| Author: | Wong, Nonthaphat |
| Title: | Exploring novel regulatory roles of long non-coding RNAs associated with myeloproliferative neoplasms |
| Advisors: | Huang, Chien-ling (HTI) Yip, Shea Ping (HTI) |
| Degree: | Ph.D. |
| Year: | 2022 |
| Subject: | Non-coding RNA Myeloproliferative disorders Chronic myeloid leukemia Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Health Technology and Informatics |
| Pages: | xxiii, 194 pages : color illustrations |
| Language: | English |
| Abstract: | Myeloproliferative neoplasms (MPNs) are a group of heterogeneous diseases which primarily include chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). CML is characterized by the presence of Philadelphia chromosome (Ph) which encodes a dysregulated tyrosine kinase. In contrast, other Ph-negative MPNs are associated with notable mutations in JAK2, MPL and CALR genes with JAK2-V617F being the most prevalent one. In recent years, accumulating evidence has demonstrated the involvement of non-coding RNAs, especially long non-coding RNAs (lncRNAs), in the pathogenesis of various human diseases. However, the functional characteristics of these non-coding elements in MPNs are still largely unknown. The present study aimed to explore novel mechanisms that account for the regulatory roles of the lncRNA- and microRNA (miRNA)-axes in classical MPNs and drug response of CML. To identify JAK2-V617F-associated lncRNAs, a qPCR array screening was performed using HEL cells. My data demonstrated that BANCR was the most downregulated lncRNA species after JAK2 inhibition. Subsequent experiments revealed its expression was specifically regulated by the JAK2-V617F signaling. Furthermore, RNA sequencing was performed to search for novel lncRNAs and explore their potential functions. Bioinformatics prediction analysis revealed putative interaction networks between the lncRNA, miRNA and mRNA in the JAK2 signaling that warrants further validation and investigation. For the investigation of CML drug resistance, a novel lncRNA named LNC000093, which showed the largest downregulation in imatinib-resistant (IMR) K562 cells, was identified by RNA sequencing. Expression studies revealed a negative correlation between LNC000093 and H19/miR-675 expression, and subsequent in silico analysis as well as luciferase reporter assays demonstrated their interaction via direct binding. Furthermore, the potential interaction of H19/miR-675 and LNC000093 with RUNX1 was investigated to reveal their interrelationship. Taken together, my findings demonstrated that LNC000093 served as a competing endogenous RNA (ceRNA) to compete for miR-675-5p and indirectly regulate RUNX1 to mediate imatinib resistance of CML. Moreover, the potential regulatory role of LNC000093 in cell differentiation was also demonstrated using the induced pluripotent stem cell (iPSC) model. LNC000093 expression was substantially increased during iPSC differentiation, and the extent of differentiation was reduced after deletion of LNC000093 by CRISPR-Cas9. An altered chromatin accessibility profile of iPSCs was also revealed by scATAC-seq data analysis. To conclude, this study has explored the molecular basis of MPNs in the non-coding area based on in vitro models that possess distinct genetic characteristics of Ph-positive or Ph-negative MPNs. Further translational research tools that can confirm the clinical relevance of my findings are required to enable the use of potential lncRNA biomarkers in the diagnosis and treatment of MPNs. |
| Rights: | All rights reserved |
| Access: | open access |
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