ABSTRACT
Objective To investigate the effects and its mechanisms of bradykinin B2 receptor (B2R) on the growth and function of human extravillous trophoblast cells (HTR-8/SVneo cells).Methods B2R expression plasmid (pcDNA3.1-B2R) was constructed and B2R-specific small interfering RNA (siRNA) was synthesized.HTR-8/SVneo cells were divided into four groups and transfected with pcDNA-3.1 (blank plasmid group),pcDNA3.1-B2R (B2R expression plasmid group),siRNA negative control and B2R-specific siRNA,respectively.Quantitative real-time reverse transcription-polymerase chain reaction and Western blot were used to detect the changes in the expression of B2R,matrix metalloproteinase-2,matrix metalloproteinase-9,cyclin D1 and vascular endothelial growth factor-A at both mRNA and protein levels in HTR-8/SVneo cells.Cell counting kit-8 and flow cytometry were used to detect cell activity and cell cycle,respectively.Cell migration assay and cell invasion assay were used to detect cell migration and invasion,respectively.Tube formation assay was used to evaluate the tube formation abilities of HTR-8/SVneo cells.All data were analyzed with t test.Results (1) Compared with the blank plasmid group,expression of B2R in HTR-8/SVneo cells in the B2R expression plasmid group were significantly increased at both mRNA (5.06±0.49 vs 1.00±0.28,t=7.226,P=0.002) and protein levels (1.34 ± 0.07 vs 1.00± 0.05,t=3.727,P=0.006).And the expression of B2R in HTR 8/SVneo cells transfected with B2R-specific siRNA were significantly reduced at both mRNA (0.34±0.05 vs 1.00±0.17,t=3.667,P=0.021) and protein levels (0.74±0.03 vs 1.00±0.05,t=4.097,P=0.006) comparing with the siRNA negative control group.(2) Compared with the blank plasmid group,HTR-8/SVneo cells being transfected with B2R expression plasmid showed a higher proliferation activity (1.50 ±0.03 vs 1.34± 0.04) promoting G0/G1 to S phase transition;compared with the siRNA negative control group,B2R-specific siRNA inhibited the proliferation of HTR-8/SVneo cells (1.06 ± 0.04 vs 1.20± 0.02) and arrested the cell cycle at G0/G 1 phase (all P＜0.05).(3) Compared with the blank plasmid group,B2R expression plasmid significantly increased the HTR-8/SVneo cell migration distance [(80.67±0.33) vs (41.33±5.24) μm],the number of cells penetrating matrigel gel (360.70 ±12.33 vs 268.70 ±14.45) and the number of cells having tube-like structures (28.20 ± 2.47 vs 14.00± 1.67),while significantly decrease was shown in these three parameters in B2R-specific siRNA group comparing with the siRNA negative control group [HTR-8/SVneo cell migration distance:(56.00±3.51) vs (87.00±1.53) μ m,number of cells penetrating matrigel gel:143.30± 12.91 vs 252.30± 17.07;number of tube-like structures:6.25±1.49 vs 15.75 ±2.02;all P＜0.05].(4) Expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 at mRNA level,and expression of cyclin D1 and vascular endothelial growth factor-A increased in the B2R expression plasmid group than in the blank plasmid group,and decreased in the B2R-specific siRNA group than in the siRNA negative control group at both mRNA and protein levels (all P＜0.05).Conclusions B2R might enhance the activity,migration,invasion and tube formation ability of human extravillous trophoblast cells through promoting the expression of matrix metalloproteinase-2,matrix metalloproteinase-9,cyclin D1 and vascular endothelial growth factor-A.
ABSTRACT
Objective To investigate the effects ofpravastatin on the expression ofmicroRNA-155 (miR-155) and the functions of lipopolysaccharide (LPS)-treated extravillous trophoblast cells.Methods In vitro cultured HTR-8/SVneo cells were divided into the following groups:control group,enhanced plasmid with green fluoscent protein (pEGFP)-miR-155 group (transfected with green fluorescent protein-tagged miR-155),LPS group (treated with 100 ng/mL of LPS),miR-155 inhibitor+LPS group,pravastatin+LPS group (treated with 100 ng/mL of LPS following pretreatment with 12.50,25.00,50.00 and 100.00 μ g/ml of pravastatin),and pravastatin+pEGFP-miR-155 group (transfected with pEGFP-miR-155 following pretreatment with 50 μ g/ml of pravastatin).Levels of miR-155 in HTR-8/SVneo cells treated with different strategies were measured by real-time polymerase chain reaction.Expression of phosphorylated JunB (p-JunB) and p-FosB proteins was analyzed by Western blotting.Migration,invasion and apoptosis of HTR-8/SVneo cells were also analyzed.All data were analyzed with t test.Results (1) Compared with the control group,HTR-8/SVneo cells in the pEGFP-miR-155 group were characterized by shorter migration distance [(274.70± 18.82) vs (181.00±8.62) μ m],less transmembrane cells [(123.00±4.36) vs (63.00±6.08)] and enhanced apoptosis [(5.40± 0.68)％ vs (9.27±0.68)％] (all P＜0.05).(2) Compared with the LPS group,the miR-155 inhibitor+LPS group showed longer migration distance of HTR-8/SVneo cells [(166.30±5.07) vs (242.00±18.07) μm,P＜0.05],more transmembrane cells [(71.67±6.12) vs (109.00±7.81),P＜0.05] and decreased cell apoptosis [(14.40±1.69)％ vs (6.23± 0.44)％,P＜0.05].(3) Expression of miR-155 at mRNA level in the LPS group was increased as compared with that of the control group (1.65 0.07 vs 0.79±0.12,P＜0.05).Compared with the LPS group,pretreatment with 12.50,25.00,50.00 and 100.00 μ g/ml of pravastatin decreased the expression of miR-155 at mRNA level [(1.14±0.10),(1.02±0.10),(0.74±0.15) and (1.140.02)],especially at the concentration of 50 μμ g/ml (all P＜0.05).(4) Expression ofp-JunB and p-FosB proteins in the control,LPS and pravastatin (50 μ g/ml)+LPS groups were (0.33 ±0.06) vs (0.37±0.07),(1.22±0.20) vs (0.80±-0.13),and (0.31 ±0.02) vs (0.21 ±0.05),respectively,showing higher expression level in both p-JunB and p-FosB proteins in the LPS group compared with that of the other two groups (all P＜0.05).(5) Compared with the LPS group,the pravastatin (50 μμ g/ml)+LPS group showed increased migration distance [(166.30±5.07) vs (246.80± 13.42) μ m,P＜0.05],increased numbers of transmembrane cells [(71.67 ± 6.12) vs (95.33 ± 2.73),P＜0.05] and decreased cell apoptosis [(14.40± 1.69) vs (6.05 ± 0.35)％,P＜0.05].(6) Compared with the pEGFP-miR-155 group,the pravastatin (50.00.00 μμ g/mL)+pEGFP-miR-155 group showed longer migration distance [(197.50± 13.86) vs (275.80± 13.63) μ m,P＜0.05],more transmembrane cells [(52.67±5.49) vs (125.00±6.66),P＜0.05] and lower rate of cell apoptosis [(8.90± 1.00) vs (5.05±0.35)％,P＜0.05].Conclusions Pretreatment of extravillous trophoblast cells with pravastatin can protect them from apoptosis and loss of migratory and invasive abilities through inhibiting the activation of AP-1 and down-regulating the expression of miR-155,which may be a mechanism that inhibits the development of preeclampsia.