[Construction of eukaryotic expression plasmid expressing siRNA targeting HLA-G gene and detection of its specific downregulation effect].
OBJECTIVE
To investigate the effect of HLA-G siRNA on the protection of trophoblast cells from NK cell lysis, an HLA-G-targeting siRNA (small interfering)-expressing plasmid was constructed.


METHODS
HLA-G siRNA plasmid, pSuppressor-U6-neo-HLA-G, was constructed and transfected to JEG-3 cells. The level of messenger RNA and protein of HLA-G in the transfected cells was detected by RT-PCR and Western blot. NK-92MI cells were co-cultured with pSuppressor-U6-neo-HLA-G transfected JEG-3 cells. The cytotoxicity of NK-92MI cells against the transfected cells was analyzed by LDH releasing assay.


RESULTS
Downregulation of HLA-G in HLA-G siRNA plasmid transfected JEG-3 cells was confirmed by RT-PCR and Western blot. Compared with JEG-3 cells without HLA-G without HLA-G siRNA plasmid transfection, the transfected JEG-3 cells showed higher lytic activity.


CONCLUSIONS
HLA-G siRNA can increasing NK cytolysis against JEG-3 cells. HLA-G plays a role in preventing of trophoblast cells from NK cytotoxicity.
Chen
Jian-hui
JH
Department of Obstetrics and Gynecology, Tangdu Hospital, Xi'an 710038, China. jhch0904@163.com
Yao
Yuan-qing
YQ
Hou
Kai-bo
KB
Lei
Ying-feng
YF
Yin
Wen
W
D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D015536:Down-Regulation; D006680:HLA Antigens / Q000235:genetics* / Q000378:metabolism; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007694:Killer Cells, Natural / Q000166:cytology / Q000502:physiology; D010957:Plasmids / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014327:Trophoblasts / Q000166:cytology
D006680:HLA Antigens; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I; D034741:RNA, Small Interfering