The Functions Of Zebrafish MiR-125c In Cellular Hypoxia Response And Embryogenesis

The effects of hypoxia on fish including less food intake,slow growth and embryonic development and even death,therefore,dissolved oxygen in water is an important environmental factor affecting fish survival and evolution.Hypoxia-inducible factor HIF-1? is a crucial transcription factor in organism response to hypoxic stress and can controls the cellular responses to hypoxia by targeting a number of downstream genes.In recent years,it has been discovered that microRNAs?miRNAs?are non-coding small RNAs and occupy a crucial role in cell survival under low-oxygen environment.Previous evidences suggested that miR-125 c was induced in hypoxia,but its precise biological roles and the regulatory mechanism underlying hypoxia responses remained unknown.In this study,the HRE?Hypoxia response element?motifs upstream of miR-125 c were constructed into PGL3-HRE vector and the regulatory relationship between Hif-1? and miR-125 c was analyzed under hypoxia.The target of miR-125 c was verified both in vivo and in vitro.In addition,the functions of miR-125 c in cell proliferation,cell cycle,cell apoptosis and zebrafish embryogenesis were proved.The main results were as follows:1)The sequence analysis of miR-125 familyThe phylogenetic analysis revealed high homology of mi R-125 a and mi R-125 b among various teleost fishes,human and mouse and suggested that they had functional similarities.Then we characterized the mature sequence conservation of the miR-125 family in zebrafish,medaka,human and mouse by multiple sequence alignment which revealed that the seed sequences of miR-125a/b/c were fully conserved.2)Zebrafish miR-125 c is transcriptionally induced by Hif-1?Zebrafish miR-125 c was significantly induced in a time-dependent manner in response to hypoxia-simulating treatment with CoCl₂ in ZF4 cells.The promoter analysis revealed that four HRE binding sites were found in the promoter of miR-125 c.Then the four pGL3-Basic recombinants including four HREs were constructed and transfected HeLa cells,luciferase reporter assay showed that hypoxia markedly enhanced the activity of these recombinants only containing HRE1 and HRE3,indicating that HRE1 and HRE3 may play important roles in hypoxia response.Hif-1? knockdown by its siRNA was performed in ZF4 cells under hypoxic condition?CoCl₂ treatment for 12 h?,and Western blot and qRT-PCR showed that accumulation of endogenous Hif-1? was effectively inhibited,and the expression level of miR-125 c was significantly decreased.All these demonstrated that miR-125 c may be transcriptionally induced by Hif-1?.3)MiR-125 c directly targets cdc25aBioinformatics analysis revealed that zebrafish cdc25a-3'UTR contained the miR-125 c binding site.The assay in vitro showed that the luciferase activity was significantly reduced when psiCHECK?-2-cdc25a-3'UTR?wt?and miR-125 c mimics were co-transfected into HaLa cells,whereas the activity was not changed when co-transfection psiCHECK?-2-cdc25a-3'UTR?mt?and miR-125 c mimics.All these results indicated that miR-125 c specifically combined with cdc25 a 3'UTR.4)MiR-125 c negatively regulates the cdc25 a expressionIn vitro experiment showed that the cdc25 a mRNA and protein levels were clearly decreased by miR-125 c overexpression,but increased by miR-125 c inhibitor.Zebrafish embryos microinjected with mi R-125 c mimics showed ectopic expression of miR-125 c resulted in a dramatic reduction of both cdc25 a mRNA and protein levels.Furthermore,the endogenous cdc25 a mRNA and protein levels were significantly down-regulated in a time-dependent manner in response to hypoxia-simulating treatment with CoCl₂ in ZF4 cells.Therefore,mi R-125 c negatively regulated the cdc25 a expression both in vivo and in vitro.5)MiR-125 c represses cell proliferation through cell-cycle arresting and induces apoptosisCell Counting Kit-8?CCK8?assays were performed to assess the effect of miR-125 c on cell proliferation.The result showed that miR-125 c significantly suppressed the proliferation of ZF4 cells from 36 h to 60 h.The flow cytometry assay revealed that miR-125 c significantly blocked the cell cycle progression at G1 phase,induced the canonical G1/S transition regulator Cdk2 as well as G1 phase regulatory genes cdk2,cdk6,ccnd1,and decreased the expression of S phase regulatory genes cdk7 and ccnh mRNA levels.However,these genes showed contrary mRNA levels by the miR-125 c inhibitor.In addition,it was also found that ZF4 cells transfected with mi R-125 c resulted in a significant increase of the apoptosis level.Accordingly,the results of acridine orange showed that miR-125 c overexpression gave rise to a significant increase of apoptotic cells in brain,eyes and tail of zebrafish.In addition,cell apoptosis in tail was further confirmed by TUNEL assay,which could also be rescued by the miR-125 c inhibitor.6)The functions of miR-125 c during zebrafish embryogenesisIt was revealed that miR-125 c overexpression resulted in pericardial edema,severely impaired head with concomitant reduction of eyes,and abnormally curved tail.Touch-evoked Escape Behavior Assay showed that mechanosensory stimulating the tail provoked the wild-type larvae to swim out of the field of view rapidly,but miR-125 c injected larvae swam slowly in circular motions.Additionaily,histological examination revealed that the lens appeared smaller,the inner plexiform layer?IPL?in eyes became thinner and the cells in inner nuclear layers?INL?seemed clumped and irregularly arranged in mi R-125 c overexpressed larvae.Besides,miR-125 c overexpression resulted in a reduction of brain size with tightly clumped cells,smaller myotome and degenerated muscle.