RNAseq of primary human erythroid progenitor cells expressing a RHEX targeting shRNA vs. a non-targeting control shRNA NT (luciferase shRNA): Insight into RHEX's pro-erythropoietic effects. Homo sapiens

A prime goal of this study was to employ RNAseq to identify the effects of the knockdown of RHEX mRNA (and protein) on gene expression profiles in primary human erythroid progenitor cells ex vivo. An shRNA that targets RHEX-C1ORF186 and a non-targeting shRNA (shRNA-NT) were expressed using lentiviral vectors to transduce CD34-pos primary human stem cells. Cells were then cultured in a phase-I medium including SCF, IL3 and EPO (with puromycin at 3 micrograms/mL). For fully selected (GFP-pos) and exponentially growing phase-I erythroid progenitors, total RNA was isolated from three biological replicates for experimental, and control groups. RNAseq was then performed following Illumina's TruSeq-stranded-mRNA sample preparation protocol, and paired-end sequencing was completed on an Illumina NovaSeq 600. In phase-I culture, the knockdown of RHEX was observed to moderately decrease cell proliferation. Flow cytometry and cytohistological analyses did not reveal observable effects of RHEX knockdown on development during this phase. At subsequent phases, however, RHEX knockdown compromises erythroid development. RNAseq analyses of phase-I cells therefore was employed to uncover possible knockdown effects on expressed genes that inform on RHEX's molecular and cellular actions. RHEX is a novel and unique plasma membrane protein that is sharply modulated in its tyrosine phosphorylation upon erythropoietin receptor ligation. Studies to date indicate important roles for RHEX during human erythroblast growth, development and maturation.