title: Zebrafish let-7b acts downstream of hypoxia-inducible factor-1α to assist in hypoxia-mediated cell proliferation and cell cycle regulation.
abstract: OBJECTIVE
  Hypoxia-inducible factor-1α (HIF-1α) is a transcriptional regulator of cellular responses to hypoxic stress. MicroRNAs (miRNAs) play an essential role in hypoxia-mediated cellular responses. Previous studies have identified some let-7 family members as hypoxia-regulated miRNAs (HRMs). In the present study, we aimed to investigate whether zebrafish let-7b/7f contribute cellular hypoxic response in a Hif-1α-dependent manner.
  
  
  METHODS
  Stable suppression of zebrafish hif-1α was achieved by microinjection of an optimized short-hairpin RNA (shRNA) expression vector. Next-generation sequencing was conducted to characterize miRNA and mRNA expression profiles. MiRNA promoter analysis and target detection was performed by dual-luciferase assay. Quantitative real-time PCR (qRT-PCR) and western blot were used to determine the expression of let-7b/7f, Hif-1α and Foxh1. Proliferation of ZF4 cells was examined using Cell Counting Kit-8 (CCK-8) and cell cycle progression was analyzed by flow cytometry assay.
  
  
  RESULTS
  Correlation between 7 miRNAs and 76 putative targets was identified based on integrated analysis of miRNA-mRNA profiles. Let-7b and let-7f were further considered as potential HRMs, with let-7b further validated as Hif-1α up-regulated. In addition, Forkhead-box H1 (Foxh1) was confirmed as a bona fide downstream target of let-7b. Furthermore, overexpression of both let-7b and let-7f repressed cell proliferation through blocking cell cycle progression of the G1-S transition.
  
  
  CONCLUSIONS
  Our findings for the first time suggest zebrafish let-7b acts downstream of Hif-1α to assist in hypoxia-mediated cell proliferation and cell cycle regulation at least in part through the downregulation of foxh1. We also identified 4 novel potential HIF-1α-regulated miRNAs in zebrafish.
authors.affiliation: Key Lab of Freshwater Animal Breeding, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Fishery, Huazhong Agricultural University, Wuhan, China.
authors.forename: Chun-Xiao
authors.initials: CX
authors.lastname: Huang
authors.affiliation: Key Lab of Freshwater Animal Breeding, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Fishery, Huazhong Agricultural University, Wuhan, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Chen
authors.affiliation: Key Lab of Freshwater Animal Breeding, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Fishery, Huazhong Agricultural University, Wuhan, China.
authors.forename: Xin-Jie
authors.initials: XJ
authors.lastname: Wu
authors.affiliation: Key Lab of Freshwater Animal Breeding, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Fishery, Huazhong Agricultural University, Wuhan, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: He
authors.affiliation: Key Lab of Freshwater Animal Breeding, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Fishery, Huazhong Agricultural University, Wuhan, China.
authors.forename: Cui-Hong
authors.initials: CH
authors.lastname: Huang
authors.affiliation: Key Lab of Freshwater Animal Breeding, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Fishery, Huazhong Agricultural University, Wuhan, China; Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.affiliation: Key Lab of Freshwater Animal Breeding, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Fishery, Huazhong Agricultural University, Wuhan, China.
authors.forename: Wei-Min
authors.initials: WM
authors.lastname: Wang
authors.affiliation: Key Lab of Freshwater Animal Breeding, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Fishery, Huazhong Agricultural University, Wuhan, China; Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan, China. Electronic address: hbauwhl@hotmail.com.
authors.forename: Huan-Ling
authors.initials: HL
authors.lastname: Wang
mesh_terms: D000818:Animals; D002453:Cell Cycle / Q000502:physiology*; D049109:Cell Proliferation / Q000502:physiology*; D020868:Gene Silencing; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000502:physiology*; D035683:MicroRNAs / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D015027:Zebrafish
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D035683:MicroRNAs; D012333:RNA, Messenger
keywords: Cell cycle; Hif-1α; Let-7b/7f; Proliferation; foxh1; miRNA profile; shRNA