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SiRNA for specifically inhibiting ECHS1 gene expression and application thereof

A transgenic mouse and purpose technology, applied in the field of siRNA, can solve the problems of slow modeling speed, high price, poor model stability, etc., and achieve the effect of increasing lipid droplets and promoting growth

Active Publication Date: 2010-05-12
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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AI Technical Summary

Problems solved by technology

[0005] The molecular mechanism of the occurrence and development of NAFLD has not yet been clarified. At present, NAFLD is believed to be related to insulin resistance, fat storage disorder, and fat secretion inhibition. Among them, fat storage disorder is one of the hotspots in the study of the pathogenesis of NAFLD. Normal liver fatty acid oxidation and synthesis are in a dynamic balance , Once fatty acid oxidation disorder, fat accumulation in the liver, fatty liver occurs. Mitochondrial β-oxidation is the main oxidation form of fatty acid oxidation. This process can be divided into three stages: activation, transfer, and β-oxidation. The activated fatty acid is converted into acyl CoA The form of β-oxidation is transferred to the mitochondrial matrix for β-oxidation. β-oxidation undergoes four-step reactions, namely dehydrogenation, water addition, dehydrogenation and thiolysis, to generate a molecule of acetyl CoA and a new fatty acyl CoA with two less carbons. More Repeating the cycle above will gradually generate acetyl-CoA. Among them, the second step of adding water is catalyzed by ECHS1 (enoyl-CoA hydratase). The reduction of ECHS1 expression may lead to NAFLD, but there is no correlation between ECHS1 and non-alcoholic hydratase. The study of the mechanism of ECHS1 in the pathogenesis of NAFLD using RNAi technology is an important supplement to the pathogenesis of NAFLD.
[0006] The existing animal models of non-alcoholic fatty liver disease, such as ob / ob, db / db mice and Obese Zucker rats and other genetically derived spontaneous animal models, mostly need to be imported from abroad, are expensive, and require high feeding conditions , and these animal models cannot spontaneously reproduce non-alcoholic steatohepatitis due to the lack of leptin gene; while the non-alcoholic fatty liver disease animal model reproduced by feeding SD rats with a high-fat diet can reproduce non-alcoholic fatty liver disease steatohepatitis, but the molding speed is slow, generally takes about six months, and the stability of the model is not good

Method used

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  • SiRNA for specifically inhibiting ECHS1 gene expression and application thereof
  • SiRNA for specifically inhibiting ECHS1 gene expression and application thereof
  • SiRNA for specifically inhibiting ECHS1 gene expression and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1, siRNA design and synthesis

[0031] 1. Synthesis of ECHS1 siRNA

[0032]Obtain the full-length sequence of mouse ECHS1 mRNA (NM_053119.2; sequence 3 in the sequence table) from the database of the National Center for Biotechnology Information (NCBI), and design and synthesize it based on the principle of RNAi and design software. And an effective 21nt siRNA against the mouse ECSH1 gene was screened out. A BLAST comparison check was performed to ensure that there was no homology to other genes.

[0033] The sequence of ECHS1 siRNA is: 5'-GCCUUUGAGAUGACGUUAAtt-3' (sense strand) (sequence 1);

[0034] 3'-gtCGGAAACUCUACUGCAAUU-5' (antisense strand) (SEQ ID NO: 2).

[0035] Chemical synthesis was carried out by Shanghai Gemma Pharmaceutical Technology Co., Ltd. (factory address: 602, No. 1011 Halei Road, Zhangjiang, Pudong, Zip Code: 201203): using NTP as raw material, using ABI3900 nucleic acid synthesizer to chemically synthesize single-stranded RNA, and f...

Embodiment 2

[0040] Example 2, Effect of ECHS1 siRNA on the expression of ECHS1 gene in AML-12 cells in vitro

[0041] 1. Group transfection

[0042] Spread AML-12 cells evenly in a 24-well plate and divide them into 4 groups:

[0043] High-fat experimental group: containing 10% (volume percentage) inactivated newborn bovine serum, 100U / ml penicillin and 100mg / ml streptomycin, sodium oleate of 666 μ mol / L and sodium palmitate of 333 μ mol / L DMEM / F12 medium; transfect 200 pmol of ECHS1 siRNA of Example 1.

[0044] High-fat control group: containing 10% (volume percentage) inactivated newborn bovine serum, 100U / ml penicillin and 100mg / ml streptomycin, sodium oleate of 666 μ mol / L and the sodium palmitate of 333 μ mol / L DMEM / F12 medium; 200 pmol negative control siRNA of Example 1 was transfected.

[0045] Normal experimental group: DMEM / F12 medium containing 10% (volume percent) inactivated neonatal bovine serum, 100 U / ml penicillin and 100 mg / ml streptomycin; transfected with 200 pmol of...

Embodiment 3

[0051] Example 3, Effect of ECHS1 siRNA on AML-12 Cell Steatosis in Vitro

[0052] 1. Group transfection

[0053] AML-12 cells were spread evenly in 12-well plates and divided into 4 groups. Concrete grouping is with the step one of embodiment 2.

[0054] 2. The effect of ECHS1 siRNA on lipid change of AML-12 cells in vitro

[0055] The cells after step 1 were subjected to the following experiments 1 or 2 or 3, respectively.

[0056] 1. Oil red dyeing

[0057] Add 4% paraformaldehyde to a 12-well plate and fix at 4°C for 30 min, wash twice with distilled water and then add oil red O dilution (oil red O 0.5g dissolved in isopropanol 100ml saturated solution and distilled water by 3: 2 diluted, left to stand for 5-10min and filtered for use), dark stained for 10-15min, washed once with 60% ethanol and differentiated under the microscope until the interstitium was clear, washed twice with Mayer’s hematoxylin counterstained nuclei for 8 seconds, washed twice Afterwards, inver...

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Abstract

The invention discloses siRNA for specifically inhibiting ECHS1 gene expression and application thereof. The small interfering RNA is double-stranded RNA consisting of nucleotide sequences shown in a sequence 1 of a sequence table and a sequence 2 of the sequence table. The invention provides the siRNA which can specifically and efficiently inhibit the ECHS1 gene expression and modified RNA obtained by modifying the siRNA. In vitro and in vivo experiment results show that the RNA can specifically and efficiently inhibit the ECHS1 gene expression, promote the increase of free fatty acid and the deposition of triglyceride, and show the increase of lipid droplets. The siRNA can be applied to the preparation of a nonalcoholic fatty liver disease animal model and the research on a nonalcoholic fatty liver disease pathogenesis. The siRNA has important values.

Description

technical field [0001] The invention relates to a siRNA specifically inhibiting the expression of ECHS1 gene and its application. Background technique [0002] RNA interference (RNA interference, RNAi) is an evolutionarily conserved defense mechanism against the invasion of transgenes or foreign viruses. Double-stranded RNA (dsRNA) specifically degrades the mRNA in the cell, thereby resulting in the process of effectively blocking the specific gene, which is a sequence-specific post-transcriptional gene silencing (PTGS). RNAi technology has been successfully applied to the study of gene functions in various organisms. Construct siRNA for the target gene, use RNAi technology to shut down the expression of the gene, and analyze the function of the gene according to the change of the phenotype. Gene knockout technology takes a long time to permanently shut down the expression of a certain gene, while RNAi technology can controllably shut down 10 genes within 1 week or even 2 ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85
CPCA01K2207/05C12N2310/14C12N9/88A01K2267/0362A01K2217/058A01K67/0275C12N15/1137
Inventor 贺福初姜颖厉有名张雪群杨俊涛叶桦虞朝辉孙薇魏汉东
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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