Construction of ARID1A gene mutants and identification of the overexpression in hepatocellular carcinoma cells
10.3760/cma.j.issn.1003-9279.2017.04.015
- VernacularTitle: ARID1A基因突变体的构建及其在肝癌细胞中的过表达鉴定
- Author:
Hua XIAO
1
;
Qicheng LIAO
2
;
Haiyan WANG
1
;
Weilong LIU
1
;
Chi WU
1
;
Jian HUANG
1
;
Jialin LIU
1
Author Information
1. Shenzhen Third People’s Hospital, Shenzhen 518112, China
2. Gannan Medical University, Ganzhou 341000, China
- Publication Type:Journal Article
- Keywords:
ARID1A;
Functional domain;
Truncated mutation;
Overexpression;
Liver neoplasms;
Cell culture
- From:
Chinese Journal of Experimental and Clinical Virology
2017;31(4):343-347
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct the mutants of ARID1A gene, which is an important component in human chromosomal remodeling complex Switch/Sucrose Non Fermentable (SWI/SNF), and identify their overexpression in liver cancer HepG2 cells.
Methods:Overlap PCR was used to construct domain truncated mutantss pcDNA6-ARID1A/ΔARID and pcDNA6-ARID1A/ΔD UF3518 based on wild type plasmids pcDNA6-ARID1A. Lipofectionmethod was used to transfect the wild type and mutants into HepG2. Real-time PCR and western blotting were used to confirm the overexpression of ARID1A and the mutants.
Results:SDS-PAGE and sequencingresult confirmed the successful construction of pcDNA6-ARID1A/ΔARID and pcDNA6-ARID1A/ΔDUF3518. Real-time PCR and western blottingresult confirmed the overexpression of both mRNA and protein of wild type ARID1A and ARID1A/ΔARID. The mRNA levels indicated that ARID1A/Δ DUF3518 were overexpressed, but the protein levels were quite low.
Conclusions:Functional domain truncated mutants of ARID1A were successfully constructed. Overexpression of wild type ARID1A and ARID1A/ΔARID in liver cancer HepG2 cells was successful. Loss of ARID1A/ΔDUF3518 protein suggest that DUF3518 may contribute to the protein structure stability.
