25.30±0.1404

33.70±0.1351

Blank mouse lung

39.25±0.5662

33.12±0.1...

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Abstract

The present invention discloses a new precursor miRNA and applications of the new precursor miRNA in tumor treatment. Specifically the present invention provides a precursor miRNA, wherein the structure represented by a formula I exists at the 5' to 3' terminal, B1 is anti-miRNA-214-5p, B2 is a sequence substantially complementary or completely complementary to B1, B2 and C are not complementary, C is a stem-loop structural sequence, A1 and A2 respectively are none, optionally RNA sequences comprising 4-5 bases, the precursor miRNA can be processed in a host to form anti-miRNA-214, and in the anti-miRNA-214, only the anti-miRNA-214-5p is expressed while the anti-miRNA-214-3p is not expressed. The formula I is defined in the specification.

Description

technical field [0001] The present invention relates to the field of tumor treatment, in particular to a new precursor miRNA, and the method and application of the precursor miRNA for inhibiting tumor growth. Background technique [0002] microRNA is derived from the long-chain RNA initial transcription product (Pri-miRNA) with a length of about 1000bp, and the Pri-miRNA molecule is cleaved by Drosha enzyme in the nucleus to form a miRNA precursor with a stem-loop structure of about 60-80nt in length. After the precursor miRNA is transported to the cytoplasm, it is further cleaved into a double-stranded miRNA of about 18-26 nt. After the double-stranded miRNA is unwrapped, the mature miRNA enters into the RNA-induced gene silencing complex (RNA-induced silencing complex, RISC), complete or incomplete pairing with the complementary mRNA, and degrades the target mRNA or represses its expression. [0003] Although the proportion of microRNA in total cellular RNA is very small,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K31/7088A61P35/00C12N15/113C12N15/11C12N15/63
CPCC12N15/11C12N15/63C12N15/111C12N2310/113A61P35/00C12N15/113C12N2330/51A61K48/00C12N2310/14C12N2310/141C12N2310/531C12N2320/32
Inventor 张辰宇曾科陈熹张峻峰粱宏伟
Owner JIANGSU MICROMEDMARK BIOTECH
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