| Implantation of human conceptus involves invasion of trophoblast cells into the uterine epithelium and the underlying stroma that undergoes a complex process of proliferation, migration and differentiation of embryo. A typical feature of placentation in humans is the trophoblast cells wih high degree invasion to gain access to the maternal circulation in the first trimester pregnancy. An impaired endovascular trophoblast invasion has been confirmed not only associated with pre-eclampsia, fetal intrauterine growth restriction, but also the first-trimester or late miscarriage.Trophoblast cells display a very unique capability, and physiologically invade into the surrounding tissue that is similar to tumour. However, as opposed to malignant invasion, trophoblastic invasion during implantation and placentation is stringently controlled both in space and time.The human lymphocyte antigen(HLA)-G is a non-classical major histocompatibility complex (MHC) I molecule. HLA-G can participate in maternal-fetal interface immune tolerance through a variety of approaches, especially play a role in regulating immunocompetent cells, such as NK cells, T cells, antigen presenting cells, thus protect embryos from attack of immunocompetent cells and conducive to the maintenance of normal pregnancy[1]. But our previous studies found that HLA-G gene expression inhibited in JEG-3 cells leaded to invasive of cells weakened and invasive index lower significantly[2], which showed that suppressed HLA-G expression weakened invasive of trophoblast cells directly, which may lead to a series of pathological pregnancy, such as pre-eclampsia, fetal growth restriction(FGR), recurrent spontaneous abortion(RSA), etc. However, mechanisms of HLA-G and pathological pregnancy are unclear. Mitogen-activated protein kinase (MAPK) is important intracellular signalling pathways, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38MAPK involved in regulating cell proliferation, differentiation, apoptosis, adhesion, invasion and migration[3].It has been reported that p38MAPK is widely distributed in placental tissue[4], its expression may be related with pathological pregnancy closely. HLA-G expression was inhibited by RNA interference technology. Our experiments were designed to study the effects of low-expression of HLA-G on biological behaviors and the expression of p38MAPK, p-p38MAPK, JNK, p-JNK, ERK, p-ERK in the extravillous trophoblast cell line, which will illuminate a further pathogenesis of pathological pregnancy and provided new approaches to its treatments.1.Knock-down of HLA-G expression by siRNA in HTR-8/SVneo cellsHTR-8/SVneo cell line was used. The expression of HLA-G in HTR-8/SVneo cells was interfered with siRNA. In order to detect the transfection efficiency, a 100 nM concentration of fluorescent oligo was used for the analysis. About 70%-90% of cells were filled with fluorescent oligo after 6h incubation, which means the siRNA was successfully transfected into cells. RT-PCR and Western-blot analysis were applied to verify the expression of HLA-G mRNA and protein expression. HLA-G mRNA expression was inhibited significantly 48 h after transfection in the HLA-G siRNA transfected group compared with the negative siRNA transfected group or blank control group. The mRNA levels of HLA-G transfection group, negative control group and blank control group were 0.26±0.08,0.71±0.11,0.79±0.07, respectively. There was significantly different between transfection group and negative control group (P<0.01), while there was no significant difference between negative control group and blank control group(P>0.05). The mRNA inhibition rates of HLA-G transfection group, negative control group and blank control group were (69.8±6.3)%, (14.9±2.2)%,0, respectively. There was significantly different between transfection group and negative control group (P<0.01). The protein levels of HLA-G transfection group, negative control group and blank control group were 0.20±0.15,0.75±0.12,0.76±0.21, respectively.There was significantly different between transfection group and negative control group (P<0.01), while there was no significant difference between negative control group and blank control group(P>0.05). The protein inhibition rates of HLA-G transfection group, negative control group and blank control group were (81.1±14.4)%, (18.0±7.7)%,0, respectively. There was significantly different between transfection group and negative control group (P<0.01). Considering the significant change in mRNA and protein level. HLA-G expression was successfully knocked down.2. Effect of low-expression of HLA-G on the biological behaviors of HTR-8/SVneo cellsAfter HTR-8/SVneo cells were interfered for HLA-G, the effects of HLA-G on biological behaviors of trophoblast cells were investigated by Matrigel invasion assay, Brdu cell proliferation assay and AnnexinV/PI apoptosis test. HLA-G was silenced on HTR-8/SVneo cells and the number of cells that migrated to the lower surface was counted in 72h of incubation. Low expression of HLA-G substantially inhibited invasion of HTR-8/SVneo cells, which was consistent with the results of our previous study[2] that the invasion of JEG-3 cells with HLA-G suppressed was significantly lower compared with negative siRNA transfeetion and blank control groups (P<0.01). The invasion number of transfection group, negative control group and blank control group were 57±38,364±79 and 260±84, respectively, with a significant difference between