Effects and potential mechanisms of bradykinin B2 receptor on growth and biological function of human extravillous trophoblast cells

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/G1 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) μm, 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. Key words: Receptor, bradykinin B2; Trophoblasts; Cell line; Cell proliferation