Up-regulation of microRNA-135 or silencing of PCSK6 attenuates inflammatory response in preeclampsia by restricting NLRP3 inflammasome

Fig. 1. MiR-135 is down-regulated and PCSK6 and NLRP3 is up-regulated in placenta tissues of PE patients. A HE staining of clinical placenta tissues (× 200); B TUNEL staining of clinical placenta tissues (× 200); C, D Detection of IL-1β and TNF-α levels in serum by ELISA; E Detection of miR-135-3p expression by qPCR; F. Detection of miR-135-5p expression by qPCR; G Detection of PCSK6 expression by qPCR; H Detection of NLRP3 expression by qPCR; I Detection of PCSK6 and NLRP3 protein expression by Western blot analysis; n = 25. *P < 0.05 vs. the control group

Fig. 2. Establishment of H/R trophoblasts model. A Detection of miR-135, PCSK5 and NLRP3 expression in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by qPCR; B Detection of PCSK5 and NLRP3 protein expression in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by Western blot; C Detection of cell proliferation in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by colony formation assay; D Detection of cell viability in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by MTT assay; E Detection of cell apoptosis in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by flow cytometry; F Detection of Bcl-2 and Bax mRNA expression in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by qPCR; G Detection of invasion and migration in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by Transwell test; H Detection of IL-1β and TNF-α expression in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by ELISA; N = 3, *P < 0.05 vs. the control group

Fig. 3. Up-regulating miR-135 alleviates inflammatory response in H/R-treated trophoblasts. A Detection of miR-135, PCSK5 and NLRP3 expression in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by qPCR; B Detection of PCSK5 and NLRP3 protein expression in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by Western blot; C Detection of cell proliferation in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by colony formation assay; D Detection of cell viability in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by MTT assay; E Detection of cell apoptosis in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by flow cytometry; F Detection of Bcl-2 and Bax mRNA expression in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by qPCR; G Detection of invasion and migration in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by Transwell test; H Detection of IL-1β and TNF-α expression in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by ELISA; N = 3, *P < 0.05 vs. the H/R + mimic NC group

Fig. 4. MiR-135 targets PCSK6. A Prediction of the binding site between miR-135 and PCSK6’ B, C Detection of luciferase activity of HTR-8/SVneo and HPT-8 cells by dual luciferase reporter gene assay; N = 3

Fig. 5. Depletion of PCSK6 enhances growth of H/R-treated trophoblasts. A Detection of miR-135, PCSK5 and NLRP3 expression in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by qPCR; B Detection of PCSK5 and NLRP3 protein expression in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by Western blot; C Detection of cell proliferation in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by colony formation assay; D Detection of cell viability in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by MTT assay; E Detection of cell apoptosis in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by flow cytometry; F Detection of Bcl-2 and Bax mRNA expression in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by qPCR; G Detection of invasion and migration in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by Transwell test; H Detection of IL-1β and TNF-α expression in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by ELISA; N = 3, *P < 0.05 vs. the H/R + siRNA NC group

Fig. 6. Inhibition of NLRP3 relieves the inflammatory cascade of HTR8/SVneo cells after H/R. A Detection of miR-135, PCSK5 and NLRP3 expression in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by qPCR; B Detection of PCSK5 and NLRP3 protein expression in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by Western blot; C Detection of cell proliferation in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by colony formation assay; D Detection of cell viability in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by MTT assay; E Detection of cell apoptosis in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by flow cytometry; F Detection of Bcl-2 and Bax mRNA expression in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by qPCR; G Detection of invasion and migration in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by Transwell test; (H) Detection of IL-1β and TNF-α expression in H/R-treated HTR-8/SVneo and HPT-8 trophoblasts by ELISA; N = 3, *P < 0.05 vs. the H/R + OE NC group; #P < 0.05 vs. the H/R + OE-PCSK6 group