[Construction of shRNA eucharyotic expression plasmid targeted human VIGILIN and the investigation of VIGILIN's effect on hepatocarcinoma cell cycle].
OBJECTIVE
To construct a shRNA eucharyotic expression plasmid against human VIGILIN and explore possible relation between human VIGILIN and HepG2 cell cycle.


METHODS
We constructed the shRNA eucharyotic expression plasmid targeted human VIGILIN, and transfected HepG2 cells with shRNA expression plasmid pSIREN-VIG, then determined the expression of VIGILIN mRNA and protein in HepG2 cells by RT-PCR and Western-blot, analysed alteration of cell cycle using FACS.


RESULTS
The plasmid pSIREN-VIG can effectively and specifically inhibit the expression of human VIGILIN. After transfection 48 hours, the expression of VIGILIN was significantly decreased. Due to knockdown of human VIGILIN, cell cycle is impaired and cells are arrested in G2/M phase. The proportion of G2/M phase of all groups were listed as: C group (untreated wild HepG2 cells) 2.4%, M group (HepG2 cells treated with transfection reagent) 4.9%, G group (HepG2 cells transfected with pSIREN-GFP) 6.5% and V group (HepG2 cells transfected with pSIREN-VIG) 9.4%.


CONCLUSIONS
We have successfully constructed a shRNA expression plasmid which could effectively and specifically inhibit the expression of human VIGILIN.
Xie
Xiao-yan
XY
Department of Biochemistry and Molecular Biology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Wei
Ling
L
Yang
Wen-li
WL
Ge
Ya-jun
YJ
Qin
Yang
Y
D001483:Base Sequence; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle / Q000235:genetics* / Q000502:physiology; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C075197:high density lipoprotein binding protein