Abstract:

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.