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EZH2 variable spliceosome and application thereof

A technology of EZH2-L and EZH2-S, which is applied in the field of EZH2 variable shear body, can solve problems such as ventricular septal defects and dysplasia of compressed myocardium

Active Publication Date: 2021-01-22
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies have shown that knockout of EZH2 causes fatal congenital heart malformations, including dysplasia of the compressed myocardium and defects of the ventricular septum

Method used

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  • EZH2 variable spliceosome and application thereof
  • EZH2 variable spliceosome and application thereof
  • EZH2 variable spliceosome and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Isolation of neonatal rat cardiomyocytes (Neonatal rat ventricular myocytes, NRVMs)

[0036] 1. Myocardial tissue digestion

[0037] The cardiomyocytes of neonatal rats were sterilized, and the heart was taken out, the blood was washed with 1x Ads buffer solution, the great blood vessels at the bottom of the heart and the atrium of the atrial appendage were removed, and the myocardial tissue was cut to about 1mm 2 Add an appropriate amount of mixed enzyme digestion solution of 0.1% collagenase and 0.075% trypsin to digest for 20 minutes, discard the supernatant, add an appropriate amount of mixed enzyme digestion solution for digestion for 20 minutes, collect the supernatant, add a small amount of serum to the supernatant to stop mixing Enzyme digestion reaction, repeat the above digestion to collect the supernatant, and add a small amount of serum to the supernatant 5-6 times, then centrifuge the above digestion solution at 2200rpm x 3min to collect the cells...

Embodiment 2

[0042] Example 2 siRNA transfection method and establishment of phenylephrine-induced cardiomyocyte hypertrophy model

[0043] 1. Myocyte culture

[0044] On the first day, cardiomyocytes were counted and seeded into a six-well plate, with about 0.5x 10 cells per well. 6 The culture medium is 10% FBS+1% penicillin-streptomycin solution+high sugar DMEM medium. The next day, the culture medium was changed to 1% ITS+1% penicillin-streptomycin solution+high sugar DMEM medium.

[0045] 2. siRNA transfection

[0046] On the third day, siRNA transfection was performed. Before transfection, the culture medium was replaced with 1% ITS + high-sugar DMEM medium, and then siRNA solution and transfection reagent RNAiMAX solution were prepared respectively. The siRNA solution per well was 4ul siRNA + 200ul Opti-MEM medium , the transfection reagent solution per well is 6ul RNAiMAX+200ul Opti-MEM medium, after preparation, let stand at room temperature for 5 minutes, then mix the two and ...

Embodiment 3

[0058] Example 3 Detection of two alternative splicing forms EZH2-L and EZH2-S of neonatal rat cardiomyocyte EZH2

[0059] 1. RNA extraction

[0060]Rat cardiomyocytes were isolated according to Example 1, and mRNA was extracted. Add 1ml of Trizol solution to the collected cardiomyocytes, pipette repeatedly on ice until the cardiomyocytes are fully lysed, let stand for 5 minutes, add 0.2ml chloroform, shake vigorously for 15-30 seconds, let stand for 2-3 minutes, at 4°C Centrifuge at 12000rpm x 15min. Pipette the aqueous layer into a new EP tube, add 0.5ml of isopropanol, mix the liquid in the tube gently, let stand at room temperature for 10 minutes, and centrifuge at 12000rpm x 10min at 4°C. Discard the supernatant, add 1ml of pre-cooled 75% ethanol to the pellet, resuspend the pellet and wash thoroughly, and centrifuge at 12000rpm x 5min at 4°C. Discard the supernatant, dry it in the air, add an appropriate amount of DEPC water, and dissolve the RNA at 65°C. Take 2ul of...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to an EZH2 variable splicing site and an application thereof. Specific siRNA is used for knocking down two subtypes ofEZH2-L and EZH2-S respectively, and myocardial hypertrophy and heart failure models are established at the cellular level and the animal level respectively. Results show that after the EZH2-L is knocked down, the expression level of a myocardial hypertrophy pathological gene is obviously reduced, the myocardial cell area is reduced, and the heart weight is reduced; and after the EZH2-S is knockeddown, the expression level of the myocardial hypertrophy pathological gene is remarkably increased, the myocardial cell area is increased, the heart weight is increased, and the situation prompts that the EZH2-L and the EZH2-S have the effects of promoting and inhibiting myocardial hypertrophy and heart failure respectively. In addition, after the EZH2-L is knocked down, tumor cell proliferationis inhibited, and knock-down of the EZH2-S has no influence on tumor cell proliferation. The invention provides a new target and a new strategy for research and development of medicines for treating EZH2 gene related diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an EZH2 variable splicing body and its application. Background technique [0002] Epigenetic modifications can regulate chromatin state and gene expression through DNA methylation, histone modification, and chromatin remodeling without changing the DNA sequence. Polycomb group proteins (PcGs) are a group of important epigenetic regulatory proteins. PcG protein contains two core complexes, namely PRC1 complex (polycomb repressive complex 1) and PRC2 complex (polycomb repressive complex 2). The PRC2 complex specifically catalyzes the monomethylation, dimethylation and trimethylation of H3K27 sites to regulate gene transcription. The catalytic subunit EZH2 (enhancer of zeste homolog 2) of the PRC2 complex is a histone methyltransferase for histone H3 trimethylation at Lys27 (H3K27me3). EZH2 is a highly evolutionarily conserved gene with similar domains in different specie...

Claims

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Application Information

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IPC IPC(8): C12N9/10A61K45/00A61P9/04A61P9/00A61P35/00
CPCC12N9/1007C12Y201/01043A61K45/00A61P9/04A61P9/00A61P35/00
Inventor 王志华王顺郭宁宁李丽莉
Owner WUHAN UNIV
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