Chun-Xiao Huang1, Nan Chen1, Xin-Jie Wu1, Yan He1, Cui-Hong Huang1, Hong Liu2, Wei-Min Wang1, Huan-Ling Wang3. 1. 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. 2. 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. 3. 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.
Abstract
AIMS: 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. MAIN 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. KEY FINDINGS: 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. SIGNIFICANCE: 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.
AIMS: 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 zebrafishlet-7b/7f contribute cellular hypoxic response in a Hif-1α-dependent manner. MAIN 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. KEY FINDINGS: 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. SIGNIFICANCE: Our findings for the first time suggest zebrafishlet-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: Virginia Egea; Kai Kessenbrock; Devon Lawson; Alexander Bartelt; Christian Weber; Christian Ries Journal: Cell Death Dis Date: 2021-05-20 Impact factor: 8.469
Authors: Estefania Lozano-Velasco; Carlos Garcia-Padilla; Maria Del Mar Muñoz-Gallardo; Francisco Jose Martinez-Amaro; Sheila Caño-Carrillo; Juan Manuel Castillo-Casas; Cristina Sanchez-Fernandez; Amelia E Aranega; Diego Franco Journal: Int J Mol Sci Date: 2022-03-04 Impact factor: 5.923