Figure 4

Associations between the expression of MEG3 and neural lineage genes in human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) lines. (A) Repression of MEG3 was consistently correlated with downregulation of PAX6, RTN1, and DLK1 in various cell lines. The cell lines where MEG3 was not detectable, including NTU1, NTU3, H9, and iPSC lines, also displayed lower expression levels of PAX6, RTN1, and DLK1. The mRNA expression was quantified with the 2^−ΔΔCp method (using GAPDH for normalization). In the NTU1 and NTU3 hESC lines, error bars represent the standard error of the mean (SEM) generated from three biological repeats. In the H9 hESC line and the two iPSC lines, error bars represent the SEM generated from one biological sample with three technical repeats. *P <0.05, **P <0.01 with the corresponding MEG3-ON groups by Student’s t test. N.D., not detectable. (B) MEG3 knockdown assays were conducted via small hairpin RNA (shRNA) and small interfering RNA (siRNA) to examine the association between MEG3 reduction and the expression levels of neural lineage-related genes in NTU1 hESCs. In the sh-MEG3 group with MEG3 reduction, PAX6, RTN1, and DLK1 showed downregulated expression (upper panel) compared with the scramble control. PAX6 and RTN1 were also downregulated in two si-MEG3-treated groups with reduced MEG3 expression, whereas DLK1 was reduced in one siRNA-treated group compared with the scramble control (lower panel). The mRNA expression was quantified with the 2^−ΔΔCp method (using GAPDH for normalization). Error bars represent the SEM generated from one biological sample with three technical repeats each. *P <0.05, **P <0.01 with the corresponding scramble control groups by Student’s t test in shRNA experiments; one-way analysis of variance and Dunnett’s multiple comparisons test were used in siRNA experiments, with significance defined as *P <0.05 and **P <0.01. MEG3, maternally expressed gene 3.