[Construction of eukaryotic expression plasmid expressing siRNA targeting HLA-G gene and detection of its specific downregulation effect].

AIM

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.

CONCLUSION

HLA-G siRNA can increasing NK cytolysis against JEG-3 cells. HLA-G plays a role in preventing of trophoblast cells from NK cytotoxicity.