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A new precursor miRNA and its application in tumor therapy

A precursor and carrier technology, applied in the field of tumor treatment, can solve problems such as ineffectiveness of EGFR-targeted drugs

Active Publication Date: 2021-12-24
JIANGSU MICROMEDMARK BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Therefore, considering that if the EGFR and K-RAS pathways can be targeted at the same time, the upstream and downstream of the pathways can be inhibited at the same time, so that EGFR-targeted drugs can have a better therapeutic effect on patients with K-RAS mutations. Therefore, there is an urgent need for A treatment method that can target and inhibit the K-RAS gene and corresponding drugs to solve the problems that there is currently no drug specific for K-RAS mutations, and K-RAS mutations cause EGFR-targeted drugs to be ineffective

Method used

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  • A new precursor miRNA and its application in tumor therapy
  • A new precursor miRNA and its application in tumor therapy
  • A new precursor miRNA and its application in tumor therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0230] Example 1: Anti-miR-214 overexpression vector construction

[0231] 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 (RNaseIII), 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.

[0232] 1. Anti-miR-214-5p sequence

[0233] >anti-miR-214-5p

[0234] 5"-ACUGCCUGUCUGUGCCUGCUGU-3" (SEQ ID NO.: 2)

[0235] 2. Anti-miR-214 carrier

[0236] 2.1. Design and synthesis of Oligo DNA

[0237] 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

[0261] Example 2: Cell experiments verify the efficiency of overexpressing anti-miR-214

[0262] 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

[0267] Example 3: In vivo experiments verify the efficiency of overexpressing anti-miR-214

[0268] 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.

[0269] Table 5. Expression levels of anti-miR-214-5p and anti-miR-214-3p

[0270] (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±0.1290 0.1 mg plasmid mouse lung 26.37±0.37...

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Abstract

A new precursor miRNA and its application in tumor treatment are provided, wherein the 5' to 3' end of the precursor miRNA has a structure shown in formula I: B1 is anti-miRNA and / or siRNA to be expressed; B2 It is a sequence that is substantially complementary or completely complementary to B1, and B2 is not complementary to C; C is a stem-loop structure sequence; A1 and A2 are each independently none, or an optional RNA sequence consisting of 4-5 bases; Wherein, the indicated precursor miRNA can be processed into anti-miRNA and / or siRNA in the host.

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 the total RNA of cells is very sm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00C12N15/113C12N15/63A61K31/7088A61P35/00
CPCC12N2310/14C12N2310/531A61K31/713C12N15/113A61P35/00C12N2310/141C12N2330/51C12N15/11C12N15/111C12N15/63C12N2310/113A61K48/00C12N2320/32
Inventor 张辰宇曾科陈熹张峻峰梁宏伟
Owner JIANGSU MICROMEDMARK BIOTECH