FIGURE 4.

Hypermethylation of CPG island 2 in the promoter caused down‐regulation of lncRNA TBX5‐AS1:2. A, Four CpG islands were predicted in the regulatory sequence of lncRNA TBX5‐AS1:2 by MethPrimer online. B, Every CpG site in lncRNA TBX5‐AS1:2 CpG island 2 was hypermethylated in TOF cardiac tissue samples compared with NC, as detected by BSP for clones. The colour of circles for each CpG site represents methylation percentage. C, The methylated reporter construct pGL3‐Basic‐lncRNA TBX5‐AS1:2‐i2 could not be digested by MSRE whereas the unmethylated construct could be digested. D, The methylation percentage of each CpG site was significantly increased in HEK293T cells transfected with methylated pGL3‐Basic‐lncRNA TBX5‐AS1:2‐i2 according to BSP. E, Overall hypermethylation of CpG island 2 was significant in HEK293T cells transfected with methylated pGL3‐Basic‐lncRNA TBX5‐AS1:2‐i2. (F) Dual‐luciferase reporter assay showed that hypermethylation of CpG island 2 decreased the transcriptional activity of lncRNA TBX5‐AS1:2 in HEK293T cells. Values are mean ± SEM, n = 3, ***P < .0001. BSP, bisulphite sequencing PCR; HEK293T, human embryonic kidney 293; lncRNAs, long non‐coding RNAs; NC, negative control; TOF, Tetralogy of Fallot