Effect And Possible Mechanism Of Decidual NK Cells On Recurrent Spontaneous Abortion

Recurrent spontaneous abortion(RSA)is a common pregnancy complication which have concern of the whole world because it damage the physical and psychological health of fertile woman and family harmony.However,the causes of RSA are complicated and mostly unknown.Immunologic dysfunction is generally considered the most important cause leading to RSA.Whether gravid or inchoate,there is a unique combination of human leukocyte antigen(HLA)expression on extravillous trophoblasts(EVTs)and maternal leukocytes in decidual tissues at the maternal-fetal interface.This combination plays an important role in maintain the normal pregnancy.Of the HLA expression on EVT,HLA-G is the most frequently studied and plays crucial roles in inducing immune tolerance to maintain pregnancy.Many researches indicated that decrease expression of HLA-G is related with RSA.In human,approximately 40% of the decidual cells are lymphocytes and more than 70% are CD56brightCD16-NK cells which can contact with EVT directly.Recent studies have shown that dNK cells play an important role in early pregnancy.These studies demonstrated that dNK cells possess the unique ability to regulate crucial pregnancy processes,including trophoblast invasion,vascular growth and immunol tolerate.However,the regulate mechanism of dNK cells is still unknown.Killer immunoglobulin-like receptor 2DL4(KIR2DL4),a specific receptor for HLA-G,is expressed on human NK cells.Therefore,it has been suggested that NK cells expressing KIR2DL4 might be involved in the maintenance of pregnancy by recognizing HLA-G.In studying the mechanisms underlying RSA,we found that miR-133 a was greatly overexpressed in RSA villi compared to villi from induced abortion patients.Multi-software prediction and real-time PCR confirmed that miR-133 a was most likely to bind to the HLA-G 3’UTR,as established in our previous study.Therefore,this study was designed to confirm that miR-133 a negatively regulates HLA-G expression to influence dNK function via KIR2DL4 in RSA patients.Part 1: The resereach of d NK cells and recurrent spontaneous abortion.Objective: To investigate the relationship between dNK cell count and dysfunction with RSA.Methods: Decidual mononuclear cells of IA and RSA were isolated by centrifugation through Ficoll.The percentage of CD56+ dNK cells and KIR2DL4 expression on dNK cells in patients with IA and patients with RSA by flow cytometry analysis.The profile of the cytokine secretion of dNK cells in the two groups was examined using a multiplex cytokine assay after purification of dNK cells and culutured for 24 hours.The dNK culture supernatants of the two groups were harvested and co-cultured with HTR-8/SVneo cells to perform the Matrigel invasion assay.At the same time,the supernatants were co-cultured with HUVECs to perform the tube formation assay.Results: The RSA group expressed lower KIR2DL4 than did the IA group in terms of both the percentage and mean fluorescence intensity(P<0.05).However,the mRNA levels of IL-10 and VEGF in dNK cells were much lower in the RSA group(P<0.05).We confirmed that the dNK supernatants of the RSA group significantly reduced the invasive activity of HTR-8/SVneo cells compared with the IA group(P<0.05).The results of the tube formation assay also revealed that dNK supernatants of the RSA group significantly reduced the tube formation ability of HUVECs(P<0.05).Part 2: HTR-8/SVneo cells transfected with miR-133 a and HLA-G expression in HTR-8/SVneo cells after transfected with miR-133 a.Objective: To confirm that miR-133 a decrease HLA-G expression in HTR-8/SVneo cells.Methods: To the HTR-8/SVneo cells transfection of miR-133 a by lipo2000.There are three groups: control(transfection of miR-133 a negative control),overexpress group(transfection of miR-133 a mimics)and inhibit express group(transfection of miR-133 a inhibitor).To confirmed the down-regulation of HLA-G by miR-133 a using western blot.Results: Increased HLA-G expression in HTR-8/SVneo cells after transfected with miR-133 a inhibitor.Decreased HLA-G expression in HTR-8/SVneo cells after transfected with miR-133 a mimics(P<0.05).Part 3: Influence in the pro-invasive and pro-angiogenesis ability of dNK Cells affected with miR-133 a.Objective: To investigate whether decreased HLA-G by miR-133 a contributes to impaired functions of dNK cells.Methods: Fresh dNK cells were co-cultured with HTR-8/SVneo cells transfected with miR-133 a.We co-cultured dNK cells with HTR-8/SVneo cells plus anti-KIR2DL4 antibodies or isotypes.The cell culture medium was harvested,and the cytokines were analyzed,co-cultured with HTR-8/SVneo cells to perform the Matrigel invasion assay.At the same time,the supernatants were co-cultured with HUVECs to perform the tube formation assay.Results: The results of the assay showed that dNK cells co-cultured with HTR-8/SVneo cells transfected with miR-133 a mimics significantly decreased the levels of IL-8,IP-10 and VEGF(P<0.05).The Transwell assay showed that the dNK cells co-cultured with HTR-8/SVneo cells transfected with miR-133 a mimics significantly reduced the migration ability of the HTR-8/SVneo cells.The results showed reduced IL-8,IP-10 and VEGF in the presence of blocking antibodies for KIR2DL4(P<0.01).We found that the anti-KIR2DL4 antibodies reduced the invasive activity of HTR-8/SVneo cells compared with the control group(P<0.05).In addition,tube formation by HUVECs was observed after 6h with supernatant stimulation.The results showed the significantly reduced tube formation capacity following the blockade of KIR2DL4 by Ab(P<0.05).In conclusion,our current study showed that decreased HLA-G expression by miR-133 a impaired pro-invasion and pro-angiogenesis ability of dNK cells may via KIR2DL4 receptor.Our findings provide a possible mechanism of RSA and a basis for further study;in addition,this study may provide a drug target for therapy of RSA.