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siRNA for interfering with ZNF24 gene expression and application to inhibition of cell proliferation and migration thereof

A technology for gene expression and cell inhibition, applied in the field of molecular genetics, can solve problems such as low interference with ZNF24 gene expression, and achieve the effect of alleviating or preventing diseases

Pending Publication Date: 2019-10-08
SHANDONG INST OF PARASITIC DISEASES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there are relatively few studies on interference with ZNF24 gene expression, and targeting siRNA sequences that interfere with ZNF24 gene expression is of great significance for the development of applied research in certain cells

Method used

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  • siRNA for interfering with ZNF24 gene expression and application to inhibition of cell proliferation and migration thereof
  • siRNA for interfering with ZNF24 gene expression and application to inhibition of cell proliferation and migration thereof
  • siRNA for interfering with ZNF24 gene expression and application to inhibition of cell proliferation and migration thereof

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1: Design and synthesis of interfering RNA (siRNA) target sequence

[0036] The ZNF24 gene sequence was obtained from GenBank, and according to the siRNA design principle, three interference target sequences were designed and synthesized by screening the conserved region of the ZNF24 gene sequence, named ZNF24-siRNA-292, ZNF24-siRNA-485, and ZNF24-siRNA-575, respectively. At the same time, a nonsense sequence not targeting any gene was designed as a negative control (siFAM), and the sequence is shown in Table 1.

[0037] Table 1 siRNA sequence design and synthesis

[0038]

Embodiment 2

[0039] Example 2: Cell culture and transfection

[0040] HEK-293 cells were cultured in DMEM containing FBS and antibiotics at 37°C in 5% CO 2 Cultivate in a constant temperature incubator, and when the growth rate of the cells reaches 70-90%, they are digested and passaged with 0.25% trypsin. The day before transfection, press 4-5×10 4Cells / well were seeded on a 24-well plate, and 0.5ml DMEM medium was added to each well. For transfection, add 20 pmol of ZNF24-siRNA to 50 μl of serum-free DMEM medium, and mix gently; at the same time, dilute 1 μl of Lipofectamin 2000 reagent with 50 μl of serum-free DMEM medium, mix gently and place at room temperature for 5 minutes. Then the diluted siRNA and transfection reagent were mixed, mixed gently, and left at room temperature for 20 minutes to form the siRNA / Lipofectamin 2000 complex. Add 100 μl of the complex to the 24-well plate containing the cells, gently shake the cell culture plate back and forth, and keep the cells in 5% CO...

Embodiment 3

[0041] Embodiment 3: Detection of inhibition efficiency of siRNA

[0042] (1) Total RNA extraction

[0043] Add 1ml / well TRNzol reagent directly to the culture plate to lyse the cells, pipette several times with a sampler, and place the homogenate sample at 15-30°C for 5 minutes to completely separate the nucleic acid-protein complex. Add 0.2ml of chloroform for every 1ml of TRNzol used, cover the tube cap, shake vigorously for 15s, place it at room temperature for 3min, and then place it in a centrifuge at 4°C and centrifuge at 12,000rpm for 10-15min. At this time, the sample will be divided into three layers: yellow organic Phase, the middle layer and the upper colorless aqueous phase, the RNA is mainly in the aqueous phase, transfer the aqueous phase (about 600 μl) to a new centrifuge tube. Add an equal volume of isopropanol to the obtained aqueous phase solution, mix well, leave at room temperature for 20-30 minutes, then centrifuge at 12,000 rpm at 4°C for 10 minutes, an...

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Abstract

The invention provides siRNA for interfering with ZNF24 gene expression and application to inhibition of cell proliferation and migration thereof. Three pairs of siRNA which are ZNF24-siRNA-292, ZNF24-siRNA-485 and ZNF24-siRNA-575 are synthesized and designed, and can effectively and specifically interfere with the expression of ZNF24 so that cell proliferation and migration can be effectively inhibited. According to the siRNA for interfering with the ZNF24 gene expression and application to the inhibition of cell proliferation and migration thereof, the number of siRNA sequences which interfere with the ZNF24 gene expression is increased, and the proliferation and migration of cells are inhibited by specifically inhibiting the ZNF24 gene expression through RNA interference at the cellularlevel, so that diseases which are possibly caused by ZNF24 genes are inhibited, alleviated or prevented, and a foundation is laid for the study of the action mechanism of the ZNF24 genes in cell proliferation and migration and the future application research.

Description

technical field [0001] The invention belongs to the field of molecular genetics, and in particular relates to an siRNA for interfering with ZNF24 gene expression and its application in inhibiting cell proliferation and migration. Background technique [0002] RNA interference (RNA interference, RNAi) is a process of post-transcriptional silencing of homologous target genes mediated by double-stranded RNA. It can induce endogenous target genes by artificially introducing double-stranded RNA with the same sequence as endogenous target genes. mRNA degradation, thereby achieving the purpose of reducing gene expression. The phenomenon of RNAi is also an evolutionarily conserved defense mechanism against the invasion of transgenic or foreign viruses. Although the research time of RNA interference technology is relatively short, as an emerging gene blocking technology, it has the advantages of high specificity, high interference efficiency and simple operation, and is widely used ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/713A61P35/00
CPCA61K31/713A61P35/00C12N15/113C12N2310/141C12N2320/30
Inventor 刘功振崔勇高歌于涛寇景轩
Owner SHANDONG INST OF PARASITIC DISEASES
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