Functional Study Of Zebrafish MiR-462-731 Cluster In Cellular Adaptation To Hypoxia And Embryonic Hematopoiesis

Hypoxia,or low oxygen tension,is a unique and essential environmental stress that not noly represents the insufficient oxygen supply to cells an tissues but also occurs during a range of physiologic and pathophysiogical circumstances,including development,tissue ischemia and tumor growth.Hypoxic condition in aquatic environments is a great challenge for the development and survival of fish.Aerobic orgnisms and cells have developed complex and highly coordinated adaptive mechanisms to survive hypoxic stress.The evolutionarily conserved Hypoxia-inducible factor(HIF)signaling pathway has been recognized as a core pathway in the cellular adaptive response to hypoxic stress.MicroRNAs(miRNAs),a major class of posttranscriptional gene expression regulators,also play pivotal roles in orchestrating hypoxia-mediated cellular adaptations.On the other hand,oxygen transport in living organisms largely depends on blood circulation.MiRNAs also play significant roles in both embryonic hematopoiesis and hematological malignancy.In the present study,we identified the hypoxia-regulated miRNAs in zebrafish by the next-generation sequencing.By performing endogenous overexpression or knockdown,combined with RNA-seq analysis,we further investigated the crucial roles of teleost-specific miR-462-731 in cellular adaptations to hypoxic stress,as well as its poteintial functions during embryonic hematopoiesis,which might provide further insights into the complex adaptive mechanisms to hypoxic stress in vertebrate.The major results are as follows:1.By global miRNA expression profiling,a total of 10 potential hypoxia-regulated miRNAs(HRMs)entified,among which miR-181a and miR-150 were previously recognized as HRMs in sevearal mammalian cell lines.But,functions of the others in hypoxic responses,including teleost-specific miR-462-731,have not been reported.2.Analysis of promoter activity demonstrated that Hif-1? activates miR-462-731 transcription by binding the upstream hypoxia response element(HRE),and regulates the expression of endogenous miR-462-731 both in ZF4 cells and zebrafish embryos.Baseed on dual luciferase reporter assay and Western blot analysis,the present study confirmed that miR-462 and miR-731 target DEAD box helicase 5(ddx5)and protein phosphatase,Mg2+/Mn2+ dependent,1 Da(ppm1da)respectively.Also,miR-462-731 may induce the cell cycle arrest and repress cell proliferation by negative regulating ddx5 and ppm1da.Therefore,we suggest that miR-462-731 acts downstream of Hif-1? in the hypoxia signaling pathway and function in celluar adaptation to hypoxia via targeting ddx5 and ppm1da.3.Whole-mount in situ hybridization and Semi-qRT-PCR revealed that zebrafish miR-462-731 cluster is expressed in a consistent and ubiquitous manner in the whole embryo throughout the early developmental stages.At 48 hpf,the cluster became enriched in the brain,eye,somatic muscle and fin.From 3 dpf to 5 dpf,the signal became more prominent in the liver and digestive tract.The expression of zebrafish miR-462-731 showed no obvious tissue-specificity in adaults,with relatively higher expression detected in swim bladder,eye,gut,fin and gill.Knockdown of miR-462/miR-731 resulted in developmental defects,with related genes detected differentially expressed by RNA-Seq.In addition,significant apoptosis in brain,eye,tail and pericardium were detected in morphants.These data indicates that miR-462-731 is required for normal embryonic development and organ formation at least in part by regulating cell survival.4.Furthermore,miR-462/miR-731 morphants developed with a striking deficit of erythroid cell and blood circulation,while an ectopic myeloid expansion,along with dysregulation of a set of hematopoiesis-related.MiR-462/miR-731 morphants showed an an increased expression of hemangioblast markers etsrp and scl in the ALPM,and an enhanced specification of pu.1-expressing myeloid progenitors.It suggests that miR-462-731 acts upstream of etsrp to regulate ALPM myelopoiesis before the myeloid commitment driven by pu.1.On the other hand,we confirmed that Pu.1 activates miR-462-731 transcription by binding a teleost-conserved responsive element in its promoter,and miR-731 in turn negatively regulates pu.1.These results characterize a previously unrecognized pu.1/miR-462-731 regulatory feed back loop which may play a role in hematopoietic lineage differentiation and influence the interplay between pu.1 and gata1.5.We further performed integrated KEGG pathway enrichment analysis of putative targets of miR-462/miR-731 and differentially expressed genes in miR-462/miR-731 morpants.It was revealed that miR-462 and miR-731 may coordinatly regulate important signaling pathways,including TGF-? signaling,by targeting different genes.An apparent mild ventralization,characterized by posterior ICM expansion,was detected in miR-462/miR-731 morphants.Molecular analysis revealed an induced expression of P-Smad1/5 and alk8 as early as 70%-epiboly stage.The ICM expansion in miR-462/miR-731 morphants represents an enhanced hematopoietic specification of mesodermal progenitors that resulted from elevation of BMP signaling.Our findings from rescue experiments showed that miR-462-731 might act upstream of alk8 within the Bmp2b/7 signaling pathway and function as a novel endogenous BMP antagonist.6.Besides,miR-462-731 may regulate intersegmental vessles(ISV)formation and arteriovenous programming.Meanwhile,putative targets of miR-462-731 significantly enriched in Notch signaling pathway.Therefore,we deduce that miR-462-731 is required for Notch regulation of vescular development and may consequently affect blood circulation and downstream hematopoietic stem cell(HSC)production.However,further researches are needed to clarify the detailed regulatory role of miR-462-731 within Notch signaling.