| Author: | Lin, Wing Keung |
| Title: | Identifying JAK2V617F mediated long non-coding RNAs as potential new molecular targets for myeloproliferative neoplasms |
| Advisors: | Huang, C. L. (HTI) Yip, S. P. (HTI) |
| Degree: | DHSc |
| Year: | 2023 |
| Subject: | Blood -- Diseases Non-coding RNA Cancer -- Molecular aspects Hong Kong Polytechnic University -- Dissertations |
| Department: | Faculty of Health and Social Sciences |
| Pages: | xix, 190 pages : color illustrations |
| Language: | English |
| Abstract: | Introduction Extensive studies have focused on the downstream signaling cascades mediated by JAK2V617F mutation, a prevalent mutation in Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-MPNs). However, little is known about the role and clinical significance of long non-coding RNA (lncRNA) within the purview of this mutation. The involvement of lncRNAs in tumor development has been evidenced for years, but details of their actions in hematological malignancies have remained unexplored. In addition to their epigenetic regulatory role in the nucleus, competing endogenous RNA (ceRNA) is another crucial function in regulating the expression of microRNAs (miRNAs) and messenger RNAs (mRNAs) in the cytoplasm. The present study aimed to identify the aberrant expression of lncRNAs associated with JAK2V617F mutation and to explore any potential ceRNA interaction involving lncRNA, miRNA, and mRNA mediated by the JAK2V617F mutation. Materials and Methods Differential expression of lncRNAs and mRNAs were examined by RNA-sequencing (RNA-seq) in human erythroleukemia cell line (HEL) and ruxolitinib (RUX)-treated HEL cells (HEL-RUX). Differentially expressed lncRNAs (DElncRNAs) and Differentially expressed mRNAs (DEmRNAs) were identified (|log2Fold-change| >2, adjusted p-value <0.01). Additional DElncRNAs candidates were recruited from published PCR-array results of a similar study. The relative expression of DElncRNAs was validated by quantitative real-time PCR (qPCR). Cell fractionation and RNA isolation from different cell fractions were performed, and cytoplasmic expression of the screened DElncRNAs was determined by droplet digital PCR (DDPCR). Differentially expressed miRNA (DEmiRNA) candidates were retrieved from a published study of Janus kinase 2 (JAK2) inhibition in HEL cells (Pagano’s dataset: |log2Fold-change| >2, adjusted p-value <0.01). Published whole genome sequencing data of MPN patients were retrieved and analyzed. Genomic aberration of protein-coding and non-coding (lncRNA and miRNA) genes were identified in the dataset. All potential complementary binding sites of miRNA-lncRNA and miRNA-mRNA interactions were predicted by STarMir. Results A total of 13 DElncRNAs (eight from RNA-seq and five from published PCR-array dataset) and 109 DEmRNAs (from RNA-seq) were shown to be differentially expressed in HEL cells upon RUX treatment. Six of the 13 DElncRNAs (BANCR, LINC00261, LINC00887, NBR2, MIR646HG, and RP11-867O8.5) were selected after subsequent qPCR validation. All six selected DElncRNAs (two up-regulated and four down-regulated) were partially localized in the cytoplasm with relative abundance ranging from 38.5% to 53.2%, which were measured by DDPCR. Eight up-regulated DEmiRNAs (hsa-miR-1244, hsa-miR-1246, hsa-miR1248, hsa-miR-1290, hsa-miR-3609, hsa-miR-3653-3p, hsa-miR-3654, and hsa-miR-3916) and two down-regulated DEmiRNAs (hsa-miR-222-5p and hsa-miR-223-5p) were retrieved from Pagano’s dataset. In the whole genome sequencing (WGS) dataset analysis, two amplified and 31 deleted lncRNA genes, seven amplified and 14 deleted miRNA genes, and nine amplified and 149 deleted protein-coding (PC) genes were identified. The potential RNA-RNA interactions were predicted by STarMir. All eight up-regulated DEmiRNAs were predicted to interact with the four down-regulated DElncRNAs (BANCR, LINC00261, LINC00887, and MIR646HG). No interaction was predicted between all the down-regulated DEmiRNAs and up-regulated DElncRNAs. 73 out of 79 down-regulated DEmRNAs were predicted to interact with eight up-regulated DEmiRNAs. Four putative ceRNA networks were built for the four down-regulated DElncRNAs by combining the predicted miRNA-lncRNA and miRNA-mRNA interactions. Two ceRNA networks formed by LINC0021 and LINC00887 become the biggest and most complex networks among the rest. The results of GO analysis of PC genes cluster of ceRNA networks showed that genes are enriched in the plasma membrane, cytokine receptor activities, and cell surface receptor signaling, which greatly differ from the results of WGS gene clusters. Conclusion In this study, we explored the interactions among RNAs mediated by the aberrant JAK2V617F mutation. The relative abundance of cytoplasmic lncRNAs indicated that they might have functions and activities in the cytoplasm. The in-silico analysis revealed that four lncRNAs, eight miRNAs, and 73 mRNAs could potentially form interaction networks which uncovered the regulatory potential of the lncRNAs. Although we have not revealed the involved mechanism, the findings may provide insight into the function of lncRNA associated with JAK2 mutation. |
| Rights: | All rights reserved |
| Access: | restricted access |
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| File | Description | Size | Format | |
|---|---|---|---|---|
| 7291.pdf | For All Users (off-campus access for PolyU Staff & Students only) | 3.6 MB | Adobe PDF | View/Open |
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