Table 1.
Clinical characteristics of the pregnant women enrolled in this study.
Table 2.
The sequences of siRNA and miRNA.
Table 3.
Sequences of the primers used for plasmid construction or real-time PCR.
Figure 1.
Expression of miR-195 and ActRIIA in placentas derived from severe preeclamptic patients (PE) and gestational-week-matched normal pregnant women (Control).
A, B and C, Quantitative real time PCR to reveal pri-miR-195 (A), mature miR-195 (B) levels and mRNA expression of ActRIIA (C) in placentas from PE and Control. *, **, compared with Control, p<0.05, p<0.01. D, Western blotting to show protein levels of ActRIIA in placentas from PE and Control. Upper panel, a typical result of Western blotting. Lower panel, bar chart according to the statistical analysis based on the result of three independently repeat experiments. *, compared with Control, p<0.05.
Figure 2.
Validation of ActRIIA as target gene of miR-195 in HTR8/SVneo cells.
A. Real-time PCR to reveal change of ActRIIA mRNA level in HTR8/SVneo cells transfected with scramble siRNA (NC), miR-195 mimics (miR-195) or ActRIIA siRNA (si-ActRIIA). **, compared with NC, P<0.01. B. Western blot analysis to show change of ActRIIA protein level in HTR8/SVneo cells transfected with NC, miR-195 or si-ActRIIA. Upper panel, typical results of Western blotting; Lower panel, bar chart representing the statistical analysis by ANOVA according to three independent experiments. The density of ActRIIA was adjusted by that of GAPDH in the same blot, and the values were presented as Mean±SEM. *, compared with NC, p<0.05. C. Schematic map for generating luciferase assay constructs. The constructs containing the region complementary to the seed sequence for miR-195 in 3′UTR segment of human ActRIIA gene are shown as BD1-WT and BD2-WT, and the mutant constructs are shown as BD1-MUT and BD2-MUT with asterisks indicating the mutation sites. D. Luciferase assay in HTR8/SVneo cells transfected with BD1-WT, BD2-WT, BD1-MUT or BD2-MUT reporter construct together with miR-195 mimics (miR-195) or scramble siRNA (NC). The value of corresponding control group (NC) was set as 100, and the results were presented as Mean±SEM according to three independent experiments. **, compared with corresponding NC, p<0.01.
Figure 3.
Effect of miR-195 on cell invasion in HTR8/SVneo cells.
A and D. The expression of miR-195 in HTR8/SVneo cells transfected with miR-195 mimics (miR-195), scramble siRNA (NC), miR-195 antigomir (Anti-miR-195) or scramble antigomir (Inhibitor NC) was measured by real time PCR. The level of miR-195 was adjusted by that of U6, and the relative value was presented as Mean±SEM according to three independent experiments. *, compared with corresponding NC, p<0.05. B and E. Transwell insert assay to examine cell invasiveness transfected with NC, miR-195, siRNA for ActRIIA (si-ActRIIA), Inhibitor NC or Anti-miR-195. The value of invasion index in corresponding NC was set as 100, and the results were presented as Mean±SEM according to three independent experiments. *, compared with corresponding NC, p<0.05. C and F. Gelatin zymography to measure MMP-2 and MMP-9 productions in cells transfected with miR-195, NC, Inhibitor NC or Anti-miR-195. Upper panels, typical results of gelatin zymography. Conditioned media of HT1080 cells was included as positive control for zymography. The pro- and active forms of MMP-2 and MMP-9 were separately indicated. Lower panels, bar charts representing the statistical analysis by ANOVA according to three independent experiments. The densities of pro- and active forms of MMP-2 and MMP-9 were presented as Mean±SEM. *, compared with data in corresponding NC, p<0.05.
Figure 4.
ActRIIA rescued the effect of miR-195 on cell invasion in HTR8/SVneo cells.
HTR8/SVneo cells were transfected with miR-195 and ActRIIA alone or in combination, with scramble siRNA (NC) or pcDNA4 vector (pcDNA4) as corresponding negative control. Transwell insert assay was performed to monitor cell invasiveness (A) and Gelatin zymography was performed to measure MMP-2 and MMP-9 productions (B). The statistical analysis was carried out by ANOVA according to three independent experiments, and the values were presented as Mean±SEM. *, compared with corresponding group as indicated, p<0.05.
Figure 5.
Effect of miR-195 on protein expression of CCND1 and CCND3 in HTR8/SVneo cells.
A. Real-time PCR to reveal miR-195 level in HTR8/SVneo cells transfected with scramble siRNA (NC) or miR-195 mimics (miR-195). **, compared with NC, p<0.01. B. Western blot analysis to show change of CCND1 and CCND3 protein level in HTR8/SVneo cells transfected with NC or miR-195. Upper panels, typical results of Western blotting; Lower panels, bar charts representing the statistical analysis by ANOVA according to three independent experiments. The densities of CCND1 and CCND3 were adjusted by that of Actin in the same blot, and the values were presented as Mean±SEM.