let-7a overexpression represses HIC2 in HUDEP1 cells. (A) The expression levels of 303 predicted miRNAs targeting HIC2 in fetal and adult erythroblasts. The small RNA-seq data were obtained from published data sets.15 let-7 family members are highlighted in red. The balance between let-7 and LIN28B expression is shown for adult and fetal cells. (B) The sequence of LIN28B resistant chimeric let-7a/miR21 (clet-7a). The stem and loop structure from let-7a-1 and miR-21 are highlighted in yellow and blue, respectively. Mature let-7a-5p and miR-21-5p sequences are highlighted in red. (C) The expression levels of let-7a-5p measured by miRNA RT-qPCR upon clet-7a overexpression in HUDEP1 cells. Results are normalized to miRNAs miR-16/103a/191 and shown as mean ± SD (2 independent biological replicates with 2 technical replicates each). Other let-7 family members are shown in supplemental Figure 3. (D) HIC2, LIN28B, and BCL11A mRNA measured by qRT-PCR upon clet-7a overexpression in HUDEP1 cells. Results are normalized to AHSP and shown as mean ± SD (2 independent biological replicates with 2 technical replicates each). (E) Western blot with indicated antibodies of extracts from clet-7a overexpression in HUDEP1 cells. (F) Schematic of the construct for HIC2 3'UTR luciferase reporter assay. let-7 binding sites are highlighted in green. (G) Sequences of the 4 let-7 binding sites in HIC2 3'UTR. Mutations are highlighted in bold. (H) Luciferase reporter assay of HIC2 3'UTR in HeLa cells. Results are shown as mean ± SD (n = 3). (I) Schematic demonstrating low let-7 levels and high HIC2 expression in fetal-like HUDEP1 cells. Overexpression of a let-7 mimic leads to decreased levels of HIC2 protein and increased level of BCL11A. BS, binding site; pA, BGH polyA signal; RPM, reads per million.
