*2.3. Overexpression of LIN28 in iOTR Cells Resulted in Increased Expression of Proliferation-Associated Genes*

To determine if LIN28 overexpression will rescue the expression of proliferation-associated genes, the iOTR cells were infected with lentiviral particles to generate LIN28A knockin (AKI) or LIN28B knockin (BKI), or lentiviral particles with empty expression vector as expression vector control (EVC). Real-time PCR data showed that AKI iOTR cells had a significant increase in *LIN28A* mRNA while BKI iOTR cells had a significant increase in *LIN28B* mRNA compared to EVC (Figure 8A). The densitometric analysis of Western blots showed a significant increase in LIN28A protein in AKI and significant increase in LIN28B protein in BKI iOTR cells compared to EVC (Figure 8B). Moreover, the real-time PCR data showed that *let-7* miRNAs (*let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i*) were significantly reduced in both AKI and BKI iOTR cells compared to EVC (Figure 8C). These results suggest that increased expression of either LIN28A or LIN28B leads to reduction in *let-7* miRNAs.

**Figure 8.** LIN28A, LIN28B, and *let-7* miRNAs in LIN28A knockin (AKI) and LIN28B knockin (BKI) immortalized ovine trophoblast (iOTR) cells. (**A**) *LIN28A* and *LIN28B* mRNA in AKI and BKI iOTR cells compared to expression vector control (EVC). (**B**) Representative immunoblots for LIN28A, LIN28B, and β-actin, and densitometric analysis of immunoblotting results in AKI and BKI iOTR cells compared to EVC. (**C**) *Let-7* miRNAs in AKI and BKI iOTR cells compared to EVC (*n* = 3), \* *p* < 0.05 vs. EVC.

To determine the effect of LIN28 overexpression and reduction in *let-7* miRNAs on expression of proliferation-associated genes, real-time PCR and Western blot analysis were done. Real-time PCR showed that mRNA levels of *IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B,* and *c-MYC* were significantly increased in both AKI and BKI iOTR cells compared to EVC (Figure 9). Densitometric analysis of Western blots showed significant increase in IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, and c-MYC proteins in both AKI and BKI iOTR cells compared to EVC (Figure 10). These results suggest that the expression of proliferation-associated genes in immortalized ovine trophoblast cells is regulated by the LIN28-*let-7* axis.

**Figure 9.** *IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B,* and *c-MYC* mRNA in AKI and BKI iOTR cells compared to EVC (*n* = 3), where \* *p* < 0.05 vs. EVC and # *p* < 0.05 vs AKI.

**Figure 10.** Representative immunoblots for IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, c-MYC, and β-actin, and densitometric analysis of immunoblotting results in AKI and BKI iOTR cells compared to EVC (*n* = 3), where \* *p* < 0.05 vs. EVC and # *p* < 0.05 vs AKI.
