Role of Long non-coding RNAs in Human Induced Pluripotent Stem Cells derived Megakaryocytes: A p53, HOTAIRM1 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 critical to vascular hemostasis and wound healing. In vivo generation of MKs from Human induced pluripotent stem cells (hiPSCs)-derived CD34+ hematopoietic stem cells (hiPSCs-HSCs) could provide an alternative source of platelets for treating thrombocytopenic patients. In this approach, we developed a method to produce functional MKs from hiPSCs-HSCs under a feeder-free and xeno-free condition and minimize the risk and variation from the animal-derived products in cell cultures. We have also investigated the genome-wide expression and functional significance of long non-coding RNAs (lncRNAs) in hiPSCs-HSCs-derived MKs remains unclear to get insight into MK biology. We have performed lncRNAs expression profiling by using the Human LncProfilers{trade mark, serif} qPCR Array Kit and spotted 26 differentially regulated lncRNAs (FC {plus minus}2.0; p<0.05) in hiPSCs-HSCs-derived MKs as compared to hiPSCs-HSCs. HOTAIRM1 (HOX antisense intergenic RNA myeloid 1) was the highest upregulated (~20 fold) lncRNA in hiPSCs-HSCs derived MKs and 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 MKs maturation HOTAIRM1 may be involved in the transcriptional regulation of p53, via acting as a decoy for miR-125b (keeping them away from p53 mRNA). Thus, the interaction between HOTAIRM1, p53 and miR-125b is likely involved in controlling cell cycling (Cyclin D1), ROS production, and apoptosis to support terminal maturation of MKs. Significance Statement In vivo generation of MKs from Human induced pluripotent stem cells (hiPSCs)-derived CD34+ hematopoietic stem cells (hiPSCs-HSCs) could provide an alternative source of platelets for treating thrombocytopenic patients. We have investigated the functional significance of long non-coding RNAs (lncRNAs) in hiPSCs-HSCs-derived MKs which remains unclear and their role is important in studying the megakaryocyte biology. Our findings suggest that the regulatory role of HOTAIRM1 in p53 mediated regulation of cyclin D1 during megakaryocytopoiesis to promote MK maturation by decoying miR-125b.