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siRNA for interfering DNALI1 gene expression and application of siRNA in cell proliferation and migration inhibition

A technology of gene expression and cell inhibition, applied in the field of molecular genetics to reduce or prevent diseases

Pending Publication Date: 2019-10-08
山东省寄生虫病防治研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But up to now, there is no siRNA sequence for interfering with DNALI1 gene expression and related research or patent disclosure

Method used

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  • siRNA for interfering DNALI1 gene expression and application of siRNA in cell proliferation and migration inhibition
  • siRNA for interfering DNALI1 gene expression and application of siRNA in cell proliferation and migration inhibition
  • siRNA for interfering DNALI1 gene expression and application of siRNA in cell proliferation and migration inhibition

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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 DNALI1 gene sequence was obtained from GenBank. According to the siRNA design principle, three interference target sequences were designed and synthesized by screening the conserved region of the DNALI1 gene sequence, named DNALI1-siRNA-66, DNALI1-siRNA-426, and DNALI1-siRNA-777, 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 Cultured in a constant temperature incubator, and when the growth rate of the cells reached 70-90%, they were digested and passaged with 0.25% trypsin. The day before transfection, press 4-5×10 4 Cells / well were seeded on a 24-well plate, and 0.5ml DMEM medium was added to each well. For transfection, add 20 pmol of DNALI1-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 DNALI1-siRNAs and the 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 ...

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 an siRNA for interfering DNALI1 gene expression and application of siRNA in cell proliferation and migration inhibition. According to the siRNA for interfering DNALI1 gene expression and application of siRNA in cell proliferation and migration inhibition, three pairs of siRNAs for targetedly inhibiting DNALI1 gene expression are designed and synthesized, the three pairs of siRNAs are DNALI1-siRNA-66, DNALI1-siRNA-426 and DNALI1-siRNA-777, and expression vectors in an HEK-293 cell of the siRNAs are successfully constructed. According to the siRNA for interfering DNALI1 gene expression and application of siRNA in cell proliferation and migration inhibition, when the siRNAs enter a cell, expression of the DNALI1 gene can be effectively and specifically interfered, and thus cell proliferation and migration are effectively inhibited. According to the siRNA for interfering DNALI1 gene expression and application of siRNA in cell proliferation and migration inhibition, the blank that an siRNA sequence aiming at interfering DNALI1 gene expression does not exist is filled, DNALI1 gene expression is effectively interfered through the siRNAs, the function that diseases might caused by DNALI1 gene expression are treated or prevented is achieved, and foundation is laid for research on mode of action of DNALI1 gene in cell proliferation and migration and application inthe future.

Description

technical field [0001] The invention belongs to the field of molecular genetics, and specifically relates to an siRNA for interfering with DNALI1 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 RNAi 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 in viral in...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/713A61P35/00A61P35/04
CPCA61P35/00A61P35/04C12N15/113C12N2310/14
Inventor 刘功振崔勇于涛高歌寇景轩
Owner 山东省寄生虫病防治研究所
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