[Construction of a siRNA vector targeting human MTA1 gene and the gene-silencing effect].
OBJECTIVE
To construct an expression vector of siRNA targeting human MTA1 gene and observe its gene-silencing effect in esophageal carcinoma cells.


METHODS
The siRNA sequences targeting MTA1 gene were designed and synthesized with two complementary oligonucleotide strands. The oligonucleotide strands were annealed and recombined into pRNAT-U6.2 vector, which was identified by sequencing following transformation and amplification. The siRNA expression vector pRNAT-U6.2-MTA1 was transfected into human esophageal carcinoma EC9706 cells via liposome. RT-PCR and Western blotting were used to detect expression levels of MTA1 mRNA and protein in the transfected EC9706 cells, respectively.


RESULTS
The double-stranded oligonucleotide fragments of the siRNA targeting MTA1 gene were cloned into pRNAT-U6.2 vector, which was validated by sequence analysis. RT-PCR and Western blotting indicated that MTA1 mRNA and protein expressions were significantly decreased in the transfected cells, especially in those transfected with the siRNA targeting the sequence of GACCCTGCTGGCAGATAAA (481-499), which induced almost complete silencing of MTA1 protein expression.


CONCLUSIONS
The siRNA expression vector pRNAT-U6.2-MTA1 for silencing MTA1 gene expression in the esophageal carcinoma cells has been successfully constructed, which may facilitate further study for decreasing the invasive and metastatic potentials of malignant tumors by MTA1 gene silencing.
Yang
Song-hua
SH
College of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, China. yangsonghua0615@yahoo.com.cn
Zhao
Guo-qiang
GQ
Dong
Zi-ming
ZM
D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004938:Esophageal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D005822:Genetic Vectors / Q000235:genetics*; D006655:Histone Deacetylases / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators; D014162:Transfection
C089320:MTA1 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D015534:Trans-Activators; D006655:Histone Deacetylases