miR-126-5p protects from URSA via inhibiting Caspase-1-dependent pyroptosis of trophoblast cells

Abstract

Unexplained recurrent spontaneous abortion (URSA) is a distressing pregnancy complication that seriously threat to women's reproductive health. Trophoblast pyroptosis was involved in the occurrence of URSA, but the potential mechanism remains unclear. In this work, we found CASP1 transcription and the level of pyroptosis were significantly elevated in the villous tissues of URSA patients. Suppression of cell pyroptosis by Gasdermin-D (GSDMD) or Caspase-1 inhibitors can reduce embryo resorption rate of URSA mice, while Caspase-1 over-expression in normal pregnant (NP) mice can aggravate embryo resorption. Meanwhile, a pronounced decline in the expression of microRNA-126-5p (miR-126-5p) was found in URSA patients, which was inversely related to CASP1 expression. Over-expression of miR-126-5p restrained trophoblast pyroptosis via inhibiting Caspase-1/GSDMD signaling pathway by direct binding to 3'-UTR of CASP1. Moreover, experiments in vivo substantiated that up-regulation of miR-126-5p effectively suppressed Caspase-1-mediated pyroptosis in placental tissue and significantly reduced embryo resorption rate. Collectively, these results underscored that diminished miR-126-5p expression plays a crucial role in URSA by enhancing trophoblast pyroptosis through activating Caspase-1/GSDMD signaling pathway. As a result, miR-126-5p shows significant promise as a possible biomarker for diagnosis and treatment of URSA.

Keywords: Caspase-1; Pyroptosis; Trophoblast; Unexplained recurrent spontaneous abortion; miR-126-5p.

Fig. 1

Pyroptosis was significantly activated in the villous tissue of URSA patients. A GO pathway enrichment analysis of up-regulated genes in villous tissue of URSA patients. B Heatmap showing the up-regulated genes enriched in pyroptosis pathway. C The CASP1 mRNA levels in villous tissue of URSA patients and NP women (n = 35) were determined via qRT-PCR. D GO enrichment analysis of up-regulated proteins in villous tissue of URSA. E WB revealed the pyroptosis related proteins in villous tissue of URSA patients and NP women (n = 5). F The quantitative analyses of pyroptosis related proteins. G-H The serum contents of IL-1β/IL-18 in URSA and NP women were analyzed via ELISA (n = 35). I-J Immunofluorescence staining of Caspase-1 protein in villous tissue of URSA patients and NP women (Scale bar, 50 µm, 40 ×). K-L Immunofluorescence staining of GSDMD protein in villous tissue of URSA patients and NP women (Scale bar, 50 µm, 40 ×). ***P < 0.001, ****P < 0.0001

Fig. 2

Caspase-1 mediated pyroptosis affects embryo resorption rate. A Photograph (red arrow: absorption point) of uterus from URSA pregnant mice (n = 5). B The absorption rates were analyzed (n = 5). C Proteins related to pyroptosis in the placenta of pregnancy mice were detected by WB (n = 3). D The quantitative analyses of pyroptosis related proteins. E–F The serum contents of IL-1β/IL-18 of pregnancy mice. G Photograph (red arrow: absorption point) of uterus from NP pregnant mice tail vein injected with Flag-mCasp1 or Flag-mCasp1and DSF (n = 5). H The absorption rates were analyzed (n = 5). I Proteins related to pyroptosis in the placenta of NP pregnancy mice were detected by WB (n = 3). J The quantitative analyses of proteins related to pyroptosis. K-L The serum contents of IL-1β/IL-18 in serum of pregnancy mice. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns means no statistical difference

Fig. 3

miR-126-5p is down-regulated and negatively correlates with Caspase-1 in URSA patients. A Venn diagram displays the 3 down-regulated miRNAs that might complementary combined with CASP1 analyzed by RNA seq and TargetScan prediction. B-D The expression of miR-126-5p, miR-181c-5p and miR-181 d-5p in villous tissues of NP and URSA patients (n = 35) were detected by qRT-PCR. E Correlation analysis between CASP1 and miR-126-5p in villous tissues of URSA patients (n = 30). F ROC curve analysis of the diagnostic value of miR-126-5p in villous tissues for URSA (n = 30). G Using FISH technique to determine the localization and level of miR-126-5p in villous tissue (scale bar, 50 µm, 40 ×). *** P < 0.001, ns means no statistical difference

Fig. 4

miR-126-5p attenuates trophoblast pyroptosis by suppressing Caspase-1. A MiR-126-5p level in HTR-8/SVneo and JEG-3 was detected by qRT-PCR. B-E Cells were treated with Hcy following the modulation of miR-126-5p, The CASP1 mRNA level was analyzed in (B), Proteins associated with pyroptosis were detected by WB in (C), The quantitative analyses of proteins related to pyroptosis in (D), The contents of IL-1β/IL-18 present in the cell supernatant were assessed as shown in (E–F). G-H IF analyzed Caspase-1 expression in HTR-8/SVneo cells (Scale bar, 50 µm, 20 ×). I-J Representative images taken under the microscope of HTR-8/SVneo cells undergoing pyroptosis, arrows indicate the cell with bubbles (Scale bar, 50 µm, 20 ×). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns means no statistical difference

Fig. 5

CASP1 is a direct target of miR-126-5p. A The schematic diagram shown the binding sequence (WT) and mutation sequence (MUT) of miR-126-5p to CASP1 3'UTR. B-C The luciferase activity was determined in 293 T cells. ***P < 0.001, ns means no statistical difference

Fig. 6

miR-126-5p reduces embryo resorption rate by down-regulating Caspase-1 mediated pyroptosis in URSA mice. A-B The embryo resorption rates were measured across three groups of URSA mice, with arrows highlighting instances of embryo resorption (n = 5). C The miR-126-5p levels in the placental tissue of pregnant mice. D WB analysis was performed to assess the expression of pyroptosis-related proteins in the placental tissue of pregnant mice (n = 3). E The quantitative analyses of proteins related to pyroptosis. F-G Serum levels of IL-1β and IL-18 in pregnant mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns means no statistical difference

Fig. 7

Inhibition of miR-126-5p aggravate embryo loss by up-regulating Caspase-1 mediated pyroptosis. A-B The embryo resorption rates were measured across three groups of URSA mice, with arrows highlighting instances of embryo resorption (n = 5). C The expression levels of miR-126-5p in the placental tissue of pregnant mice. D The expression levels of pyroptosis-related proteins in the placental tissue of pregnant mice were analyzed by WB (n = 3). E The quantitative analyses of proteins related to pyroptosis. F-G The serum level of IL-1β/IL-18 in pregnancy mice. *P < 0.05, ***P < 0.001

Fig. 8

Schematic diagram of miR-126-5p mediated trophoblast pyroptosis in URSA. Downregulation of miR-126-5p in trophoblasts attenuates its inhibitory effect on Caspase-1 expression, thereby promoting trophoblast pyroptosis-mediated embryo loss and contributing to the pathogenesis of URSA