transfection group and negative control group (P<0.01). There was no significant difference between negative control group and blank control group(P>0.05). The proliferation number of transfection group, negative control group and blank control group were 0.19±0.08,0.26±0.08 and 0.27±0.09, respectively, with a significant difference between transfection group and negative control group (P<0.01). There was no significant difference between negative control group and blank control group(P>0.05). The apoptosis rates of transfection group, negative control group and blank control group were (19.5±0.47)%, (15.8±0.83)%and (16.3±0.92)%, respectively, with a significant difference between transfection group and negative control group (P<0.01). There was no significant difference between negative control group and blank control group(P>0.05). It is concluded that low-expression of HLA-G inhibited the invasion, proliferation and induced apoptosis of HTR-8/SVneo cells.3. Human leukocyte antigen-G regulates biological behaviors of trophoblast-derived cell line HTR-8/SVneo via MAPK signaling pathwaysAfter HTR-8/SVneo cells were interfered for HLA-G, the key signal transduction molecules associated with invasion were detected by Western-blot to screen the possible signal pathways. We found p38MAPK pathway might play an important role. Thereafter, we treated the HLA-G-silenced HTR-8/SVneo cells with SB203580, p38MAPK pathway inhibitor, and then the biological behaviors of the trophoblast cells were analyzed by Matrigel invasive assay, Brdu cell proliferation assay, and AnnexinV/PI apoptosis test. First, we detected the expression of the critical molecules of MAPK signaling pathways, The p-p38MAPK/p38MAPK values of the HLA-G transfection group, negative control group and blank control group were 0.74±0.04,0.47±0.09 and 0.36±0.21, respectively. HLA-G transfection group was significantly different(P<0.01) compared with the other two groups(P<0.01). Without or with SB203580,the p-p38MAPK/p38MAPK values of the HLA-G transfection group were 0.89±0.09 and 0.16±0.04, the number of negative control group were 0.76±0.08,0.14±0.03. There was significantly different between without SB203580 and with SB203580 (P<0.01). The proportion of phospho-p38MAPK to total p38MAPK in the low-expression of HLA-G is significantly lower than that of the HTR-8/SVneo trophoblast cells without SB203580 (P<0.01). The results show that low-expression of HLA-G in HTR-8/SVneo trophoblast cells significantly increases the expression of the proportion of phospho-p38MAPK to total p38MAPK (P<0.01), which suggests that low-expression of HLA-G in HTR-8/SVneo trophoblast cells were via p38MAPK signaling pathways. The p-JNK/JNK values of the HLA-G transfection group, negative control group and blank control group were 0.46±0.07,0.79±0.08 and 0.78±0.13, respectively.HLA-G transfection group was significantly different(P<0.01) compared with the other two groups(P<0.01). Without or with SB203580, the p-JNK/JNK values of the HLA-G transfection group were 0.62±0.15 and 0.61±0.21, the number of negative control group were 0.86±0.11 and 0.88±0.09. There was no significant difference between without SB203580 and with SB203580 (P>0.05). The results show that low-expression of HLA-G in HTR-8/SVneo trophoblast cells significantly inhibits the expression of the proportion of phospho-JNK to total JNK (P<0.01). The p-ERK/ERK values of the HLA-G transfection group, negative control group and blank control group were 0.89±0.13,0.88±0.21 and 0.89±0.26, respectively. HLA-G transfection group was not significantly different compared with the other two groups(P>0.05). Without or with SB203580, the p-ERK/ERK values of the HLA-G transfection group were 0.89±0.18 and 0.91±0.11, the number of negative control group were 0.87±0.17 and 0.89±0.23. There was no significant difference between without SB203580 and with SB203580 (P>0.05). We next determined whether p38MAPK signaling pathway was involved in the HLA-G-mediated suppression of human trophoblast cells biological behaviors. The matrigel-based transwell assay demonstrated the effect of low-expression of HLA-G on the invasion of human HTR-8/SVneo trophoblast cells, and the invasion of HTR-8/SVneo trophoblast cells transfected by HLA-G after treatment with SB203580,and the number of cells that migrated to the lower surface were counted. Without or with SB203580, the invasion number of transfection group were 51±13 and 90±21, which was significantly different(P<0.01). The number of negative control group were 290±52 and 298±33. There was no significant difference between without SB203580 and with SB203580 (P>0.05).Without or with SB203580, the proliferation number of transfection group were 0.22±0.09 and 0.22±0.16, which was no significant difference(P>0.05). The number of negative control group were 0.22±0.15 and 0.25±0.15. There was no significant difference between without SB203580 and with SB203580 (P>0.05) Without or with SB203580, the apoptosis rates of transfection group were (19.0±0.58)%and (13.4±0.28)%, which was significantly different(P<0.01). The number of negative control group were (10.0±0.80)%and (9.51±2.73)%. There was no significant difference between without SB203580 and with SB203580 (P>0.05). Based above the main findings, HLA-G gene may regulate biological behaviors of trophoblast cells through p38MAPK and be involved in the occurrence of pathologic pregnancy.
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