Role of microRNAs and their downstream target transcription factors in zebrafish thrombopoiesis
Abstract Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, a comprehensive study on the microRNAs and their targets has not been undertaken. Zebrafish thrombocytes could...
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| Format: | Article |
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Nature Portfolio
2023-09-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-42868-7 |
| _version_ | 1797452655738486784 |
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| author | Ayah Al Qaryoute Weam Fallatah Sanchi Dhinoja Revathi Raman Pudur Jagadeeswaran |
| author_facet | Ayah Al Qaryoute Weam Fallatah Sanchi Dhinoja Revathi Raman Pudur Jagadeeswaran |
| author_sort | Ayah Al Qaryoute |
| collection | DOAJ |
| description | Abstract Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, a comprehensive study on the microRNAs and their targets has not been undertaken. Zebrafish thrombocytes could be used as a model to study their role in megakaryocyte maturation and platelet function because thrombocytes have both megakaryocyte features and platelet properties. In our laboratory, we identified 15 microRNAs in thrombocytes using single-cell RNA sequencing. We knocked down each of these 15 microRNAs by the piggyback method and found knockdown of three microRNAs, mir-7148, let-7b, and mir-223 in adult zebrafish led to an increase in the percentage of thrombocytes. Functional thrombocyte analysis using plate tilt assay showed no modulatory effect of the three microRNAs on thrombocyte aggregation/agglutination. We also found enhanced thrombosis using arterial laser thrombosis assay in a group of zebrafish larvae after mir-7148, let-7b, and mir-223 knockdowns. These results suggested mir-7148, let-7b, and mir-223 are repressors for thrombocyte production. We then explored miRWalk database for let-7b downstream targets and then selected those that are expressed in thrombocytes, and from this list based on their role in differentiation selected 14 genes, rorca, tgif1, rfx1a, deaf1, zbtb18, mafba, cebpa, spi1a, spi1b, fhl3b, ikzf1, irf5, irf8, and lbx1b that encode transcriptional regulators. The qRT-PCR analysis of expression levels of the above genes following let-7b knockdown showed changes in the expression of 13 targets. We then studied the effect of the 13 targets on thrombocyte production and identified 5 genes, irf5, tgif1, irf8, cebpa, and rorca that showed thrombocytosis and one gene, ikzf1 that showed thrombocytopenia. Furthermore, we tested whether mir-223 regulates any of the above 13 transcription factors after mir-223 knockdown using qRT-PCR. Six of the 13 genes showed similar gene expression as observed with let-7b knockdown and 7 genes showed opposing results. Thus, our results suggested a possible regulatory network in common with both let-7b and mir-223. We also identified that tgif1, cebpa, ikzf1, irf5, irf8, and ikzf1 play a role in thrombopoiesis. Since the ikzf1 gene showed a differential expression profile in let-7b and mir-223 knockdowns but resulted in thrombocytopenia in ikzf1 knockdown in both adults and larvae we also studied an ikzf1 mutant and showed the mutant had thrombocytopenia. Taken together, these studies showed that thrombopoiesis is controlled by a network of transcription regulators that are regulated by multiple microRNAs in both positive and negative manner resulting in overall inhibition of thrombopoiesis. |
| first_indexed | 2024-03-09T15:11:47Z |
| format | Article |
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| institution | Directory Open Access Journal |
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| language | English |
| last_indexed | 2024-03-09T15:11:47Z |
| publishDate | 2023-09-01 |
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| spelling | doaj.art-7085578e49574ff680ea52073cc24b892023-11-26T13:18:54ZengNature PortfolioScientific Reports2045-23222023-09-0113111610.1038/s41598-023-42868-7Role of microRNAs and their downstream target transcription factors in zebrafish thrombopoiesisAyah Al Qaryoute0Weam Fallatah1Sanchi Dhinoja2Revathi Raman3Pudur Jagadeeswaran4Department of Biological Sciences, University of North TexasDepartment of Biological Sciences, University of North TexasDepartment of Biological Sciences, University of North TexasDepartment of Biological Sciences, University of North TexasDepartment of Biological Sciences, University of North TexasAbstract Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, a comprehensive study on the microRNAs and their targets has not been undertaken. Zebrafish thrombocytes could be used as a model to study their role in megakaryocyte maturation and platelet function because thrombocytes have both megakaryocyte features and platelet properties. In our laboratory, we identified 15 microRNAs in thrombocytes using single-cell RNA sequencing. We knocked down each of these 15 microRNAs by the piggyback method and found knockdown of three microRNAs, mir-7148, let-7b, and mir-223 in adult zebrafish led to an increase in the percentage of thrombocytes. Functional thrombocyte analysis using plate tilt assay showed no modulatory effect of the three microRNAs on thrombocyte aggregation/agglutination. We also found enhanced thrombosis using arterial laser thrombosis assay in a group of zebrafish larvae after mir-7148, let-7b, and mir-223 knockdowns. These results suggested mir-7148, let-7b, and mir-223 are repressors for thrombocyte production. We then explored miRWalk database for let-7b downstream targets and then selected those that are expressed in thrombocytes, and from this list based on their role in differentiation selected 14 genes, rorca, tgif1, rfx1a, deaf1, zbtb18, mafba, cebpa, spi1a, spi1b, fhl3b, ikzf1, irf5, irf8, and lbx1b that encode transcriptional regulators. The qRT-PCR analysis of expression levels of the above genes following let-7b knockdown showed changes in the expression of 13 targets. We then studied the effect of the 13 targets on thrombocyte production and identified 5 genes, irf5, tgif1, irf8, cebpa, and rorca that showed thrombocytosis and one gene, ikzf1 that showed thrombocytopenia. Furthermore, we tested whether mir-223 regulates any of the above 13 transcription factors after mir-223 knockdown using qRT-PCR. Six of the 13 genes showed similar gene expression as observed with let-7b knockdown and 7 genes showed opposing results. Thus, our results suggested a possible regulatory network in common with both let-7b and mir-223. We also identified that tgif1, cebpa, ikzf1, irf5, irf8, and ikzf1 play a role in thrombopoiesis. Since the ikzf1 gene showed a differential expression profile in let-7b and mir-223 knockdowns but resulted in thrombocytopenia in ikzf1 knockdown in both adults and larvae we also studied an ikzf1 mutant and showed the mutant had thrombocytopenia. Taken together, these studies showed that thrombopoiesis is controlled by a network of transcription regulators that are regulated by multiple microRNAs in both positive and negative manner resulting in overall inhibition of thrombopoiesis.https://doi.org/10.1038/s41598-023-42868-7 |
| spellingShingle | Ayah Al Qaryoute Weam Fallatah Sanchi Dhinoja Revathi Raman Pudur Jagadeeswaran Role of microRNAs and their downstream target transcription factors in zebrafish thrombopoiesis Scientific Reports |
| title | Role of microRNAs and their downstream target transcription factors in zebrafish thrombopoiesis |
| title_full | Role of microRNAs and their downstream target transcription factors in zebrafish thrombopoiesis |
| title_fullStr | Role of microRNAs and their downstream target transcription factors in zebrafish thrombopoiesis |
| title_full_unstemmed | Role of microRNAs and their downstream target transcription factors in zebrafish thrombopoiesis |
| title_short | Role of microRNAs and their downstream target transcription factors in zebrafish thrombopoiesis |
| title_sort | role of micrornas and their downstream target transcription factors in zebrafish thrombopoiesis |
| url | https://doi.org/10.1038/s41598-023-42868-7 |
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