*2.3. Fractalkine Exerts Di*ff*erent E*ff*ects on the mRNA Expression of Proliferation, Di*ff*erentiation and Invasion Regulating Genes in Mono- and Co-Cultured JEG-3 Cells*

Next, we investigated the mRNA expression levels of PR, which is responsible for differentiation and proliferation of trophoblast cells, and CX3CR1 to reveal if fractalkine provides an autoregulatory effect on its receptor expression on JEG-3 cells. The mRNA expression of PR significantly increased both at 6 h and at 24 h in the monoculture, and was also elevated at the same time points in the co-cultured JEG-3 cells, although there was a significant difference between mono- and co-cultures (Figure 3A). At 48 h, the mRNA expression of PR returned to the control level both in mono- and co-cultured JEG-3 cells (Figure 3A). *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 6 of 19

**Figure 3.** Effects of fractalkine treatments on the mRNA expressions of the implantation-related genes in mono- and co-cultured JEG-3 cells. Real-time PCR was performed with SYBR green protocol using gene specific primers. β-actin was used as house-keeping gene for the normalization. The relative expression of untreated controls was regarded as 1. The mRNA expressions of the treated cells were compared to their appropriate controls (6 h, 24 h or 48 h). **(A)** mRNA expression levels of progesterone receptor. **(B)** mRNA expression levels of fractalkine receptor. **(C)** mRNA expression levels of activin receptor 1B. **(D)** mRNA expression levels of MMP2. **(E)** mRNA expression levels of MMP9. **(F)** mRNA expression levels of SRC-1. The columns represent mean values and error bars represent standard deviation (SD) of three independent determinations (*n* = 3). The \* indicates *p* < 0.05 compared to the untreated controls. The † shows *p* < 0.05 between mono- and co-cultures. Abbreviations of fractalkine treatments: 5 ng/mL-F5; 10 ng/mL-F10; 20 ng/mL-F20. **Figure 3.** Effects of fractalkine treatments on the mRNA expressions of the implantation-related genes in mono- and co-cultured JEG-3 cells. Real-time PCR was performed with SYBR green protocol using gene specific primers. β-actin was used as house-keeping gene for the normalization. The relative expression of untreated controls was regarded as 1. The mRNA expressions of the treated cells were compared to their appropriate controls (6 h, 24 h or 48 h). (**A**) mRNA expression levels of progesterone receptor. (**B**) mRNA expression levels of fractalkine receptor. (**C**) mRNA expression levels of activin receptor 1B. (**D**) mRNA expression levels of MMP2. (**E**) mRNA expression levels of MMP9. (**F**) mRNA expression levels of SRC-1. The columns represent mean values and error bars represent standard deviation (SD) of three independent determinations (*n* = 3). The \* indicates *p* < 0.05 compared to the untreated controls. The † shows *p* < 0.05 between mono- and co-cultures. Abbreviations of fractalkine treatments: 5 ng/mL-F5; 10 ng/mL-F10; 20 ng/mL-F20.

*2.4. Western Blot Analysis of the Implantation-Related Genes Reveals Alterations Between Fractalkine Treated Mono- and Co-Cultured JEG-3 Cells*  After the mRNA expression analysis, it was unravelled that FKN influenced the expression of genes that are implicated in the implantation process. We also examined if these alterations appeared CX3CR1 significantly raised at 24 h in the monoculture (Figure 3B). The same phenomenon was found in the co-cultured cells, but in this case, fractalkine maintained the augmented CX3CR1 mRNA expression at 48 h, suggesting a longer effect of FKN on the co-cultured JEG-3 cells through CX3CR1 (Figure 3B).

at protein level, suggesting that FKN regulates implantation by controlling proliferation, differentiation and invasion related proteins of trophoblast cells. In the monocultures, PR and CX3CR1 protein levels showed delays between the elevation of The activin receptor is supposed to have a role in implantation [50], while matrix metalloproteinases MMP2 and MMP9 are the major contributors to normal implantation by increasing invasiveness [51].

mRNA and protein expressions at F10 treatment, suggesting that the lower FKN concentration has a

mRNA expression levels (Figures 3C–F and 4A), but their expressions varied by time and by FKN

We examined also whether FKN affected the expression of these genes. Interestingly, activin receptor was upregulated in the JEG-3 cells at 6 h and 24 h but its mRNA expression level did not change significantly in the co-cultured JEG-3 cells (Figure 3C), suggesting that the interaction between JEG-3 and HEC-1A cells may influence the effect of fractalkine treatment. The same result was found in case of MMP2 in both mono- and co-cultured JEG-3 cells (Figure 3D). MMP9 mRNA expression was triggered by FKN at 24 h in the monoculture; meanwhile in the co-cultured JEG-3, MMP9 expression began to elevate earlier, at 6 h and remained elevated at 24 h although this level was significantly lower than those in the monoculture (Figure 3E). These results suggest that MMP9 facilitates trophoblast invasion after binding to the endometrial cells while MMP2 expression is downregulated.

We also examined SRC-1 mRNA expression, the co-factor of PR, which is regulated by MAPK pathways and can promote cell differentiation. The expression analysis revealed that SRC-1 mRNA expression only increased at 24 h in the monocultured JEG-3 cells, and there was no significant alteration of SRC-1 mRNA level in the co-culture (Figure 3F).

It seems that in the monocultured JEG-3 cells, F10 exerted a higher effect at 6 h, while at 24 h, F20 was more effective on the gene expression. In case of the co-cultures, the action of FKN did not show concentration dependence.
