Construction Of Eukaryotic Expression Plasmid Expressing SiRNA Targeting HLA-G Gene And Detection Of Its Specific Downregulation Effect

The HLA-G gene is a kind of non-classical human leukocyte antigen (HLA) class Ib genes, located the sixth short arm of a chromosome of the human being.In contrast to classical HLA class I antigens, exhibits tissue-restricted distribution; limited polymorphism; and alternative transcription of spliced mRNAs that encode at least seven different HLA-G isoforms.The HLA-G gene expresses in vascular endothelial cells of fetus chorionic villus, amniotic fluid, proliferating cytotroblast cells of chorionic villus and epithelial tissue of thymus.It plays a role in materno-fetal tolerance maintaining normal pregnancy. For a woman in child-bearing age, the abnormal expression of HLA-G is possibly associated with certain complications of pregnancy (such as infertility, idiopathic recurrent spontaneous abortions (RSA) and gestational hypertension). The HLA-G gene can inhibit NK cell-mediated cytolysis and the lytic activity of antigen-specific of T cells, can induce the apoptosis of CD8+ CTL. It can regulate releasing of cytokine, and then influence organism immunoreaction. the investigation of the HLA-G gene functions on trophoblasts will contribute to mother-infant health and human assisted reproduction. Thus, the HLA-G gene is a significant modulin of anthropo-reproduction, the study of its functions should contribute to better understanding of human reproduction.RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing(PTGS) in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. Since Fire et.al. found the phenomenon in the nematode caenorhabditis elegans for the first time in 1998, consequently similar processes have been described for Drosophila melanogaster, trypanosome, mammals including humans. At present, RNAi has been widely applied in the research of gene function and gene therapy as an efficient tool for specific gene silencing.Objective: 1. To construct an eukaryotic expression plasmid expressing siRNA targeting HLA-G gene and detect its specific downregulation of HLA-G expression in transfected JEG-3 cells; 2. To establish a human JEG-3 cell line with the stably suppressed of HLA-G; 3. To investigate the effect of HLA-G siRNA on the protection of trophoblast cells from NK cell lysis.Methods: The first part of the study: According to algorithm for selection of functional siRNA sequences, two pieces of siRNAs targeting the HLA-G mRNA were distinguished inhibition by detecting. Hairpin siRNA templates were synthesized and was cloned into pSuppressor-U6-neo vector. The pSuppressor-U6-neo-HLA-G with siRNA targeting HLA-G gene(a kind of Eukaryotic expression plasmid) was identified by restrict endonuclease digestion and DNA sequence analysis . Then the resultant plasmids were transfected into JEG-3 cells with lipofectin2000. The inhibitory effects on HLA-G mRNA and protein expression were detected respectively by semi-quantitative RT-PCR and Western-blot.The second part: The resultant plasmid pSuppressor-U6-neo-HLA-G was transfected into JIEG-3 cells by lipofectin2000 and selected with G-418-containing culture medium. HLA-G inhibition in stable transfected cells was detected by RT-PCR and Western-blot.The third part of the study: 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: 1.The plasmid pSuppressor-U6-neo-HLA-G was constructed successfully and efficiently reduced the expression of HLA-G and conferred G-418 resistance in JEG-3 cells; 2. A human cellular model with stably suppressed HLA-G was established. Semi-quantitative RT-PCR, Western-blot and FCM all revealed a potent inhibition of the expression of HLA-G in transfected cells; 3. Compared with JEG-3 cells without HLA-G without HLA-G siRNA plasmid transfection, the transfected JEG-3 cells showed higher lytic activity;HLA-G siRNA can increasing NK cytolysis against JEG-3 cells and plays a role in preventing of trophoblast cells from NK cytotoxicity.Conclusion: We have designed an appropriate oligonucleotides to interfere exclusively with HLA-G mRNA; The successful construction of the siRNA expressing plasmid(pSil-HLA-G) will facilitate the application of RNA interference technique. For the analysis of function and mechanisms of HLA-G-induced immune regulation, a human cellular model with suppressed HLA-G was established and would be very helpful; We proved HLA-G siRNA can increasing NK cytolysis against JEG-3 cells. It lay the foundation for further investigation of HLA-G functions in maternal-fetal immune tolerance, normal pregnancy and human assisted reproduction in vitro and in vivo.