PB0355 - Knockdown screening of microRNAs in thrombocytes and their role in thrombopoiesis

Background: Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, there is limited information on their role in megakaryocyte maturation. We hypothesize that zebrafish thrombocytes could be used as a model to study their role in megakaryocyte maturation because thrombocytes have megakaryocyte features. Recently, we have identified 15 microRNAs in thrombocytes using single-cell RNA sequencing. Investigating their role in thrombocyte maturation should provide insight into thrombocyte development.

Aims: To knockdown microRNAs found in zebrafish thrombocytes by piggyback knockdown method and understand their role in thrombocyte maturation.

Methods: Piggyback knockdown ASO/VMO hybrids of each microRNA were injected into the adult fish. Blood was collected 48 hours post-injection, and the thrombocytes were labeled by DiI-C18 and quantified by flow cytometry. Likewise, we performed knockdowns in larvae and measured TTO using an arterial thrombosis assay.

Results: To evaluate the role of microRNAs in zebrafish thrombocyte maturation, we performed a knockdown of each of the 15 microRNAs and quantified the number of thrombocytes. We found that knockdown of mir-223, let-7b, and mir-7148 increased total thrombocyte percentage. We then gated young, more mature, and fully mature thrombocyte populations according to the intensity of DiI and quantified these populations. The results showed that more mature and fully mature populations increased whereas the ratio of young to more mature populations decreased. We also confirmed the increase in total thrombocytes by knockdowns in larvae using TTOs.

Conclusion(s): We found knockdown of mir-223, let-7b, and mir-7148 led to an increase in the percentage of the total, fully mature, and more mature thrombocytes, suggesting that mir-223, let-7b, and mir-7148 are repressors for thrombocyte maturation. This information should be useful in understanding downstream targets for the microRNAs that ultimately could lead to novel therapeutic targets to treat thrombocytopenia.