KRT18 regulates trophoblast cell migration and invasion which are essential for embryo implantation

Abstract

Female infertility is a worldwide concern that impacts the quality of life and well-being of affected couples. Failure of embryo implantation is a major cause of early pregnancy loss and is precisely regulated by a programmed molecular mechanism. Recent studies have shown that proper trophoblast adhesion and invasion are essential for embryo implantation. However, the potential regulatory mechanism involved in trophoblast adhesion and invasion has yet to be fully elucidated. KRT18 has been reported to play a critical role in early embryonic development, but its physiological function in embryo implantation remains unclear. In the present study, we revealed that KRT18 was highly expressed in trophoblast cells and that knockdown of KRT18 in mouse embryos inhibited embryo adhesion and implantation. In vitro experiments further showed that silencing KRT18 disturbed trophoblast migration and invasion. More importantly, we provide evidence that KRT18 directly binds to and stabilizes cell surface E-cadherin in trophoblast cells through microscale thermophoresis (MST) analysis and molecular biology experiments. In brief, our data reveal that KRT18, which is highly expressed in trophoblast cells, plays an important role in the regulation of trophoblast invasion and adhesion during embryo implantation by directly binding to E-cadherin.

Keywords: Cell invasion; Cell migration; E-cadherin; Embryo adhesion; Embryo implantation; KRT18.

Fig. 1

The expression and subcellular localization of KRT18 in preimplantation embryos. A-F Embryos at the 2-cell, 4-cell, 8-cell, morula and blastocyst stages were harvested for immunofluorescence staining. Anti-KRT18 antibody (green), phalloidin (red) and Hoechst (blue) were used to determine the expression of the corresponding proteins. A-E The expression level of KRT18 gradually increased during embryo development. KRT18 signalling was clearly observed in blastocysts trophoblasts. F A negative control experiment was performed by replacing the anti-KRT18 antibody with IgG. Scale bar, 20 µm

Fig. 2

Knockdown of KRT18 impairs mouse embryo adhesion and implantation. A-C Knockdown of KRT18 with lentivirus reduced KRT18 expression at the mRNA (A) and protein levels (B) and (C). The histogram shows the quantitative analysis of western blotting (C). D Schematics of the experimental mouse embryo adhesion and in vivo implantation process. E Representative picture of embryos treated with lentivirus for 48 h. Scale bar, 20 µm. F Representative picture of embryos that adhered to a monolayer of Ishikawa cells. Scale bar, 50 µm. G The attachment rate of the mouse embryos was plotted as a histogram. *, p < 0.05. Blastocysts treated with control or siKRT18-2 lentivirus were transferred into pseudopregnant mice. Seventy-two hours after transfer, the number of implantation sites in the uteri was counted. (H) Representative picture of implantation sites in the uteri. I The number of implantation sites in the uteri with transferred embryos treated with control or siKRT18-2 lentivirus (n = 6). The data are presented as the mean ± SE. **P < 0.01

Fig. 3

Knockdown of KRT18 inhibits trophoblast cell migration and invasion. A and B Knockdown of KRT18 with siRNAs reduced KRT18 mRNA (A) and protein (B) (C) expression. D and E Twenty-four hours after siKRT18-3 transfection, three scratches were made onto a monolayer of HTR8/SVneo cells, and images of the initial scratch and the scratch after 24 h were taken (D). Scale bar, 200 µm. The histogram shows the statistical results of the cell scratch assay (E). F and G HTR8/SVneo cell invasiveness was determined using Matrigel transwell assays after 24 h of siKRT18-3 transfection (F), and the histogram shows the statistical results (G). Scale bar, 100 µm. *, p < 0.05

Fig. 4

Knockdown of KRT18 impairs JEG-3 spheroid adhesion and outgrowth. A-C Knockdown of KRT18 with siRNAs reduced KRT18 expression at the mRNA (A) and protein levels (B). The histogram shows the quantitative analysis of western blotting (C). D After treatment with siKRT18-3 for 24 h, JEG-3 spheroids were generated and transferred onto a monolayer of Ishikawa cells. After centrifugation of the plate at 145 rpm for 10 min and removal of the spheroids that did not bind, the attachment rate was determined (the number of attached spheroids divided by the total number of input spheroids). The JEG-3 spheroid adhesion rates were plotted as a histogram. E and F The outgrowth ability of JEG-3 spheroids. The spheroids that attached to the Ishikawa cell monolayer were photographed at 1 h and 48 h after being transferred onto the Ishikawa cell monolayer (E). Scale bar, 100 µm. The histogram shows the outgrowth rate of the two groups (F). *, p < 0.05

Fig. 5

Knockdown of KRT18 impairs the expression of E-cadherin. A and B MST results of His-E-cadherin fusion protein vs. GST-KRT18 fusion protein. The fluorescence change upon switching the laser on and off at 20% intensity is shown. C HTR8/SVneo cells were treated with the control or siKRT18 siRNA. After incubation for 48 h, the cells were harvested for immunofluorescence staining and then photographed with confocal laser scanning microscopy. Anti-KRT18 antibody (green), anti-E-cadherin antibody (red), and Hoechst (blue) were used to determine the expression of the corresponding proteins. The results shown are representative of 3 independent experiments. Scale bar, 20 µm. D Quantification of mean fluorescence intensity. KRT18 and E-cadherin fluorescence was normalized with the mean fluorescence intensity of the control group. *P < 0.05