Trophectoderm-Specific Knockdown of LIN28 Decreases Expression of Genes Necessary for Cell Proliferation and Reduces Elongation of Sheep Conceptus

Abstract

LIN28 inhibits let-7 miRNA maturation which prevents cell differentiation and promotes proliferation. We hypothesized that the LIN28-let-7 axis regulates proliferation-associated genes in sheep trophectoderm in vivo. Day 9-hatched sheep blastocysts were incubated with lentiviral particles to deliver shRNA targeting LIN28 specifically to trophectoderm cells. At day 16, conceptus elongation was significantly reduced in LIN28A and LIN28B knockdowns. Let-7 miRNAs were significantly increased and IGF2BP1-3, HMGA1, ARID3B, and c-MYC were decreased in trophectoderm from knockdown conceptuses. Ovine trophoblast (OTR) cells derived from day 16 trophectoderm are a useful tool for in vitro experiments. Surprisingly, LIN28 was significantly reduced and let-7 miRNAs increased after only a few passages of OTR cells, suggesting these passaged cells represent a more differentiated phenotype. To create an OTR cell line more similar to day 16 trophectoderm we overexpressed LIN28A and LIN28B, which significantly decreased let-7 miRNAs and increased IGF2BP1-3, HMGA1, ARID3B, and c-MYC compared to control. This is the first study showing the role of the LIN28-let-7 axis in trophoblast proliferation and conceptus elongation in vivo. These results suggest that reduced LIN28 during early placental development can lead to reduced trophoblast proliferation and sheep conceptus elongation at a critical period for successful establishment of pregnancy.

Keywords: cell proliferation; gene regulation; let-7 miRNAs; placenta; trophectoderm.

Figure 1

LIN28A or LIN28B knockdown and let-7 miRNAs in day 16 sheep TE. (A) LIN28A and LIN28B mRNA in AKD (n = 5) and BKD (n = 6) day 16 TE compared to SC (n = 6). (B) Representative immunoblots for LIN28A, LIN28B, and β-actin in AKD, BKD, and SC day 16 TE, and densitometric analysis. (C) Let-7 miRNAs in AKD and BKD day 16 TE and SC. * p < 0.05 vs. SC.

Figure 2

(A) Conceptus length at day 16 after LIN28A knockdown (AKD, n = 6) and LIN28B knockdown (BKD, n = 8) compared to scramble control (SC, n = 5). (B) Representative images of day 16 sheep conceptuses for AKD, BKD, and SC day 16 TE; * p < 0.05 vs. SC.

Figure 3

IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, and c-MYC mRNA in AKD and BKD day 16 TE compared to SC (n = 5), * p < 0.05 vs. SC.

Figure 4

Representative immunoblots for IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, c-MYC, and β-actin, and densitometric analysis of immunoblotting results in AKD and BKD day 16 sheep TE compared to SC (n = 3), * p < 0.05 vs. SC.

Figure 5

LIN28A, LIN28B, and let-7 miRNAs in non-immortalized ovine trophoblast (OTR) cells. (A) LIN28A and LIN28B mRNA in OTR cells compared to day 16 sheep TE. (B) Representative immunoblots for LIN28A, LIN28B, and β-actin, and densitometric analysis of immunoblotting results in OTR cells compared to day 16 TE. (C) Let-7 miRNAs in OTR cells compared to day 16 TE (n = 3), * p < 0.05 vs. TE.

Figure 6

IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, and c-MYC mRNA in OTR cells compared to day 16 TE (n = 3), * p < 0.05 vs. TE.

Figure 7

Representative immunoblots for IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, c-MYC, and β-actin, and densitometric analysis of immunoblotting results in OTR cells compared to day 16 TE (n = 3), * p < 0.05 vs. TE.

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.

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.

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.

Figure 12

Graphical abstract. (A) In control day 9 TE, let-7 miRNAs are low because of high LIN28A and LIN28B. Because of low levels of let-7 miRNAs, IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, and c-MYC are higher leading to increased proliferation of trophoblast cells and hence conceptus elongation. (B) In LIN28A or LIN28B KD day 9 TE, let-7 miRNAs are higher. Elevated let-7 miRNAs target IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, and c-MYC, leading to reduced expression of these genes, leading to reduced proliferation of trophoblast cells and, hence, reduced conceptus elongation.