Objective: To investigate the role of human leukocyte antigen-G (HLA-G) on the invasion and the molecular mechanism involved in this cellular progress in HTR-8/SVneo cell line.
Methods: There were three groups: groups of transfection, negative control and blank control, which corresponding to treatment by HLA-G specific siRNA, negative siRNA and only lipofectamine 2000 using lipofection technology in HTR-8/SVneo cell line. The efficiency of down-regulated of HLA-G was detected by reverse transcription-polymerase chain reaction and western blot analysis in mRNA and protein level, respectively. Changes of p38 mitogen-activated protein kinases (p-p38MAPK)/p38MAPK protein levels and the cell invasion were respectively detected by western blot analysis and transwell test.
Results: (1) 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 significant difference 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 efficiencies of down-regulated of HLA-G were (69.8±6.3)%, (14.9±2.2)%, 0 in transfection group, negative control group and blank control group respectively in mRNA level. (2) In protein levels, HLA-G were 0.20±0.15, 0.75±0.12, 0.76±0.21 in transfection group, negative control group and blank control group, respectively. There was significant difference between transfection group and negative control group (P<0.01), whereas there was no significant difference between negative control group and blank control group (P>0.05). The efficiencies of down-regulated of HLA-G were (81.1±14.4)%, (18.0±7.7)%, 0 in transfection group, negative control group and blank control group respectively. (3) The invasive 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). (4) 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 compared with the other two groups (P<0.01). (5) 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 values of negative control group and blank control group were 0.76±0.08, 0.14±0.03 and 0.51±0.05, 0.03±0.01, respectively. There was significant difference between without SB203580 and with SB203580 (P<0.01). (6) Without or with SB203580, the invasive number of transfection group were 51±13 and 90±21, respectively, which was significantly different (P<0.01). The invasive number of negative control group and blank control group were 290±52, 298±33 and 290±73, 264±64, respectively, which was no significant difference between without SB203580 and with SB203580 (P>0.05).
Conclusions: HLA-G gene may regulate invasion of trophoblast-derived cell line HTR-8/SVneo via p38MAPK signaling pathway. The lower expression of HLA-G in trophoblast cells may lead to the occurrence of pathologic pregnancy.