*2.4. Overexpression of LIN28 Led to Significant Increase in Trophoblast Cell Proliferation*

The role of LIN28-*let-7* miRNA axis on the functionality of iOTR cells was determined by measuring proliferation of AKI and BKI iOTR cells compared to EVC after 4 h, 24 h, 48 h, and 72 h. The results showed that proliferation of both AKI and BKI iOTR cells was significantly increased at 24 h, 48 h, and 72 h compared to EVC (Figure 11A). Furthermore, proliferation of BKI iOTR cells was not different at 24 h but was significantly higher at 48 h and 72 h compared to AKI iOTR cells (Figure 11A). The matrigel invasion assay showed that there was no significant change in the invasion index of AKI and BKI iOTR cells compared to EVC (Figure 11B). These results suggest that increased proliferation of AKI and BKI iOTR cells is due to increased expression of proliferation-associated genes in these cells compared to EVC.

**Figure 11.** (**A**) Proliferation of AKI, BKI, and EVC iOTR cells (*n* = 4/treatment) was measured after 4 h, 24 h, 48 h, and 72 h using Quick Cell Proliferation Assay Kit. (**B**) Invasion of AKI, BKI, and EVC iOTR cells (*n* = 4) measured after 2 h, 4 h, 24 h, and 48 h using the Matrigel Invasion Assay Kit; where a, *p* < 0.05 for AKI vs. EVC; b, *p* < 0.05 for BKI vs. EVC; and c, *p* < 0.05 for BKI vs. AKI.
