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eSymposia | Advances in Cancer Immunotherapy


Megakaryoblastic Leukemia: A Study on Novel Role of Clinically Significant Long non-coding RNA Signatures in megakaryocyte development and immune regulation during Treatment with Phorbol Ester


Aug 17, 2020 12:00am ‐ Aug 17, 2020 12:00am

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Megakaryoblastic Leukemia: A Study on Novel Role of Clinically Significant Long non-coding RNA Signatures in megakaryocyte development and immune regulation during Treatment with Phorbol Ester Ravi Kumar Gutti* and Swati Dahariya Department of Biochemistry, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad-500046 (TS), India. *E-mail: guttiravi@gmail.com Background: Treatment of hematopoietic cells with phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), causes them to undergo differentiation to more mature cell types. Dami cell line differentiates into megakaryocyte-like cells. The ability of phorbol esters to induce differentiation of leukemic cells suggests that these can be used for the treatment of human leukemia. PMA-induced differentiation regulates several proteins that are essential for megakaryocytopoiesis through long-non-coding RNAs (lncRNAs) mediated regulation. Several studies show that lncRNAs play important role in the biological processes, including differentiation and development of the blood cells. However, lncRNA expression has not yet been comprehensively characterized in megakaryocytes (MKs). Method: Megakaryoblastic cell line Dami (ATCC Cat # CRL-9792) are derived from the peripheral blood of a patient with megakaryoblastic leukaemia and these cells provide perfect model to study the process of megakaryopoiesis. These cells were grown in RPMI-1640 medium supplemented with 10% FBS and 1% antibiotic-anti mycotic (Invitrogen). We created an inducible system that utilizes a versatile chemical PMA. We collected cultures expressing high CD41+ by flow cytometry. Total RNA was isolated after 7 days of PMA (100 nM) treatment using Qiagen RNeasy Mini kit using manufacturer instructions and cDNA was prepared which was further used for profiling Human LncRNAs using Profiler qPCR Array kit. Result: In this study, we found that PMA-induced differentiation induces expression of several lncRNAs, and was significantly associated with different stages of MK development. The analysis showed that 48 of 91 detected lncRNAs demonstrated significant >2-fold differential expression in response to treatment with PMA for 7 days. Among them, when the cut-off was set to 4-fold, we recognized 12 lncRNAs showing up-regulated significantly, while 2 lncRNAs were significantly down-regulated in mature MKs on day 7 when compared to dami on day 0. The significantly up-regulated 12 lncRNAs are BACE1AS (family), DISC2 (family), H19 upstream conserved 1 & 2, HOXA6as, IGF2AS (family), Malat1, NDM29, NEAT1 (family), PSF inhibiting RNA, PTENP1, SNHG5, UCA1. Furthermore, the down-regulated lncRNAs are Dio3os (family) and HOXA3as. I Conclusion: Our results suggest a differential expression of lncRNAs upon PMA treatment and their involvement in MK biology.

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