Human umbilical cord mesenchymal stem cell-derived exosome-mediated transfer of microRNA-133b boosts trophoblast cell proliferation, migration and invasion in preeclampsia by restricting SGK1

Abstract

Objective: Exosomes have been documented to function in human diseases, yet their transfer of microRNA (miRNA) in preeclampsia (PE) has seldom been reported. This study intends to discuss the role of miR-133b derived from exosomes in human umbilical cord mesenchymal stem cells (hUC-MSCs) in trophoblast cell development in PE.

Methods: Placentas from PE patients and normal pregnant women were collected. The hUC-MSCs and their exosomes were obtained and identified. Trophoblast cell HPT-8 and HTR8-S/Vneo were obtained and co-cultured with hUC-MSCs-derived exosomes that had been transfected with different miR-133b plasmids. MiR-133b and glucocorticoid-regulated kinase 1 (SGK1) expression in placental tissues and HPT-8 and HTR8-S/Vneo cells was determined. HTR8-S/Vneo and HPT-8 cell proliferation, cell cycle distribution, apoptosis rate, migration and invasion were detected.

Results: MiR-133b was down-regulated and SGK1 was up-regulated in placental tissues of PE patients. MiR-133b expression was inversely related to SGK1 expression in HTR8-S/Vneo and HPT-8 cells co-cultured with hUC-MSC-derived exosomes. Exosomes promoted HTR8-S/Vneo and HPT-8 cell proliferation, migration and invasion abilities, cell cycle entry and inhibited apoptosis. Elevated exosome-derived miR-133b from hUC-MSCs boosted HTR8-S/Vneo and HPT-8 cell proliferation, cell cycle progression, migration and invasion and limited cell apoptosis. MiR-133b targeted SGK1.

Conclusion: Collectively, we demonstrate that miR-133b is down-regulated and SGK1 is up-regulated in PE, and miR-133b derived from exosomes in hUM-MSCs facilitates trophoblast cell proliferation, migration and invasion in PE via constraining SGK1.

Keywords: Preeclampsia; SGK1; exosomes; human umbilical cord mesenchymal stem cells; microRNA-133b; trophoblast cells.

Figure 1.

Isolation of hUC-MSCs and hUC-MSC-derived exosomes. a. Observation of hUC-MSC morphology; b. Alizarin red staining of hUC-MSC osteogenic induction; c. Oil red O staining of hUC-MSC adipogenic induction; d. Identification of exosomes by TEM; e. Identification of exosomes by Nanosight; f. Detection of exosome surface antigen by flow cytometry; g. Detection of CD81, CD9 and CD63 expression by Western blot analysis; the data in the figure were measurement data expressed as mean ± standard deviation; comparison between two groups was analyzed by independent sample t-test.

Figure 2.

MiR-133b is downregulated and SGK1 is upregulated in placental tissues of PE patients; miR-133b expression is inversely related to SGK1 expression in HTR8-S/Vneo and HPT-8 cells co-cultured with hUC-MSC-derived exosomes. a. Detection of miR-133b and SGK1 mRNA expression in placental tissues of PE patients and normal pregnant women by RT-qPCR; b. Detection of SGK1 protein expression in placental tissues of PE patients and normal pregnant women by Western blot analysis; c. Detection of miR-133b and SGK1 mRNA expression in HPT-8 cells and HTR8-S/Vneo cells after normal culture and co-culture with exosomes; d. Detection of SGK1 protein expression in HPT-8 cells and HTR8-S/Vneo cells after normal culture and co-culture with exosomes; e. Detection of miR-133b and SGK1 mRNA expression of HTR8-S/Vneo cells cultured with exosomes in each group by RT-qPCR; f. Detection of SGK1 protein expression of HTR8-S/Vneo cells cultured with exosomes in each group by Western blot analysis; g. Detection of miR-133b and SGK1 mRNA expression of HPT-8 cells cultured with exosomes in each group by RT-qPCR; h. Detection of SGK1 protein expression of HPT-8 cells cultured with exosomes in each group by Western blot analysis; &, P < 0.05 vs the Blank group; *, P < 0.05 vs the mimics-NC-Exo group; #, P < 0.05 vs the inhibitors-NC-Exo group; the data in the figure were all measurement data expressed as mean ± standard deviation; comparison between two groups were analyzed by independent sample t-test, and that among multiple groups by one-way ANOVA, after which pairwise comparison was performed with Tukey’s multiple comparisons test.

Figure 3.

