Role of Long Non-Coding RNAs in Human-Induced Pluripotent Stem Cells Derived Megakaryocytes: A p53, HOX Antisense Intergenic RNA Myeloid 1, and miR-125b Interaction Study

Megakaryocytes (MKs) are rare polyploid cells found in the bone marrow and produce platelets. Platelets are small cell fragments that are essential during wound healing and vascular hemostasis. In vitro differentiation of MKs from human-induced pluripotent stem cell–derived CD34⁺ hematopoietic stem cells (hiPSC-HSCs) could provide an alternative treatment option for thrombocytopenic patients as a platelet source. In this approach, we developed a method to produce functional MKs from hiPSC-HSCs using a xeno-free and feeder-free condition and minimize the variation and risk from animal-derived products in cell culture. We have also investigated the genome-wide expression as well as functional significance of long noncoding RNAs (lncRNAs) in hiPSC-HSC–derived MKs to get insight into MK biology. We have performed lncRNAs expression profiling by using the Human LncProfilers qPCR Array Kit and identified 26 differentially regulated lncRNAs in hiPSC-HSC–derived MKs as compared with those in hiPSC-HSCs. HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) was the most highly upregulated lncRNA in hiPSC-HSC–derived MKs and phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic-differentiating K562 cells. Furthermore, we have studied the potential mechanism of HOTAIRM1 based on the interactions between HOTAIRM1, p53, and miR-125b in PMA-induced K562 cells. Our results demonstrated that during MK maturation, HOTAIRM1 might be associated with the transcriptional regulation of p53 via acting as a decoy for miR-125b. Thus, the interaction between HOTAIRM1, p53, and miR-125b is likely involved in controlling cell cycling (cyclin D1), reactive oxygen species production, and apoptosis to support terminal maturation of MKs.

SIGNIFICANCE STATEMENT

In vitro generation of megakaryocytes (MKs) from human-induced pluripotent stem cell–derived hematopoietic stem cells (hiPSC-HSCs) could provide an alternative source of platelets for treating thrombocytopenic patients. This study has investigated the functional significance of long non-coding RNAs in hiPSC-HSC–derived MKs, which remains unclear. This study's findings suggest that the regulatory role of HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) in p53-mediated regulation of cyclin D1 during megakaryocytopoiesis is to promote MK maturation by decoying miR-125b.