Circular RNA FOXP1 relieves trophoblastic cell dysfunction in recurrent pregnancy loss via the miR-143-3p/S100A11 cascade

Figures & data

Table 1. Clinicopathological features of subjects

Subject	RPL (n = 28)	Control (n = 28)	P value
Maternal Age (years)	33.99 ± 0.85	33.42 ± 1.01	0.66
Number of pregnancy loss	3.46 ± 0.12	0	<0.0001
Gestational period (weeks)	8.45 ± 0.34	8.75 ± 0.20	0.35

Figure 1. MiR–143–3p is lifted in RPL and impairs the biological functions of trophoblastic cells. (a) Relative miR–143–3p expression in placental tissues from RPL patients (n = 28) and normal pregnant women (n = 28) was detected by RT-qPCR. (b) miR–143–3p expression in HTR8/SVneo cells transfected with NC mimics, NC inhibitor, or miR–143–3p inhibitor was detected by RT-qPCR. (c) The cell viability of transfected HTR8/SVneo cells was analyzed by CCK-8 assay at different time points. (d) The apoptosis condition of transfected HTR8/SVneo cells was analyzed by flow cytometry assay. (e) The migration capability of transfected HTR8/SVneo cells was analyzed by wound healing assay. (f) The invasion capability of transfected HTR8/SVneo cells was analyzed by transwell assay. (g and h) The mRNA or protein expression level of EMT markers (E-cadherin, vimentin, and N-cadherin) in transfected HTR8/SVneo cells was determined by RT-qPCR or western blotting. Data are shown as the mean ± SD (n = 3). *P < 0.05; **P < 0.01

Figure 2. S100A11 is directly targeted by miR–143–3p in trophoblastic cells. (a) 8 candidate target mRNAs for miR–143–3p were predicted by using three miRNA databases (miRanda, miRmap, and PITA). (b) Relative S100A11 expression in placental tissues from RPL patients (n = 28) and normal pregnant women (n = 28) was detected by RT-qPCR. (c) The putative binding sites between miR–143–3p and S100A11. (d) The HTR8/SVneo cells were co-transfected with either S100A11-WT or S100A11-MUT, and miR–143–3p mimics/inhibitor or corresponding NC. Then, the relative luciferase activity was measured. (e) Pearson’s correlation analysis indicated a negative correlation between the expression of miR–143–3p and S100A11 in placental tissues from RPL patients (n = 28). (f and g) S100A11 mRNA or protein level in HTR8/SVneo cells transfected with NC mimics, miR–143–3p mimics, NC inhibitor or miR–143–3p inhibitor was detected by RT-qPCR or western blotting. Data are shown as the mean ± SD (n = 3). *P < 0.05; **P < 0.01

Figure 3. MiR–143–3p weakens trophoblastic cell functions via S100A11. (a and b) S100A11 mRNA or protein level in HTR8/SVneo cells transfected with si-NC or si-S100A11 was detected by RT-qPCR or western blotting. (c) HTR8/SVneo cells transfected with NC inhibitor, miR–143–3p inhibitor, miR–143–3p inhibitor+si-NC, or miR–143–3p inhibitor+si-S100A11, respectively. the cell viability of transfected HTR8/SVneo cells was analyzed by CCK-8 assay at different time points. (d) The apoptosis condition of transfected HTR8/SVneo cells was analyzed by flow cytometry assay. (e) The migration capability of transfected HTR8/SVneo cells was analyzed by wound healing assay. (f) The invasion capability of transfected HTR8/SVneo cells was analyzed by transwell assay. (g and h) The mRNA or protein expression level of S100A11 and EMT markers (E-cadherin, vimentin, and N-cadherin) in transfected HTR8/SVneo cells were determined by RT-qPCR or western blotting. data are shown as the mean ± SD (n = 3). *P < 0.05; **P < 0.01

Figure 4. CircFOXP1 binds to miR–143–3p in trophoblastic cells. (a) The putative binding sites between miR–143–3p and circFOXP1. (b) The HTR8/SVneo cells were co-transfected with either circFOXP1-WT or circFOXP1-MUT, and miR–143–3p mimics or NC mimics. then, the relative luciferase activity was measured. (c) Relative S100A11 expression in placental tissues from RPL patients (n = 28) and normal pregnant women (n = 28) was detected by RT-qPCR. (d) CircFOXP1 expression in HTR8/SVneo cells transfected with si-NC or si-circFOXP1 was detected by RT-qPCR. (e) CircFOXP1 expression in HTR8/SVneo cells transfected with vector or oe-circFOXP1was detected by RT-qPCR. (f) Pearson’s correlation analysis indicated a negative correlation between the expression of miR–143–3p and circFOXP1 in placental tissues from RPL patients (n = 28). (g) miR–143–3p expression in HTR8/SVneo cells transfected with si-NC, si-circFOXP1, Vector, or oe-circFOXP1 was detected by RT-qPCR. (h) Pearson’s correlation analysis indicated a positive correlation between S100A11 mRNA expression and circFOXP1 expression in placental tissues from RPL patients (n = 28). (i and j) S100A11 mRNA or protein level in HTR8/SVneo cells transfected with si-NC, si-circFOXP1, Vector, or oe-circFOXP1 was detected by RT-qPCR or western blotting. data are shown as the mean ± SD (n = 3). *P < 0.05; **P < 0.01

Figure 5. CircFOXP1/miR–143–3p/S100A11 axis regulates trophoblastic cell functions. (a) HTR8/SVneo cells transfected with vector, oe-circFOXP1, oe-circFOXP1+NC mimics, or oe-circFOXP1+ miR–143–3p mimics, respectively. The cell viability of transfected HTR8/SVneo cells was analyzed by CCK-8 assay at different time points. (b) The apoptosis condition of transfected HTR8/SVneo cells was analyzed by flow cytometry assay. (c) The migration capability of transfected HTR8/SVneo cells was analyzed by wound healing assay. (d) The invasion capability of transfected HTR8/SVneo cells was analyzed by transwell assay. (e and f) The mRNA or protein expression level of S100A11 and EMT markers (E-cadherin, vimentin, and N-cadherin) in transfected HTR8/SVneo cells were determined by RT-qPCR or western blotting. data are shown as the mean ± SD (n = 3). *P < 0.05; **P < 0.01

Data availability statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.