CHOP基因shRNA载体构建及沉默效应

Gene-silencing effect and small hairpin RNA vector construction of targeting human CCAAT/enhancer-binding protein-homologous protein gene

背景:研究认为,CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein-homologous protein,CHOP)通路是内质网应激介导细胞凋亡的3条通路之一,目前国内外对内质网应激介导细胞凋亡在胃癌方面的研究较少。目的:构建针对人CHOP基因的短发卡RNA表达载体,观察RNA干扰对人胃癌细胞系BGC823细胞CHOP基因表达的抑制作用。方法:设计CHOP的shRNA靶序列,分别合成两条互补的寡核苷酸链,退火后重组入真核表达载体pSUPER-EGFP1,转化扩增后进行序列测定。用脂质体包裹转染人胃癌细胞系BGC823细胞,采用RT-PCR和Western blotting分别检测CHOP基因mRNA和蛋白表达的变化。结果与结论:把针对CHOP基因的shRNA的双链寡核苷酸片段克隆入pSUPER-EGFP1载体,经测序分析,插入片段正确;RT-PCR和Western blot检测显示,CHOP基因的表达水平明显降低,其中以CHOP-1为靶序列的shRNA沉默作用最强,CHOP的表达抑制率约67%。

BACKGROUND： Studies have proved that CCAAT/enhancer-binding protein-homologous protein （CHOP） is one of the three pathways of endoplasmic reticulum stress mediated apoptosis. There are few studies in home and abroad on endoplasmic reticulum stress mediated apoptosis in gastric carcinoma. OBJECTIVE： To construct an expression vector of a small hairpin RNA （shRNA） targeting human CHOP gene and to observe gene-silencing effects of CHOP in human gastric carcinoma cell BGC823. METHODS： The shRNA sequences targeting CHOP gene were designed and synthesized with two complementary oligonucleotide strands. The oligonucleotide strands were annealed and recombined into pSUPER-EGFP1 vector, which was identified by sequencing following transformation and amplification. The shRNA expression vector pSUPER-EGFP1-CHOP was transfected into human gastric carcinoma cell BGC823 via liposome. Reverse transcription-PCR and Western blot were used to detect expression levels of CHOP mRNA and protein in the transfected BGC823 cells, respectively. RESULTS AND CONCLUSION： The double-stranded oligonucleotide fragments of the shRNA targeting CHOP gene were cloned into pSUPER-EGFP1 vector and validated by sequence analysis which showed that expression vector pSUPER-EGFP1- CHOP was successfully constructed. Reverse transcription-PCR and Western blot indicated that CHOP mRNA and protein expressions were significantly decreased in the transfected cells, especially in those transfected with the shRNA targeting the sequence of CHOP-1, which induced 67% silencing of CHOP expression.