The Relationship Of Altered Expression Of GRIM-19with Missed Abortion

PurposesWe examined the differential expression of GRIM-19in villous from women with missed abortion and normal pregnancies to explore the correlation between GRIM-19and missed abortion. After that, we detected apoptotic cells and micro vessel density (MVD) in both cases, and study the difference of mitochondrial membrane potential (△Ψ) and apoptosis and angiogenesis after GRIM-19has lower expression in vivo and vitro study. Explore the role of altered expression of GRIM-19on missed abortion.MethodsIn vivo study:this study involved50pregnant women diagnosed with first-trimester missed abortion as experimental group, and the control group consisted of50women in unplanned healthy pregnancy. Immunohistochemistry was used to assess the GRIM-19location in the trophoblast, western blot and real-time PCR were used to analyse levels of GRIM-19protein and mRNA in villous samples. We studied apoptotic changes in trophoblast by TdT-mediated dUTP-biotin nick end labeling (TUNEL) method, the microvessel density (MVD) was defined as the mean number of CD34+vessels per section of one villous sample. In vitro study:we also used JC-1and AnnexinV/PI measured the mitochondrial membrane potential (△Ψ) and apoptotic changes of the HTR-8/SVneo cell line response to GRIM-19knockdown by siRNA in vitro study; detected VEGF secretion changes in HTR-8/SVneo cells culture medium after GRIM-19knockdown by ELSA. Simultaneously, we examined P3、STAT3、 HIF-1α and VEGF protein, mRNA levels and location in villous samples, The relationship of the mRNA expression level of GRIM-19and their mRNA in villous samples of missed abortion patients was analyzed by spearman correlation analysis.Results1. GRIM-19is mainly expressed in cytoplasm of villous cytotrophoblast, syneytiotrophoblast and extravillous trophoblastic cell column. The protein and mRNA level in missed abortion are both significantly lower than normal pregnancies (P<0.01).2. A significant increase in the number of TUNEL-positive cells is detectable in villous from missed abortion group (P<0.01).3. Knockdown HTR-8/SVneo cells of GRIM-19give rise to a collapse of AT and increased apoptosis, and the increased percentage of low△Ψ with JC-1show a significant positive correlation with a simultaneous increase apoptotic cells (r=-0.754, P<0.01).4. P53is expressed in caryon of villous cytotrophoblast, syneytiotrophoblast and extravillous trophoblastic cell column. The missed abortion samples has a conspicuous higher percentage of P53-positive cells in syneytiotrophoblast and extravillous trophoblastic cell column (p<0.01), while no difference was observed in cytotrophoblast (p=0.458). P53protein and mRNA in villous from missed abortion are both significantly higher than those from normal pregnancies (P<0.01). In villous samples of missed abortion patients, the expression of GRIM-19mRNA was negatively correlated with the expression of P53mRNA (r=-0.37), and the relationship is significant (p=0.028).5. We find a significantly lower MVD in villous from missed abortion group as compared to that from the control group (P<0.01).6. The VEGF production in the culture medium of GRIM-19knockdown HTR-8/SVneo cells is decreased after GRIM-19knockdown48h (P<0.05), and the decrease is striking after72h (P<0.01).7. STAT3, HIF-1α and VEGF protein are mainly expressed in cytoplasm of villous cytotrophoblast, syneytiotrophoblast and extravillous trophoblastic cell column. It’s worth noting that in villous samples of missed abortion, VEGF expression significantly decrease in cytotrophoblast, while most cytotrophoblast cells in control samples are intense staining. STAT3and HIF-1α protein and mRNA in the missed abortion villous are significantly higher than those from control samples (P<0.01, P<0.05), while VEGF has a significantly lower level in the samples from missed abortion (P<0.05). The expression of GRIM-19mRNA in the missed abortion villous is negatively correlated with the expression of STAT3, HIF-1α (r=-0.21, r=-0.49), and the relationship was significant (P=0.009, P=0.003); while the expression of GRIM-19is positively correlated with that of VEGF (r=0.56), and the relationship was significant (P=0.01).ConclusionGRIM-19is essential for early pregnancy development, and the GRIM-19deficiency in the villous may be associated with missed abortion via mitochondria-mediate apoptosis and reducing angiogenesis.