Qaryoute Ayah Al, Fallatah Weam, Dhinoja Sanchi, Raman Revathi, Jagadeeswaran Pudur
University of North Texas.
Res Sq. 2023 Apr 24:rs.3.rs-2807790. doi: 10.21203/rs.3.rs-2807790/v1.
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, , and 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 , and knockdowns. These results suggested , and are repressors for thrombocyte production. We then explored miRWalk database for downstream targets and then selected those that are expressed in thrombocytes, and from this list based on their role in differentiation selected 14 genes, , and that encode transcriptional regulators. The qRT-PCR analysis of expression levels of the above genes following 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, , and that showed thrombocytosis and one gene, that showed thrombocytopenia. Furthermore, we tested whether regulates any of the above 13 transcription factors after knockdown using qRT-PCR. Six of the 13 genes showed similar gene expression as observed with knockdown and 7 genes showed opposing results. Thus, our results suggested a possible regulatory network in common with both and . We also identified that , , and play a role in thrombopoiesis. Since the gene showed a differential expression profile in and knockdowns but resulted in thrombocytopenia in knockdown in both adults and larvae we also studied an 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.
先前的研究表明，人类血小板和巨核细胞携带微小RNA，分别提示它们在血小板功能和巨核细胞发育中的作用。然而，尚未对微小RNA及其靶标进行全面研究。斑马鱼血小板可作为研究它们在巨核细胞成熟和血小板功能中作用的模型，因为血小板兼具巨核细胞特征和血小板特性。在我们实验室，我们使用单细胞RNA测序在血小板中鉴定出15种微小RNA。我们通过搭载方法敲低这15种微小RNA中的每一种，发现敲低成年斑马鱼中的三种微小RNA，即 、 和 ，会导致血小板百分比增加。使用平板倾斜试验进行的功能性血小板分析表明，这三种微小RNA对血小板聚集/凝集没有调节作用。我们还发现在敲低 、 和 后，一组斑马鱼幼虫通过动脉激光血栓形成试验显示血栓形成增强。这些结果表明 、 和 是血小板生成的抑制因子。然后我们在miRWalk数据库中探索 下游靶标，然后选择那些在血小板中表达的靶标，并基于它们在分化中的作用从该列表中选择14个基因，即 、 和 ，它们编码转录调节因子。在敲低 后对上述基因表达水平进行的qRT-PCR分析显示13个靶标的表达发生了变化。然后我们研究了这13个靶标对血小板生成的影响，鉴定出5个基因，即 、 和 表现为血小板增多，以及1个基因 表现为血小板减少。此外，我们使用qRT-PCR测试了在敲低 后 是否调节上述13种转录因子中的任何一种。13个基因中的6个显示出与敲低 时观察到的相似基因表达，7个基因显示出相反的结果。因此，我们的结果提示了一个可能与 和 共同的调控网络。我们还确定 、 和 在血小板生成中起作用。由于 基因在敲低 和 时显示出差异表达谱，但在成年和幼虫敲低 时均导致血小板减少，我们还研究了一个 突变体，发现该突变体有血小板减少。综上所述，这些研究表明血小板生成受转录调节因子网络控制，这些转录调节因子受到多种微小RNA以正负两种方式的调节，从而导致血小板生成的整体抑制。