Exosomes elevates HTR8-S/Vneo and HPT-8 cell proliferation, migration, invasion abilities, and cell cycle entry and represses apoptosis. a.) Detection of HTR8-S/Vneo and HPT-8 cell proliferation in each group by MTT assay; b. Detection of Cyclin D1 and Ki-67 protein expression of HTR8-S/Vneo and HPT-8 cells in each group by Western blot analysis; c. Detection of HTR8-S/Vneo and HPT-8 cell cycle distribution in each group by flow cytometry; d. Detection of apoptosis of HTR8-S/Vneo and HPT-8 cells in each group by flow cytometry; e. Detection of Bcl-2 and Bax protein expression of HTR8-S/Vneo and HPT-8 cells in each group by Western blot analysis; f. Detection of HTR8-S/Vneo and HPT-8 cell migration and invasion abilities by Transwell assay. *, P < 0.05 vs the Blank group; Measurement data were expressed as mean ± standard deviation and those subjected to normal distribution between two groups were compared with independent sample t-test.

Figure 4.

Elevated exosome-derived miR-133b from hUC-MSCs boosts HTR8-S/Vneo and HPT-8 cell proliferation. a) Detection of HTR8-S/Vneo cell proliferation in each group by MTT assay; b. Detection of Cyclin D1 and Ki-67 protein expression of HTR8-S/Vneo cells in each group by Western blot analysis; c. Detection of HPT-8 cell proliferation in each group by MTT assay; d. Detection of Cyclin D1 and Ki-67 protein expression of HPT-8 cells in each group by Western blot analysis; *, P < 0.05 vs the mimics-NC-Exo group; #, P < 0.05 vs the inhibitors-NC-Exo group; the data in the figure were all measurement data expressed as mean ± standard deviation; comparison among multiple groups was analyzed by one-way ANOVA, after which pairwise comparison was performed with Tukey’s multiple comparisons test.

Figure 5.

Elevated exosome-derived miR-133b from hUC-MSCs contributes to cell cycle progression and limits apoptosis of HTR8-S/Vneo and HPT-8 cells. a. Quantitative results of HTR8-S/Vneo cell cycle in each group; b. Detection of HTR8-S/Vneo cell cycle by flow cytometry; c. Quantitative results of HPT-8 cell cycle in each group; d. Detection of HPT-8 cell cycle by flow cytometry; e. Quantitative results of HTR8-S/Vneo cell apoptosis in each group; f. Detection of HTR8-S/Vneo cell apoptosis in each group by flow cytometry; g. Quantitative results of HPT-8 cell apoptosis in each group; h. Detection of HPT-8 cell apoptosis in each group by flow cytometry; i. Detection of Bcl-2 and Bax protein expression of HTR8-S/Vneo cells in each group by Western blot analysis; j. Detection of Bcl-2 and Bax protein expression of HPT-8 cells in each group by Western blot analysis; *, P < 0.05 vs the mimics-NC-Exo group; #, P < 0.05 vs the inhibitors-NC-Exo group; the data in the figure were all measurement data expressed as mean ± standard deviation; comparison among multiple groups was analyzed by one-way ANOVA, after which pairwise comparison was performed with Tukey’s multiple comparisons test.

Figure 6.

Elevated exosome-derived miR-133b from hUC-MSCs facilitates HTR8-S/Vneo and HPT-8 cell migration and invasion. a. Quantitative results of HTR8-S/Vneo cell migration in each group; b. Detection of HTR8-S/Vneo cell migration in each group by Transwell assay; c. Quantitative results of HTR8-S/Vneo cell invasion in each group; d. Detection of HTR8-S/Vneo cell invasion in each group by Transwell assay; e. Quantitative results of HPT-8 cell migration in each group; f. Detection of HPT-8 cell migration in each group by Transwell assay; g. Quantitative results of HPT-8 cell invasion in each group; h. Detection of HPT-8 cell invasion in each group by Transwell assay; *, P < 0.05 vs the mimics-NC-Exo group; #, P < 0.05 vs the inhibitors-NC-Exo group; the data in the figure were all measurement data expressed as mean ± standard deviation; comparison among multiple groups was analyzed by one-way ANOVA, after which pairwise comparison was performed with Tukey’s multiple comparisons test.

Figure 7.

MiR-133b targets SGK1. a. Prediction of the binding site of miR-133b and SGK1 at bioinformatics website; b. Validation of the regulatory relationship between miR-133b and SGK1 in HTR8-S/Vneo cells by dual-luciferase reporter gene assay; c. Validation of the regulatory relationship between miR-133b and SGK1 in HPT-8 cells by dual-luciferase reporter gene assay; the data in the figure are all measurement data, using the mean ± standard deviation form; comparison between two groups were analyzed by independent sample t-test.